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GENETIC DIVERSITY ANALYSIS OF DATE PALM (Phoenix
dactylifera L.) CULTIVARS OF PAKISTAN
BY
NADIA
A dissertation submitted to The University of Agriculture Peshawar in partial
fulfillment of the requirement for the degree of
DOCTOR OF PHILOSOPHY
(PLANT GENOMICS AND BIOTECHNOLOGY)
DEPARTMENT OF PLANT GENOMICS AND BIOTECHNOLOGY
PARC INSTITUTE OF ADVANCED STUDIES IN AGRICULTURE
NATIONAL AGRICULTURAL RESEARCH CENTRE, ISLAMABAD
THE UNIVERSITY OF AGRICULTURE, PESHAWAR, PAKISTAN
NOVEMBER, 2016
GENETIC DIVERSITY ANALYSIS OF DATE PALM (Phoenix
dactylifera L.) CULTIVARS OF PAKISTAN
BY
NADIA
A dissertation submitted to The University of Agriculture Peshawar in partial
fulfillment of the requirement for the degree of
DOCTOR OF PHILOSOPHY IN
(PLANT GENOMICS AND BIOTECHNOLOGY)
Approved by:
Dr. Aish Muhammad (Principal Scientific Officer)
Chairman Supervisory Committee
Dr. Muhammad Zeeshan Hyder (Assistant Professor)
Co-Supervisor
Dr. Ghulam Muhammad Ali (Chief Scientific Officer)
Member
Dr. Armghan Shehzad (Senior Scientific Officer)
Member
Prof. Dr. Hafeez Ur Rahman (Principal Scientific Officer)
Member
Dr. Ghulam Muhammad Ali (Chief Scientific Officer)
Chairman & Convener Board of Studies
Dr. Zahir Shah (Professor)
Rector/Dean Faculty of Crop Production
Prof. Dr. Khalid Nawab (Professor)
Director Advanced Studies & Research
DEPARTMENT OF PLANT GENOMICS AND BIOTECHNOLOGY
PARC INSTITUTE OF ADVANCED STUDIES IN AGRICULTURE
NATIONAL AGRICULTURAL RESEARCH CENTRE, ISLAMABAD
THE UNIVERSITY OF AGRICULTURE, PESHAWAR, PAKISTAN
NOVEMBER, 2016
GENETIC DIVERSITY ANALYSIS OF DATE PALM (Phoenix
dactylifera L.) CULTIVARS OF PAKISTAN
BY
NADIA
Approved by:
External Examiner 1: Prof. Dr. Chaoying He
State Key Laboratory of
Systematic and Evolutionary
Botany Institute of Botany,
The Chinese Academy of
Sciences
External Examiner 2: Aureliano Bombarely
Assistant Professor
Department of Horticulture
Virginia Tech
Latham Hall, 216
Blacksburg, VA, USA
Dedicated to my parents
TABLE OF CONTENTS
Chapter No. Title Page No.
List of Tables i
List of Figures ii
List of Appendices iii
Abbreviations iv
Acknowledgements viii
Abstract ix
I Introduction 1
II Review of Literature 9
2.1 Date palm diversity evaluation based on morphological
parameters
9
2.2 Date palm diversity evaluation based on proximate
composition of fruit
11
2.3 Date palm diversity evaluation based on simple sequence
repeat (SSR) markers
14
2.4 Date palm diversity evaluation based on other molecular
markers
19
2.5 Date palm diversity evaluation based on both
morphological and molecular markers
22
2.6 Date palm diversity evaluation based on chloroplast DNA
sequences
23
2.7 Date palm diversity evaluation based on single nucleotide
polymorphism (SNPs)
24
III Material and Methods 29
3.1 Morphological characterization 29
3.2 Proximate composition determination 31
3.2.1 Sample preparation 31
3.2.2 % moisture determination 31
3.2.3 % Ash determination 32
3.2.4 Total soluble solids (TSS) determination 32
3.2.5 Sugar content determination 33
3.2.5.1 Extraction of Sugars 33
3.2.5.2 % Reducing sugar determination 33
3.2.5.3 % Total sugar determination 33
3.3 Morphological and proximate composition analysis 34
3.4 Molecular diversity analysis 34
3.4.1 Sampling and DNA isolation 34
3.4.2 DNA quantification 35
3.4.3 Primer design / Selection 35
3.5 PCR based molecular diversity analysis 38
3.5.1 Amplification of simple sequence repeats (SSR) 38
3.5.2 Gel electrophoresis 38
3.5.2.1 Agarose gel electrophoresis 38
3.5.2.2 Polyacrylamide gel electrophoresis 38
3.5.3 SSR data analysis 39
3.6 Sequence based molecular analysis 39
3.6.1 Amplification of chloroplast DNA and SNPs 39
3.6.2 Gel electrophoresis, PCR product purification and
sequencing
40
3.6.3 Sequence based molecular analysis of chloroplast DNA 41
3.6.4 SNP typing 41
3.6.5 Submission of sequences 41
IV Results 42
4.1 Morphological and proximate analysis 42
4.2 Principal Component Analysis 45
4.3 Correlation 47
4.4 Dendrogram based on moprhologcal and proximate data 50
4.5 PCR based molecular analysis 52
4.6 Sequence based molecular analysis 55
4.6.1 Amplification of chloroplast DNA 55
4.6.2 SNP typing 56
V Discussion 64
5.1 Morphological evaluation 65
5.2 Proximate composition analysis 67
5.3 Molecular analysis 69
5.3.1 Microsatellite markers 70
5.3.2 Molecular analysis based on sequencing 72
5.3.2.1 Chloroplast DNA analysis 72
5.3.2.2 Single Nucleotide Polymorphism detection 74
VI Summary, Conclusions and Recommendations 79
References 85
Appendices 103
i
LIST OF TABLES
Table No. Title Page No.
3.1 List of Date palm cultivars from Pakistan used in this
study with their codes and sampling sites
29
3.2 Simple sequence repeat markers used in this study
showing amplification status
36
4.1 Descriptive statistics of traits of date palm cultivars
studied
43
4.2 Principal components showing proportion of variability
among date palm cultivars
46
4.3 Correlation among the variables studied and the first
seven components
48
4.4 Correlation matrix of the traits studied 49
4.5 Sequence and details of primers for chloroplast gene/gene
fragments
57
4.6 Sequence and details of primers for amplification of SNPs 58
4.7 Accession numbers of sequences of different
genes/fragments of date palm submitted to Gen Bank
59
4.8 Origin, details and Sequence of SNPs 60
ii
LIST OF FIGURES
Figure No. Title Page No.
1.1 Map of Pakistan showing date palm producing areas 1
3.1 Diagram of date palm leaf showing various traits measured 30
4.1 Dendrogram of 45 date palm cultivars studied based on
morphological and proximate composition data 51
4.2 PCR amplification of mpdCIRO25 in45 date palm cultivars
scored on 2% agarose gel
52
4.3 PCR amplification of mpdCIRO85 in 45 date palm cultivars
scored on 2% agarose gel
53
4.4 Dendrogram of 45 date palm cultivars based on SSR data
54
4.5 Maximum Parsimony (MP) Tree based on SNP data from
various date palm cultivars. 62
4.6 SNPs system for varietal identification 63
iii
LIST OF APPENDECES
Appendix No. Description Page No.
Appendix-1 Pictures of date fruit of 45 cultivars studied 103
Appendix-2 Mean data of morphological traits and proximate
compostion of date fruit
111
Appendix-3 10X TAE Stock Solution (1L) 113
Appendix-4 Bromophenol Blue 113
Appendix-5 5X TBE Stock Solution (1L) 113
Appendix-6 45 % acrylamide: Bisacrylamide Solution (100mL) 113
Appendix-7 10% Ammonium per Sulphate (1mL) 113
Appendix-8 Sequences from date palm chloroplast ribulose -1, 5-
biphosphate carboxylase large subunit (rbcL) partial
gene submitted to the GenBank
114
Appendix-9 Sequences from date palm chloroplast Atp synthase beta
subunit (atpB) partial gene submitted to the GenBank
117
Appendix-10 Sequences from date palm chloroplast geranyl geranyl
diphosphate reductase (GGR) partial gene submitted to
the GenBank
120
Appendix-11 Sequences from date palm chloroplast maturase K
(matK) partial gene submitted to the GenBank
122
Appendix-12 Sequences from date palm chloroplast 16S ribosomal
RNA (16S rRNA) partial gene submitted to the GenBank
128
Appendix-13 Sequences from date palm genomic regions harboring
SNPs submitted to the GenBank
132
Appendix-14 Accession numbers of sequences of different genes/fragments
of date palm submitted to Genbank 155
iv
LIST OF ABBREVIATIONS
% percent
(NH4)2SO4 Ammonium Sulphate
A Adenine
AD anno Domini (in the year before Lord)
AFLP Amplified Fragment Length Polymorphism
ANOVA Analysis of Variance
AOAC Association of Official Analytical Chemists
atpB atp synthase Beta subunit
BC Before Christ
C Cytocine
cm centi meter
cm3 cubic centimeter
CTAB Cetyl trimethyl ammonium bromide
DNA Deoxyribonucleic acid
dNTPs Deoxyribo nucleoside triphosphate
EDTA Ethylene diamine tetraacetic Acid
EST Express Sequence Tag
et al et alia (Latin) meaning and others
Ft Feet
G Guanine
GGR Geranyl geranyl biphosphate reductase
v
gm Gram
ha hectare
HCl Hydrochloric Acid
hrs Hours
i.e id est(Latin) meaning that is
ID3 Iterative Dichotomizer 3
ISSR Inter Simple Sequence Repeats
KCl Potassium Chloride
kg kilo gram
LSD Least Significant Difference
M Molar
MAFFT Multiple Alignment using Fast Fourier Transform
mAmp milli ampere
matK maturase K
Mb Mega basepairs
MEGA Molecular Evolutionary Genetic Analysis
MgCl2 Magnesium Chloride
MI Marker Index
min minute
Ml marker index
ML Maximum Likelihood
mM micro mole
vi
MP Maximum Parsimony
NaCl Sodium Chloride
NaOH Sodium Hydroxide
NARC National Agriculture Research Centre
ng Nano gram
ng nano gram
NIGAB National Institute for Genomics and Advanced Biotechnology
nt nucleotide
oC centigrade
oF Fahrenheit
PARB Punjab Agriculture Research Board
PC Principal Component
PCR Polymerase Chain Reaction
PIC Polymorphic Information Content
pM Pico mole
RAMPO Random Amplification Microsatellite Polymorphism
RAPD random amplified polymorphic DNA
RAPD Randomly Amplified Polymorphic DNA
rbcL Ribulose biphosphate carboxylase Larger subunit
RFLP Restriction Fragment Length Polymorphism
s second
SNP Single Nucleotide Polymorphism
vii
SNPs Single Nucleotide Polymorphism
SSR Simple Sequence Repeat
T Thymine
TAE Tris-Acetic Acid-EDTA
TBE Tris Boric Acid T-EDTA
TE Tris-EDTA
TEMED (N,N,N',N'-tetramethylethylenediamine)
ug micro gram
uL micro liter
uM micro mole
uM miro mole
V Volts
w/v weight to volume ration
viii
ACKNOWLEDGEMENTS
All praise be to Almighty ALLAH for His blessings. Glory and praise to our
last Prophet MUHAMMAD (Peace Be Upon Him), who is a source of guidance
forever.
I wish to express my gratitude to my Supervisor Dr. Aish Muhammad PSO,
PGRI, NARC for his consistent guidance, encouragement and help during the entire
course of my doctoral study.
I am really grateful to my co Supervisor Dr. Muhammad Zeeshan Hyder,
Assistant Professor Department of Biosciences, COMSATS Institute of Information
Technology, Islamabad for his valuable guidance, cooperation and support throughout
my study period. I would like to thank other members of my supervisory committee,
Dr. Armghan Shehzad, PSO, NIGAB, NARC, for his guidance, encouragement and
help, Dr. Hafeez ur Rahman, PSO, HRI, NARC, for his valuable suggestions and
Dr. Ghulam Muhammad Ali, Head of department of Plant Genomics and
Biotechnology and CSO, NIGAB, NARC for his cooperation.
I am thankful to Mr. Muhshtaq Ahmad Director, HRS Bahawalpur, Dr.
Azhar Bashir Assistant Director, HRS Bahawalpur and Mr. Malik Fayaz Ahmad in
charge date palm research substation Jhang, for their cooperation in sampling and data
acquisition.
I acknowledge Higher Education Commission (HEC) Pakistan for providing
me financial support through Indigenous scholarship for my PhD.
Special thanks are due for my friends Saira Abbas, Arshia Ameen and Safeena
Inam for their sincere help, valuable suggestions and support throughout my studies.
I am very grateful to all other friends and laboratory fellows at NARC and
CIIT for their help and support. I am also thankful to the supporting staff of the
department for their help.
I am really indebted to my whole family especially to my brother Muhammad
Asad Haroon for their love, prayers and support.
NADIA
ix
GENETIC DIVERSITY ANALYSIS OF DATE PALM (Phoenix
dactylifera L.) CULTIVARS OF PAKISTAN
NADIA AND AISH MUHAMMAD
Department of Plant Genomics and Biotechnology
PARC Institute of Advanced Studies in Agriculture,
National Agricultural Research Centre, Islamabad
The University of Agriculture, Peshawar, Pakistan
November, 2016
ABSTRACT
Date palm has a long history of cultivation and a valuable germplasm in
Pakistan with little knowledge about genetic makeup and variation among the most
important cultivars. Date palm is among the top three fruit crops of Pakistan which is
grown throughout the country except the northern highlands. This study was
conducted for evaluation of morphological, chemical and molecular diversity of date
palm cultivars of Pakistan. Important morphological parameters of fruit, leaf and
trunk of forty five locally adapted cultivars were evaluated for this purpose.
Proximate analysis of the date fruit was also carried out. Morphological traits of
trunk, leaves and spines had no significant correlation with fruit traits. Seven
components explained 81% variability in the data set by principal component
analysis. Length, weight, volume of fruit, pulp weight, total soluble solids, %
reducing sugars, % total sugar, % ash content, length and width of leaf, midrib length
with pinnae, spine number, leaf base width and perianth height largely contributed to
variability among the cultivars. Forty six simple sequence repeat markers were used
to find genetic diversity in date palm cultivars under study. Only two SSR markers
showed polymorphism with five amplicons, 24 markers showed monomorphic bands
while the remaining 20 primers did not amplify. Coefficient matrices were computed
to form clusters to assess the relationship among the studied cultivars. Dendrogram
based on morphological and proximate composition data divided the cultivars into
four clusters while due to the less number of polymorphic SSR markers the studied
cultivars were divided into two groups.
Currently characterization of commercially important varieties is made
primarily through morphological and yield parameters and to a lesser extent on
genetic analysis using RAPD markers. There is a great need to develop some genetic
x
identification system that could be used at sucker stage without relying on phenotypic
traits of adult plant such as fruit characteristics. For this purpose, a detailed genetic
analysis of seven commercially important cultivars was performed. Initially more than
3.5 kb of DNA fragments belonging to rbcL, atpB, GGR, matKand 16S rRNA genes of
date palm chloroplast genome from seven commercially important date palm cultivars
of Pakistan were sequenced. All these genomic fragments were found near identical
among the selected cultivars. Twelve DNA fragments already reported to harbor
single nucleotide polymorphisms (SNPs) in date palm nuclear genome were also
sequenced. Eight novel SNPs were also found in the sequenced fragments in addition
to those already reported. The analysis of sequencing data indicated that three
fragments have the highest marker index (MI) of 4.61, 3.61 and 2.26 and bear eight,
seven and five SNPs respectively. A SNP typing system was developed for varietal
identification of date palm cultivars which is able to distinguish not only all the seven
studied cultivars from Pakistan but also other cultivars from the world. The study
suggests, that SNPs are important markers to study closely related cultivars and in
some instances might prove superior even to sequencing of genes. An authentic
sequence based identification key for date palm germplasm in Pakistan can be
developed by extending this study to all the indigenous cultivars.
1
I. INTRODUCTION
Date palm is an important species of the palm family (Jain et al, 2011). Date
palm is distributed widely and it occurs in diverse climatic, soil and geographical
conditions. Egypt, Saudi Arabia, Iran, UAE, Algeria, Iraq, Pakistan, Oman, Tunisia
and Libya are the top ten date producing countries of the world. Date palm requires
low humidity for fruit set and high temperature (35oC) for pollen development. It has
a well-developed root system that helps it draw water from deep in the soil.
Domesticated and wild species have quite similarity in morphology and ecology but
the fruit of wild plant is small in size and not edible (El-Hadrami and Al-Khayri,
2012). Date palm has an average economic life of 40 to 50 years that may go up to
150 years (Chao and Krueger, 2007).
Date palm is a significant fruit crop in Pakistan. It covers an area of 91154 ha
that produces 537204 tons of dates (Anonymous, 2014-15). Date palm is grown in all
the four provinces (Figure 1.1). Dera Gazi Khan, Jhang, and Muzaffar Garh of
Punjab, Khairpur and Sukkur of Sindh, Dera Ismail Khan of Khyber Pakhtunkhwa
and Turbat and Panjgoor of Baluchistan are the main districts of date palm production
(Abul-Soad, 2015).
Figure 1.1: Map of Pakistan showing date palm producing areas
2
The origin of domestication of date palm, its routes of germplasm flow and its
history of breeding are not fully known yet Phoenix dactylifera L. has been identified
as a distinct species. It may have more than one origin of domestication. One in the
Middle East and the other in South Western Europe and North Western Europe. The
present agrobiodiversity of date palm may be the result of isolation and gene flow
within the species (Pintaud et al. 2011).
Date palm cultivation in Pakistan is very ancient. Evidence of dates being a
part of staple food is as old as 7000 to 6000 BC from Mehrgarh (Balochistan) which
remained one of the most important city of Neolithic era (Costantini, 1985; Kenoyer
and Heuston, 2005). The date fruits found in excavation of Moen-jo-Daro, an
archeological site of the Sindh province, suggests cultivation of date palm in Sindh
since 2000 BC (Jandan, 1974). It is believed that Alexander the Great also brought
date palm in Indo-Pak subcontinent around 326 BC (Pasha et al., 1972 and Nixon,
1951), while some believe that dates were present here before Alexander, as his army
fed on dates while passing through Makran coast of Balochistan (Qasim and Naqvi,
2012). Mohammed Bin Qasim is also believed to have introduced dates from Arabian
Peninsula to Sindh around 712 A.D (Jatoi, 2010). In 1910-12 British Indian
Government brought offshoots of Arabian cultivars like Halawy, Zahidi, Khudrawy,
Diayri and Sayer which were brought from Iraq and planted in Multan and Muzaffar
Garh. (Milney, 1918).
Date palm is a tall monocotyledonous, dioecious domesticated plant. It is
diploid (2n=2x=36) in nature (Jain et al, 2011). Dates are rich in nutrients and are
economical in production and preservation. Carbohydrate content of dates is 44-88%.
Dates have 14 types of fatty acids, proteins, six types of vitamins and high content of
dietary fiber. Thus dates may be used to improve the nutrition especially amino acid
and mineral content of our normal food that we eat every day (Al-Shahib and Marshall,
2003). The edible portion of the date fruit is its pulp which is an important aspect of
fruit quality (Iqbal et al., 2012). Date fruit has good antioxidant potential and can be
used to produce novel natural antioxidant as well as a good flavoring agent that can be
used in various food products (Anjum et al., 2012).
3
Diversity is the variability of a specie. Plants show variation in yield,
vegetative traits and morphological properties of fruits and seeds in response to
environmental changes (Jahromi et al., 2007 and Odewale et al., 2012). Variation in a
population, breeding line or germplasm accessions can be analyzed on the basis of
pedigree data, agronomic traits, morphological attributes, biochemical data and DNA
based molecular data. Assessment of genetic diversity is important for genetic
variability analysis of the cultivars, identification of the parental combination which
may provide maximum diversity for selection, introduction of the desirable genes
from diverse origin into the existing germplasm and in the identification of varieties
for their protection (Mohammadi and Parasana. 2003).
Phoenix dactylifera L. is different from other plants with respect to genetic
diversity due to several reasons. Firstly it is vegetatively propagated with long life
which is able to pass its genes to many generations. Secondly a single female can be
selected on the basis of phenotype for further cloning. Thirdly due to cross breeding,
heavy selection and transportation to long distances its history is more complicated
(AlMssallem et al, 2013). Genetic variation among the cultivars is important for
development of the improved varieties (Khanam et al., 2012).
Commercial cultivars of date palm have been disseminated by offshoots from
oasis situated in the center of origin and diversity palm in lower Mesopotamia and
eastern Arabia. Cultivars propagated by offshoots are almost similar. Adapted
cultivars are a result of human and natural selection. Human selection is based mainly
on fruit traits and resistance to biotic and abiotic stresses. While noncommercial
cultivars propagated through seeds have also gone through natural selection (Jaradat,
2011). In date palm cultivars there exist a high degree of variation especially in the
reproductive characters so cultivars are usually identified on the basis of fruit
characteristics (Hammadi et al., 2011). The constant change in traits as a result of
interaction between the genotypes and environment necessitates the regular update of
morphological properties of plants. This knowledge can be used for assessment of
genetic diversity for crop improvement and to design equipment to be used for
sorting, grading, cleaning and packing of fruit after harvest (Jahromi et al., 2007 and
Odewale et al., 2012). Phenotypic markers of date palm like leaves, leaflets and
spines (Elhoumaizi et al., 2002), offshoots and inflorescence along with isozyme
4
markers (Azequor et al., 2002; Salem et al., 2008; Hammadi et al., 2009), fruit
morphological traits (Markhand et al., 2010), inflorescence, fruit and leaf along with
molecular markers (Hammadi et al., 2011; Al-Khalifah et al., 2012; Haider et al.,
2015), and seeds (Naqvi et al., 2015) have been used for characterization of date
palm germplasm.
Biochemical analysis of fruit is important in various aspects for example,
antinutritional factors and physicochemical characters are evaluated for determination
of suitability of the dates for direct consumption or processing (Iqbal et al; Nadeem et
al., 2011) Carbohydrates, fat, protein, vitamins and salts are determined to prove it as
a source of balanced diet that satisfy the daily requirement of these components.
Chemical composition of fruit is important for fruit physiological and technological
ability studies (Hasnaoui et al. 2012). Flavonoid profile and isozyme banding pattern
can be used as an early test to screen tissue culture derived clones for genetic stability.
Isozyme analysis is for cultivar identification at an early stage in the date palm life
cycle (Saker et al. 2002). It is important to generate qualitative data of the fruit to be
helpful for processors, exporters and consumers (Nadeem et al., 2011). Quality of the
date fruit changes with cultivar and depend on climatic conditions and farming
practices (Hasnaoui et al., 2012).
Molecular markers or DNA markers have been in use since past three decades.
The DNA profiles that give information about the genotype, they screen the whole
genome and show variation in both the coding and noncoding region and hence give
information about polymorphism. DNA marker can be automated that make their use
very efficient for selection by the plant breeders (Jehan and Lakhanpaul, 2006).
Molecular marker technology can be applied to identify commercial varieties and the
knowledge of genome polymorphism based on DNA analysis may be used in
breeding program. Techniques used for detection of molecular markers are AFLP,
RAPD, RFLP and SSR. DNA markers provide information on the similarity or
diversity of various clones and varieties which can be hardly differentiated on
morphological basis. DNA markers may prove helpful in managing plant accessions
and also help in breeding programs (Ahmed and Al-Qaradawi, 2009). Genetic
diversity measurements can be used for increasing genetic variability in populations
5
that have high level of genetic distance as well as to introduce the foreign germplasm
(Elmeer et al., 2011).
Since plastid genes are transferred mostly from the mother line so
identification of maternal lines is possible by sequencing of plastid genes (Khew and
Chia, 2011). Ribulose biphosphate carboxylase larger subunit (rbcL) is a chloroplast
gene of 1400bp in length encoding ribulose 1, 5 biphosphate carboxylase oxygenase.
matK encodes maturase needed for photosynthetic like activities of the chloroplast. It
is a 1500bp gene located within the trnk intron (Barthet and Hilu, 2007). Nucleotide
substitution rate indicators have suggested rbcL to be least conserved, followed by
atpB and 18S in seven genera of Lardizabalaceae along with three outgroup taxa in a
study of their utilization in phylogenetic studies (Hoot et al., 1995). Data of
chloroplast DNA like rbcL and atpB being abundant and having simple substitution
properties makes it the most reliable for estimating the phylogeny of Palms (Hahn,
2002). Resolution power of matK is better than other regions like rpoC, trnH-psbA,
rbcL, atpF-atpH (Burgess et al, 2011). The consortium for barcode of life in 2009
suggested rbcL and matK along with additional region, according to the requirement,
as a barcode of life to help in diversity studies (Patwardhan et al., 2014). Genetic
distance among the cultivars determined by matK ranged from 0.00-0.72 thus matK
alone or in combination has the potential to distinguish among the cultivars (Enan and
Ahmad, 2014). Intergeneric and interspecific nucleotide distances determined by
matK were higher (0-10.9% and 0-52.5% respectively) as compared to 0-3.2% and 0-
17.9% determined by rbcL for xerothermic plant of the Central Europe (Heise et al.,
2015).
Simple sequence repeats (SSRs) are segments of repeated DNA sequence in
higher eukaryotic genome. They can detect length variation with the help of
Polymerase Chain Reaction (PCR) and may be used as highly informative genetic
markers (Powell et al., 1996). SSR markers are popular for the analysis of plant
diversity because these are PCR based co-dominant markers which show high allelic
diversity at different loci (Elmeer et al., 2011). Simple sequence repeats which are
abundantly present throughout the higher eukaryotic genomes are highly polymorphic
than other genetic markers. These are helpful in identification of cultivars, analysis of
6
pedigree, germplasm characterization and genetic mapping study (Billote et al.,
2004).
SNPs are the third generation of molecular markers. SNPs are more stable and
have high fidelity of inheritance as compared to other marker systems (Gupta et al.,
2001). Single nucleotide polymorphism is the type of polymorphism that occur among
DNA samples with respect to single base. Where the least frequent allele has
abundance of at least 1%. In DNA molecule theoretically four possible nucleotides
are involved but actually only two of these four possibilities have been observed at a
particular site in a population. Thus SNPs are largely biallelic, this makes them less
informative but their abundance in the genomes overcome this deficiency as more
number of loci can be examined for polymorphism. Limited data is available on SNPs
in plants because of the SNPs development requires huge cost. SNPs in the coding
sequence may result in the morphological polymorphism or an association with some
trait. Similarly SNPs present near the coding region can also be used in marker
assisted selection (Jehan and Lakhanpaul, 2006).
Over the years many varieties have been transplanted to areas other than the
native regions where they may have been adapted and cultivated with different names,
thus same variety may have different names in different areas where it is grown or
even two genetically different varieties may have the same name, making selection of
needed variety difficult (Abdullah and Gamal, 2010). As compared to other crops date
palm is less researched in terms of molecular genetics. Molecular markers have been
developed but these are not enough for sufficient diversity assessment. So there is a
need to increase the number of DNA markers in date palm (Zhao et al., 2012). The
dominance of the elite cultivars may pose a threat to the genetic erosion of the date
palm crop. This situation may cause the cultivars of medium or low fruit quality to
disappear thus a system is needed for the assessment of genetic diversity and the
conservation of existing germplasm (Powell et al., 1996). Clonally propagated
offshoots of the date palm cultivars are difficult to distinguish by the farmers (Zehdi
et al., 2004).
Genetic characterization is important for the identification of varieties and
conservation of germplasm. Such information is also useful for plantation of different
varieties to avoid devastation by biotic stresses and to have diverse parental
7
combination to create variability (Jubrael et al., 2005). Date palm is prone to the loss
of genetic diversity due to continuous use of offshoots from few genetic material of
the gene pool. Therefore to evaluate and preserve the genetic diversity are important
for germplasm conservation of date palm (Zhao et al., 2012).
Morphological traits of the tree have been used for assessment of variation in
the germplasm but these traits are greatly influenced by the environmental factors and
developmental stages of the plant (Elhoumaizi et al, 2002; Ahmed and AlQaradawi,
2009). Moreover date palm has a long generation cycle and it takes quite long to
appear reproductive traits. Therefore any trait that may help in the early detection of
cultivar, its sex and/or resistance to some disease or pest will save the time and
resources of the breeders by quick selection of the plant with desirable traits (Zhao et
al., 2012).
Previously, there are several studies to type various commercially important
germplasm in Pakistan, mostly based on morphological or yield parameters
(Markhand et al, 2010., Iqbal et al, 2012., Bashir et al, 2014., Haider et al. and Naqvi
et al. 2015) or biochemical composition of the date fruit (Jamil et al, 2010., Haider et
al, 2013., Iqbal et al. and Nadeem et al. 201I., Iqbal et al.2012). Comparatively few
investigations have been made for characterization using genetic fingerprinting
however, those are restricted to RAPD markers (Raza et al. 2006., Mirbahar et al.
2014) or typing through one or two genes (Akhtar et al. 2014). It is already known
that morphological and biochemical markers are limited in number and are affected
by environmental factors and growth stage of the plant which reduce their reliability
in assessment of diversity and characterization of the germplasm (Elhoumaizi et al,
2002; Ahmed and AlQaradawi, 2009). This necessitates the use of genetic
characterization, utilizing DNA markers, gene sequencing or SNPs genotyping which
can be reproducibly employed independently to any stage of the plant growth and are
not affected by environmental factors. A combination of morphological, biochemical
and molecular characterization of the date palm cultivar can better assess the level of
diversity and relationship among the cultivars.
8
The objectives of this study therefore are the:
1. Development of reliable identification system for germplasm characterization
in date palm
2. Assessment of genetic diversity in local date palm cultivars.
9
II. REVIEW OF LITERATURE
Date palm is a diploid, dioecious plant which has a long history of cultivation.
Date palm culture is important for its nutritive, economic and social value in the arid
and semiarid environments of the world. Being out crossing in nature the tree presents
immense diversity in cultivars with separate male and female plants. Female trees are
mainly important for their nutritious fruits. Date palm cultivars have been identified
by the growers on the basis of morphology of the tree and fruits when no breeding
programs based on science existed.
The crop has been investigated in various aspects and literature covers a wide
variety of work done in this regard but this review focuses on the analysis of
morphological traits of date palm, proximate composition of its fruit, use of
chloroplast DNA for barcoding of plant species and molecular analysis based on DNA
markers.
2.1 Date palm diversity evaluation based on morphological parameters
Rizk and El-Sharabsy (2007) suggested a set of descriptors for
characterization of date palm to be helpful in diversity studies, establishment of gene
bank and to conserve the specie. They collected different parameters of
characterization, management, ethno botany, ecogeography and passport data. These
parameters were derived from 55 date palm trees grown in Egypt. These descriptors
provide a standard set of information for evaluation and characterization of date palm.
Salem et al. (2008) studied morphological variation in Mauritanian date palm
cultivars using vegetative characteristics. They studied 12 ecotypes of date palm on
the basis of eighteen vegetative traits selected from standard descriptors set for date
palm. The data were analyzed using principal component analysis to identify the
parameters that are distinguishing among the studied ecotypes. Fourteen traits were
found to be discriminating that clustered the similar ecotype together.
Markhand et al. (2010) evaluated the quality of different Pakistani dates.
Fruits of 85 varieties of Pakistani date were analyzed on the basis of shape and size of
fruit, its color at khalal stage, height and color of the perianth, edible stage, fruit type,
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split of the seed and micropyle position. Most of the Pakistani varieties belong to semi
dry group. ‘Aseel’ variety is considered predominant and commercial varieties in
Pakistan.
Sakr et al. (2010) identified eight important Egyptian cultivars of date palm by
the physical (length, weight, diameter, pulp and seed weight and their color), chemical
(moisture, and dry matter content, titratable acidity, and total soluble solids, reducing
sugars) and anatomical properties like number and thickness of different layers of
pericarp. They concluded that Samany cultivar had the highest content of total soluble
solids, while ‘Zaghloul’ had the highest value of reducing sugar, titratable acidity and
total phenol contents.
Al-Khalifa et al. (2012) characterized 14 elite cultivars of date palm from
Saudi Arabia on the basis of morphological characters like length width ration, shape
and color of the fruit, fruit base width, length and % area covered by fruit cap in
correlation with the RAPD markers. They found that fruit shape is the most
influenced trait by the genetic variation.
In order to find the genetic relationship among date palm accessions, to detect
polymorphism in their characteristics and to look for important traits that may help in
their classification some important physical and mechanical dimensions of date fruit
were determined by Odewale et al. (2012). They used a multivariate technique called
principal component analysis to study the genetic diversity even at interspecific level.
They rendered this information to be useful for reduction in time needed for selection
as stock breeders from large population.
Farag et al. (2012) investigated fruit characteristics of date palm variety
‘Zaghloul’ as affected by the metaxenic influence of two different pollinators as well
as difference in the fruit characteristics of pollinated and non-pollinated fruits. They
observed an increase in the values of physical dimensions of fruit, total soluble solids,
reducing and non reducing sugars, vitamin C, crude fiber and anthocyanin content
while a decrease in the chlorophyll a and b content. They concluded that different
pollinator sources had metaxenic effect on fruit parameters.
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Abul Soad et al. (2013) assessed three Saudi Arabian date palm varieties
namely Ajwa, Safawi and Ruthana in Khairpur (Pakistan) on the basis of vegetative
traits of leaves, leaflets and thorns, flowering traits of spathes and spikes and
morphological traits of flesh and seed. They observed the fruit quality to be the same
as that at the place of origin. They concluded that climatic conditions of Khairpur are
suitable for growth of exotic date palm cultivars.
Bashir et al. (2014) found the effect of strand thinning on fruit parameters like
length and weight and diameter of fruit, total soluble solids yield, stone pulp ration,
and fruit drop of Kur variety of date palm in Bahawalpur, Pakistan. They reported that
50% strand thinning resulted in maximum fruit weight, length, width and yield and
smallest stone pulp ratio.
Haider et al. (2015) characterized sixteen date palm cultivars morphologically
to determine their similarity level. They found clear differences in the germplasm of
Pakistan with respect to trunk length and diameter, total number of leaves, rachis
length, length and width of leaflets, leaflet number per side, length and grouping of
spines. Cultivars of same origin were found to be closely associated. They concluded
that quantitative and qualitative characteristics can be successfully used for
assessment of phenotypic characterization.
Naqvi et al. (2015) studied twenty exotic date palm cultivars grown in
Pakistan on the basis of nineteen morphological traits of seed using multivariate
analysis techniques like correlation index, cluster analysis and principal component
analysis. They found all the quantitative traits to be highly divergent among the
studied germplasm. They have also reported heterogeneity among the cultivars of the
same origin.
2.2 Date palm diversity evaluation based on proximate composition of fruit
Nadeem et al. (2011) studied the textural profile and phenolic content of
twenty one varieties of date palm at tamar stage. They observed that Dhaki variety
had the maximum fruit weight, length, volume and fresh weight. Aseel Sindh variety
was followed by Dhaki in the fruit characteristics, they recommended Aseel Sindh,
Dhaki and Halawi to be suitable for direct eating because of their good fruit weight,
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volume, size and nutritional properties. Other varieties possessing high sugar and low
moisture content were proposed to be suitable for processing. In another study they
investigated the same varieties for anti-nutritional factors like Tannin, Phytate and
Oxalate. The highest mean content of the Tannin in the studied varieties ranged from
0.22±0.01% -0.87±0.01% on dry weight basis. Aseel (0.22±0.01%), Dhaki
(0.25±0.01%) and Halawi (0.28±0.01%) had the lowest tannin content. Phytate
content was found to be in the range of 0.77±0.01%-0.30±0.01% for Dora desi and
Dora variety respectively. Oxalate content varied from 3.63±0.01% to 6.4±0.01%.
They concluded that date varieties from Pakistan had low level of antinutrients like
phytate, oxalate and tannin and thus were rendered suitable for consumption or
processing. In third study they evaluated the quality of some Pakistani date varieties
with respect to their physicochemical characteristics at ‘tamar’ stage. Highest values
of fruit attributes were recorded for Dhaki variety. They found the edible/nonedible
ratio in the range of 1.94-14.50. Dora variety had the highest peak force of puncture
which shows firmness of fruit. Crude protein and fiber were found to be in the range
1.57-3.5% and 2.65-4.55% respectively. Karbalain was found to have highest sugar
content (73.92%). While Desi Basry had the minimum mean sugar value (59.03%).
Due to good physical properties Dhaki was proposed to be good for processing.
Iqbal et al. (2011) studied physicochemical characteristics of fruits of six date
palm cultivars i.e Azadi, Dhakki, Gulistan, Khudrawi, Zahidi and Shakri at various
developmental stages under the agro climatic conditions of D. I. Khan. They observed
significant differences in the physicochemical characteristics of the studied cultivar,
maximum fruit length (5.092cm), diameter (3.065cm), weight (23.36g) and pulp
weight (21.58g) were observed for Dhaki cultivar at its kimri stage while the moisture
content was found to be maximum in Shakri (85.05%) at this stage. Maximum
reducing and total sugar were recorded for Dhaki at Tamr stage. Khudrawi was found
to be the most astringent (1.46%) at Rutab stage. Dhakki was found to have maximum
yield (96.00kg/ Palm). In another study conducted in 2012 they assessed the periodic
growth and development of fruit of five date palm cultivars namely Dhaki, Gulistan,
Khudrawi, Shakri and Zaidi-1. Highest fruit weight, length and pulp weight were
recorded in Dhaki cultivar followed by Gulistan collected throughout the growing
season at 15 days interval. Similarly highest fruit yield/plant was recorded in Dhaki
(97.59 kg) followed by Gulistan (86.75 kg/ plant). Thus Dhaki cultivar of date palm
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was found to have the best fruit size and yield among all the studied cultivars
followed by Gulistan and Khudrawi.
Hasnaoui et al. (2011) studied fourteen date palm cultivars from Morocco,
Tunisia, and Algeria with respect to their proximate composition, water activity and
CIELAB parameters. Sugar was the major component of all the studied varieties on
the basis of dry matter (75.56g/100g dry matter) followed by moisture (7.2-31.9%),
protein (1.9-3.3g/100g dry matter), fat (0.1-0.44g/100g dry matter) and ash (1.8-
3.9g/100g dry matter). Early maturing varieties were found to have greater moisture
activity as compared to late maturing varieties. Proximate composition of date fruit of
different varieties from different origin had no significant difference. In an another
study conducted in 2012 they analyzed the chemical composition and functional
characteristics of fruit fiber concentrate in five cultivars of date palm namely Assiane,
Aziza bouzid, Boufeggous, Boufeggous gharas and Mejhoul-1 from Figureuig oasis.
Total sugar and total dietary fiber were found to be in high concentration in the date
flesh while the dietary fiber concentrate had high concentration of total dietary fiber,
protein and ash and low concentration of fat, total phenolic and flavonoid content.
The dietary fiber had high water holding, and oil holding capacity and emulsion
stability and was found to have antiradical capacity too. They suggested on the basis
of their study that date fruit is a potential source of sugar, fiber and phenolic
compounds, dietary fiber concentrates can be obtained from it as a potential source of
valuable nutrients.
Suleiman et al. (2012) studied the physical, chemical and microbiological
properties of five Sudanese date palm cultivars, their results showed significant
differences in the physical properties of date fruit but chemical properties of all the
studied cultivars were almost the same. No significant difference was found in the
microbiological properties of these studied cultivars.
Haider et al. (2013) characterized some Pakistani date palm cultivars on the
basis of biochemical traits and observed fruit maturity effect on the radical scavenging
capacity. They also determined the total phenolic content, specific activity of
antioxidant enzymes, sugar profiles and assessed the soluble protein content, total
phenolic contents and antioxidant enzymes. Total phenolic contents were found to be
higher at khalal stage while their values decreased from Rutab to Tamar stage. The
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variation in biochemical characteristics of the date fruit was concluded to be
dependent upon the type of cultivar and fruit developmental stage.
Taain (2013) studied physicochemical and physiological characteristics of Um
Aldehin variety of date palm in Iraq at different developmental stages. They collected
the fruit throughout the growing season till khalal stage. A gradual increase in length,
diameter, volume and fresh weight of the fruit was observed. Moisture content, total
titratable acidity and fatty compounds were found to decrease with increase in total
soluble solids. Total and reducing sugar content also increased with advancement
from rutab to tamar stage.
Aleid et al. (2014) studied the physicochemical characteristics and microbial
loads of four date palm cultivars from Saudi Arabia. They found the moisture content
and insect damage level to be within the codex standards for all the four studied
cultivars. Upper limit of moisture content is 26% for single sugar dates and 30% for
two sugar dates and no lower limit for moisture level is suggested but one of the
cultivars ‘Ambara’ had highly significant mould and yeast count than recommended
standards for packaging of whole dates. Weight of pitted and unpitted single date fruit
was found to be higher than the recommended international standards. They found
fruit color to be an important feature being affected by cultivar. Thus they suggested
the fruit color to be included in the technical regulation and standards for date
grading.
2.3 Date palm diversity evaluation based on simple sequence repeat (SSR)
markers
Powel et al. (1996) in a comparative study of RFLP, RAPD, AFLP and SSR
markers for germplasm analysis of the soybean found that the expected
heterozygosity determined by SSR markers was the highest (0.60) while highest value
of multiplex ratio (19) was determined by AFLP markers. They found that similarity
matrices based on RFLP, AFLP and SSRs are highly correlated showing that there is
similarity between the assays, while correlation of data obtained by RAPD markers
was lower as compared to other marker systems. They proposed a universal matrix
called marker index to represent the amount of information for a marker system. In
this study the SSR markers detected the highest polymorphism while RAPD and
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AFLP detected the lowest level and the RFLP detected the intermediate level of
polymorphism. They found (AC)n SSRs to be more informative than (AG)n SSRs.
Billotte et al. (2004) constructed microsatellite library rich in (GA)n repeats
and characterized 16 SSR loci in date palm. These SSR markers were the first to be
published for date palm and are available for identification of cultivars, analysis of
pedigree, diversity analysis of germplasm and genetic mapping studies.
Zehdi et al. (2004) assessed the genetic diversity of 49 date palm cultivars of
Tunisia using fourteen microsatellite loci. They fingerprinted all the 49 accessions and
were able to distinguish the cultivars using three loci i.e mpdCIRO78, mpdCIRO85
and mpdCIRO25.
Elshibli and Korpelainen (2008) investigated the genetic diversity in 60 date
palm accessions from Sudan including both males and females using 16 SSR primers
developed by Billote et al. (2004). Eight Moroccan accessions were used as reference
material for the study. The SSR primers detected 343 alleles ranging from 14 for
mpdCIRO35 to 44 for mpdCIRO63. Cultivars from Morocco group showed
significant variation as compared to Sudan group. Weak clustering association
showed the effect of methods of pollination and selection of the cultivars on the
genetic structure for most of the accessions from both the Sudan and Morocco.
AlRuqaishi et al. (2008) used microsatellite markers for genetic diversity
screening of date palm genotypes of Oman which were obtained by somatic
embryogenesis. Twenty one cultivars from Bahrain, Iraq and Oman were genotyped
using 10 SSR primers. This study resulted in unique fingerprints for the important
genotypes of date palm from Oman. This study also showed that if different explants
of the same genotype are used for somatic embryogenesis, will give the same
fingerprint.
Elshibli and Korpelainen (2009) characterized fruit of 15 date palm cultivars
from Sudan on the basis of their chemical and morphological traits. They also
investigated the morphological and DNA polymorphism of the mother trees. They
observed a significant difference in the plant height, number and length of the pinnae
and number of spines among the cultivars. Parameters like fruit weight and size, flesh
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weight and size of seeds, sugar content and titratable acidity were also found to be
significantly different among the studied cultivars. The sixteen SSR primers pairs
resulted in 167 alleles with a mean of 10.4 alleles per locus for primers mpdCIRO15
and mpdCIRO63 ranging from 7-62 respectively.
Jhonson et al. (2009) used thirty seven SSR primer pairs developed from oil
palm sequences to screen eighteen date palm varieties obtained from USDA-ARS
National clonal germplasm repository for citrus and date, Riverside, CA, USA. Three
of the tested primers were found to identify all the studied varieties clearly.
Ahmed and Al Qaradawi (2009) used SSR markers for molecular
characterization of phylogenetic relationships of the date palm cultivars of Qatar.
They tested 16 primer pairs. Ten primers produced the expected bands in most of the
genotypes while six primers did not amplify clear bands. They selected 4 cultivars for
initial screening of the primers and those that resulted in clear bands were used to
amplify all the 15 Qatari date palm. They found the studied cultivars to be interrelated
although they were agronomically divergent.
Akkak et al. (2009) isolated and screened forty one microsatellite loci from
two libraries of Phoenix dactylifera L. They used a set of thirty one date palm
cultivars for characterization and evaluation of seventeen screened markers. High
polymorphism was measured in the studied samples. Most of these markers were also
found to be effective in fourteen other species of phoenix.
In 2010 Hamweih et al. developed 1000 microsatellite markers through the
date palm genome. They found dinucleotide repeats to be the most frequent (52442
motifs) type of microsatellite repeats with GA as the most common (48.7%) among
them. They suggested that 1090 new SSR markers could be developed from these
motifs to be used for genetic study and diversity analysis of the crop.
Elmeer et al. (2011) assessed the genetic diversity of eleven date palm
cultivars selected from different areas of Qatar using thirty new microsatellite primer
pairs. 33.4% of the primers tested generated polymorphic banding pattern, 43.3%
amplified monomorphic bands and 23.3% did not amplify the expected bands. They
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observed 77 alleles with a mean of 7.7 alleles per locus. The average gene diversity
was found to be 8. The studied cultivars were highly divergent.
Hammadi et al. (2011) studied twenty six date palm cultivars from Tunisia
using morphological and DNA markers. They used microsatellite loci developed by
Billote et al. (2004) to characterize the diversity level and genetic relationship among
semi soft, soft and semi dry fruits of date palm in Tunisia. They scored 36 alleles with
an average of 7.2 alleles per locus. Observed (0.3-0.8) and expected heterozygocity
(0.8) were found to be quite high showing that Tunisian date palm cultivars have high
genetic diversity. Cluster analysis based on vegetative features grouped the 26
cultivars on the basis of fruit consistency and maturity periods. They found a
significant differentiation of semi soft fruit subpopulation with soft and semidry fruit
subpopulations.
Khierallah et al. (2011) evaluated genetic diversity among 30 date palm
cultivars from Iraq using 22 SSR markers developed by Billotte (2004) and Akkak
(2009). They scored 188 alleles at 22 loci with average heterozygocity of 0.503 and
genetic distance varying from 0.171 to 0.938 among the cultivars showing high
polymorphism but cultivar identification still remained a question.
Arabnehzad et al. (2012) constructed two SSR enriched libraries comprising
of (AG)n and (AAG)n repeat motifs and designed 25 primer pair, 22 of which were
able to differentiate among 16 date palm cultivars from Iran , Iraq and Africa. Cluster
analysis successfully differentiated the African cultivars from Iraqi and Iranian date
palms.
Bodian et al (2012) analyzed genetic diversity of date palm cultivars from
Morroco using 18 SSR markers developed by Billotte et al (2004) and Akkak et al
(2009). They screened 128 date palm samples and obtained 107 alleles with 96.11%
polymorphic loci. Although a high heterozygocity was calculated in general but male
samples were found to have no genetic distance with the female samples. They used
ALFwin software for scoring, DAR win 5.0 for dendrogram formation and GenAles
6.3 for calculation of genetic distance.
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Elmeer et al. (2012) reported the use of 254 microsatellite markers for sex
determination in immature date palm. Twenty two out of 254 microsatellite loci were
found to identify 75% of the male plants. The primer mpdCIRO48 reoccurred in 12
male samples but not in 117 female samples they suggested that SSR markers have
the potential to be used in sex discrimination of date palm at an early stage of
development. Three out of 14 primers (mpdCIRO35, mpdCIRO44, mpdCIRO90)
could not differentiate between male and female while the rest of 11 primers could
identify 22 loci in the male samples only.
Zhao et al. (2012) analyzed EST sequences of date palm genome data base
and identified 4,609 ESTs containing simple sequence repeats. They found that
trinucleotide motifs were the most common (69.75). The percentage of dinucleotide
and tetra nucleotide were (9.6%) and 10.4% respectively. They designed 4,697 primer
pairs for EST-SSR markers from computational data. Twenty of primer pairs were
randomly selected from the designed primers to check the polymorphism of twelve
date palm cultivars. Thus showing that date palm EST sequences exhibit a good
resource for developing gene based markers to be used for diversity study.
Raachi et al. (2014) typed eighteen date palm cultivars from Libiya using 16
SSR loci. The resulting 110 alleles showed a high polymorphism among the cultivars.
They also developed a varietal identification key with the help of only three SSR loci
identifying 23 alleles.
Elmeer and Matatt (2015) used 14 SSR primers developed by Billotte et al.
(2004) for genetic diversity assessment of 12 cultivars from Qatar. Ninety four alleles
were detected with band size of 104 to 330bp. Gene diversity ranged from 0.39 to
0.86 showing high genetic diversity among the Qatari date palm collection.
Yusuf el al. (2015) used six SSR markers for investigating the diversity
among fourteen Nigerian and Saudi date palm cultivars. They were able to
characterize the fourteen cultivars with only two markers mpdCIRO25 and
mpdCIRO50. They did not find any gender specific allele with SSR markers. 83.3%
polymorphism was detected among the studied cultivars which shows high diversity.
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2.4 Date palm diversity evaluation based on other molecular markers
Cao and Chao (2002) determined polymorphism in 21 date palm cultivars in
California using AFLP technique with fluorescent labeled primers. They obtained 328
polymorphic bands using four primers sites. The AFLP markers efficiently identified
the studied date palm cultivars.
Diaz et al. (2003) applied the amplified fragment length polymorphism
technique for identification of palm varieties. Date palm varieties Boufagous, Madjool
and E528 from Spain were analyzed in this study. 310 AFLP bands were scored using
five primer combinations. They used the obtained AFLP markers for comparison and
identification of vitro plants of palm.
Adawy et al. (2005) analyzed fourteen date palm accessions from different
locations in Egypt. These accessions were from six different cultivars. Sixteen AFLP
primer combinations were used for this analysis. AFLP generated a total of 651
amplicons representing 45.8% polymorphism. The genetic similarity and relationship
were estimated according to Dice coefficient. Data obtained from RAPD’s and ISSR’s
previously obtained on same fourteen accessions were combined with AFLP to
generate more effective data in detecting high level of polymorphism. The results
indicated that AFLP is more effective in detecting high level of polymorphism.
El-Assar et al. (2005) collected and studied forty-seven samples of date palm
(Phoenix dactylifera L.) from eight locations in Egypt, using four sets of amplified
fragment length polymorphism (AFLP) markers with near infrared fluorescence
labeled primers. These samples belonged to 21 named accessions and 9 of unknown
pedigrees. A total of 350 bands were scored and 233 (66.6%) were polymorphic.
Twenty-seven Egyptian accessions and ‘Medjool’ and ‘Deglet Noor’ accessions from
California could be classified into the major cluster. This major cluster may represent
a major group of date palm germplasm in North Africa. There were four other
clusters, each containing one or two accessions. The variety ‘Halawy’ and one
accession of unknown provenance were most likely from hybridization between two
clusters. Six groups of accessions which had the same names, revealed similar but not
identical AFLP profiles suggesting these accessions might have derived from
seedlings rather than through clonal offshoot propagation.
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Jubrael et al. (2005) used AFLP markers for discrimination among 18 Iraqi
date palm cultivars and to assess their genetic relationship. They scored 122
polymorphic loci with 17.4 average loci per primer combination. They used
P101(accg/M95(aaaa), P74(ggt)/M95(aaaa), P73(ggg/M95(aaaa) and
P100(aacc)/M95(aaaa). Any of these four combination can successfully identify all
the varieties.
ElHoumaizi et al. (2006) confirmed Medjool as a landrace variety of date
palm in Morocco through genetic analysis using AFLP markers. They investigated 66
accessions of Medjool from Morocco, one from California and seven different
varieties of date palm from Egypt and California. They obtained 402 bands with four
sets of AFLP markers out of which 217 were polymorphic. Moroccan accessions of
Medjool had 79% genetic similarity. Their results supported the hypothesis that
Medjool is a landrace variety. This confirmation is important for germplasm
collection and preservation.
Saker et al. (2006) assessed the genetic variation in tissue culture derived date
palm offshoots using RAPD and AFLP methods. They used 37 RAPD primers and 13
AFLP primer combinations. RAPD primers showed no polymorphism while AFLP
showed 2.6, 0.79 and 1% genetic variation in tissue cultured offshoots of three
cultivars i.e Sakkoty, Gondila and Bertamoda. The genetic stability of the tissue
cultured derived dry date palm cultivars was confirmed by low genetic variation.
Al-Khalifa (2006) tissue cultured 19 popular date palm cultivars from Saudi
Arabia. Out of 140 OPERON primers screened for reproducible and polymorphic
DNA amplification pattern 42 were selected for DNA fingerprinting. All the 19
genotypes revealed a unique profile with the 42 primers and showed an average of
more than 50% similarity indicating narrow genetic diversity. Among 19 cultivars
Mowakil and Khalas showed maximum similarity, 12 formed couples and the rest
showed various percentages of similarity with either to one of the couples or to more
than one couple.
Rawashdeh and Amri (2006) used RAPD markers to characterize five date
palm varieties namely Tabarzar, Zagloul. Mkfazy, Barhee and Nabt saif. Thirty
primers were found to be polymorphic among which seven were highly polymorphic.
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Based on the results all the varieties were different based on RAPD technique
showing the efficiency of this technique in the study of genetic diversity analysis of
date palm.
Raza et al. (2006) analyzed genetic diversity among six female date palm
cultivars namely Aseel, Fusly, Khupra, Kurblan, Punjmail and Pathri and one male,
by RAPD markers. 9 out of 17 primers used for this study revealed polymorphic
bands. Out of 75 amplified fragments 65 were polymorphic.
Rani et al. (2007) studied genetic diversity among 40 date palm genotypes
using 29 RAPD markers. A total of 223 amplified bands were polymorphic. Cluster
analysis by UPGMA subprogram of NTSYS-PC grouped forty genotypes into 2 major
clusters. Zaidi genotype Z2 was out grouped and showed to be the most diverse
among all.
Abdullah and Gamal (2010) applied three types of markers such as protein
RAPD-PCR and ISSR on four important cultivars in Saudi Arabia i.e Med 3002b1,
Sugay1b1, Khalasb1 and Sukkarib. Intervarietal variation was investigated using five
RAPD and five ISSR markers were also applied to assess the genetic polymorphism.
cluster analysis by UPGMA showed two main clusters, sukhari b1 in cluster A and
the other three in cluster B. most of the cultivars had the narrow genetic diversity as
already expected. The result of the analysis can be used for the selection of the
possible parents to generate mapping population.
Kheriallah et al. (2011) used AFLP for evaluation of genetic diversity among
18 date palm varieties from Iraq. They obtained 83 polymorphic AFLP fragments by
the use of six primer combinations with an average of 13.8 polymorphic bands per
primer pair. All the primer pairs helped in differentiation of date palm varieties
showing the efficiency of AFLP technique for assessment of genetic diversity in date
palm. Their results also showed large genetic diversity among the studied date palm
cultivars.
Soumaya et al. (2011) assessed the genetic diversity and relationships of date
palm cultivars from Tunisia. They used random amplified microsatellite
polymorphism and amplified fragment length polymorphism techniques for this
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purpose. Forty date palm cultivars were assessed using six AFLP and 18 RAMPO
combinations. Their results showed large and continuous genetic diversity among the
studied cultivars. AFLP proved to be more informative than the RAMPO method due
to the greater number of markers per assay in AFLP with 0.7 PIC value and 50.54
marker index. They also found good congruence between the two marker systems on
the basis of Pearson and Spearman correlation.
Marsafari and Mehrab (2013) studied the genomic profile of 15 date palm
cultivars native to south and southwest of Iran. They used 14 ISSR and 10 RAPD
primers to determine the genetic affinity and allelic diversity. 92.4% and 95.67% of
the polymorphism was represented by ISSR and RAPD primers respectively. This
study proved the efficiency of ISSR and RAPD for determining the genetic
relationship of the date palm cultivars.
Mirbahar et al. (2014) assessed genetic diversity and population genetics
relationship of twenty five date palm cultivars from Pakistan using RAPD markers.
Average genetic diversity among the studied cultivars was calculated to be 79.4%.
They concluded that a high genetic diversity exists among the studied cultivars,
Mazawati and Halawi were found to be closely related having similarity index of
0.95%.
2.5 Date palm diversity evaluation based on both morphological and molecular
markers
Eissa et al. (2009) characterized nine Egyptian date palm cultivars on the basis
of morphological and molecular markers. They selected eight RAPD, four ISSR
markers and seventy seven phenotypic traits for identification of nine cultivars. They
found fruit shape, weight, length and color leaf traits as distinguishing but common
among most of the Egyptian cultivars. RAPD and ISSR data were used to form
dendrograms on the basis of similarity matrices using SPSS software. Both RAPD
and ISSR primers were found to be reliable for cultivar identification.
Elshibli and Korpalainen (2010) evaluated date palm germplasm from Sudan
on the basis of morphological, chemical and molecular markers. They used 16 SSR
primers to characterize 37 date palm cultivars from Sudan. Expected heterozygocity
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among the cultivars was found to be 0.841. Clustering resulted in weak relationship
among the studied cultivars. Morphological and chemical analysis divided the
cultivars into soft and dry types while the results of SSR analysis was not comparable
with those of morphological and chemical traits. SSR analysis could not differentiate
among the 37 date palm cultivars. The difference exhibited by morphological and
chemical attributes may be due to the effect of environment or interaction of genotype
and environment.
Hammadi et al. (2011) studied the diversity in Tunisian date palm cultivars
using molecular markers. The use of reliable and stable vegetative features on 26
cultivars showed clusters characterized also by fruit traits such as consistency and
maturity period. Microsatellites also support this statement and it was carried out by
using markers with high polymorphism. These results suggest that continental
Tunisian date palm cultivars are not a unique population which is in opposition with a
unique one ancestral date palm population and this result is the first to be published in
date palm.
Ibrahim et al. (2014) characterized genetic diversity in date palm cultivars on
the basis of morphological variability and molecular markers. They used
morphological traits and molecular markers like RAPD, SSR and AFLPs to determine
the genetic relationship among the cultivars. These technologies resulted in different
values of polymorphism and separate markers for each cultivars. SSRs were found to
be the best for genetic diversity assessment with unique DNA markers for the studied
cultivars. But the three technologies resulted in somewhat different dendrograms.
2.6 Date palm diversity evaluation based on chloroplast DNA sequences
Kress et al. (2007) evaluated a global plant DNA barcode system using nine
putative barcode including both coding and noncoding regions either alone or in
combination in 48 genera, taking two species per genera. They found 88%
discrimination when trnH-psbA region was used in combination with a coding region
like rbcL. Thus they suggested this combination of noncoding trnH-psbA as two locus
global barcode for land plants being universal in nature and having the species
discriminating ability.
24
AlQurainy et al. (2011) assessed eight Saudi date palm cultivars on the basis
of their chloroplast DNA sequence of rpoB and psbA-trnH for molecular signature.
They sequenced a combined matrix of 1147 characters out of which 173 were variable
sites. Their results showed that these sequences can be used as molecular signatures at
seedling stage for trading and farming.
Enan and Ahmad (2012) analyzed the matK and rpoC1 markers, as suggested
by the consortium for the barcode of life plant working group, for identification of
date palm cultivars. They amplified matK and rpoC1 genes in 11 date palm cultivars
for establishing molecular phylogram using MEGA 5 software. They found matK to
be more informative than rpoC1, thus concluding that matK alone or in combination
with rpoC1 can determine genetic variation in date palm.
Akhtar et al. (2014) analyzed fifteen date palm cultivars from Sindh province
of Pakistan on the basis of Rps14 gene of chloroplast. They found very little genetic
distance (0.001), low average evolutionary divergence (0.008) and low nucleotide
diversity (0.007) thus concluding that the studied date palm varieties have high degree
of similarity.
Heise et al. (2015) represented triple barcode data set based on trnL intron,
matK and plastid rbcL for xerothermic plants of the central Europe. They analyzed
126 xerothermic plant species for this purpose. Their database contains rbcL and trnL
barcodes for 117 species, and matK barcodes for 96 species. They were able to
identify the plants up to specie level with 89.6% rbcL, 96.4% matK and 98.4% trnL
barcodes. Their database has application in phylogeography, biodiversity and
conservation.
2.7 Date palm diversity evaluation based on single nucleotide polymorphism
(SNPs)
Date palm has a typical chloroplast genome with little rearrangement and gene
loss or gain. High-throughput sequencing technology facilitates the identification of
intravarietal variations in chloroplast genomes among different cultivars. The date
palm chloroplast genome is 158,462bp in length. Seventy eight SNPs as major
intravarietal polymorphisms were identified by Yang et al. (2010) after extracting
25
369,022 chloroplast sequencing reads from their whole-genome-shotgun data by
putting together an assembly and validating it with intensive PCR-based verification,
coupled with PCR product sequencing. The first publicly available draft genome of
date palm was assembled for Khalas variety of date palm by Al Dous et al. (2011).
More than 3.5 million polymorphic sites were identified by sequencing of eight other
cultivars, including females of the Deglet Noor and Medjool varieties and their
backcrossed males. Germano and klein (1989) identified 13 chloroplast and 12
nuclear SNPs that were able to differentiate among three species of Picea (spruce).
Osman et al. (2003) identified 51 SNPs from Eurychoma longifolia, a medicinal
plant. These SNPs reflect the geographic origin of each species and can differentiate
among natural populations. Jones et al. (2007) while comparing the SSR and SNP
marker technologies for genotypic analysis of maize (Zea mays) reported that the
quality and quantity of marker data provided by SNPs is better the SSRs.
Varshney et al. (2007) assessed the potential of three different types of
markers i.e Expressed Sequence Tag (EST) derived simple sequence repeat markers,
EST derived Single Nucleotide Polymorphism (SNP) and Amplified Fragment Length
Polymorphism (AFLP) for genotyping of wild, cultivated and elite populations of
barley (Hordium vulgare). Their results showed that SSR markers had the highest
(0.593) polymorphic information content value while AFLP had the highest marker
Index and multiplex ratio. The highest effective marker index (0.468) was calculated
for AFLP markers by SSR (0.442) then by SNP (0.341). Their study proved that for
characterizing and conserving the gene bank material, SNP markers are the best while
for analysis of diversity and fingerprinting AFLP and SSR are suitable.
Heinz (2007) found that chloroplast genomes evolve slowly and many primers
for PCR amplification and analysis of chloroplast sequences can be used across a
wide array of genera. The database described is designed to serve as a resource for
researchers who are working on the poorly described chloroplast genomes, whether
for large or small scale DNA sequencing projects, to study molecular variation or to
investigate chloroplast evolution.
Yang et al. (2010) after extracting 369,022 cp sequencing reads from their
whole-genome-shotgun data, they put together an assembly and validated it with
intensive PCR-based verification, coupled with PCR product sequencing. The date
26
palm chloroplast genome is 158,462bp in length. They identified 78 SNPs as major
intravarietal polymorphisms. Date palm has a typical cp genome similar to that of
tobacco that is with little rearrangement and gene loss or gain. High-throughput
sequencing technology facilitates the identification of intravarietal variations in cp
genomes among different cultivars.
Al Dous et al. (2011) assembled a draft genome for a Khalas variety of date
palm, the first publicly available resource of its type for a member of the order
Arecales. The ~380 Mb sequence, spanning mainly gene-rich regions, includes
>25,000 gene models and is predicted to cover ~90% of genes and ~60% of the
genome. Sequencing of eight other cultivars, including females of the Deglet Noor
and Medjool varieties and their backcrossed males, identified >3.5 million
polymorphic sites, including >10,000 genic copy number variations. A small subset of
these polymorphisms can distinguish multiple varieties. They identified a region of
the genome linked to gender and found evidence that date palm employs an XY
system of gender inheritance.
AlMssalem et al. (2013) reported 605.4 Mb genome assembly covering 90%
of the genome and 96% of the genes for ‘Khalas’ which is an elite cultivar of date
palm. Their analysis showed that genome wide duplication has occurred in date palm.
They also discovered that chromosomal regions has low density of single nucleotide
polymorphism and most of the genes for sugar metabolism and stress resistance are
found in this region. They have also shown through the transcriptomic data that
development of date fruit and ripening is related to sugar metabolism.
Sabir et al. (2014) used the whole mitochondrial and plastid genomes
sequences to see the single nucleotide polymorphism in date palm and to assess the
use of this technique for cultivar characterization. They sequenced the mitochondrial
and plastid genomes of nine Saudi date palm cultivars. Sixty million 100bp reads
were generated from total genomic DNA. They used Illumina Hi Seq 2000 platform
for sequencing to identify the SNPs and aligned the sequences separately to the
published reference genomes. They identified cultivar specific SNPs for eight out of
nine cultivars under study. They suggested the use of nuclear SNPs for molecular
characterization of date palm.
27
Mathew et al. (2015) performed genotyping by sequencing of seventy female
date palm cultivars from all the growing regions. Four phoenix species were also
included in this study. They generated up to 65000 SNPs genotyping data from leaves
and fruits of the collected samples. Their analysis suggested that date palm shared
genetic origin of the North Africa and Arabian Gulf. They found genomic regions
having geographically segregating SNPs. They also found some date palm cultivars
like Zayaki, Gorakh and Barani to be falling in the western date palm group. They
concluded that Pakistani cultivars may have resulted from the elite Medjoul cultivar
being pollinated by local pollinators as seed propagated date palms are commonly
used in Pakistan that has resulted in the mixed genetic makeup of the cultivars.
Reproducibility, accuracy, codominance, high polymorphism and low cost are
the characteristics of a good marker system. Although SSRs are commonly being used
for most of the crops these days, there are some problems in their use viz correct
sizing of SSR bands because of the electrophoresis artifacts, unequal allele
amplification by PCR, null alleles may result if mutation occurs in the SSR primer
binding site and size homoplasy which means that the alleles are of equal size but
they may not necessarily have the same sequence (Jones et al. 2007). A phylogenetic
study based on only one gene or marker shows the evolution of that specific marker
and such interpretation may be misleading because the other genes may show
different evolution rate if horizontal gene transfer phenomenon is also involved
(Patwardhan, 2014).
Pintaud et al. (2010) used 16 SSR markers and one dodecanucleotide plastid
minisatellite to genotype 308 accessions that belonged to 12 species of genus phoenix.
Their results showed high polymorphism with SSR nuclear loci and five haplotypes at
the minisatellite locus, all individuals of the same species were grouped together.
They concluded that domesticated date palm originated from wild population of the
Phoenix dactylifera and the other species may have a local genetic contribution. This
study shows the importance of SSR markers for evaluation of taxonomy up to species
level but sequence based phylogenetic analysis is needed to detect the sufficient level
of variation (Pintaud et al., 2010). Morphological based phylogenetic approach is as
important as molecular analysis based method as the structure of basic biomolecules
of all organism is similar and morphological characters of an organism are the
28
illustration of its genome, protein, transcriptome profiles. Thus the combination of the
two methods gives strength to the phylogenetic relationship of the organism
(Patwardhan et al., 2014).
Hence it is concluded that PCR based molecular markers have been
successfully used for genetic diversity analysis of the date palm cultivars and in some
cases identification keys have also been developed for few cultivars but recent
disclosure of the date palm genome and advances in molecular biological techniques
necessitates the development of an authentic identification key for date palm cultivars
based on knowledge of the date palm genome sequence.
29
III. MATERIALS AND METHODS
This research work was carried out at National Institute for Genomics and
Advanced Biotechnology (NIGAB), National Agriculture Research Center (NARC),
Islamabad and Plant Biochemistry and Biotechnology Laboratory, Biosciences
Department, COMSATS, Islamabad during 2012-2015. This study was conducted to
analyze the diversity of date palm cultivars grown in Pakistan as measured by
morphology of leaves, trunk and fruit, proximate composition of fruit and DNA
markers.
3.1 Morphological characterization
Forty five date palm cultivars grown at Date Palm Research Farm, Jhang and
Horticulture Research Station, Bahawalpur (Punjab), Pakistan were selected and
properly tagged for this study (Table 3.1).
Table 3.1: Date palm cultivars from Pakistan used in this study with their codes
and sampling sites
Samples from Date palm Research Farm
Jhang
Samples from Horticulture
Research Station Bahawalpur
Cultivar Code Cultivar Code Cultivar Code
Akhrot DP-01 Daanda DP-17 Sanduri DP-30
Dhaki DP-02 Begum Jhangi DP-18 Makhi DP-31
Aseel DP-03 Peela Dora DP-19 Dhady DP-32
Halawi DP-04 Shamran DP-20 Kur DP-33
Qantar DP-05 Rachna DP-21 Haleni DP-34
Makran DP-06 Saib DP-22 Eedal shah DP-35
Chohara DP-07 Zerdo DP-23 Sufaidah DP-36
Zaidi DP-08 Shado DP-24 Taar wali DP-37
Berahmi DP-09 Peeli sunder DP-25 Fasli DP-38
Neelum DP-10 Khudrawi DP-26 Basra wali DP-39
Zirin DP-11 Wahan wali DP-27 Pathri DP-40
Kohraba DP-12 Angoor DP-28 Kupra DP-41
Kozananbad DP-13 Champa kali DP-29 Shakri DP-42
Karbaline DP-14 - - Baidhar DP-43
Jansohar DP-15 - - Gajjar wali DP-44
Kokna DP-16 - - Halwain DP-45
30
The trees were of same age and well maintained with uniform cultural
practices. Data were taken in triplicates. Each replication was represented by a single
tree of the cultivar selected randomly for sampling. Seventeen morphological
parameters from standard descriptors set for date palm by Rizk and Sharabasy (2007)
were selected for this study. The parameters studied were trunk diameter, leaf length
and width, leaf base width, spine and pinnae number, midrib length, length of midrib
with spines, length of midrib with pinnae, length of the top pinnae. Length, weight,
volume and diameter of fruit, pulp weight, seed weight and perianth height were also
measured.
Data were scored on mature leaves from second whirl below the canopy of
selected trees for leaf length and width, midrib length, midrib length with spines,
midrib length with pinnae and length of the top pinnae. Leaf length (LL) was
measured from base of the leaf to the top of the last pinnae, leaf width (LW) was
measured at the middle of the leaf across the pinnae, midrib length (ML) was
measured from the first spine at the base of the leaf to the last pinnae at the top.
Midrib length with pinnae (MLP) was measured from first pinnae at the base to the
last pinnae at the tip. Midrib length with spines (MLS) was measured from first spine
at the base of the leaf to the last spine towards the pinnae. Leaf base width (LBW)
was measured at the base of the leaf. Number of pinnae and spines on the frond were
also counted (Figure 3.1).
Figure 3.1: Digram of date palm leaf showing various traits measured
LL: Leaf Length; MLS: Midrib length with spines; LBW: Leaf Base Width; MLP:
Midrib length with Pinnae; LTP: Length of the top pinnae; LW: Leaf width Adapted
from Salem et al., 2008
31
Trunk circumference was measured at a height of 4ft above the ground and
then diameter was calculated with formula:
Diameter = Circumference/𝛑 (Powell, 2005)
Fruit (500g) from every replication of each cultivar under study were collected
from July to September at khalal stage in properly abeled sampling bags and were
brought to the laboratory (Appendix 1). Fruit of all the three replicates of each
cultivar were mixed separately and twenty dates were chosen randomly from each
cultivar for data acquisition. Fruits were thoroughly rinsed with tap water to remove
dirt and foreign material and were air dried. Digital Vernier calipers (Model: CD-
6”CSX Mitutoyo Digimatic Caliper, Japan) was used to determine length from the top
of the perianth to the base of the fruit. Diameter was measured at the mid along the
length of fruit. Perianth height was measured by removing the perianth from fruit and
placing vertically between the jaws of digital Vernier calipers. Fruit, pulp and seed
weight were measured with top load digital balance. Water displacement method was
used to determine fruit volume. Individual fruits were dipped in graduated cylinders
containing water. The difference in initial and final volume of water was measured as
volume of the fruit. The mean fruit morphological data were averaged over 20
replicates. Fruits were photographed using 18.0 mega pixel camera of Cannon (EOS
550D, EF-S 18-135 IS kit, Japan)
3.2 Proximate composition determination
3.2.1 Sample preparation
After recording data for morphological traits, the fruits were chopped finely
with cutter and were immediately subjected to moisture and ash determination.
Remaining samples were kept in properly labelled plastic bags at 4oC for further
analysis.
3.2.2 %Moisture determination
Method number 923.03 described by Horwitz and Latimar (2007) was
followed for determination of moisture content of fruit at khalal stage. Finely chopped
pitted sample (20gm) was taken in a pre weighed empty moisture dish, the sample
32
was heated in oven at 710C for 72hrs. The sample was then cooled to room
temperature in desiccator and weighed again. Moisture content was determined by the
formula:
% moisture =
Where
w1 =initial weight of moisture dish and sample
w2= weight of sample
w3= final weight of moisture dish and sample
3.2.3 %Ash determination
Ash content of date fruit was determined using method number 925.10
described by Horwitz and Latimar (2007). Pitted chopped fruit sample (20 gm) was
taken in pre weighed ash crucible. Sample was heated to 5850F in furnace for
overnight. After cooling in dessicator, the sample was weighed again. Ash content
was determined by the formula:
% ash =
Where
w1=weight of empty crucible
w2= weight of sample
w3=final weight of crucible+sample
3.2.4 Total soluble solid (TSS) determination
Total soluble solids were measured with digital Abbe Refractometer (ATAGO
3T) by putting 2-3 drops of the fruit extract (obtained by squeezing 5 dates from each
sample) on the prism of refractometer and recording the reading in oBrix.
33
3.2.5 Sugar content determination
Lane and Eynon method described by Kirk and Sawyer (1991) was used to
determine sugars as follows:
3.2.5.1 Extraction of Sugar
Finely chopped and pitted sample of date fruit (10gm) was taken in a beaker
and 75 ml of water and ethanol each were added to it. This mixture was boiled for 1hr
on flux system. After 1hr the balls of flux system were removed to let ethanol
evaporate. When the volume reached to 100ml, the sample was removed from flux
system, allowed to cool and then filtered. The volume was made up to 100ml with
distilled water. The solution was neutralized using NaOH and HCl. The solution was
divided into two halves of 50ml each for determination of total and reducing sugars.
3.2.5.2 % reducing sugars determination
To determine % reducing sugars, 1-2 ml of sample solution was taken in a
conical flask. Fehling solution A (5ml) and B (5ml) were added to it and the solution
was boiled for 2 minutes. After boiling 2-3 drops of methylene blue were added to it.
The color of the solution turned blue. Sample sugar solution was added through
pipette till the color of solution turned brick red. Total volume of sample solution
used was noted and put in formula to calculate reducing sugars:
% reducing sugar = 0.051 x 100 x 100
10 x X
Where
0.051=Fehling’s factor
100=volume make up
100=for percentage
10=sample weight
X=titer value
3.2.5.3 %Total sugar determination
For total sugar determination 5gm citric acid was added to the remaining 50
ml solution and the volume was made up to 100ml by distilled water, boiled for
10min and titrated as above recording the volume of the sugar solution used. Total
sugar content was determined by the formula:
34
50= initial volume of sugar solution taken
200=final volume of sugar solution
3.3 Morphology and proximate composition analysis
Basic statistics including mean, standard deviation and range of morphological
and proximate composition data were determined using statistical software Minitab
version 16 (Table 4.1). The morphological and proximate data obtained were
analyzed to find the characteristics contributing to the total variability. Principal
component analysis and correlation tools of Minitab version 16 statistical software
were used for this purpose. (Table 4.2, 4.3 & 4.4). Morphological and proximate data
were converted to interval data and similarity matrices were computed using
Euclidean coefficient. These matrices were then used to draw dendrogram using
Numerical Taxonomy and Multivariate Analysis System NTSYSpc version 2.10 by
Unweighted Pair Group Arithmatic Average (UPGMA) method (Sneath & Sokal
1973) under SAHN subprogram of the software (Figure 4.1).
3.4 Molecular diversity of the date palm
3.4.1 Sampling and DNA Isolation
Soft immature leaves from the suckers of trees tagged for morphological
analysis were sampled in properly labelled sampling bags. Leaf samples were brought
to the laboratory and preserved at -20oC till DNA extraction.
The DNA from frozen leaves of date palm was extracted using modified
CTAB method (Hyder et al., 2011). Approximately 0.5 g of leaf was pulverized in
liquid nitrogen using a pestle and mortar in 1.5 mL CTAB buffer (100 mM Tris-HCl
pH 8.0, 2% (w/v) CTAB, 20 mM EDTA, 1.4 M NaCl, and 1% (v/v) β-
mercaptoethanol) and was incubated at 65°C for 45 minutes in water bath. The
supernatant was extracted with an equal volume of phenol: chloroform: isoamyl
alcohol (24:24:1) and then by chloroform. DNA was precipitated by adding 0.1
volume of 3 M potassium acetate and an equal volume of isopropanol to the
supernatant, followed by incubation for five minutes at room temperature. The
35
resultant pellet was washed with 70% (v/v) ethanol and dissolved in 100 μL of Tris-
EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing RNase (100
μg/mL) and was stored at -20°C until used.
3.4.2 DNA Quantification
The extracted DNA was checked on 1% agarose gel and quantified on
BioSpec Nano Spectrophotometer (Shimadzu, Japan). Gel was prepared by dissolving
1gm agarose in 100ml TAE (1X) buffer in a beaker in microwave oven, it was
allowed to cool up to 60oC (bearable warmth). Ethidium bromide (3ul of 0.5ug/ml
solution) was added to it. The gel was then poured into casting tray fixed with combs
and allowed to solidify. On polymerization the gel was placed in gel tank (containing
1X TAE buffer) and combs were removed. DNA samples were loaded into the wells
after mixing with loading dye (bromophenol blue). Gel was run in 1X TAE buffer at
100volts and 300mAmp current for 20 minutes. After running the gel it was checked
in Alpha Innotech Gel Doc System using Alpha view software version 1.0.1.14
(Alpha Innotech Coorporation). Only those samples having good quality of DNA
were retained for further analysis. DNA was quantified by putting 1ul of each sample
on the target of BioSecp nano spectrophotometer and concentration of DNA was
noted from the system.
3.4.3 Primer Design/Selection
For amplification of simple sequence repeats, forty six SSR primers (Table
3.2), 16 primers developed by Billotte et al., (2004) and 30 primers developed by
Elmeer et al., (2011) were selected. For molecular characterization 1270bp of 16S
rRNA, ~1515bp atp synthase beta subunit (atpB), ~560bp of ribulose bisphosphate
carboxylase large subunit (rbcL) gene, geranyl geranyl biphosphate reductase gene
(GGR) and ~1450bp of maturase K (matK) gene from date palm chloroplast, were
chosen. Primers (Table 4.5) were designed manually on the above mentioned date
palm sequences (Appendix 2) available online (www.ncbi.nlm.nih.gov/). For Single
Nucleotide Polymorphism (SNP) typing, primers (Table 4.6) were designed on SNPs
reported by Al-Dous et al. (2011) using primer3 program of JustBio software
available online (http://www.justbio.com/index.php?page=primer).
36
Table 3.2: Simple sequence repeat markers used in this study showing
amplification status.
Primer Forward sequence Reverse Sequence Repeat
motif
Expected
Allele
size(bp)
*Amplification
status
DP150 CTGCGCCAATCTAAACCATT GCAAATTGCAACAAATCCTTG (GAA)9 177 +
DP151 TTGCTGGTTGAAATGGTGTT GCAACAGATGCTCTTGCTCA (AC)37 168-186 -
DP152 ACGAGTTTTTGGGAGAGCAA GCAAGTTGCCAACATTCTTGT (TAT)8 224 -
DP153 TCATCACAGGCAATGGCTAA GCAGATGGCCATTGAACC (TCA)9 204 +
DP154 ACACACACACACCGCGAAT GCATGTGAGGCGCATATCTA (AC)19 249 +
DP155 CCCCCTCTCTCTCTCTCTCTC GCCAAGAGGATTGGAGATTG (TC)51 200 -
DP156 TGTGTGTGTGTGTGTGTGTGA GCCATTGTTTGTGTGGACTG (GA)17 221 -
DP157 TGGACAATGACACCCCTTTT GCCCACACAACAACCTCTCT (TC)19 180-244 -
DP158 TCATTGGCTAATCCACACACA GCCTTGTGGTCATGAGAGGT (GA)29 204 -
DP159 AGCTCCAATTTGCTGCAGAG GCTGACCTGGAGTCCAAAAC (TC)27 156-172 +
DP160 AAGAGCGACAATCATGACCA GGAAATTGAAGGGCATCTTG (GAAA)5 108-136 -
DP161 TGGTTGCTGCTTATCTGCTG GGAGGGAACCGAGAGAGAGA (CT)13 211 -
DP162 TGGACTTCAAGAAGTGCGAAT GGCAGTCACATTTTGCTTCA (TACA)9 183 +
DP163 GTGCGTGTGTGTGTGTGTGT GGCTGTTTGGGTTCGTACTG (GA)19 215 -
DP164 GGACCAAGAACCGACAGTTG GGGAAGGTGAGGTGGTATGA (ATAG)6 200 +
DP165 AAGCATCCTATGGCTTTGACA GGGCTGTATGTGATGCATTG (AATA)5 222 -
DP166 CAATTTCTTCTCGCCTGGAG GGGGTTTCTTTTCCTTCTGC (GAAA)5 210 +
DP167 ACATCCAATGGCATCCAAAT GGGTTTCCAGGTTTTCTTCTC (GAAA)6 243 +
DP168 GCAGCAAAGCCCTTAGGC GGTGTTATGTGCAGCCAATG (CAT)8 163-175 +
DP169 GCATGGACTTAATGCTGGGTA GGTTTTCCTGCCAACAACAT (AAT)12 129-223 +
DP170 TCTTTGGGCTTACGACAACC GTATGGCCCAAGATGCAGAT (AGGG)5 195-227 +
DP171 GTGGGAGTAGCGAGGTATGG GTCCGGCACTTTAGGAAGTT (TTC)10 197-218 -
37
DP172 GTGTTTGGGCCTATTTCCT GTCCTCCTCCTCCTCTGTCC (AGG)11 199-235 -
DP173 GTGTTTGGGCCTATTTCCT GTGTTTGGGCCTATTTCCT (TC)27 198 -
DP174 GTGTTTGGGCCTATTTCCT GTGTTTGGGCCTATTTCCT (CGTG)5 187 -
DP175 GTGTTTGGGCCTATTTCCT GTGTTTGGGCCTATTTCCT (CA)19 196-274 -
DP176 GTGTTTGGGCCTATTTCCT GTGTTTGGGCCTATTTCCT (CAA)9 198 +
DP177 TTCCTTGGGCTCACTTCAAC TAACATGCCAGCAAAGGTGA (AGGC)6 216 +
DP178 AGTTTGTCAGGCCATTTGGT TACATGTGCGTATCGGGAGA (TC)19 186 -
DP179 GGTTAGCCATCCAAAAGTGG TATGTAGCCTCCACCGCATC (ATTT)5 183 +
mPdCIR010 ACCCCGGACGTGAGGTG CGTCGATCTCCTCCTTTGTCTC (GA)22 114-236 +
mPdCIR015 AGCTGGCTCCTCCCTTCTTA GCTCGGTTGGACTTGTTCT (GA)15 104-150 +
mPdCIR016 AGCGGGAAATGAAAAGGTAT ATGAAAACGTGCCAAATGTC (GA)14 104-198 +
mPdCIR025 GCACGAGAAGGCTTATAGT CCCCTCATTAGGATTCTAC (GA)22 192-244 ++
mPdCIR032 CAAATCTTTGCCGTGAG GGTGTGGAGTAATCATGTAGTAG (GA)19 248-310 +
mPdCIR035 ACAAACGGCGATGGGATTAC CCGCAGCTCACCTCTTCTAT (GA)15 154-198 +
mPdCIR044 ATGCGGACTACACTATTCTAC GGTGATTGACTTTCTTTGAG (GA)19 250-330 -
mPdCIR048 CGAGACCTACCTTCAACAAA CCACCAACCAAATCAAACAC (GA)32 108-198 -
mPdCIR050 CTGCCATTTCTTCTGAC CACCATGCACAAAAATG (GA)21 114-208 +
mPdCIR057 AAGCAGCAGCCCTTCCGTAG GTTCTCACTCGCCCAAAAATAC (GA)20 214-284 +
mPdCIR063 CTTTTATGTGGTCTGAGAGA TCTCTGATCTTGGGTTCTGT (GA)17 100-216 -
mPdCIR070 CAAGACCCAAGGCTAAC GGAGGTGGCTTTGTAGTAT (GA)17 154-230 +
mPdCIR078 TGGATTTCCATTGTGAG CCCGAAGAGACGCTATT (GA)13 106-184 +
mPdCIR085 GAGAGAGGGTGGTGTTATT TTCATCCAGAACCACAGTA (GA)29 110-201 ++
mPdCIR090 GCAGTCAGTCCCTCATA TGCTTGTAGCCCTTCAG (GA)26 108-202 -
mPdCIR093 CCATTTATCATTCCCTCTCTTG CTTGGTAGCTGCGTTTCTTG (GA)16 150-188 +
+ means monomorphic bands; ++ means polymorphic bands; - means No amplification
38
3.5 PCR based molecular diversity analysis
3.5.1 Amplification of simple sequence repeats (SSRs)
SSR polymorphism was detected by polymerase chain reaction. PCR was
carried out in 96 well veriti®thermal cycler of Applied Biosystems. Initially ten
samples were selected randomly for amplification of all SSR markers. PCR reaction
mixture contained about 25ng DNA template, Taq buffer (75mM Tris-HCl, 20mM
(NH4)2SO4, 0.01% (v/v) Tween, 3mM MgCl2, 0.2mM of dNTPs mix, 1.0 units Taq
DNA Polymerase (recombinant) (Fermentas, UAB Lithuania), and 10 pM of each
primer. PCR protocol for SSR primers developed by Billote et al. (2004) was an
initial denaturation of 5min at 95oC followed by 35 cycles of denaturation at 94oC for
30sec, annealing temperature of 48-60oC depending on the primer for 90 sec,
extension temperature of 72oC for 90sec, a final extension temperature of 7min and a
final hold at 4oC (Ahmad and Al-Qaradawi, 2009). PCR program for SSR primers
developed by Elmeer et al. (2011) was initial denaturation of 95oC for 10min, 35
cycles of denaturation for 30 sec at 95oC, annealing at primer dependent temperature
for 30sec and extension of 1min at 72oC followed by 1 cycle of final extension at
72oC for 7min.
3.5.2 Gel Electrophoresis
3.5.2.1 Agarose gel electrophoresis
PCR product with SSR primers was run on 2% agarose gel in TAE buffer for
35 min at constant current of 300mAmp and voltage at 100V (as mentioned above).
SSR bands were measured with Alpha Innotech Gel Doc System using Alpha view
software version 1.0.1.14 (Alpha Innotech Coorporation). PCR amplification and gel
electrophoresis were repeated for all the forty five samples with primers which
appeared to be polymorphic in initial screening with ten samples.
3.5.2.2 Polyacrylamide gel electrophoresis
PCR product with SSR primers was also run on polyacrylamide gel for better
resolution of bands. Glass plates were cleaned with Kim wipe after washing with
detergent and fixed in the apparatus. The apparatus was checked for leakage using
39
distilled water. After setting the apparatus 8% polyacrylamide gel was formed by
mixing 7.11 ml of 45% acrylamide: bisacrylamide solution with 4ml of 10X TBE
buffer and 28.61 ml of double distilled water. 280ul of 10% Ammonium per sulphate
(APS) solution and 14ul TEMED ((N,N,N',N'-tetramethylethylenediamine) were
added to the solution just before pouring in the glass plates. Combs were inserted
immediately after pouring the gel in the glass plates and gel was allowed to
polymerize for 30 minutes. After solidification gel caster was fixed in the gel tank
containing 1X TBE running buffer. The same buffer was also poured between the
glass plates. Combs were removed carefully and 15ul of PCR product mixed with 5ul
of bromophenol blue dye was loaded in the wells. 10-300bp DNA ladder was also
loaded in one well. Electrodes were fixed and gel was run at 60volts and 300mAmp
current for 4 hrs. Gel was stained in ethidium bromide solution after running and
visualized in Gel Documentation System as mentioned for agarose gel.
3.5.3 SSR data analysis
Each polymorphic band with SSR markers on the gel was considered as an
allele. Bands on gel were scored as present (1) or absent (0) to form binary data
matrix. Data were then computed with NTSYSpc version 2.10j software to produce
similarity matrix using Jaccard’s coefficient to detect the polymorphism among the
cultivars on the basis of size of alleles. The matrix was then computed to form
dendrogram (Figure 4.4).
3.6 Sequence based molecular analysis
3.6.1 Amplification of chloroplast DNA and SNPs
For sequence based molecular evaluation a subset of seven samples was
selected. The selected cultivars were Dhaki, Aseel, Halawi, Qantar, Hamin wali,
Kupra and Shakri. The genes of ribulose bisphosphate carboxylase large subunit
(rbcL), atp synthase beta subunit (atpB), geranyl geranyl biphosphate reductase
(GGR), maturase K (matK) and 16S rRNA from date palm chloroplast genome were
amplified from seven samples. PCR reaction for chloroplast genes and for
amplification of SNPs contained about 50 ng DNA template, Taq buffer (10mM Tris-
HCl, pH 8.8, 50mM KCl and 1.0 % (v/v) Nonidet P40) 1.5mM MgCl2, 200μM of
40
each dNTPs, 5.0 units Taq DNA Polymerase (recombinant) (Fermentas, UAB
Lithuania), and 25pM of each primer. The thermal profile for all primer sets included
pre-PCR denaturation at 96 °C for 3 min followed by 35cycles of denaturing at 96°C
for 30 s, annealing at 53 °C for 30s and extension at 72°C for 1 min, and a final
extension at 72 °C for 20min.
3.6.2 Gel electrophoresis, PCR product purification and sequencing
The PCR products were analyzed using 1% agarose gel and purified using
Wizard® SV Gel and PCR Clean-up system (Cat#A9282, Promega, Madison, USA)
following manufacturer’s instructions. PCR product was transferred to the eppendorf
tubes and vortexed slightly. Membrane binding solution (50ul) was added to each
eppendorf tube and centrifuged for 1 min. when the kit was opened for the first time,
75ml of ethanol was added to the membrane binding solution before use. Mixture of
PCR product and membrane binding solution were transferred to the SV mini column
assembly marked the same as eppendorf tubes and incubated at room temperature for
1 min and then centrifuged for 1 min at 14800rpm. Flow through was discarded from
the collection tube. Mini columns were inserted in the collection tubes. Membrane
wash solution (700ul) was added to the mini columns and centrifuged at 14800 rpm
for 1 min. Flow through was discarded and mini columns were again inserted into the
collection tubes. Wash step was repeated with 500ul of membrane wash solution and
centrifuged for 1 min. Flow through was discarded and empty collection tubes and
column assemblies were centrifuged for 1 min and then incubated at room
temperature for few min. Minicolumns were transferred to the clean eppendorf tubes.
Nuclease free water (40ul) was added to the mini columns and centrifuged for 1 min.
Again 30ul of the nuclease free water was added and centrifuged for 1 min. The
purified DNA was in the Eppendorf tubes containing mini columns. Minicolumns
were discarded and DNA was stored at -20oC.The purified PCR product was
sequenced commercially using DNA Sequencing Services (Macrogen, Inc. Seoul,
Korea).
41
3.6.3 Sequence based molecular analysis of Chloroplast DNA
The sequence of both strands of every fragment of chloroplast DNA amplified
from each sample was assembled separately using DNA Dragon, DNA Sequence
Contig Assembler Software version 1.5.2 (www.sequentix.de) and when found
identical in the triplicate samples of each cultivar was represented by a single
sequence and used for sequence analysis. The consensus sequences for each cultivars
for chloroplast gene/fragment along with sequences available for corresponding
gene/fragment for other cultivars reported in GenBank were aligned in ClustalW
Sequence Alignment program (Thompson et al., 1994) implemented in the Molecular
Evolutionary Genetics Analysis Program (MEGA) version 6 (Tamura et al., 2007).
3.6.4 Single Nucleotide Polymorphism typing
For phylogenetic analysis, the sequences of all twelve SNPs in diploid state
from the same cultivar were combined into a single concatemeric sequence, which
were further used to generate alignments in MAFFT version 6.847b
(mafft.cbrc.jp/alignment/software). The corresponding SNPs sequence data for
Arabian, Qatari, Californian and North African cultivars was obtained from report of
Al-Dous and colleague (Al-Dous et al., 2011) and website (http://qatar-weill. cornell.
edu/ research/datepalmGenome/download.html). The sequence alignments were used
to identify best nucleotide substitution model and construct maximum likelihood
(ML) and maximum parsimony (MP) tree (Figure 4.5) in Molecular Evolutionary
Genetic Analysis (MEGA) version 6 (Tamura et al., 2013). An Iterative Dichotomiser
3 (ID3) decision-tree learning algorithm (Quinlan, 1986) was used to identify the
minimum set of SNPs required to discriminate among all the 16 date palm cultivars
SNPs data set (Figure 4.6). To reduce the number of fragments which could be
minimally typed, an empirical stepwise forward feature i.e., fragments selection
strategy was used in which the fragments bearing the highest number of SNPs were
given precedence over the other fragments.
3.6.5 Submission of Sequences
The sequences were submitted to National Centre for Biotechnology
Information (NCBI) Gen Bank database (Table 4.7).
42
IV. RESULTS
This is the first ever study of diversity evaluation of date palm varieties grown
in Pakistan on combined phenotypic, proximate composition and molecular basis.
4.1 Morphological and Proximate Analysis
The present study was conducted to evaluate the genetic and morphological
variation in forty five date palm cultivars of Pakistan (Table 3.1) and to find traits that
can discriminate among the cultivars.
Mean data scored on mature leaves of selected trees of all cultivars under
study for leaf length and width, midrib length, midrib length with spines, midrib
length with pinnae and length of the top pinnae, leaf base width and number of pinnae
and spines on the frond is shown in Appendix 3.
Range, Mean and standard deviation of the values for the observed traits were
calculated. The standard deviation in data showed large variation among
characteristics of cultivars studied (Table 4.1).
Descriptive statistics of the data collected showed that trunk diameter ranged
from 39 cm to 83.3 cm with a mean value of 52.2 cm. Maximum trunk diameter was
recorded for Gajjarwali (83.3cm) followed by Haleni (64.8cm) and Shamran (61.3cm)
while minimum trunk diameter was recorded for Taarwali (39.0 cm) followed by
Pathri (39.8 cm) and Sanduri (42 cm). Trunk diameter of 28 cultivars ranged from 50-
64.86 cm while the remaining 16 cultivars had diameter of less than 50 cm. Among
the studied forty five cultivars maximum leaf length (420 cm) was recorded in
cultivar Sufaidah followed by Dhady (418 cm) and Gajjar wali (412 cm) while Fasli
had the minimum leaf length followed by Makhi (271 cm) and Hamin wali (273 cm)
respectively.
Cultivar Saib had the maximum leaf width (108 cm) followed by Sufaidah
(103cm) and Basra wali (102 cm) respectively while minimum value of leaf width
was recorded in Berahmi (54cm) followed by Shado (64.3 cm) and Zerdo (65.3 cm)
respectively. Shado and Champa kali were found to have minimum value of leaf base
43
width (5cm) followed by Qantar, Makran and Zaidi (5.33 cm) while Baidhar had the
maximum value of leaf base width (10.3 cm) among the studied cultivars followed by
Dhady (9.67 cm) and Gajjarwali (9 cm).
Table 4.1: Descriptive statistics of traits of date palm cultivars studied
Variables Range Mean St. Dev
Trunk diameter (cm) 39-83.3 52.2 7.6
Leaf length (cm) 244-420 340.9 39.2
Leaf width (cm) 54-108 82.5 11.2
Leaf base width (cm) 5-10.3 7.01 1.2
Spine number 9.3-35.3 19.8 5.5
Midrib length with spines (cm) 31-114.6 66.9 17.8
Midrib length (cm) 222.6-395.3 314.7 39.4
Pinnae number 138.3-243 182.1 24.8
Midrib length with pinnae (cm) 71.6-313.3 234 50.5
Length of top pinnae (cm) 16.6-43.3 25.7 5.3
Fruit weight (gm.) 4.7-26.3 10.9 4
Fruit volume (cm3 ) 4-26 11.0 4.2
Fruit diameter (cm) 1.5-3.2 2.2 0.3
Fruit length (cm) 2.6-5.4 3.6 0.5
Pulp weight (gm.) 3-26.1 9.5 4.1
Seed weight (gm.) 0.7-1.8 1.1 0.2
Perianth height (mm) 0.5-5.6 2.6 1.2
% Moisture 45.2-83 65.2 8.2
TSS (Brix) 14-52 32.9 7.4
% Reducing Sugars 9.8-38.1 23.3 6.1
% Total Sugars 12.8-46.2 27.5 7
% Ash 0.6-4.5 3 0.7
44
Dhakki had the minimum number of spines (24.33) followed by Dhady (9.33)
and Rachna (11.33) while Makran had the highest number of spines (35.33) followed
by Begum Jhangi (31) and Shado (30). Maximum Midrib length with spines was
recorded in Begum Jhangi (114.6 cm) followed by Makran (105 cm) and Aseel (100
cm) respectively. Sufaidah had the maximum midrib length (395.3 cm) followed by
Dhady (385.67 cm) and Gajjar wali (373.33 cm) while minimum midrib length was
recorded in cultivar Zirin (222.6 cm) followed by Fasli (223 cm) and Makhi (237 cm).
Highest number of pinnae were recorded in Zaidi (243) followed by Rachna (234.33)
and Angoor (232.33) while Makhi had the lowest number of pinnae (138.33) among
the studied cultivars followed by Pathri (141.67) and Fasli (145.67). Champakali had
the maximum midrib length with pinnae (313.3 cm) followed by Peeli Sunder (306.33
cm) and Chohara (298.7 cm) while minimum midrib length with pinnae were
recorded in Qantar (71.67 cm) followed by Akhrot (115.33 cm) and Aseel (120.67
cm). Length of the top pinnae was maximum in Saib (44.33 cm) followed by
Gajjarwali (38.67 cm) and Shakri (36 cm) while Kozanabad had minimum length of
top pinnae (16.66 cm) followed by Haleni (17.33 cm) and Jansohar (17.67 cm).
Dhaki had the maximum fruit weight (26.36 gm) followed by Chohara (20.27
gm) and Saib (16.5 gm) while minimum fruit weight was recorded for Shado (94.76
gm) followed by Angoor (5.66 gm) and Zerdo (5.96 gm). Maximum fruit volume was
recorded in Dhaki (26 cm3) followed by Chohara (19.66 cm3) and Dhady (18.66 cm3)
while Shado had the minimum fruit volume (4 cm3) followed by Sanduri (5.5 cm3)
and Neelum (6 cm3). Makran had the highest fruit diameter (3.25 cm) followed by
Dhaki (2.94 cm) and Saib (92.83 cm) while Shado had the minimum diameter (1.53
cm) followed by Zerdo (1.62 cm) and Angoor (1.66 cm). Maximum fruit length was
recorded in Dhaki (5.45 cm) followed by Chohara (4.68 cm) and Zirin (4.61 cm)
while Angoor had minimum fruit length (2.69 cm) followed by Akhrot (2.69 cm) and
Halwain (2.93 cm). Maximum pulp weight was recorded for Dhaki (26.1 gm)
followed by Chohara (17.22 gm) and Jansohar (15.76 gm) while minimum pulp
weight was recorded for Shado (3.06 gm) followed by Angoor (3.7 gm) and Begum
Jhangi (4.53 gm). Maximum seed weight were recorded in Zirin (1.86 gm) followed
by Gajjar wali (1.73 gm) and Basra wali (1.63 gm) while minimum seed weight was
recorded in Haleni (0.7 gm) followed by Jansohar and Baidhar (0.8). Angoor had the
heighest perianth (5.61mm) followed by Rachna (5.6 mm) and Peeli Sunder (4.8 mm)
45
while Chohara had the smallest (0.5mm) perianth height followed by Shakri (0.67
mm) and Zerdo (0.93 mm) respectively. Peeli sunder had the highest moisture content
(83.08%) at ‘khalal’ stage among the studied cultivars followed by Jansohar (79.58%)
and Haleni (76.42%) respectively while Shakri had minimum moisture content
(45.2%) followed by Hamin wali (47.37%) and Kozanabad (48.83%) respectively.
Highest value of total soluble solids was recorded in Hamin wali (52 oBrix) followed
by Taar wali (50 oBrix) and Zaidi (48 oBrix) respectively, while minimum TSS were
recorded in Peeli Sunder (14 oBrix) followed by Halawi (20 oBrix) and Jansohar (20
oBrix) respectively. Hamin wali had the maximum content of reducing sugars
(38.16%) followed by Taar wali (38.28%) and Zaidi (35.22%) respectively while
Peeli sunder had the least content of reducing sugars (9.87%) followed by Neelum
(12.21%) and Haleni (14.92%) respectively. Total sugar content was found to be
maximum in Taar wali (46.29%) followed by Hamin wali (42.08%) and Zaidi
(40.44%) respectively while minimum total sugar (12.86%) was recorded in Peeli
Sunder followed by Neelum (15.22%) and Jansohar (16.31%) respectively. Ash
content of Neelum was maximum (4.3%) among the studied cultivars at ‘khalal’ stage
followed by Gajjar wali and Peeli Sunder (4.22%) while minimum content of ash was
recorded in Daanda (0.64%) followed by Saib (2.12%) and Shakri (2.26%)
respectively (Table 4.1 and Appendix 3).
4.2 Principal Component Analysis (PCA)
Morphological and proximate composition data were subjected to Principal
Component Analysis. Principal components (PCs) with Eigen values of one or greater
than one were retained as important for analysis of data. The first seven components
had eigen values equal to or greater than one and contributed 81% variability in the
data (Table 4.2).
46
Table 4.2: Principal components (PCs) showing Eigen values and proportion of
variability among date palm cultivars.
PCs Eigen value Proportion of
variability Cumulative
1 5.48 0.24 0.24
2 3.85 0.17 0.42
3 2.73 0.12 0.54
4 1.78 0.08 0.63
5 1.56 0.07 0.70
6 1.26 0.05 0.75
7 1.15 0.05 0.81
The first component (PC1) had an Eigen value of 5.48 and contributed 24%
variability in the data (Table 4.2). Five variables including fruit weight (0.356), fruit
volume (0.353), pulp weight (0.352), fruit length (0.269) and total soluble solids
(0.274) had higher absolute values of coefficient than other variables in this
component (Table 4.3).
The second component (PC2) had an Eigen value of 3.85 and explained 17 %
variability in the data (Table 4.2). In PC2 the three variables i.e % reducing sugars
(0.327), % total sugars (0.322) and % ash (-0.329) had greater weight than other
variables (Table 4.3). PC3 with Eigen value of 2.73 explained 12% variability in the
data and represented two variables, leaf length (0.391) and midrib length with pinnae
(0.301) having the higher values of coefficient than other variables in this component.
Eigen value of PC4 is 1.78 and thus represented one variable. The variable having
highest coefficient value in this component is the spine number (0.443) being
responsible for causing variance in this component. The eigne value of PC 5 is 1.56
and represents one variable mainly responsible for variance of this component. Leaf
width has the highest coefficient (0.470) and is therefore responsible for variability of
this component. Eigen value of PC6 is 1.26 it also represents one variable. The
variable with highest value of coefficient in this component is leaf base width (0.516 )
causing variation in data. Eigen value of PC7 is 1.15 represented one variable.
Perianth height (0.696) having the highest coefficient is responsible for variation of
this component (Table 4.2 and 4.3).
47
4.3 Correlation
Correlation matrix of the date palm traits studied is represented in Table 4.4. A
strong positive correlation (r=0.95) was found between midrib length and leaf length.
Midrib length with spines had a strong positive correlation (r=0.64) to spine number.
Similarly volume, diameter and length of fruit alongwith pulp weight has strong
positive correlation (r=0.96, r=0.67, r=0.70 and r=0.96 respectively) with fruit weight.
Fruit diameter, its length and pulp weight were found to have positive correlation
(r=0.69, r=0.96, and r=0.96 respectively) with fruit volume. Fruit length and pulp
weight were positively correlated (r=0.51 and r=0.69 respectively) to fruit diameter.
Pulp weight had a positive correlation (r=0.68) with fruit length. Total soluble solids,
% reducing sugars and % total sugars were negatively correlated with % moisture
content of date fruit (r=-0.71, r=-0.70 and r= -0.79 respectively). Reducing sugars and
% total sugars were positively correlated with total soluble solids (r=0.90 and r=0.91
respectively). Total sugars was also positively correlated with % reducing sugars
(r=0.93).
48
Table 4.3: Correlation among the variables studied and the first seven
components
Variables PC1 PC2 PC3 PC4 PC5 PC6 PC7
Trunk diameter (cm) 0.203 -0.108 -0.280 0.008 -0.120 0.256 -0.189
Leaf length (cm) 0.268 -0.095 -0.391 0.080 0.015 -0.175 0.065
Leaf width (cm) 0.087 0.113 -0.202 0.380 0.470* 0.042 0.130
Leaf base width (cm) 0.089 0.115 -0.018 0.337 0.094 -0.516* -0.282
Spine number -0.016 -0.170 -0.194 -0.443* 0.387 0.022 -0.050
Midrib length with spines (cm) 0.022 -0.217 -0.127 -0.380 0.399 -0.233 -0.063
Midrib length (cm) 0.257 -0.127 -0.058 0.039 -0.034 -0.290 0.081
Pinnae number 0.081 -0.058 0.049 -0.371 -0.252 0.037 0.289
Midrib length with pinnae (cm) 0.140 0.049 -0.301* 0.129 -0.296 -0.155 -0.069
Length of top pinnae (cm) 0.070 0.041 -0.176 0.285 0.428 0.425 -0.039
Fruit weight (gm.) 0.356* 0.227 0.103 -0.054 -0.057 -0.019 0.096
Fruit volume (cm3) 0.353* 0.236 0.105 -0.029 -0.021 -0.007 0.074
Fruit diameter (cm) 0.229 0.249 0.174 -0.127 0.236 -0.003 0.261
Fruit length (cm) 0.269* 0.251 -0.026 -0.156 -0.091 0.129 -0.256
Pulp weight (gm.) 0.352* 0.219 0.125 -0.066 -0.004 -0.019 0.070
Seed weight (gm.) 0.029 0.259 -0.249 -0.095 -0.072 0.442 -0.246
Perianth height (mm) -0.088 -0.080 -0.137 0.219 -0.106 0.138 0.696*
% Moisture 0.223 -0.322 0.183 0.035 0.007 0.052 0.064
TSS (0Brix) -0.274* 0.279 -0.173 -0.057 0.029 0.103 -0.012
% Reducing Sugars -0.266 0.327* -0.173 -0.037 -0.018 -0.021 0.046
% Total Sugars -0.263 0.322* -0.209 -0.028 -0.015 -0.151 0.027
% Ash 0.010 -0.329* -0.114 0.214 -0.161 0.149 -0.236
*Variables with greater weightage in a principal component
49
Table 4.4: Correlation matrix of the traits studied
T. Dia LL LW LBW SN MLS ML PN MLP LTP F. Wt F. Vol F. Dia F. Len Pp. Wt S. Wt P. Ht %M TSS
% Red.
Sugars
%
T.Sugars
LL 0.48
LW 0.12 0.35
LBW 0.01 0.21 0.23
SN 0.13 0.12 0.02 -0.21
MLS 0.08 0.24 -0.04 -0.09 0.64*
ML 0.45 0.93* 0.27 0.19 0.13 0.27
PN 0.35 0.44 -0.21 -0.23 0.31 0.13 0.44
MLP 0.31 0.46 0.11 0.14 -0.07 -0.11 0.48 0.20
LTP 0.20 0.23 0.52 0.07 0.07 -0.01 0.08 -0.06 0.04
F. Wt 0.21 0.31 0.14 0.23 -0.19 -0.17 0.27 0.10 0.21 0.03
F. Vol 0.19 0.33 0.16 0.24 -0.21 -0.17 0.27 0.05 0.18 0.09 0.96*
F. Dia -0.03 0.04 0.23 0.06 -0.06 -0.03 0.06 -0.05 -0.02 0.11 0.67* 0.69*
F. Len 0.25 0.26 0.08 0.08 -0.10 -0.07 0.22 0.07 0.29 0.04 0.70* 0.69* 0.51*
Pp. Wt 0.20 0.28 0.15 0.22 -0.16 -0.13 0.24 0.06 0.17 0.06 0.96* 0.96* 0.69* 0.68*
S. Wt 0.21 0.14 0.11 -0.05 0.01 -0.18 -0.01 0.17 0.17 0.25 0.18 0.19 0.04 0.47 0.13
P. Ht -0.01 0.06 0.13 -0.16 -0.08 -0.09 0.02 0.14 0.05 0.04 -0.18 -0.21 -0.15 -0.35 -0.24 -0.06
%M 0.20 0.26 -0.11 -0.09 0.09 0.16 0.31 -0.03 -0.04 -0.01 0.17 0.20 0.14 0.01 0.21 -0.39 -0.02
TSS -0.24 -0.27 0.08 0.01 0.04 -0.11 -0.26 -0.01 -0.02 -0.05 -0.34 -0.30 -0.11 -0.09 -0.32 0.26 0.08 -0.71*
% R. Sugars -0.29 -0.33 0.05 -0.01 -0.08 -0.21 -0.33 -0.00 -0.01 0.01 -0.27 -0.24 -0.05 -0.04 -0.26 0.33 0.10 -0.70* 0.90*
% T.Sugars -0.30 -0.23 0.09 0.05 -0.09 -0.16 -0.24 0.01 0.01 -0.05 -0.27 -0.24 -0.08 -0.08 -0.27 0.29 0.06 -0.79* 0.91* 0.93*
%Ash 0.32 0.21 -0.06 -0.07 0.06 0.07 0.25 -0.01 0.06 0.04 -0.29 -0.28 -0.47 -0.15 -0.26 -0.17 0.13 0.44 -0.21 -0.31 -0.31
Correlation significance (<0.05). Abbreviations: TD; Trunk diameter, LL; Leaf length, LBW; Leaf base width, SN; Spine number, MLS; Midrib length with spines, ML; Midrib length, PN; Pinnae number, MLP; Midrib length
with pinnae, LTP; Length of top pinnae, F. Wt; Fruit weight, F. Vol; Fruit volume, F. Dia; Fruit diameter, F. Len; Fruit length, Pp Wt; Pulp weight, S. Wt; Seed weight, P. Ht; Perianth Height.
50
4.4 Dendrogram based on morphological and proximate data
The dendrogram based on combined morphological and proximate data
(Figure 4.1) showed that dissimilarity level among the forty five date palm cultivars
ranged from 4.23 to 6.08.
This dendrogram divided the forty five cultivars into two main clusters.
Cluster1 included six cultivars in which Aseel and Halawi paired together and were
closely related to Jansohar and distantly related to Dhadi. Chohara was paired with
Peelisunder in this cluster. Cluster 2 is further divided into two sub clusters. Sub
cluster 2.1 had 10 cultivars mostly paired together. Qantar and Begum Jhangi,
Neelum and Zerdo, Shado and Angoor, Sanduri and Halwain were paired together and
closely related to each other while distantly related to Wahanwali and Taarwali in the
same cluster. Sub cluster 2.2 was further divided into two groups. One subgroup 2.2/1
had 16 cultivars including Haleni and Kupra paired together and closely related to
Kozanabad and distantly related to Zahidi. Baidhar and Champakali were paired
together and closely related to Shamran and distantly related Makran. Berahmi and
Zirin paired together with closely related Khurdrawi and distantly related Kohraba
and Daanda clustered at the same point. Rachna and Saib were paired together while
Dhaki was placed in the same cluster at a distance from the rest of the cultivars.
Second subgroup 2.2/2 of sub cluster 2 had thirteen cultivars grouped mostly in pairs.
The first sub cluster had only three cultivars Saib and Akhrot paired together and
closely related to Makhi. The second sub cluster had Kerbaline and Peeladora paired
together with closely related Kokna and distantly related Eedalshah and Basrawali,
the last two were paired together. The other paired cultivars of this cluster were
Sufaidah and Gajjarwali and Kur and Fasli paired together and closely related to
Shakri.
51
Figure 4.1: Dendrogram of 45 date palm cultivars studies based on
morphological and proximate composition data
The Dendrogram was computed from similarity matrix using Jaccard’s coefficient
with NTSYSpc ver. 2.10j
Cluster 1
Cluster 2
Cluster 3
Cluster 4
52
4.5 PCR based Molecular analysis
DNA markers that can discriminate among the genotypes are called
polymorphic markers and these are important for diversity studies. While those
markers that cannot differentiate among the genotypes are called monomorphic. A
total of forty six SSR markers (Table 3.2) were used in this study to find genetic
variation in forty five date pam cultivars. Initially fifteen samples were screened for
amplification by these markers. Twenty four markers produced monomorphic bands
while twenty markers did not amplify the tested samples therefore these makers were
not considered for further study. Only two out of forty six SSR markers showed
polymorphism with amplification of five amplicons in total. Out of thirty markers
developed by Elmeer et al. (2011) thirteen markers amplified to give monomorphic
bands and the remaining seventeen either did not amplify or gave invalid
amplification with our samples. Only two of the 16 microsatellite markers
mpdCIRO25 and mpdCIRO85 (developed by Billotte et al. 2004) produced
polymorphic bands within the expected range. Ten markers produced monomorphic
bands in the expected range while four of these primers failed to amplify. Thus twenty
six markers produced a total of twenty nine scorable and five polymorphic
amplicons/bands. Therefore only the two polymorphic markers were used for
assessment of genetic relationship in all the samples (Figure 4.2 and 4.3).
Figure 4.2: PCR amplification of mpdCIRO25 in45 date palm cultivars scored on
2% agarose gel M=100bp plus DNA ladder (Thermo Scientific)
53
Figure 4.3: PCR amplification of mpdCIRO85 in 45 date palm cultivars scored on
2% agarose gel M=100bp plus DNA ladder (Thermo Scientific)
These two primers produced clear bands with all the genotypes except Akhrot,
Dhaki, Qantar and Kohraba. SSR primer mpdCIRO25 produced two alleles. The sizes
of alleles were 190bp and 200bp respectively. 190bp allele was scored in the cultivars
Chohara, Zaidi, Berahmi, Neelum, Zirin, Jaman, Kozanabad, Kerbaline, Jansohar,
Kokna, Daanda, Begum Jhangi, Peeladora, Rachna, Zerdo, Shado, Peelisunder,
Khudrawi, and Wahan wali while the rest of cultivars produced band 200bp. The
primer mpdCIRO85 amplified three alleles of the sizes 185bp, 190bp and 390bp
respectively in the studied cultivars. Allele of 185bp was scored in cultivar Shado
while Aseel, Halawi, Makran, Chohara, Zaidi, Neelum, Zirin, Jaman, Kozanabad,
Kerbaline, Jansohar, Kokna, Daanda, Begum Jhangi, Peeladora, Saib Zerdo,
Peelisunder, Wahanwali, Shamran, Sanduri, Khudrawi, Makhi, Dhady, Kur,
Haminwali, amplified band of 190bp. A band of 390bp was scored in cultivars Zirin,
Jaman, Kerbaline, Jansohar, Daanda, Saib and Shado while Haleni, Eedalshah,
Sufaidah, Taarwali, Fasli, Basrawali, Pathri, Kupra, Shakri, Zirin, Baidhar, Gajjarwali
and Halwain amplified both alleles of 190bp and 390bp. Bands were scored either as
present (1) or absent (0) to produce binary data in NTSYSpc 1.20j computer software.
The binary data generated were used to produce similarity matrix using Jaccard’s
coefficient ranging from 0.25 to 1.00. The similarity matrix was then used to produce
tree plot to show relatedness among the studied varieties. The 45 cultivars were
divided into two main clusters (Figure 4.4).
54
Figure 4.4: Dendrogram of 45 date palm cultivars computed from similarity matrix using Jaccard’s coefficient with NTSYSpc ver. 2.10j
based on SSR data
Cluster 1
Cluster 2
55
Cluster 1 grouped Aseel, Halawi, Makran, Halwain, Shakri, Angoor, Sanduri,
Makhi, Dhady and Kur clustered at one point and closely related to Haleni, Baidhar,
Kupra, Fasli, Sufaidah, Pathri, Gajjarwali, Basrawali, Eedalshah and Taarwali which
were clustered at the other point. Saib and Champakali were grouped in the same
cluster but at a distance from the rest of the cultivars of this group. Cluster 2
contained the genotypes Chohara, Zaidi, Neelum, Kozanabad, Kokna, Wahanwali,
Peelisunder, Zerdo, Shamran, Peela Dora, Begum Jangi clustered at one point and
closely related to Zirin, Kerbaline, Jansohar, and Daanda clustered at another point.
Berahmi, Khudrawi and Rachna were grouped in another sub cluster. Shado was
grouped in the same cluster but at a distance from the rest of the cultivars.
4.6 Sequence based molecular analysis
4.6.1 Amplification of chloroplast DNA
A subset of seven cultivars from forty five date palm cultivars under study was
randomly selected for sequence based molecular analysis. The selected cultivars were
Dhaki, Aseel, Halawi, Qantar, Hamin wali, Kupra and Shakri. Approximately 550bp
from ribulose bisphosphate carboxylase large subunit (rbcL) gene and atp synthase
beta subunit (atpB) gene each, 300bp of gene fragment of geranyl geranyl
biphosphate reductase (GGR), 1450bp of maturase K (matK) and 1kb of 16S rRNA of
date palm chloroplast genome from each cultivar were sequenced using primers
specially designed for this purpose (Table 4.5) and submitted to GenBank (Table 4.7).
Sequence analysis revealed a near complete identity of these genes among all studied
cultivars in Pakistan. The matK, rbcL, atpB and 16S rRNA have a complete identity to
reference date palm genome of Khalas and among the studied seven cultivars from
Pakistan, while GGR has a single synonymous SNP (A>G) present at 627nt in codon
209 in Qantar, Hamin wali, Khupra and Shakri, while rest was identical to reference
Khalas genome. The sequencing of chloroplast genes has not enabled us to find
genetic differences among cultivars grown in Pakistan to be identified genetically.
56
4.6.2 Single Nucleotide Polymorphism typing
The date palm genome has been sequenced recently and in a pioneering work
Al-Dous and colleagues (Al-Dous et al., 2011) reported some SNPs which can
discriminate their nine studied cultivars and hold the potential to discriminate
cultivars in other regions of the world. After finding near identity in sequenced
chloroplast genes we focused to type these reported SNPs in date palm nuclear
genome and developed specific primers against each SNP (Table 4.6) and sequenced
about 500bp regions flanking particular SNPs from Pakistani cultivars (Appendix
VIII). Detailed sequences have been submitted to the GenBank of NCBI (Table 4.7).
The sequence analysis of the fragments harboring SNPs revealed not only
reported SNPs but also additional novel SNPs sites found in the nuclear genomes of
Pakistani date palm cultivars (Table 4.8). The novel SNPs found included SNP 3.2 at
13,760nt in PDK_30s1000301 and was heterozygous in Shakri where it was “AG”.
SNP 9.1 (A>G) and 9.2 (C>G) were found at 48,503nt and 48,385nt respectively in
PDK-30s1000201 in Halawi cultivar. SNP 10.2 (A>G) was found in Aseel, Qantar
and Kupra at 5,485nt while SNP10.5 (G>A) was found only in Dhaki at 5,536nt in
PDK_30s999171. In PDK_30s998171 two novel SNPs i.e. SNP17.1 (C>T) and
SNP17.3 (A>G) at 5,074nt and 5,163nt respectively were found in Dhaki cultivar. In
Kupra a homozygous SNP 21.2 (T>C) was found at position 6,050nt in
PDK_30s997901. The SNP 32.1 (A>G) was found in Dhaki, Qantar, Hamin wali and
Shakri at 6,856nt in PDK_30s929471. Majority of the novel SNPs were found in
Dhaki variety. Among the fragments we sequenced, PDSNP32, PDSNP10 and
PDSNP17 contained 8, 7 and 5 SNPs respectively and bear the highest marker index
(MI) value of 4.61, 3.61 and 2.26 respectively.
The phylogenetic analysis of complete data set of SNPs from Table 10
revealed two groups i.e. one of Pakistani cultivars and second of all other cultivars
(Figure 4.5) from study of Al-Dous and colleagues (Al-Dous et al., 2011) and one
Pakistani cultivar Qantar.
57
Table 4.5: Sequence and details of primers for chloroplast gene/gene fragments
Primers Primer Sequences Ta (0C) Coordinates of
the primers (nt)
Reference sequence to specify
the coordinates of primers
matK F 5’-ATGGAAGAATTACAAGGATATTTAGA-3’
R 5’-AAGTCTCATCACGTCAACAAACCAATT-3’ 53
1714-1740
3282-3256 NC_013991.2
atpB F 5’-GAAAATTATGTGATAATTATGAGAAC-3’
R 5’-TCATTTCTTCAATTTGCTCTCCACTTC-3’ 53
56146-56133
54670-54644 NC_013991.2
GGR F 5’-CCAAGTCATCAATGGCCTCT-3’
R 3’-GACTACGACTACGCCATCGC-5’ 60
230757-230776
230966-230947 NW_008246734.1
rbcL F 5’-TTGACTTATTATACTCCTGACTATGA-3’
R 5’-TAAGAATCGATCTCTCCAACGCATAA-3’ 53
56993-57019
57583-57557 NC_013991.2
16 S rRNA F 5’-ACGGGTGAGTAACGCGTAAG-3’
R 5'-CTTCCAGTACGGCYACCTTG-3’ 52
103643-103662
105019-105001 NC_013991.2
58
Table 4.6: Sequence and details of primers for amplification of SNPs
SNP Primers Primer Sequences Scaffold ID Coordinates of the primers (nt) Coordinates of sequences
used in phylogenetic analysis
PDSNP03 F 5’-TGTAAAACGACGGCCAGTGGATGAGAGCATATACTGAATGAAGG-’3
R 5’- CAGGAAACAGCTATGACCAGACATGTGTTTGGGTATCCTAGAGC-3’ PDK_30s1000301
13,624-13,649
14,148-14,123 13,652-13947
PDSNP05 F 5’-TGTAAAACGACGGCCAGTCCTCCTCCCCTTCAATTCG-3’
R 5’-CAGGAAACAGCTATGACCCGGTGTTTCTGCCTTTTCG-3’ PDK_30s1000401
43,380-43,398
43,912-43,498 43,445-43,856
PDSNP06 F 5’-TGTAAAACGACGGCCAGTCATGCTTTACCCCCAAAGG-3’
R 5’-CAGGAAACAGCTATGACCTGCATTCAGGAGTTCAACG-3’ PDK_30s999911
6,471-6,489
6,978-6,996 6,605-6,920
PDSNP07 F 5’-TGTAAAACGACGGCCAGTGGCACCATTGAGGACTTTGG-3’
R 5’-CAGGAAACAGCTATGACCGCCGGTTGCTCTCTAGATCC-3’ PDK_30s999931
9,301-9,320
9,876-9,857 9,383-9,810
PDSNP09 F 5’-TGTAAAACGACGGCCAGTGAAGCTTGTGGAGGCATCG-3’
R 5’-CAGGAAACAGCTATGACCAGCTGCTTGATGTCAATTCC-3’ PDK_30s1000201
48,118-48,136
48,794-48,775 48,355-48,675
PDSNP10 F 5’-TGTAAAACGACGGCCAGTACTTTGTGGCATTTGGTTCG-3’
R 5’-CAGGAAACAGCTATGACCGCTTGTCAGACAGCAATTAGG-3’ PDK_30s999171
5,123-5,142
5,703-5,683 5,219-5,571
PDSNP14 F 5’-TGTAAAACGACGGCCAGTGTTCCAAGGAGGGAACAAGC-3’
R 5’-CAGGAAACAGCTATGACCAGTGCAAGACATGCCAAAGG-3’ PDK_30s998691
40,437-40,456
41,094-41,075 40,648-40,944
PDSNP17 F 5’-TGTAAAACGACGGCCAGTGCCGAACTAGCCTCCATACC-3’
R 5’-CAGGAAACAGCTATGACCTGCTTGACCCAACTTCAACC-3’ PDK_30s998171
4,859-4,878
5,358-5,339 5,039-5,314
PDSNP20 F 5’-TGTAAAACGACGGCCAGTCCTGGCCTGTAGTCTCATAGGG-3’
R 5’-CAGGAAACAGCTATGACCAATTATGTGACACGACGACACC-3’ PDK_30s998061
8,342-8,363
8,939-8,918 8,594-8,845
PDSNP21 F 5’-TGTAAAACGACGGCCAGTCTTGGCTCCTCCAGTTCACC-3’
R 5’-CAGGAAACAGCTATGACCCACAGGATTTGTGCGTGTCC-3’ PDK_30s997901
5,799-5,818
6,361-6,342 5,869-6,151
PDSNP23 F 5’-TGTAAAACGACGGCCAGTGTCACCCACATGCTGTCTCG-3’
R 5’-CAGGAAACAGCTATGACCAAAGTATGCCAATGCGAAGG-3’ PDK_30s997701
12,543-12,562
13,043-13,024 12,633-12,908
PDSNP32 F 5’-TGTAAAACGACGGCCAGTTGAAGGAGCAAAGGAGATGG-3’
R 5’-CAGGAAACAGCTATGACCTGCTGTGTCAAACTCGGAAG-3’ PDK_30s929471
6,756-6,775
7,441-7,422 6,839-7,092
Note: The M13 forward primer sequence (5’-TGTAAAACGACGGCCAGT-3’) is added at the 5’ end of each forward primer used to amplify SNP fragment and is
underlined. The M13 reverse primer sequence (5’- CAGGAAACAGCTATGACC-3’) is also added at the 5’ end of each reverse primer used to amplify SNP fragment and is
wave underlined.
59
Table 4.7: Accession numbers sequences of different genes/fragments of date palm submitted to Gen Bank
Geographical origin Accession numbers
Cultivars Sampling site Country of origin matK GGR RBCL atpB 16S rRNA SNP03 SNP05 SNP06 SNP07 SNP09 SNP10 SNP11 SNP12 SNP14 SNP17 SNP20 SNP21 SNP23 SNP32
Dhaki Jhang Pakistan KT803890 KT983259 KT803883 KT781683 KT983365 KT983266 KT983273 KT983280 KT983287 KT983294 KT983301 KT983308 KT983315 KT983322 KT983329 KT983336 KT983343 KT983350 KT983357
Aseel Jhang Pakistan KT803889 KT983260 KT803882 KT781682 KT983364 KT983267 KT983274 KT983281 KT983288 KT983295 KT983302 KT983309 KT983316 KT983323 KT983330 KT983337 KT983344 KT983351 KT983358
Halawi Jhang Iraq KT803891 KT983261 KT803884 KT781684 KT983366 KT983268 KT983275 KT983282 KT983289 KT983296 KT983303 KT983310 KT983317 KT983324 KT983331 KT983338 KT983345 KT983352 KT983359
Qantar Jhang Pakistan KT803892 KT983262 KT803885 KT781685 KT983367 KT983269 KT983276 KT983283 KT983290 KT983297 KT983304 KT983311 KT983318 KT983325 KT983332 KT983339 KT983346 KT983353 KT983360
Hamin wali Bahawalpur Pakistan KT803893 KT983263 KT803886 KT781686 KT983368 KT983270 KT983277 KT983284 KT983291 KT983298 KT983305 KT983312 KT983319 KT983326 KT983333 KT983340 KT983347 KT983354 KT983361
Kupra Bahawalpur Pakistan KT803894 KT983264 KT803887 KT781687 KT983369 KT983271 KT983278 KT983285 KT983292 KT983299 KT983306 KT983313 KT983320 KT983327 KT983334 KT983341 KT983348 KT983355 KT983362
Shakri Bahawalpur Pakistan KT803895 KT983265 KT803888 KT781688 KT983370 KT983272 KT983279 KT983286 KT983293 KT983300 KT983307 KT983314 KT983321 KT983328 KT983335 KT983342 KT983349 KT983356 KT983363
Note: The data of every gene and fragment sequenced in this study was obtained from three individual plants of each variety. When sequences were found identical in all
individual plants then a single reference sequence was deposited under the name of that variety in GenBank,
60
Table 4.8: Origin, details and Sequence of SNPs
Details Varieties and their geographical origin
Pakistan Saudi
Arabia
Qatar California North
Africa
Fragment SNPs Scaffold ID Location PIC MI Ref. Seq. Dhaki Aseel Halawi Qantar Hamin
wali
Kupra Shakri Khls Khlt Alr Khls
X
Khls
F1
Khls
BC2
DN
BC5
Mjd
BC4
Mjd DN
PDSNP03 SNP3 PDK-30s1000301 13,864 0.68 1.49 GG GG GG GG TT GG TT TT GT TT GT GT GT GT GT TT TT
SNP3.1 13,689 0.68 TT TT TT TT CC TT CC CC CT CC CT CT CT CT CT CC CC
SNP3.2* 13,760 0.13 GG GG GG GG GG GG GG AG GG GG GG GG GG GG GG GG GG
PDSNP05 SNP5 PDK-30s1000401 43,587 0.61 1.83 AA AA AA AA AA AA AA AA AA AT AT TT AT AT AT TT AT PDSNP06 SNP6 PDK-30s999911 6,749 0.59 1.13 TT GG GG GG TT GG GG GG GT TT GT GG GT GT GG GT GG
SNP6.1 6,718 0.54 GG GG GG GG GG GG GG AA GG AG GG GG AG AG GG AG AA
PDSNP07 SNP7 PDK-30s999931 9,547 0.6 2.41 TT TT TT TT TT TT TT TT TT CT TT CT CC CC CT CC CC
SNP7.1 9,541 0.6 TT CC TT TT TT TT TT TT TT CT TT CT CC CC TT CC CC SNP7.2 9,714 0.55 CC CC CC CC TT CC CC TT CC CT CC CT CC CT CT CC CC
SNP7.3 9,750 0.66 TT TT TT TT GG TT TT GG TT GT TT GT GG GG GT GG GG
PDSNP09 SNP9 PDK-30s1000201 48,503 0.67 0.93 AA AA GG AA GG AA GG AA AG AG AA AG AA AG GG AG AA
SNP9.1* 48,385 0.13 AA AA AA GG AA AA AA AA AA AA AA AA AA AA AA AA AA SNP9.2 48,478 0.13 CC CC CC GG CC CC CC CC CC CC CC CC CC CC CC CC CC
PDSNP10 SNP10 PDK-30s999171 5,408 0.67 3.61 TT TT CC CC CC CC CC CC CT TT TT T T CT CT CT CT CC
SNP10.1 5,293 0.5 TT TT AA TT AA TT AA TT TT TT TT TT AT TT TT TT AT
SNP10.2* 5,485 0.32 AA AA GG AA GG AA GG AA AA AA AA AA AA AA AA AA AA SNP10.3 5,498 0.67 AA GG AA GG AA GG GG GG AG AA AA AG AA AG AA AG AA
SNP10.4 5,512 0.69 AA AA GG GG GG GG GG AA AG AA AA AG AG AG AA AG AG
SNP10.5* 5,536 0.13 GG AA GG GG GG GG GG GG GG GG GG GG GG GG GG GG GG
SNP10.6 5,562 0.63 GG AA AA GG AA GG AA GG AG AA AA GG AA AG AG AG AA PDSNP14 SNP14 PDK_30s998691 40,767 0.63 1.89 GG GG GG GG GG GG AA AA GG GG AG GG AG AA AG AA AG
SNP14.1 40,757 0.63 TT TT TT TT TT TT CC CC TT TT CT TT CT CC CT CC CC
SNP14.2 40,909 0.63 CC CC CC CC CC CC TT TT CC CC CT CC CT TT CT TT CT PDSNP17 SNP17 PDK_30s998171 5,216 0.67 2.26 TT TT TT CC TT TT TT TT CT CC TT CT CC CT CT CT CC
SNP17.1* 5,074 0.13 CC TT CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC
SNP17.2 5,129 0.67 CC TT TT CC TT TT TT TT CT CC TT CT CC CT CT CT CC
SNP17.3* 5,163 0.13 AA GG AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA SNP17.4 5,242 0.66 TT TT TT CC TT TT TT TT CT CT TT CT CC CT CT CT CC
PDSNP20 SNP20 PDK_30s998061 8,628 0.6 0.6 TT AA AA AA AA TT AA AA AT AT AA TT AT AA AT AA TT
PDSNP21 SNP21 PDK_30s997901 6,144 0.69 1.45 AA AA GG GG AA GG GG GG AG AA AG AG AA AG AG GG AA
SNP21.1 5,876 0.63 GG AA GG GG AA AA AA AA AG AA AG AG AA AG AG GG AA SNP21.2* 6,050 0.13 TT TT TT TT TT TT CC TT TT TT TT TT TT TT TT TT TT
PDSNP23 SNP23 PDK_30s997701 12,737 0.67 0.8 AA GG AA AA GG AA AG GG AG GG AG AA AG AG AG GG AG
SNP23.1 12,714 0.8 AA AA AA AA AA AA AA GG AA AA AA AA AA AA AA AA AA
PDSNP32 SNP32 PDK_30s929471 6,880 0.45 4.61 GG AA GG GG AA GG GG GG AA AA AA AA AA AA AA AA AA SNP32.1* 6,856 0.67 AA GG AA AA GG GG AA GG AG GG AG AG GG AG AG AA GG
SNP32.2 6,859 0.67 AA GG AA AA GG GG AA GG AG GG AG AG GG AG AG AA GG
SNP32.3 6,890 0.4 GG AA GG GG GG GG GG GG GG GG GG GG AA AG GG GG AA
SNP32.4 6,917 0.67 CC AA CC CC AA AA CC AA AC AA AC AC AA AC AC CC AA SNP32.5 6,956 0.68 TT CC TT TT CC CC TT CC CT CT CT CT CC CT CT TT CC
SNP32.6 7,053 0.4 GG CC GG GG GG GG GG GG GG GG GG GG CC GC GG GG CC
SNP32.7 7,074 0.67 AA GG AA AA GG GG AA GG AG GG AG AG GG AG AG AA GG
Note: The sequence of each SNP is depicted in diploid state using two nucleotides. * indicates novel SNP sites found in this study. Polymorphic Information Content (PIC)
and Marker Index (MI) are calculated according to Powel et al. (1996) for the fragment based on all SNPs present in that fragment. The sequence of reference genome (Ref.
Seq.) and varieties other than Pakistani origin are reported by Al-Dous and colleagues (2011). Khls is Khalas, DN is Deglet Noor, Mjd is Medjool, Khlt is Khalt and Alr is
Alrijal
61
The SNPs data reported in this study for Pakistani and along with the data
from Arabian, Qatari, Californian and North African cultivars reported by Al-Dous
and colleagues (Al-Dous et al., 2011) was subjected to Iterative Dichotomiser 3 (ID3)
decision-tree learning algorithm to find out the minimum number of SNPs needed to
discriminate among the studied cultivars. The fragments PDSNP32 and PDSNP10 got
the highest priority containing minimum SNPs sufficient to fully discriminate among
all the cultivars. Based on this data a typing scheme was generated and depicted
through schematic representation (Figure 4.6). In total, seven SNPs, four from
PDSNP10 and three from PDSNP32 fragments could type all sixteen date palm
cultivars with unique SNPs signature.
62
Figure 4.5: Maximum Parsimony (MP) Tree based on SNP data from
various date palm cultivars.
The data of SNPs from a single variety was merged in concatemers and these
concatemeric sequences from all varieties were aligned through MAFFT
version 6.847b and used in phylogenetic analysis using MEGA version 6. Due
to smaller data set with high polymorphism, the bootstrap values obtained are
low. The cultivars belonging to two groups are depicted by parenthesis marks.
The bar represents substitutions per site.
63
Figure 4.6: SNPs system for varietal identification.
The information of SNPs from PDSNP10 and PDSNP32 fragments can discriminate
completely all cultivars analyzed in current study with unique SNP signatures. For
example Qantar have (SNP10.4: GG, SNP32.7: GG, SNP10.2: GG) signature, while
Deglet Noor have (SNP10.4: AG, 497 SNP32.7: GG, SNP10: CC) signature.
64
V. DISCUSSION
Date palm (Phoenix dactylifera L.), a dioecious ever green woody tree with
very long productive life, belongs to family Arecaceae and has a genome size of
about 700Mb with a diploid genome of 18 pairs of chromosomes (Al-Dous et al.,
2011; Al-Mssallem et al., 2013). Date palm is an important fruit crop of Pakistan after
citrus and mango. It is an important export commodity. Date palm is unique in the
sense that it has the largest number of varieties among the fruits (Afzal, 2005). In
Pakistan more than 300 varieties of date palm exist (Jamil et al., 2010). Globalization
has necessitated the need of standardized information about date palm germplasm for
characterization, evaluation and conservation (Rizk and Sharabasy, 2006).
Genetic diversity in date palm may be the result of dissemination of the
germplasm with human migration, human selection and clonal propagation
(Chaludvadi et al. 2014). Date palm cultivars may differ due to the genotypes that
arise from seed (Al Salih and Hussain, 1980; Afzal, 2005) or due to the cytological
differences (AlDous et al., 2001). A cultivar is domesticated only when it shows
characteristics that are better than their wild parents or ancestors (Johnson et al.,
2013). Due to its long generation cycle little plant breeding has been practiced in date
palm. Mostly traditional varieties are used that are propagated vegetatively from
suckers or offshoots (Chaludvadi et al., 2014). The cultivated date palm is propagated
through offshoots and also by micro propagation. Being cross pollinated it is highly
heterozygous but that depends upon the type of male plant used for pollination and the
fact that how the parent itself was originated (Chaludvadi et al., 2014). Introduction of
new germplasm from other countries and the local hand pollination system has
brought recombination in genotypes and has caused genetic variation (Arabnehzad,
2012).
The date palm cultivars with distinct fruit shapes, colors, sizes and commercial
importance have been traditionally identified through plant morphology and fruit
characteristics. Later with the advancement in biochemical analysis, fruit proximate
analysis also complimented the identification process. However, the data for these
characteristics can only be acquired at adult fruiting stage and has the propensity to be
affected by environmental factors (Zehdi et al., 2004; Jehan and Lakhanpaul, 2006).
65
The need for stable identification systems applicable at planting stage requires the
utilization of genetic markers.
Numerous studies have been undertaken for analysis of genetic diversity in
date palm cultivars using SSR markers (Billotte et al., 2004; Elmeer et al. 2011;
Elshibli & Korpelainen, 2008, 2009; Zehdi et al., 2004; Zhao et al., 2012) and gene
sequencing, and have successfully revealed the existence of genetic diversity among
cultivars but a universal system for cultivar identification has not yet been developed,
primarily because of low marker density (Mathew et al., 2015) and smaller nucleotide
differences among cultivars.
To our knowledge this is the first study on genetic diversity analysis of date
palm cultivars grown in Pakistan utilizing morphological, chemical and molecular
approach.
5.1 Morphological evaluation
Descriptive statistics has revealed that the studied forty five cultivars grown in
Pakistan (Table 3.1) possess significant variation with respect to the traits studied
(Table 4.1). Based on the Eigen values of the principal components (Table 4.2) and
their correlation with the studied traits (Table 4.3), ten phenotypic and four
biochemical traits were rendered to be important for characterization of local date
palm cultivars. These traits include weight, volume and length of date fruit. Pulp
weight, total soluble solids, % reducing sugar, % total sugar, % ash, leaf length,
midrib length with pinnae, spine number, leaf width, leaf base width and perianth
height (Table 4.3). Descriptive statistics of the trunk diameter (Table 4.1) showed
variation among the cultivars but principal component analysis has not mentioned it to
be one of the traits that showed variability among the data set or as a marker of
varietal identification. El-Merghany and Al-Daen (2014) while evaluating date palm
cultivars grown under Toshky conditions found no significant difference in trunk
diameter of the studied cultivars. Similarly in an attempt to compare vegetative
Barhee cultivar of date palm and its two seedlings strains with respect to vegetative
morphological traits El-kosary et al (2009) found slight variation in the trunk girth
that was non-significant. In contrast Elsafi (2012) found trunk aspect to have the
highest percent and cumulative variation in the 116 studied date palm accessions in
66
Sudan. Although varieties differ in their trunk diameter to various degrees but this is
not a good criterion for discrimination unless the trunk has a big difference (Afzal,
2005).
Leaf length of the date palm cultivars under study ranged from 244cm-420cm.
The maximum leaf length (470cm) recorded for date palm cultivars from Saudi
Arabia by Alwusaibai et al. (2014). Principal component analysis has shown leaf
length to be one of the characteristics contributing to variability of the data set of the
studied cultivars. This is in accordance with Elhoumaizi et al. (2002) and Salem et al.
(2008) who also found leaf length to be an important discriminant for date palm
cultivars from Morocco and Mauritania respectively. Leaf width ranged from 54cm-
108cm and statistically it is among the traits causing variability. Salem et al. (2008)
also found it to be important discriminant among the cultivars. Leaf base width was
found to be 5cm-10.3cm and is also a measure of variation among the studied
cultivars. This is in contrast to Merghany and AlDaen (2014) who found no
significant difference in leaf base width of date palm cultivars under Toshki
conditions. Spine number of the studied date palm cultivars ranged from 9.33-35.33
and according to our analysis spine number is a varietal discriminant criterion. Afzal
(2005) has mentioned thorns or spines of date palm to be important for date palm
cultivars characterization. Hammadi et al. (2011) found spine number as useful for
determination of maturity period and fruit consistency characteristics as this character
is less affected by the environmental factors. Less number of spines and more leaflets
area were used as distinguishing characters among date palm cultivars grown in
different regions (AlWusaibai et al., 2014).
Highest fruit weight (26.50gm) and volume (26.00cm3) was measured in
Dhaki. This is in accordance with (Nadeem et al., 2011) who recorded highest weight
and volume for Dhaki among 21 date palm varieties of Pakistan. Fruit weight of
Dhaki cultivar was more than the maximum fruit weight (12.78gm) recorded for
Sudanese date fruit cultivar by (AlYahyai, 2008). Fruit length and diameter of the
date palm cultivars of Pakistan were recorded to be in the range of 2.4cm-6.0cm and
1.5cm-6.5cm respectively (Markhand et al., 2010).
67
Fifty five percent of the date palm varieties from Qatar and 88% from Kuwait
were classified correctly on the basis of their fruit traits (Jaradat, 2014). Fruit
characteristics like length, reducing and non reducing sugar were significantly
different in the studied cultivar belonging to soft and dry types of dates of Toshky
(Merghany and AlDaen, 2014). Farag et al. (2012) found that fruit characteristics of
Zaghloul cultivar of date palm are influenced by the type of pollinator used for
pollination of the mother tree.
Information about the qualitative and quantitative properties of fruit and its
chemical composition is of equal importance to date processors and traders because
these qualities determine the end use of the raw material. Consumers are concerned
about the taste and nutritional value of the date fruit only (Saleem, 2005). Physical
properties are important to sort the fruits and to determine how many of these fruits
will be placed in a package for transportation. This information is also helpful in
machines for sorting, cleaning and kernel removal (Jahromi, 2008).
Morphological traits are the visible marks of genes and give clue about the
genotype but most of morphological traits being qualitative in nature are controlled by
many genes and thus environment has a great effect on such traits (Jehan and
Lakhanpaul, 2006). Germplasm characterization and diversity evaluation needs a
large set of morphological data that is most likely to be influenced by the environment
(Zehdi et al., 2004). Time, effort and labor are required to score data so these
limitations make their use restricted. (Jehan and Lakhanpaul, 2006). Morphological
traits are affected by many factors so they don’t represent genetic makeup correctly
moreover they are limited in number. These are dominant in nature because of
interaction of dominant and recessive alleles. These markers do not represent the non
coding region which make up 95% of the higher plants genome (Jehan and
lakhanpaul, 2006).
5.2 Proximate composition analysis
Nutritional and medicinal values of date fruit depend upon chemical
composition (Tang et al, 2013). Chemical composition of date fruit changes with
variety, environment, stage of development and the post harvest conditions.
Maximum moisture content (83.08%) was found for Peeli Sunder having minimum
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value of TSS (140 Brix). Ash percent is a measure of the nutritive quality of the food
(Pearson, 1996). Ash content was found to range from 0.64 to 4.54%. Ash content
varied from 1.82%-2.87% in eight date palm cultivars studied by Jamil et al.
(2010).Maximum moisture content was found to be 83.08 % for Peelisunder while
Iqbal et al. (2011) recorded maximum moisture content (67.38%) of date fruit at
‘khalal’ stage for Dhaki variety.
Herchi et al. (2014) while analyzing the chemical composition of fruit and
seed of date palm found that moisture content (9.23-11.17%) was the major
component of date fruit after carbohydrates (78.69-83.46g/100g dry matter) while ash
content varied from 1.18-1.64g/100g dry matter). Shaba et al. (2015) while
investigating the nutritional content of date palm fruit, found the ash content in the
range of 1.88±0.03% and moisture content in the range of 1.16±0.16%. Hamza et al.
(2014) while evaluating the proximate composition variability in the date palm
cultivars from Nigeria found ash content (22.1-25.7) more than the moisture content
(20.3-25.0) after the major component of carbohydrates. Yahaya et al. (2015) studied
the proximate composition and fruit weight of fresh date fruit varieties in wet season
of Nigeria and found that moisture content is directly related to perishability of the
fruit. The high moisture content leads to a short storage life and vice versa and will
also be prone to microbial attack. Ash content was found to have non-significant
difference among the studied varieties with maximum value of 1.80±0.01. Mohamed
et al. (2014) while studying the chemical composition, antioxidant capacity and
mineral extractability of six date palm cultivars from Sudan found significant
difference in these varieties with respect to their chemical composition. Moisture and
ash content were found to be in the range of 8.78-10.68 and 1.96-2.50 respectively.
Guido et al. (2011) while investigating the maturity stage effect on the physico
chemical composition and volatile components of the date palm fruit at three
developmental stages found that total sugar and ash content increase with
developmental stage while weight and moisture decreased. They found chemical
composition and variation in it to be a varietal dependent phenomenon. According to
Merghany and Al-Daen (2014) total sugar, reducing sugar and non reducing sugar
were significantly different in the studied cultivars belonging to soft and dry types of
dates.
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Correlation study among the traits of date palm showed that vegetative traits
were positively correlated with other vegetative traits but had no significant
correlation with fruit traits. Similarly some fruit traits were found to be positively
correlated with other fruit traits but fruit traits had no significant correlation with
vegetative traits (Table 4.4). Positive correlation between leaf length and midrib
length and pinnae number and pinnated part length was also reported by Elhoumaizi
et al. (2002) for Moroccan date palm cultivars and this can be explained as the long
leaf can have a long midrib with greater number of pinnae thus accounting for a
greater pinnated part length. Saker et al. (2010) studied eight date palm cultivars with
respect to their anatomical, morphological and phytochemical values and found the
studied cultivars to be varying in these traits.
Al-khalifa et al. (2012) in their study of identification of fourteen date palm
cultivars by morphological and molecular markers (RAPD) concluded that genetic
variation has strongly influenced the fruit shape of the studied cultivars. Hammadi et
al. (2012) while investigating the genetic diversity in Tunisian date palm through
ISSR markers and fruit characterization found that fruit consistency which is an
important characteristic of date fruit having association with genetic markers.
Morphological and molecular phylogenetic study of the family Palmea by Uhl et al.
1995 and Baker et al, (2000) have resulted in different patterns suggesting that only
morphological data is not enough to study the phylogenetic relationship within the
family. Palms have been identified to have slow rate of molecular evolution to date
(Hahn, 2002).
5.3 Molecular analysis
Characterization of the cultivars is important for conservation and sustainable
use of plant genetic resources. Botstien et al. (1980) reported the use of molecular
marker technique to detect DNA polymorphism. Gregor Mendal gave the concept of
molecular markers based on phenotype in nineteenth century (Gurteg Singh, 2014).
Molecular markers have a proven efficiency in genetic diversity assessment of date
palm cultivar for example RAPD and ISSR markers were found to be helpful in
discrimination of the date palm cultivars by Mitra et al. (2011). The genome assembly
of Phoenix dactylifera reported by AlMasselem et al. (2013) is better in precision and
contiguity, thus allowing inter and intra specific comparative studies and finding the
70
evolutionary context of the date palm as Phoenix dactylifera is a cross pollinating
crop and its recessive alleles are in heterozygous state so more and more varieties
should be sequenced to quantitative traits loci. Morphological and biochemical
attributes of date fruit should be studied at molecular level to provide basis for
improvement of yield and quality of date (AlMasselem et al., 2013). Yousif et al.
(2014) characterized Iraqi date palm cultivars on the basis of molecular markers and
analyzed their relationship. They proved the ability of the RAPD markers to assess
diversity among the studied cultivars. The polymorphism detected by RAPD markers
ranged from 0-85.7%. Ahmad and AlHadidi (2014) used eighteen ISSR markers for
evaluation of genetic diversity and relationship among fifteen cultivars from Qatar.
They found that although the cultivars were different in their agronomic traits but
genetically they were interrelated.
5.3.1 Microsatellite markers
Simple sequence repeat (SSR) markers, the repeated DNA stretches in genome of
higher eukaryotes were first reported by Hamada et al. in 1982. Polymorphism in
simple sequence repeats results from polymerase slippage in DNA replication or
unequal crossing over (Levensin and Gutman, 1987). SSR are remarkable being
multiallelic, codominant, relatively abundant, requiring small amount of DNA for
detection and are easily detectable through PCR. These markers are transferable
across the labs in the form of primers to be used as a common standard for research
(Powell, 1996). Elmeer and Mattat (2015) suggested SSR as useful markers to be used
in date palm certification process for suckers, in vitro plantlets or any plant material at
an early stage.
Microsatellite DNA markers developed by Billotte et al. (2004) and Elmeer et
al. (2011) were used for assessment of genetic diversity among forty five date palm
cultivars grown in Pakistan (Table 3.2). Fifteen samples were randomly selected for
initial screening of amplification by these primers. Of the thirty markers developed by
Elmeer et al. (2011) sixteen markers either did not amplify or gave improper
amplification in the tested samples, while each of the remaining fourteen primers
amplified single monomorphic band. Our results are in contrast to Elmeer et al.
(2011) who developed and tested these thirty SSR markers in date palm cultivars from
Qatar. SSR primers DP159, DP168, DP169, DP170 each produced single
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monomorphic amplicon while these were reported to be highly polymorphic by
Elmeer et al. (2011) similarly markers DP160, DP171, DP172, DP175 reported to be
polymorphic (Elmeer et al, 2011) did not amplify in our initial screening. Of the 16
markers developed by Billotte et al. (2004) 10 markers mpdCIRO10, mpdCIRO15,
mpdCIRO16, mpdCIRO32, mpdCIRO35, mpdCIRO50, mpdCIRO57, mpdCIRO70,
mpdCIRO78 and mpdCIRO93 produced single monomorphic amplicons in our tested
samples while mpdCIRO44, mpdCIRO48, mpdCIRO63 and mpdCIRO90 did not give
amplification. This is in contrast to work of earlier researchers like Zehdi et al. (2012)
differentiated a total of 137 alleles with a mean of 9.71 with these SSR markers.
Alleles ranged from 5 for locus mpdCIRO16 and mpdCIRO35 to 16 for mpdCIRO78.
They developed an identification key based on five loci mpdCIRO78, mpdCIRO85,
mpdCIRO70, mpdCIRO93 and mpdCIRO50. This is in accordance with Billotte et al.
(2004), Ahmad and Al-Qaradawi (2009), Pintaud et al. (2010) where mpdCIRO44
showed erratic amplification. Similarly mpdCIRO48 cannot be amplified in the
previous experiments by Zehdi et al. (2004), Henderson et al. (2006) and Pintaud et
al. (2010). Only two markers mpdCIRO25 and mpdCIRO85 produced polymorphic
bands within the expected range. Thus twenty six markers produced a total of twenty
nine scorable and five polymorphic amplicons/bands. Therefore only the two
polymorphic markers were used for assessment of genetic relationship in all the
samples (Figure 4.2 and 4.3). Elmeer and Mattat (2015) used 14 SSR markers
developed by Billotte et al. (2004) for assessment of diversity in 59 accessions of 12
date palm cultivars from Qatar. They were able to obtain 3 alleles for mpdCIRO90
and 11 for mpdCIRO10 and mpdCIRO15 while mpdCIRO63 did not amplify. Our
results are in contrast to Elshibli and Korpalainen (2009) who obtained 343 alleles by
the use of 16 SSR markers in 68 accessions whereby mpdCIRO35 detected 14 alleles
and mpdCIRO63 detected 44 alleles in date palm germplasm from Sudan. Ahmed and
Al-Qaradawi (2009) were able to produce 44 alleles with mpdCIRO16, mpdCIRO25,
mpdCIRO32, mpdCIRO35, mpdCIRO50, mpdCIRO57, mpdCIRO78, mpdCIRO85,
mpdCIRO90 and mpdCIRO93 while mpdCIRO10, mpdCIRO15, mpdCIRO32,
mpdCIRO35, mpdCIRO63 and mpdCIRO70 did not amplify clear bands. They
obtained three alleles with mpdCIRO16 and 6 alleles with mpdCIRO32. Elshibli and
Korpalainen (2009) used SSR markers for genetic diversity analysis of the 15 date
palm cultivars from Sudan and observed 7 alleles for mpdCIRO15 and mpdCIRO16
and 22 for mpdCIRO63 and genetic distance of 0.69 to 3.49. Zehdi et al. (2004) was
72
able to fingerprint 49 accessions with 14 SSR markers developed by Billotte et al.
(2004) with scoring of 100 alleles. They obtained 4 alleles with mpdCIRO16 and
mpdCIRO10 alleles with mpdCIRO78. Hammadi et al. (2011) studied 26 Tunisian
cultivars and found mpdCIRO10, mpdCIRO15, mpdCIRO32, mpdCIRO70 and
mpdCIRO93 to be highly polymorphic. PCR reaction for mpdCIRO57 failed while
studying 12 species of genus phoenix for their morphological and molecular diversity
(Rivera, 2008). Raachi et al. (2013) developed an identification key for date palm
cultivars from Libya based on only three multi locus SSR markers that amplified 23
alleles.
Reproducibility, accuracy, codominance, high polymorphism and low cost are
the characteristics of a good marker system. Although SSRs are commonly being used
for most of the crops these days, there are some problems in their use viz correct
sizing of SSR bands because of the electrophoresis artifacts, unequal allele
amplification by PCR, null alleles may result if mutation occurs in the SSR primer
binding site and size homoplasy which means that the alleles are of equal size but
they may not necessarily have the same sequence (Jones et al. 2007).
5.3.2 Molecular analysis based on sequencing
5.3.2.1 Chloroplast DNA analysis
In an attempt to develop an identification system for important cultivars grown
in Pakistan, we started with the sequencing of rbcL, atpB, GGR, matK and 16S rRNA
genes of date palm chloroplast genome from Dhaki, Aseel, Halawi, Qantar,
Haminwali, Shakri and Kupra cultivars. We found a complete identity in sequenced
fragments (Table 4.5) except for GGR gene where a single synonymous SNP (A>G)
was present at nucleotide 627 of GGR codon 209 in Qantar, Hamin wali, Kupra and
Shakri, suggesting a lack of divergence in these genes in the studied cultivars.
Chloroplast DNA is commonly used for phylogenetic studies in plants because it is
believed to be slow evolving and conserved in terms of nucleotide substitution thus
making it suitable for phylogenetic studies (Patwardhan, 2014). Hoot (1995) while
assessing the utility of atpB gene sequences in resolving phylogenetic relationships
sequenced atpB gene for seven genera of family Lardizabalaceae. They compared the
phylogenetic tree generated by atpB sequences with the ones produced by nuclear 18S
73
rDNA and rbcL from chloroplast and with the tree obtained by the combined
sequences of all the atpB, rbcL and 18S rDNA. They found 18S to be the most
conserved, followed by atpB and rbcL respectively moreover tree produced by 18S
was strongly supported than those by atpB or combination of these three of rbcL
sequence information.
In animals only one gene, chytochrom oxidase 1 (CO1) is enough for
phylogenetic studies but plants lack such a universal barcode. The consortium for
barcode of life has suggested that the combination of rbcL and matK may be used
along with some other suitable region in plants (Patwardhan, 2014). The previous
studies suggested that the genes of ribulose bisphosphate carboxylase large subunit
(rbcL), atp synthase beta subunit (atpB), (Hahn, 2002; Hoot et al., 1995) and
maturase K (matK) (Burgess et al., 2011; Heise et al., 2015) could be utilized for
studying diversity among various plant species (Patwardhan et al., 2014) and cultivars
(Enan & Ahmed, 2014). rbcL is a single copy gene in the chloroplast genome, which
is 1428bp long and is found in all the plants. While the matK (approximately 1500bp
is located within the introns of trnk and is involved in the splicing of type II introns
from RNA transcript (Patwardhan, 2014). A phylogenetic study based on only one
gene or marker shows the evolution of that specific marker and such interpretation
may be misleading because the other genes may show different evolution rate if
horizontal gene transfer phenomenon is also involved (Patwardhan ,2014). AlQurainy
et al. (2011) assessed eight Saudi date palm cultivars on the basis of their chloroplast
DNA sequence of rpoB and psbA-trnH for molecular signature. They sequenced a
combined matrix of 1147 characters out of which 173 were variable sites. Their
results showed that these sequences can be used as molecular signatures at seedling
stage for trading and farming. Enan and Ahmad (2012) analyzed the matK and rpoC1
markers, as suggested by the consortium for the barcode of life plant working group,
for identification of date palm cultivars. They amplified matK and rpoC1 genes in 11
date palm cultivars for establishing molecular phylogram using MEGA 5 software.
They found matK to be more informative than rpoC1, thus concluding that matK
alone or in combination with rpoC1 can determine genetic variation in date palm.
Akhtar et al. (2014) analyzed fifteen date palm cultivars from Sindh province of
Pakistan on the basis of Rps14 gene of chloroplast. They found very little genetic
distance (0.001), low average evolutionary divergence (0.008) and low nucleotide
74
diversity (0.007) thus concluding that the studied date palm varieties have high degree
of similarity. Heise et al. (2015) represented triple barcode data set based on trnL
intron, matK and plastid rbcL for xerothermic plants of the central Europe. They
analyzed 126 xerothermic plant species for this purpose. Their database contains rbcL
and trnL barcodes for 117 species, and matK barcodes for 96 species. They were able
to identify the plants up to specie level with 89.6% rbcL, 96.4% matK and 98.4% trnL
barcodes. Their database has application in phylogeography, biodiversity and
conservation. Kress et al. (2007) evaluated a global plant DNA barcode system using
nine putative barcode including both coding and noncoding regions either alone or in
combination in 48 genera, taking two species per genera. They found 88%
discrimination when trnH-psbA region was used in combination with a coding region
like rbcL. Thus they suggested this combination of noncoding trnH-psbA as two locus
global barcode for land plants being universal in nature and having the species
discriminating ability. Phylogenetic signal of matK is powerful enough to make it an
important gene for systematic and evolutionary studies in plants (Barthet and Hilu,
2007). Combining molecular and morphological data increased the resolution of palm
phylogenies as compared to the previous one but still inconsistencies existed between
resolution of molecular and morphological data and that of nuclear and chloroplast
DNA. But estimates of phylogenies based on cp DNA was rendered to be the most
reliable (Hahn, 2002). Rate of substitution in plant mitochondrial genome is very low
than that of animals. Thus a single barcode system for plant would be difficult and
multiple loci would be needed in plants for identification of the unfamiliar species,
taxonomic and ecological exploration and forensic analysis of plant parts. rbcL is the
most characterized plastidic coding region in the genebank and is thus good and
provides basis for comparison with other plastid genes.
5.3.2.2 Single Nucleotide Polymorphism detection
We genotyped few of the recently reported SNPs by Al-Dous and colleague
(Al-Dous et al., 2011). Interestingly, the SNP sites previously reported polymorphic
were also polymorphic in Pakistani cultivars. In addition, eight novel SNPs sites were
also found in the sequenced fragments from Pakistani cultivars. These typed SNPs lie
in various contigs in assembled genome of date palm. Few of these SNPs such as
PDSNP03, PDSNP06, PDSNP09, PDSNP10, PDSNP14 and PDSNP17 are present in
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coding genes including kinesin-1 like protein (PSS1), signal peptidase complex
subunit 3, carboxyl-terminal-processing peptidase 1, neutral alpha-glucosidase C,
myb-related protein (308-like) and pollen specific protein (C13 like) respectively. The
fragment PDSNP10, is a part of neutral alpha-glucosidase C gene and bears 7 SNPs
with marker index of 3.61, while fragment PDSNP17 belongs to pollen-specific
protein and bears 5 SNPs with marker index of 2.26, suggesting significant
divergence in these gene in Pakistani cultivars. Majority of these SNPs are
synonymous and do not alter the amino acid of their respective genes except SNP10.1
and SNP14.1 that caused non synonymous substitution in respective amino acids.
SNP10.1 has resulted in change of Serine to Proline in all the cultivars except Aseel
while SNP14.2 has resulted in change of Valine to Alanine in cultivars Kupra and
Shakri. The rest of SNPs lie in non-coding or poorly annotated regions of the date
palm genome including a noteworthy fragment PDSNP32 which harbor 8 SNPs with
the highest marker index of 4.61 (Table 4.8).
A phylogenetic analysis of these SNPs data and corresponding sequences from
Saudi Arabian, Qatari, Californian and North African cultivars (Table 4.8) suggests
two subgroups (Figure. 4.5) one containing Pakistani cultivars except for Qantar
which is grouped with rest of the cultivars. Though the splitting of groups and various
other nodes received weak bootstraps support owing to smaller data set with high
polymorphism in SNPs, in a recent study however, based on about 65000 SNPs in
date palm genome, Mathew and colleagues (Mathew et al., 2015) have shown
splitting of few Pakistani cultivars in Eastern group comprising of those cultivars
which originated in Arabian Gulf, and in Western group of cultivars having origin in
North Africa. Although they studied date palm cultivars other than those included in
this study, presence of dates with distinct origins in Pakistan nevertheless, becomes
established. The tree suggests that based on typed SNPs data, Pakistani cultivars
might have quite different genetic makeup which points out towards their long geo-
restricted propagation in this region and origin from a different germplasm except
from Qantar which is similar to a Qatari male cultivar Khalt.
In this study a total of forty two SNPs sites were typed including thirty four
already reported and eight novel SNPs sites in twelve fragments located across
various contigs in data palm nuclear genome (Table 4.8). The data of these SNPs in
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Pakistani and various other cultivars of date palm were used to develop a typing
strategy for cultivar identification (Figure 4.6) by subjecting this data to Iterative
Dichotomiser 3 (ID3) decision-tree learning algorithm (Quinlan, 1986) to identify
minimum set of SNPs required to discriminate among all cultivar using minimum
number of DNA fragments. The resultant typing strategy (Figure. 4.6) uses seven
SNPs, four from PDSNP10 and three from PDSNP32 fragments to type all sixteen
date palm cultivars enabling us in distinguishing them with unique SNPs signatures.
This strategy though currently based on data from 16 samples, has a great flexibility
to be expanded to accommodate other cultivars and may provide a foundation for
developing a universal system for identification of date palm cultivars around the
world. Typing system based on SNPs would be more versatile because SNPs are more
stable with high fidelity of inheritance as compared to other marker systems including
SSR, RAPD and AFLP (Gupta et al., 2001) which sometimes are quite ineffective in
case of cultivars with similar genetic background. In addition, these systems also
suffer with problems such as correct sizing of bands due to electrophoretic artifacts,
unequal allele amplification by PCR, null alleles may result if mutation occurs in
primer binding sites and size homoplasy which means that the alleles are of equal size
but they may not necessarily have the same sequence (Jones et al., 2007). SNPs
typing has been used for identification of species of Picea (Germano & Klein, 1999),
Eurychoma longifolia (Osman et al., 2003) genotype of maize (Zea mays) (Jones et
al., 2007), identification of Australian barely lines (Hayden et al., 2010), genotyping
of grapevine (Cabezas et al., 2011) and genotyping for varietal identification of
soyabean (Yadav et al., 2015). Recently, typing of about 13000 to 65000 SNPs using
next generation sequencing, from seventy female date palm cultivars from all major
growing regions of the world, have suggested the North Africa and Arabian Gulf as
two genetic origins of modern date palm cultivars (Mathew et al., 2015). Typing of all
SNPs present in the genome using expensive next generation sequencing seems albeit
more authentic and rigorous but practically quite challenging for less developed
countries and sometime provides comparable information which could be obtained by
typing only few SNPs such as those thirty two SNPs suggested by Al-Dous and
colleague (Al-Dous et al., 2011) or even less typed in this study.
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We used locus specific PCR amplification method for SNP typing in the
studied cultivar whereby locus specific PCR primers were synthesized from the
available genomic sequence and PCR amplification of samples from different
varieties was done. Sequence differences in the sequenced PCR product were used for
diversity analysis. Moreover alignment among the available genomic sequences
through computer software were also conducted. Jones et al. (2007) while comparing
the SSR and SNP marker technologies for genotypic analysis of maize (Zea mays)
reported that the quality and quantity of marker data provided by SNPs is better than
SSRs.
Mathew et al. (2015) while surveying the date palm cultivars across the
genome found two main subpopulations of date palm, one from North Africa and
another from Arabian Gulf. In their study they found some date palm cultivars like
Zayaki, Gorakh and Barani to be falling in the western date palm group. They
concluded that Pakistani cultivars may have resulted from the elite Medjoul cultivar
being pollinated by local pollinators. Seed propagated date palms are commonly used
in Pakistan that has resulted in the mixed genetic makeup of the cultivars.
Cultivated date palm has descendant from wild relatives or feral plants in
warm regions of the world. Johnson (2010) has mentioned that Munier in a map has
shown two distinct directions of distribution of date palm. One from Mesopotamia to
Arabian Peninsula and east of Pakistan and India. The other from Egypt to North
Africa and Sudan (Johnson, 2010). Although an identification key has been developed
for 101 accessions of date palm by Zehdi et al. (2012) using only five loci but those
loci proved to be monomorphic in our tested samples.
Here we report a SNPs typing strategy for cultivar identification which could
be adopted to develop a universal identification system for date palm cultivars. This
strategy is not much expensive and data could be generated by sequencing few DNA
fragments from the nuclear genome of date palm.
Molecular, morphological and isozyme markers have resulted in different
grouping of the cultivars (Elhomaizi et al., 2002; Bndiab et al., 1993; Sedra et al.,
1998). The reason may be that morphological traits are controlled by many genes and
are affected by the environment too. Morphological studies are however important for
78
the selection of cultivars and improvement of crop (Elhoumaizi et al., 2002).
Morphological based phylogenetic approach is as important as molecular analysis
based method as the structure of basic biomolecules of all organisms is similar and
morphological characters of an organism are the illustration of its genome, protein,
and transcriptome profiles. Thus the combination of the two methods gives strength to
the phylogenetic relationship of the organism (Patwardhan et al., 2014). Organisms
which are similar phenotypically may be quite different in their molecular and
biochemical characteristics. Thus determination of the phylogenetic relationship is
very difficult as organisms show immense diversity shown by the molecular,
biochemical and morphological characters (Patwardhan et al., 2014). Hammadi et al.
(2011) found no correlation between molecular and morphological data. The reason
may be the SSR variation is based on variation in DNA sequence only but
morphological traits are affected by the environment.
In current study forty five cultivars of date palm grown in Pakistan were
successfully characterized on the basis of their morphological traits of trunk, leaves,
spines, fruit and seed and proximate composition of the date fruit at khalal stage.
Morphological characters of fruit and its proximate composition appeared to be
important for characterization of the local cultivars and these were successfully
grouped into four clusters but these characters were not sufficient for identification of
individual cultivar. Similarly SSR markers appeared to be mostly monomorphic in the
studied germplasm showing their genetic similarity in the studied region. Likewise
genes and gene fragments from chloroplast region showed almost complete identity to
each other and to the reference genome of data palm cultivar ‘Khalas’. Only SNPs
data were able to identify individual cultivars of date palm thereby providing an
authentic way of identification and discrimination among the cultivars. This system
can be applied at an early stage of growth of the plant and thus time and resources can
be saved.
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VI. SUMMARY, CONCLUSIONS & RECOMMENDATIONS
Date palm is an important fruit crop of Pakistan and it has a long history of
cultivation and a valuable germplasm in the country. More than 300 cultivars of date
palm are grown in the country. It is an important export commodity. Date palm is a
monocotyledonous, dioecious, perennial woody plant of the Arecaceae family. It is
diploid in nature with 18 pairs of chromosomes. Date palm is mainly grown for its
edible fruit which is very rich in nutrients. Date palm is unique in the sense that it has
the highest number of cultivars among fruits in the world.
Diversity in plants is analyzed on the basis of the agronomic characteristics,
morphological characteristics, biochemical attributes and DNA based assays.
Diversity analysis is important for determination of genetic variability of the cultivars,
for selection of parental combination to provide maximum diversity and for
identification of varieties for their protection. Date palm cultivars presents high
degree of variation in their traits. Date palm cultivars have been characterized by
phenotypic markers like leaves, leaflets, spines, offshoots and inflorescence, fruit
morphological traits, isozyme markers and molecular markers. Knowledge of the
qualitative data of fruit is important for processors, exporters, and consumers. Quality
of the date fruit changes with cultivar and depend on climatic conditions and farming
practices.
Molecular marker technology can also be applied for identification of
commercial varieties and to know about the genome polymorphism based on DNA
analysis. Techniques used for detection of molecular markers are AFLP, RAPD,
RFLP, and SSR. Simple sequence repeats (SSR) markers are used to detect length
variation with the help of PCR and are being used as highly informative genetic
markers. SSR markers are codominant markers that depict high allelic diversity at
different loci and are thus helpful in identification of the cultivars.
Date palm is mostly propagated through offshoots of the mother plant and
since plastid genes are transferred mostly from mother line so identification is
possible by sequencing of plastid genes. Chloroplast genes like rbcL (Ribulose
biphosphate carboxylase larger subunit (1400bp), matK (1500bp) encoding maturase
80
required for photosynthetic like activity of chloroplast and atpB have been previously
used for resolving the phylogeny of palm at various levels. Single nucleotide
polymorphism markers are the third generation of molecular markers that occur
among DNA samples with respect to single base. SNPs are biallelic in nature and
found abundantly in the genome.
Although date palm is economically and socially important crop but it is less
researched in terms of genetic characterization. Characterization is important for the
identification of varieties, for conservation of the germplasm and to have diverse
parental combination to create variability.
Most of the research done on date palm previously was based on
morphological or yield parameters or biochemical composition of the date fruit. Few
investigations have been made for genetic characterization using RAPD markers or
typing of one or two genes. Morphological or biochemical markers have the
limitations of being limited in number and affected by the environment and also these
markers are dependent upon the stage of the plant growth. These limitations reduce
their reliability in assessment of diversity and characterization of the germplasm. This
necessitates the use of genetic characterization with the use of DNA markers, gene
sequencing or SNPs typing that can be employed to any stage of plant and are not
affected by the environment. A combination of morphological, biochemical and
molecular characterization of the date palm cultivar can better assess the level of
diversity and relationship among the cultivars. So this study was conducted with the
objectives of development of a reliable identification system for germplasm
characterization in date palm and for assessment of genetic diversity in local date
palm cultivars.
This research work was conducted at National Institute for Genomics and
Advanced Biotechnology, National Agriculture Research Center and Plant
Biochemistry and Biotechnology laboratory of Biosciences department, COMSATS
Islamabad, during 2012-15. Forty five date palm cultivars from Date Palm Research
Farm Jhang, and Horticulture Research Station, Bahawalpur, Pakistan were selected
for this study. Seventeen morphological parameters of trunk, leaf and fruit were
selected for this study. The studied traits included trunk diameter, leaf length and
width, leaf base width, spine and pinnae number, midrib length, length of midrib with
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spines, length of midrib with pinnae, length of the top pinnae, l ength, weight, volume
and diameter of fruit, pulp weight, seed weight and perianth height. Ash content (%),
moisture content (%), total sugar (%), reducing sugar (%) and total soluble solids of
fruit at khalal stage were also determined.
Morphological and proximate composition date were subjected to statistical
analysis using Minitab version 16. Basic statistics including mean, standard deviation
and range of the data were calculated and multivariate analysis tools of principal
component analysis and correlation were used to analyze data. Morphological and
proximate data were used to draw dendrogram using NTSYSpc version 2.10 by
UPGMA method under SAHN subprogram of the software.
For molecular diversity evaluation both PCR based and sequencing based
markers were used. PCR based analysis was done through forty six already reported
SSR markers. DNA was extracted from fresh leaves of the selected forty five cultivars
using modified CTAB method. DNA was quantified on agarose gel as well as on nano
spectrophotometer. PCR amplification of the DNA samples were carried out with
selected SSR primers and the PCR product was confirmed and bands were scored on
agarose gel. For sequencing based DNA analysis seven cultivars from the forty five
date palm cultivars were selected on the basis of their commercial importance
.Sequencing based DNA analysis consisted of two parts. In first part chloroplast genes
of atpB, rbcL, matK, GGR and 16S rRNA from the subset of seven cultivars were
amplified using primers designed manually on the sequences of these genes available
in data base. PCR product was confirmed on agarose gel, purified and sequenced
commercially through MACROGEN, Korea. In second part of the sequence based
molecular analysis single nucleotide polymorphisms were typed by the use of primers
designed to amplify the genomic regions which were reported to harbor SNPs.
Principal component analysis showed that length, weight, volume of fruit,
pulp weight, total soluble solids, % reducing sugars, % total sugar, % ash content,
length and width of leaf, midrib length with pinnae, spine number, leaf base width and
perianth height contributed 81% variability among the cultivars. Morphological traits
of trunk, leaves and spines had no significant correlation with fruit traits. Only two
out of forty six SSR markers showed polymorphism with amplification of five
amplicons, 24 markers amplified monomorphic bands while the remaining 20 primers
82
did not amplify. Coefficient matrices were computed to form clusters on
morphological, proximate composition and molecular basis to assess the relationship
among the studied cultivars. Dendrogram based on SSR data divided the studied
cultivars in four clusters.
All these genomic fragments from chloroplast region were found near
identical among the selected cultivars. Twelve DNA fragments already reported to
harbor single nucleotide polymorphisms (SNPs) in date palm nuclear genome were
sequenced and in addition to thirty four already reported SNPs sites, eight novel SNPs
sites were also found in the sequenced fragments. The analysis of these SNPs
indicated that three fragments have the highest marker index (MI) of 4.61, 3.61 and
2.26 and bear eight, seven and five SNPs respectively. A SNP typing system was
developed for varietal identification of data palm cultivars. The system is able to
distinguish not only all the seven studied cultivars from Pakistan but also other
cultivars from the world. The study suggested, that SNPs might be important markers
to study closely related cultivars and in some instances might prove superior even to
sequencing of genes. Further, the strategy we employed to study SNPs in date palm,
could be used to identify closely related cultivars and germplasm found in Pakistan
and elsewhere in the world.
83
Conclusions
On the basis of the results obtained we conclude that:
1. Principal Component Analysis has selected 14 traits i.e length, weight, volume
of fruit, pulp weight, total soluble solids, % reducing sugars, % total sugar, %
ash content, length and width of leaf, midrib length with pinnae, spine number,
leaf base width and perianth height, out of 22 traits studied as important for
characterization of the date palm cultivars grown in Pakistan
2. Strong correlation was found among fruit traits and among the vegetative traits
separately but no significant correlation was found between vegetative and
fruit traits.
3. The dendrogram generated divided the cultivars into four distinct clusters on
the basis of morphological traits and proximate composition of the date fruit..
4. Most of the SSR primers reported to be polymorphic in previous studies could
not eerxplain sufficiently the morphological and chemical diversity in date
palm germplasm of Pakistan. The dendroram generated divided the cultivars
into two groups.
5. Sequence analysis of different gene fragment from chloroplast genome of date
palm have shown similarity in the studied cultivars.
6. SNPs are important markers to study closely related cultivars and in some
instances might prove superior even to sequencing of genes.
7. We report a SNPs typing strategy for cultivar identification which could be
adopted to develop a universal identification system for date palm cultivars.
84
Recommendations
Therefore we recommend that
1. Only those parameters that contributed largely to the variation among the date
palm cultivars should be used for characterization of date palm germplasm
from Pakistan.
2. As the SSR markers used in this study could not explain the diversity clearly
so more SSR markers should be used for diversity analysis of the date palm
cultivars of Pakistan.
3. As date palm is propagated through suckers so these markers are expected to
amplify the same alleles in each plant of the variety in question irrespective of
their origin, sampling site or age.
4. The strategy we employed to study SNPs in date palm, could be used to
identify closely related cultivars and germplasm found in Pakistan and
elsewhere in the world.
5. Molecular signatures through sequencing of SNPs could be developed for date
palm cultivars grown in Sindh and Baluchistan to encompass all the date palm
germplasm of Pakistan.
85
REFERENCES
Abdulla, M. and O. Gamal. 2010. Investigation on molecular phylogeny of some date
palm (Phoenix dactylifera L.) cultivars by protein, RAPD and ISSR markers
in Saudi Arabia. Aust. J. Crop Sci. 4: 23-28
Abul-Soad A. A., S. M. Mahdi, and G. S. Markhand. 2015. Date Palm Status and
Perspective in Pakistan. p. 153-205. In: Al-Khayri, J. M., S. M. Jain and D.V.
Johnson (eds). Date Palm Genetic Resources and Utilization: Vol. 2: Asia and
Europe. Dordrecht, Springer Netherlands
Adawy, S. S., E. H. A. Hussein, S. E. M. E. Ismail, and H. A. El-Itriby. 2005.
Genomic diversity in date palm (Phoenix dactylifera L.) as revealed by AFLPs
in comparison to RAPDs and ISSRs. Arab J. of Biotechnol. 8: 99-114
Afzal, M. M. 2005. Khajoor: Mutalia, Mushahidaat aur Tajerbaat. Urdu Bazar Lahore,
Pakistan: Al Faisal Nashran aur Tajiran e kutub
Ahmed, T. A. and S. Al-Hadidi. 2014. Molecular characterization of date palm
(Phoenix dactylifera L.) using Inter Simple Sequence Repeat (ISSR) markers.
In: Zaid, A., and G. A. Alhadrami, (eds). Proceedings of the Fifth International
Date Palm Conference. Abu Dhabi, UAE. March 16-18
Ahmed, T. A., and A. Al-Qaradawi. 2009. Molecular phylogeny of Qatari date palm
genotypes using simple sequence repeats markers. Biotechnology. 8: 126-131
Akhtar, W., A. Rasheed, Z. K. Shinwari, S. M. S. Naqvi, and T. Mahmood, 2014.
Genetic characterization of different Pakistani date palm varieties. Pak. J. Bot.
46: 2095-2100
Akkak, A., V. Scariot, D. T. Marinoni, P. Boccacci, C. Beltramo, and R. Botta. 2009.
Development and evaluation of microsatellite markers in Phoenix dactylifera
L. and their transferability to other Phoenix species. Biol Plantarum. 53: 164-
166
86
Al-Dous, E. K., B. George, M. E. Al-Mahmoud, M. Y. Al-Jaber, H. Wang, Y. M.
Salameh, E. K. Al-Azwani, S. Chaluvadi, A. C. Pontaroli, J. DeBarry, V.
Arondel, J. Ohlrogge, I. J. Saie, K. M. S. Elmeer, J. L. Bennetzen, R. R.
Kruegger and J. A. Malek. 2011. De novo genome sequencing and
comparative genomics of date palm (Phoenix dactylifera). Nat. Biotechnol.
29: 521-527
Aleid, S. M., B. H. Hassan, S. A. Almaiman, S. H. Al-Kahtani, and S. M. Ismail,
2014. Microbial loads and physicochemical characteristics of fruits from four
Saudi date palm tree cultivars: conformity with applicable date standards. J.
Food and Nutr Sci. 5: 316-327
Al-Khalifah, N. S. 2006. Micro propagation and DNA fingerprinting of date palm
trees of Saudi Arabia. Association of Agricultural Research Institutions in the
Near East and North Africa, AARINENA publication, Jordan
Al-Khalifah, N. S., E. Askari, and A. S. Khan. 2012. Molecular and morphological
identification of some elite varieties of date palms grown in Saudi Arabia.
Emir. J. Food Agric. 24: 456-461
Al-Mssallem, I. S., S. Hu, X. Zhang, Q. Lin, W. Liu, J. Tan, X. Yu, J. Liu, L. Pan, T.
Zhang, 2013. Genome sequence of the date palm (Phoenix dactylifera L.) Nat.
Commun. 4: 2274
Al-Qurainy, F., S. Khan, F. M. Al-Hemaid, M. A. Ali, M. Tarroum and M. Ashraf.
2011. Assessing molecular signature for some potential date (Phoenix
dactylifera L.) cultivars from Saudi Arabia, based on chloroplast DNA
sequences rpoB and psbA-trnH. Int. J. Mol. Sci. 12(10): 6871-6880
Al-Ruqaishi, I. A., M. Davey, P. Alderson, and S. Mayes. 2008. Genetic relationships
and genotype tracing in date palm (Phoenix dactylifera L.) in Oman based on
microsatellite markers. Plant Genet. Resour. 6: 70-72
Al-Shahib, W. and R. J. Marshall. 2003. The fruit of the date palm: Its possible use as
the best food for the future. Int. J. Food Sci. Nutr. 54: 247-259
87
Al-Wusaibai N., A. Ben Abdallah, M. Al-Husseini, H. Al-Hajji, H. Al-Salman, K.
Kurshed, A. Oihabi and M. El-Bellaj. 2014. Morphological characterization of
Saudi Arabian date palm cultivars based on vegetative and reproductive traits.
In: Zaid, A., and G. A. Alhadrami, (eds). Proceedings of the Fifth International
Date Palm Conference Abu Dhabi, UAE. March 16-18
Al-Yahyai, M. 2008. Arbuscular mycorrhizal fungal communities associated with
date palms in a traditional and a modern experimental plantation and with
desert plants in the adjacent natural habitats in Southern Arabia. PhD Thesis,
University of Basel, Switzerland
Anjum, F. M., S. I. Bukhat, A. H. El-Ghorab, M. I. Khan, M. Nadeem, S. Hussain,
and M. S. Arshad. 2012. Phytochemical characteristics of date palm (Phoenix
dactylifera L.) fruit extracts. Pak. J. Food Sci. 22: 117-127
Anonymous. 2015. Fruit, vegetables and condiments statistics of Pakistan, 2014-15.
Ministry of National Food Security and Research. Economic wing, Islamabad
Arabnezhad, H., M. Bahar, H. R. Mohammadi, and M. Latifian. 2012. Development,
characterization and use of microsatellite markers for germplasm analysis in
date palm (Phoenix dactylifera L.). Sci Hortic-Amsterdam. 134: 150-156
Azeqour, M., K. Majourhat, and M. Baaziz, 2002. Morphological variations and
isoenzyme polymorphism of date palm clones from in vitro culture
acclimatized and established on soil in South Morocco. Euphytica. 123: 57-66
Baker, W. J., J. Dransfield, and T. A. Hedderson. 2000. Phylogeny, character
evolution, and a new classification of the Calamoid palms. Syst Bot. 25: 297-
322
Barthet, M. M. and K. W. Hilu. 2007. Expression of matK: functional and
evolutionary implications. Am. J. Bot. 94: 1402-1412
88
Bashir, M., M. Ahmad, F. Altaf, and K. Shabir. 2014. Fruit quality and yield of date
palm (Phoenix dactylifera L.) as affected by strand thinning. J. Anim. Plant
Sci. 24: 951-954
Billotte, N., N. Marseilla, P. Brottier, J. L. Noyer, J. P. Jacquemoud-Collet, C.
Moreau, T. Couvreur, M. H. Chevallier, J. C. Pintaud, and A. M. Risterucci.
2004. Nuclear microsatellite markers for the date palm (Phoenix dactylifera
L.): characterization and utility across the genus Phoenix and in other palm
genera. Mol. Ecol. Notes. 4: 256-258
Bodia, A., M. A. Elhoumaizi, K. N .Ndir, A. Hasnoui, M. Nachtigall, P. Wehling.
2012. Genetic diversity analysis of date palm (Phoenix dactylifera L.)
cultivars from Figuig oasis (Morocco) using SSR markers. Int. J. Sci. Adv.
Technol. 2: 96-104
Botstein, D., R. L. White, M. Skolnick and R. W. Davis. 1980. Construction of a
genetic linkage map in man using restriction fragment length
polymorphisms. Am. J. Hum. Genet. 32: 314-331
Burgess, K. S., A. J. Fazekas, P. R. Kesanakurti, S. W. Graham, B. C. Husband, S. G.
Newmaster, D. M. Percy, M. Hajibabaei, and S. C. H. Barrett. 2011.
Discriminating plant species in a local temperate flora using the rbcL+matK
DNA barcode. Methods Ecol. Evol. 2: 333-340
Cabezas, J. A., J. Ibáñez, D. Lijavetzky, D. Vélez, G. Bravo, V. Rodríguez, I.
Carreño, A. M. Jermakow, J. Carreño, L. Ruiz-García, M. R.Thomas, J. M.
Martinez-Zapater. 2011. A 48 SNP set for grapevine cultivar identification.
BMC Plant Biol. 11: 1-12
Cao, B. R. and C-C. T. Chao. 2002. Identification of date cultivars in California using
AFLP markers. HortScience. 37: 966-968
89
Chaludvadi, S.R., S. Khanam, M. A. Aly and J. L. Bennetzen. 2014. Genetic diversity
and population structure of native and introduced date palm (Phoenix
dactylifera) germplasm in the United Arab Emirates. Trop Plant Biol. 7: 30-41
Chao, C.T. and R. R. Krueger. 2007. The date palm (Phoenix dactylifera L.):
overview of biology, uses, and cultivation. HortScience. 42: 1077-1082
Costantini, L. 1985. Considerazioni su alcuni reperti di palma da dattero e sul centro
di origine e l'area du coltivazione della Phoenix dactylifera L. Rome: Istituto
Italiano per il Medio ed Esremo Oriente, Serie Orientale Roma
Diaz, S., C. Pire, J. Ferrer and M. J. Bonete. 2003. Identification of Phoenix
dactylifera L. varieties based on amplified fragment length polymorphism
(AFLP) markers. Cell Mol. Biol. Letters. 8: 891-900
Eissa, E., A. A. El-Razek, S. El-Sharabasy, and R. Rizk, 2009. Morphological and
molecular genetic characterization of soft date palm (Phoenix dactylifera L.)
cultivars in Egypt. Egypt J.Genet. Cytol. 38: 269-284
El Hadrami, A. and J. M. Al-Khayri. 2012. Socioeconomic and traditional importance
of date palm. Emir. J. Food Agric. 24: 371-385
El-Assar, A. M., R. R. Krueger, P. S. Devanad and C. T. Chao. 2005. Genetic analysis
of Egyptian date (Phoenix dactylifera L.) accessions using AFLP markers.
Genet. Resour. Crop Ev. 52:601-607
Elhoumaizi, M. A, P. S. Devanand, J. Fang and C-CT. Chao. 2006. Confirmation of
`Medjool' date as a landrace variety through genetic analysis of `medjool'
accessions in Morocco. J. Am. Soc. Hortic. Sci. 131: 403-407
Elhoumaizi, M.A., M. Saaidi, A. Oihabi, and C. Cilas, 2002. Phenotypic diversity of
date palm cultivars (Phoenix dactylifera L.) from Morocco. Genet. Resour.
Crop Ev. 49: 483-490
90
El-Kosary, S. 2009. Comparison study on Barhee cultivar and two strains of Barhee
palm seedling trees. Egypt J. App Sci. 24: 768-783
Elmeer K., and I. Mattat. 2012. Marker-assisted sex differentiation in date palm using
simple sequence repeats. 3 Biotech 2: 241-247
Elmeer, K., and I. Mattat, 2015. Genetic diversity of Qatari date palm using SSR
markers. Genet. Mol. Res. 14: 1624-1635
Elmeer, K., H. Sarwath, J. Malek, M. Baum and A. Hamwieh. 2011. New
microsatellite markers for assessment of genetic diversity in date palm
(Phoenix dactylifera L.). 3 Biotech. 1: 91-97
El-Merghany, S., and E. M. A. Z. El-Daen. 2014. Evaluation of some date palm
cultivars grown under Toshky conditions. In: Zaid, A., and G. A. Alhadrami,
(eds). Proceedings of the Fifth International Date Palm Conference. Abu
Dhabi, UAE. March 16-18, 2014
Elsafi, M. 2012. Study on the on-farm diversity of local date palm (Phoenix
dactylifera L.) genetic resources grown in Northern region of Sudan. PhD
dissertation. Swedish University of Agricultural Sciences, Sweden
Elshibli, S. and H. Korpelainen, 2008. Microsatellite markers reveal high genetic
diversity in date palm (Phoenix dactylifera L.) germplasm from Sudan.
Genetica. 134: 251-260
Elshibli, S. and H. Korpelainen. 2009. Biodiversity of date palms (Phoenix dactylifera
L.) in Sudan: chemical, morphological and DNA polymorphisms of selected
cultivars. Plant Genet Resour. 7: 194-203
Elshibli, S., and H. Korpelainen. 2010. Identity of date palm (Phoenix dactylifera L.)
germplasm in Sudan: from morphology and chemical characters to molecular
markers. Acta Hortic. 859: 143-153
91
Enan M. and A. Ahmed 2014. DNA barcoding based on plastid matK and RNA
polymerase for assessing the genetic identity of date (Phoenix dactylifera L.)
cultivars. Genet. Mol. Res. 13: 3527-3536
Enan, M. and A. Ahmed. 2014. DNA barcoding based on plastid matK and RNA
polymerase for assessing the genetic identity of date (Phoenix dactylifera L.)
cultivars. Genet. Mol. Res. 13: 3527-3536
Farag, K., A. Elsabagh, and H. ElAshry. 2012. Fruit characteristics of “Zaghloul” date
palm in relation to metaxenic influences of used pollinator. Am. Eurasian J.
Agric. Environ. Sci. 12: 842-855
Germano, J. and A. S. Klein. 1999. Species-specific nuclear and chloroplast single
nucleotide polymorphisms to distinguish Picea glauca, P. mariana and P.
rubens. Theor. Appl. Genet. 99:37-49
Guido, F., S. E. Behija, I. Manel, Z. Nesrine, F. Ali, H. Mohamed, H. A. Noureddine,
and A. Lotfi. 2011. Chemical and aroma volatile compositions of date palm
(Phoenix dactylifera L.) fruits at three maturation stages. Food Chem. 127:
1744-1754
Gupta, P., J. Roy, and M. Prasad. 2001. Single nucleotide polymorphisms: a new
paradigm for molecular marker technology and DNA polymorphism detection
with emphasis on their use in plants. Curr. Sci. 80: 524-535
Gupta, P., J. Roy, M. Prasad. 2001. Single nucleotide polymorphisms: a new
paradigm for molecular marker technology and DNA polymorphism detection
with emphasis on their use in plants. Curr. Sci. 80: 524-535
Hahn, W. J. 2002. A molecular phylogenetic study of the Palmae (Arecaceae) based
on atpB, rbcL, and 18S nrDNA sequences. Syst. Biol. 51: 92-112
92
Haider, M. S., I. A. Khan, M. Jaskani, S. A. Naqvi, M. Hameed, M. Azam, A. A.
Khan, and J. C. Pintaud. 2015. Assessment of morphological attributes of date
palm accessions of diverse agro-ecological origin. Pak. J. Bot. 47: 1143-1151
Haider, M. S., I. A. Khan, S. A. Naqvi, M. Jaskani, R. W. Khan, M. Nafees, M. Pasha,
and I. Pasha. 2013. Fruit developmental stages effects on biochemical
attributes in date palm. Pak. J. Agr. Sci. 50: 577-583
Hamada, H., M. G. Petrino and T. Kakunaga. 1982. A novel repeated element with Z-
DNA-forming potential is widely found in evolutionarily diverse eukaryotic
genomes. Proceedings of the National Academy of Sciences of the United
States of America 79: 6465-6469
Hammadi, H., G. G. Vendramin and A. Ali. 2011. Microsatellite diversity among
Tunisian date palm (Phoenix dactylifera L.) subpopulations. Pak. J. Bot. 43:
1257-1264
Hammadi, H., M. A. Benabderrahim, M. Elbekkay, G. Ferdaous, T. Triki, and A.
Ferchichi. 2012. Investigation of genetic variation in Tunisian date palm
(Phoenix dactylifera L.) cultivars using ISSR marker systems and their
relation with fruit characteristics. Turk J. Biol. 36: 449-458
Hammadi, H., M. Elbekkay, M. B. Abederrahim and A. F. Ali. 2011. Molecular and
morphological analyses of date palm (Phoenix dactylifera L.) subpopulations
in southern Tunisia. Span. J. Agric. Res. 9: 484-493
Hammadi, H., R. Mokhtar, E. Mokhtar and F. Ali. 2009. New approach for the
morphological identification of date palm (Phoenix dactylifera L.) cultivars
from Tunisia. Pak. J. Bot. 41: 2671-2681
Horwitz, W. and G. W. Latimer. 2007. Official methods of analysis of AOAC
International. AOAC International. Gaithersburg, USA
93
Hamwieh, A., J. Farah, S. Moussally, K. Al-Sham'aa, K. Almer, H. Khierallah, S.
Udupa, S. Lababidi, J. A. Malek, M. Aaouine and M. Baum. 2010.
Development of 1000 microsatellite markers across the date palm (Phoenix
dactylifera L.) Genome. In: Zaid, A. and Alhadrami, G. A. (eds). Proceedings
of 4th International Date Palm Conference. Abu Dhabi, UAE.15-17 March,
2010
Hasnaoui, A., Elhoumaizi, M. A., C. Borchani, H. Attia and S. Besbes. 2012. Physico-
chemical characterization and associated antioxidant capacity of fiber
concentrates from Moroccan date flesh. Indian J. Sci Technol. 5: 2954-2960
Heise, W., W. Babik, D. Kubisz and L. Kajtoch. 2015. A three‐marker DNA
barcoding approach for ecological studies of xerothermic plants and
herbivorous insects from central Europe. J. Linn. Soc., Bot. 177: 576-592
Herch, W., H. Kallel and S. Boukhchina, 2014. Physicochemical properties and
antioxidant activity of Tunisian date palm (Phoenix dactylifera L.) oil as
affected by different extraction methods. Food Sci. Technol. 34: 464-470
Hoot, S. B., A. Culham and P. R. Crane. 1995. The utility of atpB gene sequences in
resolving phylogenetic relationships: Comparison with rbcL and 18S
ribosomal DNA sequences in the Lardizabalaceae. Ann. Missouri Bot. 82:
194-207
Hyder, M. Z., S. H. Shah, S. Hameed and S. M. S. Naqvi. 2011. Evidence of
recombination in the Banana bunchy top virus genome. Infect Genet Evol. 11:
1293-1300
Ibrahim, I. A., M. H. Hashem, A. A. Hemeida, M. M. Hassan and A. Maksoud. 2014.
Characterization of genetic diversity of date palm (Phoenix dactylifera L.)
cultivars collected from New Valley Governorate (El-Kharga and Dakhleh)
based on morphological variability and molecular markers. Life Sci. J. 11:
879-889
94
Iqbal, M., M. Munir and M. Niamatullah. 2011. Physio-chemical characteristics of
date palm (Phoenix dactylifera L.) cultivars at various maturity stages under
environmental conditions of Dera Ismail Khan. J. Agric. Res. 49: 249-259
Iqbal, M., U. U. Rahman and M. Niamatullah. 2012. Periodic growth and
development of fruits of different date cultivars grown under the agro climatic
conditions of D.I. Khan. Pak. J. Sci. 64: 259-264
Jahromi, M. K., A. Jafari, A. Keyhani, R. Mirasheh and S. Mohtasebi. 2007. Some
physical properties of date fruit (cv. Lasht). Agricultural Engineering
International: CIGR eJournal. (http:// www. cigrjournal. org /index.php/
Ejounral/article/view/ 887)
Jahromi, M. K., S. Mohtasebi, A. Jafari, R. Mirasheh and S. Rafiee. 2008.
Determination of some physical properties of date fruit (cv. Mazafati). J.
Agric. Technol. 4: 1-9
Jain, S. M., J. Al-Khayriand and D.V. Johnson. 2011. Date palm biotechnology.
Springer, New York, USA
Jamil, M. S., R. Nadeem, M. A. Hanif, M. A. Ali and K. Akhtar. 2010. Proximate
composition and mineral profile of eight different unstudied date (Phoenix
dactylifera L.) varieties from Pakistan. Afr. J. Biotechnol. 9: 3252-3259
Jandan, D.M. 1974. Studies of some characters of important varieties of date palm
(Phoenix dactylifera L.) grown in Khairpur. M.Sc Thesis. University of Sindh.
Jamshoro, Pakistan.
Jaradat, A. A. 2011. Biodiversity of date palm. In: Encyclopedia of Life Support
Systems: Land Use, Land Cover and Soil Sciences. Eolss Publishers, Oxford,
UK
Jaradat, A. A. 2014. Synthesis and assessment of date palm genetic diversity studies.
Emir. J. Food Agric. 26: 934-952
95
Jatoi, M.A., Z. Markhand, and N. Solangi. 2009. Dates in Sindh: facts and figures. p.
59-70. In: Proceedings of the international date seminar. Shah Abdul Latif
University, Khairpur, Sindh, Pakistan. 28 July 2009
Jehan, T. and S. Lakhanpaul. 2006. Single nucleotide polymorphism (SNP) –Methods
and applications in plant genetics: A review. Indian J. Biotechnol. 5: 435-459
Johnson, C., T. Cullis, M. Cullis and C. Cullis. 2009. DNA markers for variety
identification in date palm (Phoenix dactylifera L.). J. Hortic Sci. Biotechnol.
84: 591-594
Johnson, D., J. Al-Khayri, and S. Jain. 2013. Seedling date palms (Phoenix dactylifera
L.) as genetic resources. Emir. J. Food Agric. 25: 809-830
Jones, E., H. Sullivan, D. Bhattramakki and J. Smith. 2007. A comparison of simple
sequence repeat and single nucleotide polymorphism marker technologies for
the genotypic analysis of maize (Zea mays L.). Theor Appl Genet. 115: 361-
371
Jubrael, J. M. S., S. M. Udupa and M. Baum. 2005. Assessment of AFLP-based
genetic relationships among date palm (Phoenix dactylifera L.) varieties of
Iraq. J. Am. Soc. Hortic Sci. 130: 442-447
Kenoyer, J. M. and K. Heuston. 2005. The Ancient South Asian World. Oxford
University Press
Khanam, S., A. Sham, J. L. Bennetzen and M. A. Aly. 2012. Analysis of molecular
marker-based characterization and genetic variation in date palm (Phoenix
dactylifera L.). Aust. J. Crop Sci. 6: 1236-1244
Khew, GS-W. and T. F. Chia. 2011. Parentage determination of Vanda Miss Joaquim
(Orchidaceae) through two chloroplast genes rbcL and matK. AoB Plants.
(doi:10.1093/aobpla/plr018)
96
Khierallah, H., S. Bader, M. Baum and A. Hamwieh. 2011. Assessment of genetic
diversity for some Iraqi date palms (Phoenix dactylifera L.) using Amplified
Fragment Length Polymorphisms (AFLP) markers. Afr. J. Biotechnol. 10:
9570-9576
Khierallah, H.S.M., S.M Bader, M. Baum and A. Hamwieh. 2011. Genetic diversity
of Iraqi date palms revealed by microsatellite polymorphism. J. Am. Soc.
Hortic. Sci. 136: 282-287
Kirk, R. S. and ssR. Sawyer. 1991. Pearson’s Composition and analysis of foods.
Longman scientific and technical, United Kingdom
Kress, W. J. and D. L. Erickson. 2007. A two-locus global DNA barcode for land
plants: The coding rbcL gene complements the non-coding trnh-psbaspacer
region. PLoS ONE 2(6): e508. (doi:10.1371/journal.pone.0000508)
Levinson, G. and G. A. Gutman. 1987. Slipped-strand mispairing: a major mechanism
for DNA sequence evolution. Mol. Biol. Evol. 4: 203-221
Markhand, G. S., A. A. Abul-Soad, A. A. Mirbahar and N. A. Kanhar. 2010. Fruit
characterization of Pakistani dates. Pak. J. Bot. 42: 3715-3722
Marsafari, M. and A. A. Mehrabi. 2013. Molecular identification and genetic diversity
of Iranian date palm (Phoenix dactylifera L.) cultivars using ISSR and RAPD
markers. Aust J. Crop Sci. 7:1160-1166
Mathew L.S., M. A. Seidel, B. George, S. Mathew, M. Spannagl, G. Haberer, M. F.
Torres, E. K. Al-Dous, E. K. Al-Azwani and I. Diboun. 2015. A genome-wide
survey of date palm cultivars supports two major subpopulations in Phoenix
dactylifera. G3: Genes Genom Genet. 5: 1429-1438
Milney, D. 1918. The date palm (Phoenix dactylifera) and its cultivation in Punjab.
Tacker, Spink and Co. Colcutta, India
97
Mirbahar, A. A., G. S. Markhand, S. Khan and A. A. Abul-Soad. 2014. Molecular
characterization of some Pakistani date palm (Phoenix dactylifera L.) cultivars
by RAPD markers. Pak. J. Bot. 46: 619-625
Mitra, C., P. Kharb, S. Uppal and S. Jain. 2011. Genetic diversity analysis in date
palm (Phoenix dactylifera L.): a comparative assessment using ISSR and
RAPD marker assays. J. Hortic. Sci. Biotechnol. 86: 398-402
Mohamed, R., Fageer, A. S., Eltayeb, M. M., & Mohamed Ahmed, I. A. (2014).
Chemical composition, antioxidant capacity, and mineral extractability of
Sudanese date palm (Phoenix dactylifera L.) fruits. Food Sci. Nutr. 2: 478-
489.
Mohammadi, S. A. and B. M. Prasanna. 2003. Analysis of genetic diversity in crop
plants: Salient statistical tools and considerations. Crop Sci. 43: 1235-1248
Nadeem, M., Salim-ur-Rehman, F. M. Anjum and I. A. Bhatti. 2011. Textural profile
analysis and phenolic content of some date palm varieties. J. Agric. Res. 49:
525-539
Naqvi, S. A., I. A. Khan, J. C. Pintaud, M. J. Jaskani and A. Ali. 2015. Morphological
characterization of Pakistani date palm (Phoenix dactylifera L.) genotypes.
Pak. J. Agric. Sci. 52: 645-650
Nixon, R.W. 1951. Fruit thinning experiment with the Medjoool and Barhee varieties
of dates. Dates Growers Inst Report. 28:14-17
Odewale, J.O., C. D. Ataga, G. Odiowaya, A. Hamza, A. Collins, and M. N. Okoye.
2012. Multivariate analysis as a tool in the assessment of physical properties
of fruits (Phoenix dactylifera L) in Nigeria. Plant Sciences Feed. 2: 138-146
98
Osman, A., B. Jordan, P.A Lessard, N. Muhammad, M. R. Haron, N. M. Raffin, A. J.
Sinskey, C. Rha, D. E. Housman. 2003. Genetic diversity in Eurychoma
longifolia, inferred from single nucleotide polymorphism. Plant Physiol. 131:
1294-1301
Pasha, S., A. Hussain, and I. Gajani. 1972. Date palm of Sindh. Punjab fruit J. 33: 9-
14
Patwardhan, A., S. Ray and A. Roy. 2014. Molecular Markers in Phylogenetic
Studies-A Review. J. Phylogenetics Evol. Biol. 2:131 (doi:10.4172/2329-
9002.1000131)Pintaud, J-C., B. Ludeña, F. Aberlenc-Bertossi, S. Zehdi, M.
Gros-Balthazard, S. Ivorra, J. F. Terral, C. Newton, M. Tengberg, S.
Abdoulkader, A. Daher, M. Nabil, I. Hernández, M. A. González-Pérez, P.
Sosa, S. Santoni, S. Moussouni, F. Si-Dehbi and N. Bouguedoura. 2013.
Biogeography of the date palm (Phoenix dactylifera L., Arecaceae): Insights
on the origin and on the structure of modern diversity. Acta Hortic. 994, 19-38
(doi: 10.17660/ActaHortic.2013.994.1)
Pintaud, J-C., S. Zehdi, T. Couvreur, S. Barrow, S. Henderson, F. Aberlenc-Bertossi,
J. Tregear and N. Billotte. 2010. Species delimitation in the genus Phoenix
(Arecaceae) based on SSR markers, with emphasis on the identity of the date
palm (Phoenix dactylifera L.). p. 267-286. In: Seberg O., G. Petersen, A.
Barfod and J. Davis. (éds.). Diversity, phylogeny and evolution in the
monocotyledons. Arhus University Press, Denmark
Powell, D. C. 2005. Available at: http://www.fs.usda.gov (accessed 6/27/15)
Powell, W., G. C. Machray and J. Provan. 1996. Polymorphism revealed by simple
sequence repeats. Trends Plant Sci. 1: 215-222
Powell, W., M. Andre, M. Hanafey, J. Vogel, S. Tingey, and A. Rafalski. 1996. The
comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for
germplasm analysis. Mol. Breed. 2: 225-238
99
Qasim, M. and S. A. Naqvi. 2012. Dates: production, processing, food and medicinal
values. p. 341-349. In: Manickavasagan, A., M. Mohamed Essa and E. Sukumar
(eds). A fruit from heaven. CRC Press, Taylor and Francis Group,UK
Quinlan, J. R. 1986. Induction of decision trees. Mach Lear. 1: 81-106
Racchi, M. L., A. Bove, A. Turchi, G. Bashir, M. Battaglia and A. Camussi. 2014.
Genetic characterization of Libyan date palm resources by microsatellite
markers. 3 Biotech. 4: 21-32
Rani, R., P. Kharb, S. Uppal, S. Dhillon, P. Batra and V. Chowdhury. 2007. Genetic
divergence analysis in date palm (Phoenix dactylifera L.) genotypes using
RAPD markers. Haryana J. Hortic Sci. 36: 101-103
Rawashdeh, I. and A. Amri. 2006. Genetic characterization of date palm varieties
using RAPD markers. Jordan J. Agric Sci. 2: 234-242
Raza, G., D. Raza, , I. A. Khan, and Khatri A. 2006. Genetic diversity analysis of
local date palm (Phoenix dactylifera L.) cultivars of Pakistan through
molecular marker (RAPD). Int. J. Biol. Biotechnol. 3: 673-676.
Rivera, D., C. O. de Castro, E. Carreno, C. Inocencio, F. Alcaraz, S. Rios, J. Palazon,
L. Vazquez and E. Laguna. 2008. Morphological systematics of date-palm
diversity (Phoenix, Arecaceae) in Western Europe and some preliminary
molecular results. Acta Hortic. 799: 97-104
Rizk, R. M. and S. F. El-Sharabasy 2006. A descriptor for date palm (Phoenix
dactylifera L.) characterization and evaluation in genebank. American
Eurasian J. Agric. and Environ. Sci. 1:133-145
Saker, M. M., S. A. Kawshity, M. K. El-Bahr, A. S. Fahmy. 2002. Flavonoid content
and isozyme analysis of tissue culture-derived date palm clones. Arab J. of
Biotechnol. 5: 207-216
100
Saker, M. M., S. S. Adawy, A. A. Mohamed, and H. A. El-Itriby. 2006. Monitoring of
cultivar identity in tissue culture-derived date palms using RAPD and AFLP
analysis. Biol Plantarum. 50: 198-204
Sakr, M. M., I. A. Zeid, A. E. Hassan, A. I. Baz and W. M. Hassan. 2010.
Identification of some date palm (Phoenix dactylifera) cultivars by fruit
characters. Indian J. Sci Technol. 3: 338-343
Saleem, S. A. 2005. Aspects of ripening of Dhakki dates (Phoenix dactylifera L.) and
post harvest stability employing hurdle technology. PhD thesis. Gomal
University, D. I. Khan, Pakistan
Salem, A. O. M., S. Rhouma, S. Zehdi, M. Marrakchi, and M. Trifi. 2008.
Morphological variability of Mauritanian date-palm (Phoenix dactylifera L.)
cultivars as revealed by vegetative traits. Acta Bot. Croat. 67: 81-90
Shaba, E., M. Ndamitso, J. Mathew, M. Etsunyakpa, A. Tsado and S. Muhammad
2015. Nutritional and anti-nutritional composition of date palm (Phoenix
dactylifera L.) fruits sold in major markets of Minna Niger State, Nigeria. Afr.
J. Pure Appl Chem. 9: 167-174
Singh, G. 2014. Molecular characterization in fruit crops-a review. Int. J. Agric Sci.
Vet. Med. 2:81-101
Sneath, P. H. A. and R. R. Sokal. 1973. Numerical taxonomy, the principles and
practice of numerical classification. W. H. Freeman and company, San
Francisco, USA
Soumaya, R. C., B. Ghada, D. D. Sonia, Z. A. Salwa and M. Trifi. 2011. Molecular
research on the genetic diversity of Tunisian date palm (Phoenix dactylifera
L.) using the Random Amplified Microsatellite Polymorphism (RAMPO) and
Amplified Fragment Length Polymorphism (AFLP) methods. Afr. J.
Biotechnol. 10: 10352-10265
101
Sulieman, A. M. E., I. A. A. Elhafise, and A. M. Abdelrahim. 2012. Comparative
study on five Sudanese date (Phoenix dactylifera L.) fruit cultivars. Food Nutr.
Sci. 3: 1245-1251
Taain, D. A. 2013. Study on physico-chemical and physiological characteristics of
date palm fruits (Phoenix dactylifera L.) cv. um-aldehin. Pak. J. Agric. Sci.
50: 1-5
Tamura, K., G. Stecher, D. Peterson, A. Filipski and S. Kumar. 2013. MEGA6:
Molecular Evolutionary Genetics Analysis Version 6.0. Mol. Biol. Evol. 30:
2725-2729
Thompson, J. D., D. G. Higgins, T. J. Gibson. 1994. CLUSTAL W: Improving the
sensitivity of progressive multiple sequence alignment through sequence
weighting, position-specific gap penalties and weight matrix choice. Nucleic
Acids Res. 22: 4673-4680
Uhl, N. W., J. Dransfield, J. I. Davis, M. A. Luckow, K. S. Hansen and J. J. Doyle.
1995. Phylogenetic relationships among palms: Cladistic analyses of
morphological and chloroplast DNA restriction site variation. p. 623–661. In:
Monocotyledons: systematics and evolution. P. Rudall, P. J. Cribb, D. F.
Cutler and C. J. Humphries. (eds). Royal Botanic Gardens, Kew, London
Yahaya, S., C. Omokhudu, I. Koloce, A. Hamza and M. Abdullahi.2015. Proximate
composition and fruit weight of fresh date fruits (Phoenix dactylifera L.)
varieties in wet season of Nigeria. Pak. J. Nutr. 14: 834-842
Yusuf, A., A. Culham, , W. Aljuhani, C. Ataga, A. Hamza, J. Odewale and L.
Enaberue. 2015. Genetic diversity of Nigerian date palm (Phoenix dactylifera)
germplasm based on microsatellite markers. Int. J. Biosci Biotechnol. 7: 121-
132
102
Zehdi, S., E. Cherif, S. Rhouma, S. Santoni, A. S. Hannachi and J. C. Pintaud. 2012.
Molecular polymorphism and genetic relationships in date palm (Phoenix
dactylifera L.): The utility of nuclear microsatellite markers. Sci Hort.
Amsterdam. 148: 255-263
Zehdi, S., H. Sakka, A. Rhouma, A. O. M. Salem, M. Marrakchi and M. Trifi. 2004.
Analysis of Tunisian date palm germplasm using simple sequence repeat
primers. Afr. J. Biotechnol. 3:215-219
Zhao, Y., R. Williams, C. S. Prakash and G. He. 2012. Identification and
characterization of gene-based SSR markers in date palm (Phoenix dactylifera
L.). BMC Plant Biol. 12: 137
103
APPENDIX
Appendix I: Date fruit of 45 cultivars
Aseel
Halawi
Qantar
Makran
Akhrot
Dhaki
104
Chohara
Zahidi
Berahmi
Zirin
Kohraba
Kozanabad
105
Karbaline
Jansohar
Daanda
Begum Jangi
Peela Dora
Shamran
106
Rachna
Saib
Zerdo
Shado
PeeliSundar
Khudrawi
107
HaminWali
Angoor
Champa Kali
Sanduri
Makhi
Dhady
108
Kur
Haleni
Eedal Shah
Sufaidah
Taarwali
Fasli
109
Basra Wali
Pathri
Kupra
Shakri
Gajjarwali
Baidhar
110
Halwain
111
Appendix II: Mean data of morphological traits and proximate compostion of date fruit
Variety T. Dia LL LW LBW SN MLS ML PN MLP LTP F. Wt F. Vol F. Dia F. Len Pp. Wt S Wt P. Ht %moist TSS
% R
Sugars
% T.
Sugars %ash
Akhrot 50.42 284 86 6.33 15.33 43.33 256.33 165 115.33 28 9.23 10.33 2.52 2.76 9.13 0.93 2.85 69.48 32 24.2 28.66 2.94
Dhakki 56.58 385 94.7 8 24.33 57.43 355.33 189.67 295 27.67 26.36 26 2.94 5.45 26.1 1.46 2 62.06 32 20.51 24.77 2.56
Aseel 45.43 344 68.7 6.33 26.33 100 322.33 211.67 120.67 21.33 15.46 14.66 2.58 3.53 12.9 1 3.54 76.23 24 15.69 18.44 3.4
Halawi 46.07 339 75.7 7.33 17.33 75.33 320 172.67 146.33 23.66 13.7 14 2.26 4.36 11.93 1.3 0.94 73.53 20 17.33 19.04 2.98
Qantar 54.88 353 90.7 5.33 20.66 59.67 323.33 198.67 71.67 28.33 10.4 10 2.26 3.28 9.43 1 2.8 74.21 30 18.12 21.25 3.55
Makran 50.95 323 86.3 5.33 35.33 105 318.33 177.33 211.67 24.33 9.83 10 3.25 4.15 8.33 1.13 2.43 74.59 32 22.49 25.73 2.8
Chohara 59.87 373 73.3 8.33 20.66 63.67 351 206 298.7 18 20.27 19.66 2.56 4.68 17.22 1.35 0.53 61.43 36 24.07 30.06 3.07
Zaidi 49.78 347 72 5.33 22.66 53.67 328.33 243 275.33 20.5 9.73 12 2.36 3.6 10.63 1.33 2.27 63.34 48 35.22 40.44 2.87
Berahmi 57.01 306 54 6.33 14.33 59.66 284.67 176.67 223.67 23.17 12.3 12.66 2.15 4.48 10.03 1.26 0.95 67.14 28 20.2 23.26 2.42
Neelum 57.85 340 68.7 7.67 21.66 69.67 310.67 179 242 29 6.1 6 1.86 3.26 4.9 1.03 1.94 76.15 26 12.21 15.22 4.3
Zirin 55.73 311 78.7 6 16.67 57.33 222.67 169.67 229.33 26.83 13.93 14.33 2.22 4.61 11.73 1.86 3.51 62.5 32 24.33 26.5 2.66
Kohraba 53.29 352 84 7.33 14.66 66.67 327.67 189.33 258 23.33 10.56 11.66 2.12 4.13 11 0.9 1.37 62.32 30 22.45 26.54 3.43
Kozanabad 54.46 339 79 7 9.33 52 324 191.33 268.67 16.66 12.6 11.33 2.24 3.41 9.46 1.06 3.04 48.83 30 18.8 32.7 2.4
Karbaline 57.74 370 90.3 7.33 19 68.33 346.3 188.67 266.3 21.33 11.53 10 2.15 4.09 9.46 1.2 1.78 62.51 40 27.22 32.66 3.24
Jansohar 50.84 306 70 7.33 12.33 69 294.67 168.67 230 17.67 14.7 15 2.62 3.52 15.76 0.8 2.43 79.58 20 15.13 16.31 2.92
Kokna 54.77 386 77.3 8 24.33 94.33 362.67 196 275.33 22.67 10.1 10 2.16 3.65 8.1 1.03 1.77 69.31 30 17.33 25.56 3.19
Daanda 52.01 362 84.7 7.33 29.66 64.83 333.67 224.67 262 25.5 12.7 12 2.43 3.58 11.16 1.4 1.72 63.62 28 22.43 24.74 0.64
Begum Jhangi 60.51 370 91 6 31 114.6 347.33 191.67 230.67 26.66 6 6 1.74 3.56 4.53 1.06 2.88 60.55 28 19 22.15 3.32
Peela Dora 54.56 351 92 5.67 24.33 81 319 162.33 233.67 32.67 8.66 10 2.14 3.46 8.7 1.23 2.14 65.44 36 18.65 23.96 3.01
Shamran 61.35 341 84.7 6 20.33 58.67 315 187.67 256.33 26 10.66 12 2.14 4.12 8.7 1.26 2.7 68.44 32 23.4 24.41 3.83
Rachna 51.69 366 77.7 6.67 11.33 38.33 341 234.33 294 22.67 14.2 12.33 2.44 3.54 9.66 1.2 5.6 62.8 26 20.88 23.25 2.28
Saib 52.86 377 108 6 18.66 79 330.7 197.33 250 44.33 16.5 18 2.83 3.87 14.9 1.13 2.99 66.54 28 21.86 25.07 2.12
Zerdo 52.54 343 65.3 6 21.33 61 324.33 193.67 256.67 23.66 5.96 6 1.62 3.24 4.63 1.3 0.93 72.66 22 15.9 18.71 4.54
112
Shado 51.48 319 64.3 5 30 71.67 300.33 208.33 228.33 21 4.76 4 1.53 3.12 3.06 1.06 3.9 66.83 38 24.66 28.01 3.97
PeeliSundar 58.38 400 87 7 18.66 58.33 369 175.67 306.33 32.33 15.73 16.33 2.57 3.94 15.16 0.86 4.8 83.08 14 9.87 12.86 4.22
Khudrawi 57.32 302 73 6 15.33 67.67 310 184 240.67 25.66 14.13 14 2.48 4.08 12.3 1.23 1.5 62.21 38 29.95 33.17 2.77
Haminwali 43.1 273 72.7 6 15.67 58 256 182.67 200.3 25.5 6.13 6 2.71 4.36 5.43 1.43 2.85 47.37 52 38.16 42.08 2.35
Angoor 54.03 358 71.7 7 24 78 337 232.33 257.3 23.33 5.66 6 1.66 2.69 3.7 0.93 5.61 64.76 38 27.8 31.45 3.48
Champa kali 56.16 337 88 5 18.33 36.67 312.3 166.67 313.3 31.33 11.53 10.33 2.29 3.7 8.84 1.2 1.7 72.51 28 23.37 25.15 3.58
Sanduri 42.04 331 90.7 7 14 41 308 171.67 247.33 22.67 7.41 5.5 1.9 3.45 5.09 0.91 4.13 61.52 40 30.08 33.4 3.3
Makhi 42.36 271 98.3 8.67 14.33 31 237 138.33 193.33 30 9.54 10.16 2.28 3.51 8.03 1.23 2.95 58.75 36 24.82 28.21 3.07
Dhady 51.49 418 86.7 9.67 9.333 38.33 385.67 164 238.3 30.33 14.3 18.66 2.43 4.14 12.46 1.46 2.8 68.88 36 27.18 32.19 3.11
Kur 45.44 354 74.3 6.33 21.33 88 285 184.67 183.33 23.33 9.36 10 2.27 3.37 9.23 1.3 1.95 61.23 34 27.55 33.68 2.39
Haleni 64.86 342 86.7 8.33 20 70 324.7 152.67 218.7 17.33 13.12 14 2.66 3.42 12.13 0.7 2.46 76.42 30 14.92 16.62 2.69
Eedal shah 44.37 358 91.7 8.33 18.66 70.33 330 155.33 252.33 27.67 10.29 10 2.28 3.53 8.03 1.03 1.84 67.51 30 20.91 26.15 2.57
Sufaidah 49.68 420 103 7.67 22 88 395.3 184.67 270.67 24.66 8.69 9 2.09 3.73 7.33 1.13 2.07 68.2 36 20.73 32.03 3.93
Taarwali 39.06 273 80.7 8 22.33 65.67 246.33 160.33 184 23 7.35 7 1.98 2.99 5.52 1.03 3.65 50.2 50 37.28 46.29 2.36
Fasli 47.66 244 86 7 22 47 223 145.67 170.33 21.67 8.65 8.5 1.98 3.1 7.1 1.19 2.18 57.5 36 26.85 32.28 3.09
Basra wali 44.59 361 102 7 18 65.67 336 158 265 25.33 9.75 10.16 2.28 3.55 7.53 1.63 4.06 54.76 40 28.6 33.53 2.46
Pathri 39.8 289 72.3 7 18.66 76.67 276.33 141.67 260 26 7.53 7.5 2.04 2.96 6.86 1.02 3.51 68.51 34 23.67 26.55 3.35
Kupra 48.62 317 85.7 7 16 62 290.7 150.67 251 26.66 11.16 12 2.53 3.76 10.16 1.1 2.27 59.47 40 32.44 36.08 2.41
Shakri 48.41 321 80 8.67 25.67 74 285 189.33 202 36 8.91 9.16 2.23 3.16 7.76 1.2 0.67 45.2 40 27.87 34.5 2.26
Baidhar 44.8 330 85.3 10.3 18.67 74 303.7 160 235 26.33 12.3 12 2.46 3.75 11.23 0.8 1.13 72.64 30 23.55 25 2.92
Gajjarwali 83.33 412 101 9 22.67 71.67 373.33 218 274.67 38.67 10.08 9 2.05 3.75 8.83 1.73 3.81 61.63 36 27.8 31.13 4.22
Halwain 52.76 344 79.7 6.67 17.66 85.67 320 154.67 227 24.33 6.41 6.16 1.83 2.93 5.34 1.03 4.03 64.49 36 27.27 31.07 3.45
Note: T.Dia=Trunk diameter (cm), LL=Leaf Length (cm), LW= Leaf Width (cm),LBW=Leaf Base Width (cm), SN=Spine Number, MLS= Midrib Length with Spines(cm), Midrib Length(cm) , Pinnae Number,
Midrib Length with Pinnae(cm),Length of Top Pinnea(cm), Fruit Weight (gm), Fruit volume(cm3), Fruit Diameter (cm), Fruit Length (cm), Pulp Weight (gm), Seed Weight (gm), Perianth Height(mm), % Moisture,
TSS= Total Soluble Sugars (Brix), % Reducing Sugars,% Total Sugars,%ash
113
Appendix III: 10XTAE Stock Solution (1L)
Tris base 48.4 g
Glacial acetic acid (17.4 M) 11.4 mL of
EDTA (0.5 M EDTA, pH 8) 20 mL
Sterilized distilled water Volume up to 1000 mL
Appendix IV: Bromophenol Blue
Sucrose 4g
Bromophenol Blue 0.02g
H2O 10mL
Appendix V: 5X TBE Stock Solution (1L)
Tris Base (M.W= 121.14 gm) 54g
Boric Acid 27.5 g
EDTA (0.5M) 20 mL
Distilled Water 1000mL
Appendix VI: 45 % acrylamide: Bisacrylamide Solution (100mL)
Acrylamide 44g
Bisacrylamide 1g
Distilled water 100mL
Appendix VII: 10% Ammonium per Sulphate (1mL)
Ammonium per Sulphate 100mg
Distilled water 1mL
114
Appendix VIII: Sequences from date palm chloroplast ribulose -1, 5-biphosphate
carboxylase large subunit (rbcL)partial genesubmitted to the GenBank
>Phoenix dactylifera chloroplast ribulose -1, 5-biphosphate carboxylase large subunit
rbcL partial gene of cultivar Dhaki from Pakistan (Accession # KT803883)
GAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACC
CGGAGTTCCGCCTGAGGAAGCAGGGGCAGCGGTAGCTGCCGAATCTTCTA
CTGGTACATGGACAACTGTGTGGACTGATGGACTTACCAGTCTTGATCGTT
ACAAAGGACGATGCTACCACATCGAAACCGTTGTAGGGGAGGAAAATCA
ATATATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTT
ACTAACATGTTTACTTCCATTGTGGGTAATGTATTTGGTTTCAAAGCCCTA
CGAGCTCTACGTCTGGAGGATCTGCGAATTCCCACTTCTTATTCCAAAACT
TTCCAAGGCCCGCCTCATGGCATCCAAGTTGAAAGAGATAAGTTGAACAA
GTATGGTCGGCCTCTATTGGGATGTACTATTAAACCAAAATTGGGATTATC
CGCAAAGAACTACGGTAGAGCGGTTTATGAATGTCTACGCGGTGGACTTG
ATTTTACCAAGGATGATGAAAACGTGAACTCAC
>Phoenix dactylifera chloroplast ribulose -1, 5-biphosphate carboxylase large subunit
rbcL partial gene of cultivar Aseel from Pakistan(Accession # KT803882)
GAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACC
CGGAGTTCCGCCTGAGGAAGCAGGGGCAGCGGTAGCTGCCGAATCTTCTA
CTGGTACATGGACAACTGTGTGGACTGATGGACTTACCAGTCTTGATCGTT
ACAAAGGACGATGCTACCACATCGAAACCGTTGTAGGGGAGGAAAATCA
ATATATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTT
ACTAACATGTTTACTTCCATTGTGGGTAATGTATTTGGTTTCAAAGCCCTA
CGAGCTCTACGTCTGGAGGATCTGCGAATTCCCACTTCTTATTCCAAAACT
TTCCAAGGCCCGCCTCATGGCATCCAAGTTGAAAGAGATAAGTTGAACAA
GTATGGTCGGCCTCTATTGGGATGTACTATTAAACCAAAATTGGGATTATC
CGCAAAGAACTACGGTAGAGCGGTTTATGAATGTCTACGCGGTGGACTTG
ATTTTACCAAGGATGATGAAAACGTGAACTCAC
>Phoenix dactylifera chloroplast ribulose -1, 5-biphosphate carboxylase large subunit
rbcL partial gene of cultivar Halawi from Pakistan(Accession # KT803884)
GAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACC
CGGAGTTCCGCCTGAGGAAGCAGGGGCAGCGGTAGCTGCCGAATCTTCTA
CTGGTACATGGACAACTGTGTGGACTGATGGACTTACCAGTCTTGATCGTT
ACAAAGGACGATGCTACCACATCGAAACCGTTGTAGGGGAGGAAAATCA
ATATATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTT
ACTAACATGTTTACTTCCATTGTGGGTAATGTATTTGGTTTCAAAGCCCTA
CGAGCTCTACGTCTGGAGGATCTGCGAATTCCCACTTCTTATTCCAAAACT
TTCCAAGGCCCGCCTCATGGCATCCAAGTTGAAAGAGATAAGTTGAACAA
GTATGGTCGGCCTCTATTGGGATGTACTATTAAACCAAAATTGGGATTATC
115
CGCAAAGAACTACGGTAGAGCGGTTTATGAATGTCTACGCGGTGGACTTG
ATTTTACCAAGGATGATGAAAACGTGAACTCAC
>Phoenix dactylifera chloroplast ribulose -1, 5-biphosphate carboxylase large subunit
rbcL partial gene of cultivar Qantar from Pakistan(Accession # KT803885)
GAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACC
CGGAGTTCCGCCTGAGGAAGCAGGGGCAGCGGTAGCTGCCGAATCTTCTA
CTGGTACATGGACAACTGTGTGGACTGATGGACTTACCAGTCTTGATCGTT
ACAAAGGACGATGCTACCACATCGAAACCGTTGTAGGGGAGGAAAATCA
ATATATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTT
ACTAACATGTTTACTTCCATTGTGGGTAATGTATTTGGTTTCAAAGCCCTA
CGAGCTCTACGTCTGGAGGATCTGCGAATTCCCACTTCTTATTCCAAAACT
TTCCAAGGCCCGCCTCATGGCATCCAAGTTGAAAGAGATAAGTTGAACAA
GTATGGTCGGCCTCTATTGGGATGTACTATTAAACCAAAATTGGGATTATC
CGCAAAGAACTACGGTAGAGCGGTTTATGAATGTCTACGCGGTGGACTTG
ATTTTACCAAGGATGATGAAAACGTGAACTCAC
>Phoenix dactylifera chloroplast ribulose -1, 5-biphosphate carboxylase large subunit
rbcL partial gene of cultivar Haminwali from Pakistan(Accession # KT803886)
GAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACC
CGGAGTTCCGCCTGAGGAAGCAGGGGCAGCGGTAGCTGCCGAATCTTCTA
CTGGTACATGGACAACTGTGTGGACTGATGGACTTACCAGTCTTGATCGTT
ACAAAGGACGATGCTACCACATCGAAACCGTTGTAGGGGAGGAAAATCA
ATATATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTT
ACTAACATGTTTACTTCCATTGTGGGTAATGTATTTGGTTTCAAAGCCCTA
CGAGCTCTACGTCTGGAGGATCTGCGAATTCCCACTTCTTATTCCAAAACT
TTCCAAGGCCCGCCTCATGGCATCCAAGTTGAAAGAGATAAGTTGAACAA
GTATGGTCGGCCTCTATTGGGATGTACTATTAAACCAAAATTGGGATTATC
CGCAAAGAACTACGGTAGAGCGGTTTATGAATGTCTACGCGGTGGACTTG
ATTTTACCAAGGATGATGAAAACGTGAACTCAC
>Phoenix dactylifera chloroplast ribulose -1, 5-biphosphate carboxylase large subunit
rbcL partial gene of cultivar Kupra from Pakistan(Accession # KT803887)
GAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACC
CGGAGTTCCGCCTGAGGAAGCAGGGGCAGCGGTAGCTGCCGAATCTTCTA
CTGGTACATGGACAACTGTGTGGACTGATGGACTTACCAGTCTTGATCGTT
ACAAAGGACGATGCTACCACATCGAAACCGTTGTAGGGGAGGAAAATCA
ATATATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTT
ACTAACATGTTTACTTCCATTGTGGGTAATGTATTTGGTTTCAAAGCCCTA
CGAGCTCTACGTCTGGAGGATCTGCGAATTCCCACTTCTTATTCCAAAACT
TTCCAAGGCCCGCCTCATGGCATCCAAGTTGAAAGAGATAAGTTGAACAA
GTATGGTCGGCCTCTATTGGGATGTACTATTAAACCAAAATTGGGATTATC
116
CGCAAAGAACTACGGTAGAGCGGTTTATGAATGTCTACGCGGTGGACTTG
ATTTTACCAAGGATGATGAAAACGTGAACTCAC
>Phoenix dactylifera chloroplast ribulose -1, 5-biphosphate carboxylase large subunit
rbcL partial gene of cultivar Shakri from Pakistan(Accession # KT803888)
GAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACC
CGGAGTTCCGCCTGAGGAAGCAGGGGCAGCGGTAGCTGCCGAATCTTCTA
CTGGTACATGGACAACTGTGTGGACTGATGGACTTACCAGTCTTGATCGTT
ACAAAGGACGATGCTACCACATCGAAACCGTTGTAGGGGAGGAAAATCA
ATATATTGCTTATGTAGCTTATCCTTTAGACCTTTTTGAAGAAGGTTCTGTT
ACTAACATGTTTACTTCCATTGTGGGTAATGTATTTGGTTTCAAAGCCCTA
CGAGCTCTACGTCTGGAGGATCTGCGAATTCCCACTTCTTATTCCAAAACT
TTCCAAGGCCCGCCTCATGGCATCCAAGTTGAAAGAGATAAGTTGAACAA
GTATGGTCGGCCTCTATTGGGATGTACTATTAAACCAAAATTGGGATTATC
CGCAAAGAACTACGGTAGAGCGGTTTATGAATGTCTACGCGGTGGACTTG
ATTTTACCAAGGATGATGAAAACGTGAACTCAC
117
Appendix IX: Sequences from date palm chloroplast Atp synthase beta subunit
(atpB) partial genesubmitted to the GenBank
>Phoenix dactylifera chloroplast Atp synthase beta subunit atpB partial gene of
cultivar Dhaki from Pakistan (Accession # KT781683)
TCGGCGGAGCTACTCTCGGACGAATTTTCAACGTTCTTGGGGAACCTGTTG
ATAATTTAGGTCCTGTAGATACTCGTACAACATCTCCTATTCATAGATCTG
CGCCTGCCTTTATACAGTTAGATACGAAATTATCAATCTTTGAAACAGGGA
TTAAAGTGGTGGATCTTTTAGCTCCTTATCGCCGTGGAGGAAAAATCGGA
CTATTTGGGGGAGCTGGAGTGGGTAAAACAGTACTCATCATGGAATTGAT
CAATAACATTGCCAAAGCTCATGGAGGCGTATCCGTATTTGGCGGAGTAG
GCGAACGTACTCGTGAAGGAAATGATCTTTACATGGAAATGAAAGAATCC
GGAGTGATTAATGAAAAAAATATTGCGGAATCAAAAGTAGCTCTAGTCTA
TGGTCAAATGAATGAACCGCCGGGAGCTCGTATGAGAGTTGGTTTAACTG
CCCTAACCATGGCGGAATATTTCC
>Phoenix dactylifera chloroplast Atp synthase beta subunit atpB partial gene of
cultivar Aseel from Pakistan (Accession # KT781682)
GTGATTGACACGGGAGCTCCTCTAAGTGTTCCAGTCGGCGGAGCTACTCTC
GGACGAATTTTCAACGTTCTTGGGGAACCTGTTGATAATTTAGGTCCTGTA
GATACTCGTACAACATCTCCTATTCATAGATCTGCGCCTGCCTTTATACAG
TTAGATACGAAATTATCAATCTTTGAAACAGGGATTAAAGTGGTGGATCT
TTTAGCTCCTTATCGCCGTGGAGGAAAAATCGGACTATTTGGGGGAGCTG
GAGTGGGTAAAACAGTACTCATCATGGAATTGATCAATAACATTGCCAAA
GCTCATGGAGGCGTATCCGTATTTGGCGGAGTAGGCGAACGTACTCGTGA
AGGAAATGATCTTTACATGGAAATGAAAGAATCCGGAGTGATTAATGAAA
AAAATATTGCGGAATCAAAAGTAGCTCTAGTCTATGGTCAAATGAATGAA
CCGCCGGGAGCTCGTATGAGAGTTGGTTTAACTGCCCTAACCATGGCGGA
ATATTTCCGGGATGTTAATGAACAAGACGTGCTTCTATTCATCGAC
>Phoenix dactylifera chloroplast Atp synthase beta subunit atpB partial gene of
cultivar Halawi from Pakistan (Accession # KT781684)
ATGGGCTGATGAGAGGAATGGAAGTGATTGACACGGGAGCTCCTCTAAGT
GTTCCAGTCGGCGGAGCTACTCTCGGACGAATTTTCAACGTTCTTGGGGAA
CCTGTTGATAATTTAGGTCCTGTAGATACTCGTACAACATCTCCTATTCAT
AGATCTGCGCCTGCCTTTATACAGTTAGATACGAAATTATCAATCTTTGAA
ACAGGGATTAAAGTGGTGGATCTTTTAGCTCCTTATCGCCGTGGAGGAAA
AATCGGACTATTTGGGGGAGCTGGAGTGGGTAAAACAGTACTCATCATGG
AATTGATCAATAACATTGCCAAAGCTCATGGAGGCGTATCCGTATTTGGC
GGAGTAGGCGAACGTACTCGTGAAGGAAATGATCTTTACATGGAAATGAA
AGAATCCGGAGTGATTAATGAAAAAAATATTGCGGAATCAAAAGTAGCTC
TAGTCTATGGTCAAATGAATGAACCGCCGGGAGCTCGTATGAGAGTTGGT
118
TTAACTGCCCTAACCATGGCGGAATATTTCCGGGATGTTAATGAACAAGA
CGTGCTTCTATTCATCGAC
>Phoenix dactylifera chloroplast Atp synthase beta subunit atpB partial gene of
cultivar Qantar from Pakistan (Accession # KT781685)
TCGGCGGAGCTACTCTCGGACGAATTTTCAACGTTCTTGGGGAACCTGTTG
ATAATTTAGGTCCTGTAGATACTCGTACAACATCTCCTATTCATAGATCTG
CGCCTGCCTTTATACAGTTAGATACGAAATTATCAATCTTTGAAACAGGGA
TTAAAGTGGTGGATCTTTTAGCTCCTTATCGCCGTGGAGGAAAAATCGGA
CTATTTGGGGGAGCTGGAGTGGGTAAAACAGTACTCATCATGGAATTGAT
CAATAACATTGCCAAAGCTCATGGAGGCGTATCCGTATTTGGCGGAGTAG
GCGAACGTACTCGTGAAGGAAATGATCTTTACATGGAAATGAAAGAATCC
GGAGTGATTAATGAAAAAAATATTGCGGAATCAAAAGTAGCTCTAGTCTA
TGGTCAAATGAATGAACCGCCGGGAGCTCGTATGAGAGTTGGTTT
>Phoenix dactylifera chloroplast Atp synthase beta subunit atpB partial gene of
cultivar Hamin wali from Pakistan (Accession # KT781686)
TCGGCGGAGCTACTCTCGGACGAATTTTCAACGTTCTTGGGGAACCTGTTG
ATAATTTAGGTCCTGTAGATACTCGTACAACATCTCCTATTCATAGATCTG
CGCCTGCCTTTATACAGTTAGATACGAAATTATCAATCTTTGAAACAGGGA
TTAAAGTGGTGGATCTTTTAGCTCCTTATCGCCGTGGAGGAAAAATCGGA
CTATTTGGGGGAGCTGGAGTGGGTAAAACAGTACTCATCATGGAATTGAT
CAATAACATTGCCAAAGCTCATGGAGGCGTATCCGTATTTGGCGGAGTAG
GCGAACGTACTCGTGAAGGAAATGATCTTTACATGGAAATGAAAGAATCC
GGAGTGATTAATGAAAAAAATATTGCGGAATCAAAAGTAGCTCTAGTCTA
TGGTCAAATGAATGAACCGCCGGGAGCTCGTATGAGAGTTGGTTTAACTG
CCCTAACCATGGCGGAATATTTCCGGGATGTTAATGAACAAGACGTGCTT
CTATTCATCGACAATA
>Phoenix dactylifera chloroplast Atp synthase beta subunit atpB partial gene of
cultivar Kupra from Pakistan (Accession # KT781687)
GTGATTGACACGGGAGCTCCTCTAAGTGTTCCAGTCGGCGGAGCTACTCTC
GGACGAATTTTCAACGTTCTTGGGGAACCTGTTGATAATTTAGGTCCTGTA
GATACTCGTACAACATCTCCTATTCATAGATCTGCGCCTGCCTTTATACAG
TTAGATACGAAATTATCAATCTTTGAAACAGGGATTAAAGTGGTGGATCT
TTTAGCTCCTTATCGCCGTGGAGGAAAAATCGGACTATTTGGGGGAGCTG
GAGTGGGTAAAACAGTACTCATCATGGAATTGATCAATAACATTGCCAAA
GCTCATGGAGGCGTATCCGTATTTGGCGGAGTAGGCGAACGTACTCGTGA
AGGAAATGATCTTTACATGGAAATGAAAGAATCCGGAGTGATTAATGAAA
AAAATATTGCGGAATC
119
>Phoenix dactylifera chloroplast Atp synthase beta subunit atpB partial gene of
cultivar Shakri from Pakistan (Accession # KT781688)
ATGGGCTGATGAGAGGAATGGAAGTGATTGACACGGGAGCTCCTCTAAGT
GTTCCAGTCGGCGGAGCTACTCTCGGACGAATTTTCAACGTTCTTGGGGAA
CCTGTTGATAATTTAGGTCCTGTAGATACTCGTACAACATCTCCTATTCAT
AGATCTGCGCCTGCCTTTATACAGTTAGATACGAAATTATCAATCTTTGAA
ACAGGGATTAAAGTGGTGGATCTTTTAGCTCCTTATCGCCGTGGAGGAAA
AATCGGACTATTTGGGGGAGCTGGAGTGGGTAAAACAGTACTCATCATGG
AATTGATCAATAACATTGCCAAAGCTCATGGAGGCGTATCCGTATTTGGC
GGAGTAGGCGAACGTACTCGTGAAGGAAATGATCTTTACATGGAAATGAA
AGAATCCGGAGTGATTAATGAAAAAAATATTGCGGAATCAAAAGTAGCTC
TAGTCTATGGTCAAATGAATGAACCGCCGGGAGCTCGTATGAGAGTTGGT
TTAACTGCCCTAACCATGGCGGAATATTTCCGGGATGTTAATGAACAAGA
CGTGCTTCTATTCATCGACAATATTTTTCGTTTCGTCCAAGCAGGATCAGA
AGTATCCGCCTTATTAGGGAG
120
Appendix X: Sequences from date palm chloroplast geranyl geranyl diphosphate
reductase (GGR) partial genesubmitted to the GenBank
>Phoenix dactylifera chloroplast geranyl geranyl diphosphate reductase partial gene
of cultivar Dhaki from Pakistan (Accession # KT983259)
ATCGGCATGGTCCGCCGCGAGGTCCTCGACGCCTACCTCCGCGACCGCGC
CGCCGAAGCCGGCGCCCAAGTCATCAATGGCCTCTTCCTCCACCTCGACC
CGCCGGAGTCCGGCGAGGGGCCCTACCGCCTGCACTACAACCTCTACGAC
AGGGGCAGACCCTCCGCCGCCGGCGATCGCCAGACAGTCGAGGTCGACG
CCGTCGTCGGCGCCGACGGCGCCAACTCCCGCGTCGCCAAGTCCATCGGC
GCCGGCGACTACGACTACGCCATCGCATTCCAGGCAA
>Phoenix dactylifera chloroplast geranyl geranyl diphosphate reductase partial gene
of cultivar Aseel from Pakistan(Accession # KT983260)
ATCGGCATGGTCCGCCGCGAGGTCCTCGACGCCTACCTCCGCGACCGCGC
CGCCGAAGCCGGCGCCCAAGTCATCAATGGCCTCTTCCTCCACCTCGACC
CGCCGGAGTCCGGCGAGGGGCCCTACCGCCTGCACTACAACCTCTACGAC
AGGGGCAGACCCTCCGCCGCCGGCGATCGCCAGACAGTCGAGGTCGACG
CCGTCGTCGGCGCCGACGGCGCCAACTCCCGCGTCGCCAAGTCCATCGGC
GCCGGCGACTACGACTACGCCATCGCATTCCAGGCAA
>Phoenix dactylifera chloroplast geranyl geranyl diphosphate reductase partial gene
of cultivar Halawi from Pakistan (Accession # KT983261)
ATCGGCATGGTCCGCCGCGAGGTCCTCGACGCCTACCTCCGCGACCGCGC
CGCCGAAGCCGGCGCCCAAGTCATCAATGGCCTCTTCCTCCACCTCGACC
CGCCGGAGTCCGGCGAGGGGCCCTACCGCCTGCACTACAACCTCTACGAC
AGGGGCAGACCCTCCGCCGCCGGCGATCGCCAGACAGTCGAGGTCGACG
CCGTCGTCGGCGCCGACGGCGCCAACTCCCGCGTCGCCAAGTCCATCGGC
GCCGGCGACTACGACTACGCCATCGCATTCCAGGCAA
>Phoenix dactylifera chloroplast geranyl geranyl diphosphate reductase partial gene
of cultivar Qantar from Pakistan (Accession # KT983262)
ATCGGCATGGTCCGCCGCGAGGTCCTCGACGCCTACCTCCGCGACCGCGC
CGCCGAAGCCGGCGCCCAAGTCATCAATGGCCTCTTCCTCCACCTCGACC
CGCCGGAGTCCGGCGAGGGGCCCTACCGCCTGCACTACAACCTCTACGAC
AGGGGCAGACCCTCCGCCGCCGGCGATCGCCAGACGGTCGAGGTCGACG
CCGTCGTCGGCGCCGACGGCGCCAACTCCCGCGTCGCCAAGTCCATCGGC
GCCGGCGACTACGACTACGCCATCGCATTCCAGGCAA
121
>Phoenix dactylifera chloroplast geranyl geranyl diphosphate reductase partial gene
of cultivar Hamin wali from Pakistan (Accession # KT983263)
ATCGGCATGGTCCGCCGCGAGGTCCTCGACGCCTACCTCCGCGACCGCGC
CGCCGAAGCCGGCGCCCAAGTCATCAATGGCCTCTTCCTCCACCTCGACC
CGCCGGAGTCCGGCGAGGGGCCCTACCGCCTGCACTACAACCTCTACGAC
AGGGGCAGACCCTCCGCCGCCGGCGATCGCCAGACGGTCGAGGTCGACG
CCGTCGTCGGCGCCGACGGCGCCAACTCCCGCGTCGCCAAGTCCATCGGC
GCCGGCGACTACGACTACGCCATCGCATTCCAGGCAA
>Phoenix dactylifera chloroplast geranyl geranyl diphosphate reductase partial gene
of cultivar Kupra from Pakistan (Accession # KT983264)
ATCGGCATGGTCCGCCGCGAGGTCCTCGACGCCTACCTCCGCGACCGCGC
CGCCGAAGCCGGCGCCCAAGTCATCAATGGCCTCTTCCTCCACCTCGACC
CGCCGGAGTCCGGCGAGGGGCCCTACCGCCTGCACTACAACCTCTACGAC
AGGGGCAGACCCTCCGCCGCCGGCGATCGCCAGACGGTCGAGGTCGACG
CCGTCGTCGGCGCCGACGGCGCCAACTCCCGCGTCGCCAAGTCCATCGGC
GCCGGCGACTACGACTACGCCATCGCATTCCAGGCAA
>Phoenix dactylifera chloroplast geranyl geranyl diphosphate reductase partial gene
of cultivar Shakri from Pakistan (Accession # KT983265)
ATCGGCATGGTCCGCCGCGAGGTCCTCGACGCCTACCTCCGCGACCGCGC
CGCCGAAGCCGGCGCCCAAGTCATCAATGGCCTCTTCCTCCACCTCGACC
CGCCGGAGTCCGGCGAGGGGCCCTACCGCCTGCACTACAACCTCTACGAC
AGGGGCAGACCCTCCGCCGCCGGCGATCGCCAGACGGTCGAGGTCGACG
CCGTCGTCGGCGCCGACGGCGCCAACTCCCGCGTCGCCAAGTCCATCGGC
GCCGGCGACTACGACTACGCCATCGCATTCCAGGCAA
122
Appendix XI: Sequences from date palm chloroplast maturase K (matK)partial
genesubmitted to the GenBank
>Phoenix dactylifera chloroplast maturase K matK partial gene of cultivar Dhaki
from Pakistan (Accession # KT803890)
GAGTATATTTACACATTTGTTCATGATCGTGGTTTAAATAGTTCGGTTTTTT
ACGAATCCACGGAAATTTTTGGTTATGACAATAAATCTAGTTCAGTACTTG
TGAAACGTTCAATTATTCGAATGTATCAACAGAATTATTTGATTTATTCGG
TTAATGATTCTAACCAAAATCGATTTGTTGGGCACAACAATTATTTTTATT
TTCATTTTTATTCTCAGATGATATTGGAAGGTTTTGCAGTCATTGTGGAAA
TTCCATTCTTGCTGCGATTAGTATCTTCCCTCGAAGAAAAAAAAATACCAA
AATCTCAGAATTTGAATTTACGATCTATTCATTCAACATTTCCCTTTTTGGA
GGACAAATTATCGCATTTAAACTATGTGTCAGATATACTAATACCTTATCC
CATCCATCTGAAAATCTTGGTTCAAATCCTTCAATTCTGGATCCAAGATGT
TCCTTCTTTACATTTATTGCGATTCTTTCTTCACGAATATCATAATTGGAAT
AGTCTTATTACTCCGAATAATTCTATTTTTCTTTTTTCAAAAGAAAATAAA
AGAGTATTTCGGTTCCCATATAATTCTTATGTATCTGAATGCGAATTTGTA
TTAGTTTTTCTTCGTAAACAATCTTCTTATTTACGATTAACATCTTCTGGAG
CTTTTCTTGAGCGAACACATTTCTATGGAAAAATAGAACATCGTATAGTAG
TGCGCCGTAATTATTTTCAGAAGACCCTATGGTTCTTCAAGGATCCCTTCA
TGCATTATGTTCGATATCAAGGAAAAGCAATTCTGGTTTCAAAGGGGACT
CATCTTCTGATGAAGAAATGGAAATGTTACCTTGTCAATTTTTGGCAATAT
TATTTTCACTTTTGGTCTCAACCGTACAGGATCCATATAAACCAATTATCA
AACTGTTCTTTCTATTTTCTAGGTTATCTTTCAAGTGTACTAATAAATCCTT
CGGCGGTAAGGAATCAAATGCTAGAGAATTCATTTCTAATAGATACTGTT
ATTAAAAAATTCGATACCAGAGTCCCAGTTATTACTCTTATTGGATCATTG
TCTAAAGCTAAATTTTGTACCGTATTGGGGCATCCTATTAGTAAGCCGATC
TGGACCGATTTATCAGATTGCGATATTATTGATCGATTTGGTCGGATATGT
AGAAATCTTTCTCATTATCACAGTGGATCCTCAAAAAAACAGAGTTTGTAT
CGAATAAAGTATATACTTCGATTTTCGTGTGCTAGAACTTTGGCTCGTAAA
CATAAAAGTACGGTACGCGCTTTTTTGCAAAGATTAGGTTCGGGATTATTA
GAAGAATTTTTTATGAAAGAAGAACAAGTTGTTTCTTTGATCTTCCCAAAA
ACAACTTCTTTTTCTTTACATGAATCGCATATAGAACGTATTTGGTATTTG
GAT
>Phoenix dactylifera chloroplast maturase K matK partial gene of cultivar Aseel from
Pakistan (Accession # KT803889)
GAGTATATTTACACATTTGTTCATGATCGTGGTTTAAATAGTTCGGTTTTTT
ACGAATCCACGGAAATTTTTGGTTATGACAATAAATCTAGTTCAGTACTTG
TGAAACGTTCAATTATTCGAATGTATCAACAGAATTATTTGATTTATTCGG
TTAATGATTCTAACCAAAATCGATTTGTTGGGCACAACAATTATTTTTATT
TTCATTTTTATTCTCAGATGATATTGGAAGGTTTTGCAGTCATTGTGGAAA
TTCCATTCTTGCTGCGATTAGTATCTTCCCTCGAAGAAAAAAAAATACCAA
123
AATCTCAGAATTTGAATTTACGATCTATTCATTCAACATTTCCCTTTTTGGA
GGACAAATTATCGCATTTAAACTATGTGTCAGATATACTAATACCTTATCC
CATCCATCTGAAAATCTTGGTTCAAATCCTTCAATTCTGGATCCAAGATGT
TCCTTCTTTACATTTATTGCGATTCTTTCTTCACGAATATCATAATTGGAAT
AGTCTTATTACTCCGAATAATTCTATTTTTCTTTTTTCAAAAGAAAATAAA
AGAGTATTTCGGTTCCCATATAATTCTTATGTATCTGAATGCGAATTTGTA
TTAGTTTTTCTTCGTAAACAATCTTCTTATTTACGATTAACATCTTCTGGAG
CTTTTCTTGAGCGAACACATTTCTATGGAAAAATAGAACATCGTATAGTAG
TGCGCCGTAATTATTTTCAGAAGACCCTATGGTTCTTCAAGGATCCCTTCA
TGCATTATGTTCGATATCAAGGAAAAGCAATTCTGGTTTCAAAGGGGACT
CATCTTCTGATGAAGAAATGGAAATGTTACCTTGTCAATTTTTGGCAATAT
TATTTTCACTTTTGGTCTCAACCGTACAGGATCCATATAAACCAATTATCA
AACTGTTCTTTCTATTTTCTAGGTTATCTTTCAAGTGTACTAATAAATCCTT
CGGCGGTAAGGAATCAAATGCTAGAGAATTCATTTCTAATAGATACTGTT
ATTAAAAAATTCGATACCAGAGTCCCAGTTATTACTCTTATTGGATCATTG
TCTAAAGCTAAATTTTGTACCGTATTGGGGCATCCTATTAGTAAGCCGATC
TGGACCGATTTATCAGATTGCGATATTATTGATCGATTTGGTCGGATATGT
AGAAATCTTTCTCATTATCACAGTGGATCCTCAAAAAAACAGAGTTTGTAT
CGAATAAAGTATATACTTCGATTTTCGTGTGCTAGAACTTTGGCTCGTAAA
CATAAAAGTACGGTACGCGCTTTTTTGCAAAGATTAGGTTCGGGATTATTA
GAAGAATTTTTTATGAAAGAAGAACAAGTTGTTTCTTTGATCTTCCCAAAA
ACAACTTCTTTTTCTTTACATGAATCGCATATAGAACGTATTTGGTATTTG
GAT
>Phoenix dactylifera chloroplast maturase K matK partial gene of cultivar Halawi
from Pakistan (Accession # KT803891)
GAGTATATTTACACATTTGTTCATGATCGTGGTTTAAATAGTTCGGTTTTTT
ACGAATCCACGGAAATTTTTGGTTATGACAATAAATCTAGTTCAGTACTTG
TGAAACGTTCAATTATTCGAATGTATCAACAGAATTATTTGATTTATTCGG
TTAATGATTCTAACCAAAATCGATTTGTTGGGCACAACAATTATTTTTATT
TTCATTTTTATTCTCAGATGATATTGGAAGGTTTTGCAGTCATTGTGGAAA
TTCCATTCTTGCTGCGATTAGTATCTTCCCTCGAAGAAAAAAAAATACCAA
AATCTCAGAATTTGAATTTACGATCTATTCATTCAACATTTCCCTTTTTGGA
GGACAAATTATCGCATTTAAACTATGTGTCAGATATACTAATACCTTATCC
CATCCATCTGAAAATCTTGGTTCAAATCCTTCAATTCTGGATCCAAGATGT
TCCTTCTTTACATTTATTGCGATTCTTTCTTCACGAATATCATAATTGGAAT
AGTCTTATTACTCCGAATAATTCTATTTTTCTTTTTTCAAAAGAAAATAAA
AGAGTATTTCGGTTCCCATATAATTCTTATGTATCTGAATGCGAATTTGTA
TTAGTTTTTCTTCGTAAACAATCTTCTTATTTACGATTAACATCTTCTGGAG
CTTTTCTTGAGCGAACACATTTCTATGGAAAAATAGAACATCGTATAGTAG
TGCGCCGTAATTATTTTCAGAAGACCCTATGGTTCTTCAAGGATCCCTTCA
TGCATTATGTTCGATATCAAGGAAAAGCAATTCTGGTTTCAAAGGGGACT
CATCTTCTGATGAAGAAATGGAAATGTTACCTTGTCAATTTTTGGCAATAT
124
TATTTTCACTTTTGGTCTCAACCGTACAGGATCCATATAAACCAATTATCA
AACTGTTCTTTCTATTTTCTAGGTTATCTTTCAAGTGTACTAATAAATCCTT
CGGCGGTAAGGAATCAAATGCTAGAGAATTCATTTCTAATAGATACTGTT
ATTAAAAAATTCGATACCAGAGTCCCAGTTATTACTCTTATTGGATCATTG
TCTAAAGCTAAATTTTGTACCGTATTGGGGCATCCTATTAGTAAGCCGATC
TGGACCGATTTATCAGATTGCGATATTATTGATCGATTTGGTCGGATATGT
AGAAATCTTTCTCATTATCACAGTGGATCCTCAAAAAAACAGAGTTTGTAT
CGAATAAAGTATATACTTCGATTTTCGTGTGCTAGAACTTTGGCTCGTAAA
CATAAAAGTACGGTACGCGCTTTTTTGCAAAGATTAGGTTCGGGATTATTA
GAAGAATTTTTTATGAAAGAAGAACAAGTTGTTTCTTTGATCTTCCCAAAA
ACAACTTCTTTTTCTTTACATGAATCGCATATAGAACGTATTTGGTATTTG
GAT
>Phoenix dactylifera chloroplast maturase K matK partial gene of cultivar Qantar
from Pakistan (Accession # KT803892)
GAGTATATTTACACATTTGTTCATGATCGTGGTTTAAATAGTTCGGTTTTTT
ACGAATCCACGGAAATTTTTGGTTATGACAATAAATCTAGTTCAGTACTTG
TGAAACGTTCAATTATTCGAATGTATCAACAGAATTATTTGATTTATTCGG
TTAATGATTCTAACCAAAATCGATTTGTTGGGCACAACAATTATTTTTATT
TTCATTTTTATTCTCAGATGATATTGGAAGGTTTTGCAGTCATTGTGGAAA
TTCCATTCTTGCTGCGATTAGTATCTTCCCTCGAAGAAAAAAAAATACCAA
AATCTCAGAATTTGAATTTACGATCTATTCATTCAACATTTCCCTTTTTGGA
GGACAAATTATCGCATTTAAACTATGTGTCAGATATACTAATACCTTATCC
CATCCATCTGAAAATCTTGGTTCAAATCCTTCAATTCTGGATCCAAGATGT
TCCTTCTTTACATTTATTGCGATTCTTTCTTCACGAATATCATAATTGGAAT
AGTCTTATTACTCCGAATAATTCTATTTTTCTTTTTTCAAAAGAAAATAAA
AGAGTATTTCGGTTCCCATATAATTCTTATGTATCTGAATGCGAATTTGTA
TTAGTTTTTCTTCGTAAACAATCTTCTTATTTACGATTAACATCTTCTGGAG
CTTTTCTTGAGCGAACACATTTCTATGGAAAAATAGAACATCGTATAGTAG
TGCGCCGTAATTATTTTCAGAAGACCCTATGGTTCTTCAAGGATCCCTTCA
TGCATTATGTTCGATATCAAGGAAAAGCAATTCTGGTTTCAAAGGGGACT
CATCTTCTGATGAAGAAATGGAAATGTTACCTTGTCAATTTTTGGCAATAT
TATTTTCACTTTTGGTCTCAACCGTACAGGATCCATATAAACCAATTATCA
AACTGTTCTTTCTATTTTCTAGGTTATCTTTCAAGTGTACTAATAAATCCTT
CGGCGGTAAGGAATCAAATGCTAGAGAATTCATTTCTAATAGATACTGTT
ATTAAAAAATTCGATACCAGAGTCCCAGTTATTACTCTTATTGGATCATTG
TCTAAAGCTAAATTTTGTACCGTATTGGGGCATCCTATTAGTAAGCCGATC
TGGACCGATTTATCAGATTGCGATATTATTGATCGATTTGGTCGGATATGT
AGAAATCTTTCTCATTATCACAGTGGATCCTCAAAAAAACAGAGTTTGTAT
CGAATAAAGTATATACTTCGATTTTCGTGTGCTAGAACTTTGGCTCGTAAA
CATAAAAGTACGGTACGCGCTTTTTTGCAAAGATTAGGTTCGGGATTATTA
GAAGAATTTTTTATGAAAGAAGAACAAGTTGTTTCTTTGATCTTCCCAAAA
125
ACAACTTCTTTTTCTTTACATGAATCGCATATAGAACGTATTTGGTATTTG
GAT
>Phoenix dactylifera chloroplast maturase K matK partial gene of cultivar Hamin
wali from Pakistan (Accession # KT803893)
GAGTATATTTACACATTTGTTCATGATCGTGGTTTAAATAGTTCGGTTTTTT
ACGAATCCACGGAAATTTTTGGTTATGACAATAAATCTAGTTCAGTACTTG
TGAAACGTTCAATTATTCGAATGTATCAACAGAATTATTTGATTTATTCGG
TTAATGATTCTAACCAAAATCGATTTGTTGGGCACAACAATTATTTTTATT
TTCATTTTTATTCTCAGATGATATTGGAAGGTTTTGCAGTCATTGTGGAAA
TTCCATTCTTGCTGCGATTAGTATCTTCCCTCGAAGAAAAAAAAATACCAA
AATCTCAGAATTTGAATTTACGATCTATTCATTCAACATTTCCCTTTTTGGA
GGACAAATTATCGCATTTAAACTATGTGTCAGATATACTAATACCTTATCC
CATCCATCTGAAAATCTTGGTTCAAATCCTTCAATTCTGGATCCAAGATGT
TCCTTCTTTACATTTATTGCGATTCTTTCTTCACGAATATCATAATTGGAAT
AGTCTTATTACTCCGAATAATTCTATTTTTCTTTTTTCAAAAGAAAATAAA
AGAGTATTTCGGTTCCCATATAATTCTTATGTATCTGAATGCGAATTTGTA
TTAGTTTTTCTTCGTAAACAATCTTCTTATTTACGATTAACATCTTCTGGAG
CTTTTCTTGAGCGAACACATTTCTATGGAAAAATAGAACATCGTATAGTAG
TGCGCCGTAATTATTTTCAGAAGACCCTATGGTTCTTCAAGGATCCCTTCA
TGCATTATGTTCGATATCAAGGAAAAGCAATTCTGGTTTCAAAGGGGACT
CATCTTCTGATGAAGAAATGGAAATGTTACCTTGTCAATTTTTGGCAATAT
TATTTTCACTTTTGGTCTCAACCGTACAGGATCCATATAAACCAATTATCA
AACTGTTCTTTCTATTTTCTAGGTTATCTTTCAAGTGTACTAATAAATCCTT
CGGCGGTAAGGAATCAAATGCTAGAGAATTCATTTCTAATAGATACTGTT
ATTAAAAAATTCGATACCAGAGTCCCAGTTATTACTCTTATTGGATCATTG
TCTAAAGCTAAATTTTGTACCGTATTGGGGCATCCTATTAGTAAGCCGATC
TGGACCGATTTATCAGATTGCGATATTATTGATCGATTTGGTCGGATATGT
AGAAATCTTTCTCATTATCACAGTGGATCCTCAAAAAAACAGAGTTTGTAT
CGAATAAAGTATATACTTCGATTTTCGTGTGCTAGAACTTTGGCTCGTAAA
CATAAAAGTACGGTACGCGCTTTTTTGCAAAGATTAGGTTCGGGATTATTA
GAAGAATTTTTTATGAAAGAAGAACAAGTTGTTTCTTTGATCTTCCCAAAA
ACAACTTCTTTTTCTTTACATGAATCGCATATAGAACGTATTTGGTATTTG
GAT
>Phoenix dactylifera chloroplast maturase K matK partial gene of cultivar Kupra
from Pakistan (Accession # KT803894)
GAGTATATTTACACATTTGTTCATGATCGTGGTTTAAATAGTTCGGTTTTTT
ACGAATCCACGGAAATTTTTGGTTATGACAATAAATCTAGTTCAGTACTTG
TGAAACGTTCAATTATTCGAATGTATCAACAGAATTATTTGATTTATTCGG
TTAATGATTCTAACCAAAATCGATTTGTTGGGCACAACAATTATTTTTATT
TTCATTTTTATTCTCAGATGATATTGGAAGGTTTTGCAGTCATTGTGGAAA
TTCCATTCTTGCTGCGATTAGTATCTTCCCTCGAAGAAAAAAAAATACCAA
126
AATCTCAGAATTTGAATTTACGATCTATTCATTCAACATTTCCCTTTTTGGA
GGACAAATTATCGCATTTAAACTATGTGTCAGATATACTAATACCTTATCC
CATCCATCTGAAAATCTTGGTTCAAATCCTTCAATTCTGGATCCAAGATGT
TCCTTCTTTACATTTATTGCGATTCTTTCTTCACGAATATCATAATTGGAAT
AGTCTTATTACTCCGAATAATTCTATTTTTCTTTTTTCAAAAGAAAATAAA
AGAGTATTTCGGTTCCCATATAATTCTTATGTATCTGAATGCGAATTTGTA
TTAGTTTTTCTTCGTAAACAATCTTCTTATTTACGATTAACATCTTCTGGAG
CTTTTCTTGAGCGAACACATTTCTATGGAAAAATAGAACATCGTATAGTAG
TGCGCCGTAATTATTTTCAGAAGACCCTATGGTTCTTCAAGGATCCCTTCA
TGCATTATGTTCGATATCAAGGAAAAGCAATTCTGGTTTCAAAGGGGACT
CATCTTCTGATGAAGAAATGGAAATGTTACCTTGTCAATTTTTGGCAATAT
TATTTTCACTTTTGGTCTCAACCGTACAGGATCCATATAAACCAATTATCA
AACTGTTCTTTCTATTTTCTAGGTTATCTTTCAAGTGTACTAATAAATCCTT
CGGCGGTAAGGAATCAAATGCTAGAGAATTCATTTCTAATAGATACTGTT
ATTAAAAAATTCGATACCAGAGTCCCAGTTATTACTCTTATTGGATCATTG
TCTAAAGCTAAATTTTGTACCGTATTGGGGCATCCTATTAGTAAGCCGATC
TGGACCGATTTATCAGATTGCGATATTATTGATCGATTTGGTCGGATATGT
AGAAATCTTTCTCATTATCACAGTGGATCCTCAAAAAAACAGAGTTTGTAT
CGAATAAAGTATATACTTCGATTTTCGTGTGCTAGAACTTTGGCTCGTAAA
CATAAAAGTACGGTACGCGCTTTTTTGCAAAGATTAGGTTCGGGATTATTA
GAAGAATTTTTTATGAAAGAAGAACAAGTTGTTTCTTTGATCTTCCCAAAA
ACAACTTCTTTTTCTTTACATGAATCGCATATAGAACGTATTTGGTATTTG
GAT
>Phoenix dactylifera chloroplast maturase K matK partial gene of cultivar Shakri
from Pakistan (Accession # KT803895)
GAGTATATTTACACATTTGTTCATGATCGTGGTTTAAATAGTTCGGTTTTTT
ACGAATCCACGGAAATTTTTGGTTATGACAATAAATCTAGTTCAGTACTTG
TGAAACGTTCAATTATTCGAATGTATCAACAGAATTATTTGATTTATTCGG
TTAATGATTCTAACCAAAATCGATTTGTTGGGCACAACAATTATTTTTATT
TTCATTTTTATTCTCAGATGATATTGGAAGGTTTTGCAGTCATTGTGGAAA
TTCCATTCTTGCTGCGATTAGTATCTTCCCTCGAAGAAAAAAAAATACCAA
AATCTCAGAATTTGAATTTACGATCTATTCATTCAACATTTCCCTTTTTGGA
GGACAAATTATCGCATTTAAACTATGTGTCAGATATACTAATACCTTATCC
CATCCATCTGAAAATCTTGGTTCAAATCCTTCAATTCTGGATCCAAGATGT
TCCTTCTTTACATTTATTGCGATTCTTTCTTCACGAATATCATAATTGGAAT
AGTCTTATTACTCCGAATAATTCTATTTTTCTTTTTTCAAAAGAAAATAAA
AGAGTATTTCGGTTCCCATATAATTCTTATGTATCTGAATGCGAATTTGTA
TTAGTTTTTCTTCGTAAACAATCTTCTTATTTACGATTAACATCTTCTGGAG
CTTTTCTTGAGCGAACACATTTCTATGGAAAAATAGAACATCGTATAGTAG
TGCGCCGTAATTATTTTCAGAAGACCCTATGGTTCTTCAAGGATCCCTTCA
TGCATTATGTTCGATATCAAGGAAAAGCAATTCTGGTTTCAAAGGGGACT
CATCTTCTGATGAAGAAATGGAAATGTTACCTTGTCAATTTTTGGCAATAT
127
TATTTTCACTTTTGGTCTCAACCGTACAGGATCCATATAAACCAATTATCA
AACTGTTCTTTCTATTTTCTAGGTTATCTTTCAAGTGTACTAATAAATCCTT
CGGCGGTAAGGAATCAAATGCTAGAGAATTCATTTCTAATAGATACTGTT
ATTAAAAAATTCGATACCAGAGTCCCAGTTATTACTCTTATTGGATCATTG
TCTAAAGCTAAATTTTGTACCGTATTGGGGCATCCTATTAGTAAGCCGATC
TGGACCGATTTATCAGATTGCGATATTATTGATCGATTTGGTCGGATATGT
AGAAATCTTTCTCATTATCACAGTGGATCCTCAAAAAAACAGAGTTTGTAT
CGAATAAAGTATATACTTCGATTTTCGTGTGCTAGAACTTTGGCTCGTAAA
CATAAAAGTACGGTACGCGCTTTTTTGCAAAGATTAGGTTCGGGATTATTA
GAAGAATTTTTTATGAAAGAAGAACAAGTTGTTTCTTTGATCTTCCCAAAA
ACAACTTCTTTTTCTTTACATGAATCGCATATAGAACGTATTTGGTATTTG
GAT
128
Appendix XII: Sequences from date palm chloroplast 16S ribosomal RNA (16S
rRNA) partial genesubmitted to the GenBank
>Phoenix dactylifera chloroplast 16S ribosomal RNA partial gene of cultivar Dhaki
from Pakistan (Accession # KT983365)
GGTTGCTAATACCCCGTAGGCTGAGGAGCAAAAGGAGGAATCCGCCCGA
GGAGGGGCTCGCGTCTGATTAGCTAGTTGGTGAGGCAATAGCTTACCAAG
GCGATGATCAGTAGCTGGTCCGAGAGGATGATCAGCCACACTGGGACTGA
GACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAAT
GGGCGAAAGCCTGACGGAGCAATGCCGCGTGGAGGTAGAAGGCCCACGG
GTCGTGAACTTCTTTTCTCGGAGAAGAAGCAATGACGGTATCTGAGGAAT
AAGCATCGGCTAACTCTGTGCCAGCAGCCGCGGTAAGACAGAGGATGCAA
GCGTTATCCGGAATGATTGGGCGTAAAGCGTCTGTAGGTGGCTTTTCAAGT
CCGCCGTCAAATCCCAGGGCTCAACCCTGGACAGGCGGTGGAAACTACCA
AGCTGGAGTACGGTAGGGGCAGAGGGAATTTCCGGTGGAGCGGTGAAAT
GCGTAGAGATCGGAAAGAACACCAACGGCGAAAGCACTCTGCTGGGCCG
ACACTGACACTGAGAGACGAAAGCTAGGGGAGCAAATGGGATTAGATAC
CCCAGTAGTCCTAGCCGTAAACGATGGATACTAGGCGCTGTGCGTATCGA
CCCGTGCAGTGCTGTAGCTAACGCGTTAAGTATCCCGCCTGGGGAGTACG
TTCGCAAGAATGAAACTCAAAGGAATTGAC
>Phoenix dactylifera chloroplast 16S ribosomal RNA partial gene of cultivar Aseel
from Pakistan (Accession # KT983364)
CCCGTAGGCTGAGGAGCAAAAGGAGGAATCCGCCCGAGGAGGGGCTCGC
GTCTGATTAGCTAGTTGGTGAGGCAATAGCTTACCAAGGCGATGATCAGT
AGCTGGTCCGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCA
GACTCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAATGGGCGAAAGCC
TGACGGAGCAATGCCGCGTGGAGGTAGAAGGCCCACGGGTCGTGAACTTC
TTTTCTCGGAGAAGAAGCAATGACGGTATCTGAGGAATAAGCATCGGCTA
ACTCTGTGCCAGCAGCCGCGGTAAGACAGAGGATGCAAGCGTTATCCGGA
ATGATTGGGCGTAAAGCGTCTGTAGGTGGCTTTTCAAGTCCGCCGTCAAAT
CCCAGGGCTCAACCCTGGACAGGCGGTGGAAACTACCAAGCTGGAGTAC
GGTAGGGGCAGAGGGAATTTCCGGTGGAGCGGTGAAATGCGTAGAGATC
GGAAAGAACACCAACGGCGAAAGCACTCTGCTGGGCCGACACTGACACT
GAGAGACGAAAGCTAGGGGAGCAAATGGGATTAGATACCCCAGTAGTCC
TAGCCGTAAACGATGGATACTAGGCGCTGTGCGTATCGACCCGTGCAGTG
CTGTAGCTAACGCGTTAAGTATCCCGCCTGGGGAGTACGTTCGCAAGAAT
GAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGG
TTTAATTCGATGCAAAGCGAAGAACCTTACCGGGGCTTGACATGCCGCGA
ATCCTCTTGAAAGAGAGGGGTGCCTTCGGGAACGCGGACACAGGTGGTGC
ATGGCTGTCGTCAGCTCGTGCCGTAAGGTGTTGGGTTAAGTCCCGCAACG
AGCGCAACCCTCGTGTTTAGTTGCCACCGTTGAGTTTGGAACCCTGAACAG
ACTGCCGGTGATAAGCCGGAGGAAGGTGAGGATGACGTCAAGTCATCATG
129
CCCCTTATGCCCTGGGCGACACACGTGCTACAATGGCCGGGACAAAGGGT
CGCGATCCCGCGAGGGTGAGCTAACTCCAAAAACCCGTCCTCAGTTCGGA
TTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCCG
GTCAGCCATACGGCGGTGAATTCGTTCCCGGGCCTTGTACACACCGCCCG
TCACACTATGGGAGCTGGCCATGCCCGAAGTCGTTACCTTAACCGCAAGG
AGGGGGA
>Phoenix dactylifera chloroplast 16S ribosomal RNA partial gene of cultivar Halawi
from Pakistan (Accession # KT983366)
GGTTGCTAATACCCCGTAGGCTGAGGAGCAAAAGGAGGAATCCGCCCGA
GGAGGGGCTCGCGTCTGATTAGCTAGTTGGTGAGGCAATAGCTTACCAAG
GCGATGATCAGTAGCTGGTCCGAGAGGATGATCAGCCACACTGGGACTGA
GACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAAT
GGGCGAAAGCCTGACGGAGCAATGCCGCGTGGAGGTAGAAGGCCCACGG
GTCGTGAACTTCTTTTCTCGGAGAAGAAGCAATGACGGTATCTGAGGAAT
AAGCATCGGCTAACTCTGTGCCAGCAGCCGCGGTAAGACAGAGGATGCAA
GCGTTATCCGGAATGATTGGGCGTAAAGCGTCTGTAGGTGGCTTTTCAAGT
CCGCCGTCAAATCCCAGGGCTCAACCCTGGACAGGCGGTGGAAACTACCA
AGCTGGAGTACGGTAGGGGCAGAGGGAATTTCCGGTGGAGCGGTGAAAT
GCGTAGAGATCGGAAAGAACACCAACGGCGAAAGCACTCTGCTGGGCCG
ACACTGACACTGAGAGACGAAAGCTAGGGGAGCAAATGGGATTAGATAC
CCCAGTAGTCCTAGCCGTAAACGATGGATACTAGGCGCTGTGCGTATCGA
CCCGTGCAGTGCTGTAGCTAACGCGTTAAGTATCCCGCCT
>Phoenix dactylifera chloroplast 16S ribosomal RNA partial gene of cultivar Qantar
from Pakistan (Accession # KT983367)
GGTTGCTAATACCCCGTAGGCTGAGGAGCAAAAGGAGGAATCCGCCCGA
GGAGGGGCTCGCGTCTGATTAGCTAGTTGGTGAGGCAATAGCTTACCAAG
GCGATGATCAGTAGCTGGTCCGAGAGGATGATCAGCCACACTGGGACTGA
GACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAAT
GGGCGAAAGCCTGACGGAGCAATGCCGCGTGGAGGTAGAAGGCCCACGG
GTCGTGAACTTCTTTTCTCGGAGAAGAAGCAATGACGGTATCTGAGGAAT
AAGCATCGGCTAACTCTGTGCCAGCAGCCGCGGTAAGACAGAGGATGCAA
GCGTTATCCGGAATGATTGGGCGTAAAGCGTCTGTAGGTGGCTTTTCAAGT
CCGCCGTCAAATCCCAGGGCTCAACCCTGGACAGGCGGTGGAAACTACCA
AGCTGGAGTACGGTAGGGGCAGAGGGAATTTCCGGTGGAGCGGTGAAAT
GCGTAGAGATCGGAAAGAACACCAACGGCGAAAGCACTCTGCTGGGCCG
ACACTGACACTGAGAGACGAAAGCTAGGGGAGCAAATGGGATTAGATAC
CCCAGTAGTCCTAGCCGTAAACGATGGATACTAGGCGCTGTGCGTATCGA
CCCGTGCAGTGCTGTAGCTAACGCGTTAAGTATCCCGCCTGGGGAGTACG
TTCGCAAGAATGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGT
GGAGCATGTGG
130
>Phoenix dactylifera chloroplast 16S ribosomal RNA partial gene of cultivar Hamin
wali from Pakistan (Accession # KT983368)
GGTTGCTAATACCCCGTAGGCTGAGGAGCAAAAGGAGGAATCCGCCCGA
GGAGGGGCTCGCGTCTGATTAGCTAGTTGGTGAGGCAATAGCTTACCAAG
GCGATGATCAGTAGCTGGTCCGAGAGGATGATCAGCCACACTGGGACTGA
GACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAAT
GGGCGAAAGCCTGACGGAGCAATGCCGCGTGGAGGTAGAAGGCCCACGG
GTCGTGAACTTCTTTTCTCGGAGAAGAAGCAATGACGGTATCTGAGGAAT
AAGCATCGGCTAACTCTGTGCCAGCAGCCGCGGTAAGACAGAGGATGCAA
GCGTTATCCGGAATGATTGGGCGTAAAGCGTCTGTAGGTGGCTTTTCAAGT
CCGCCGTCAAATCCCAGGGCTCAACCCTGGACAGGCGGTGGAAACTACCA
AGCTGGAGTACGGTAGGGGCAGAGGGAATTTCCGGTGGAGCGGTGAAAT
GCGTAGAGATCGGAAAGAACACCAACGGCGAAAGCACTCTGCTGGGCCG
ACACTGACACTGAGAGACGAAAGCTAGGGGAGCAAATGGGATTAGATAC
CCCAGTAGTCCTAGCCGTAAACGATGGATACTAGGCGCTGTGCGTATCGA
CCCGTGCAGTGCTGTAGCTAACGCGTTAAGTATCCCGCCTGGGGAGTACG
TTCGCAAGAATGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGT
GGAGCATGTGGTTTAATTCGATGCAAAGCGAAGAACCTTACCGGGGCTTG
ACATGCCGCGAATCCTCTTGAAAGAGAG
>Phoenix dactylifera chloroplast 16S ribosomal RNA partial gene of cultivar Kupra
from Pakistan (Accession # KT983369)
GGTTGCTAATACCCCGTAGGCTGAGGAGCAAAAGGAGGAATCCGCCCGA
GGAGGGGCTCGCGTCTGATTAGCTAGTTGGTGAGGCAATAGCTTACCAAG
GCGATGATCAGTAGCTGGTCCGAGAGGATGATCAGCCACACTGGGACTGA
GACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAAT
GGGCGAAAGCCTGACGGAGCAATGCCGCGTGGAGGTAGAAGGCCCACGG
GTCGTGAACTTCTTTTCTCGGAGAAGAAGCAATGACGGTATCTGAGGAAT
AAGCATCGGCTAACTCTGTGCCAGCAGCCGCGGTAAGACAGAGGATGCAA
GCGTTATCCGGAATGATTGGGCGTAAAGCGTCTGTAGGTGGCTTTTCAAGT
CCGCCGTCAAATCCCAGGGCTCAACCCTGGACAGGCGGTGGAAACTACCA
AGCTGGAGTACGGTAGGGGCAGAGGGAATTTCCGGTGGAGCGGTGAAAT
GCGTAGAGATCGGAAAGAACACCAACGGCGAAAGCACTCTGCTGGGCCG
ACACTGACACTGAGAGACGAAAGCTAGGGGAGCAAATGGGATTAGATAC
CCCAGTAGTCCTAGCCGTAAACGATGGATACTAGGCGCTGTGCGTATCGA
CCCGTGCAGTGCTGTAGCTAACGCGTTAAGTATCCCGCCTGGGGAGTACG
TTCGCAAGAATGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGT
GGAGCATGTGGTTTAATTCGATGCAAAGCGAAGAACCTTACCGGGGCTTG
ACATGCCGCGAATCCTCTTG
131
>Phoenix dactylifera chloroplast 16S ribosomal RNA partial gene of cultivar Shakri
from Pakistan (Accession # KT983370)
GGTTGCTAATACCCCGTAGGCTGAGGAGCAAAAGGAGGAATCCGCCCGA
GGAGGGGCTCGCGTCTGATTAGCTAGTTGGTGAGGCAATAGCTTACCAAG
GCGATGATCAGTAGCTGGTCCGAGAGGATGATCAGCCACACTGGGACTGA
GACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAAT
GGGCGAAAGCCTGACGGAGCAATGCCGCGTGGAGGTAGAAGGCCCACGG
GTCGTGAACTTCTTTTCTCGGAGAAGAAGCAATGACGGTATCTGAGGAAT
AAGCATCGGCTAACTCTGTGCCAGCAGCCGCGGTAAGACAGAGGATGCAA
GCGTTATCCGGAATGATTGGGCGTAAAGCGTCTGTAGGTGGCTTTTCAAGT
CCGCCGTCAAATCCCAGGGCTCAACCCTGGACAGGCGGTGGAAACTACCA
AGCTGGAGTACGGTAGGGGCAGAGGGAATTTCCGGTGGAGCGGTGAAAT
GCGTAGAGATCGGAAAGAACACCAACGGCGAAAGCACTCTGCTGGGCCG
ACACTGACACTGAGAGACGAAAGCTAGGGGAGCAAATGGGATTAGATAC
CCCAGTAGTCCTAGCCGTAAACGATGGATACTAGGCGCTGTGCGTATCGA
CCCGTGCAGTGCTGTAGCTAACGCGTTAAGTATCCCGCCTGGGGAGTACG
TTCGCAAGAATGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGT
GGAGCATGTGGTTTAATTCGATGCAAAGCGAAGAACCTTACCGGGGCTTG
ACATGCCGCGAATCCTCTTGAAAGAGAGGGGTGCCTTCGGGAACGCGGAC
ACAGGTGGTGCATGGCTGTCGTCAGCT
132
Appendix XIII: Sequences from date palm genomic regions harboring SNPs
submitted to the GenBank
>Phoenix dactylifera nuclear genomic sequence of SNP-03 of cultivar Dhaki from
Pakistan (Accession # KT983266)
AGTAAAATGATTATAACTTCTTTTTGAAAGTTTCTATTTATTCCGATCAAG
TAGACTGCGGCCCACTATTTCAGCTCATATTGCTGATTTAAGATCAAACAT
CAGCTCGTGAGTTCTTTTGCAAGTGGTCTAGAAGAATTACCAGGACGTACT
TTCAATTCCATTGGACTGGATAAATTGATTTAAGGGAAAGGGTTAGATTG
GTTGTCTATGTTGCAAAACCCTTGATATTCATGAAGGGATTTAGATACTAT
AAGGTCCAATGTTTGAAAGCTTGGAACAAGTTTGGTTTATAT
>Phoenix dactylifera nuclear genomic sequence of SNP-03 of cultivar Aseel from
Pakistan (Accession # KT983267)
AGTAAAATGATTATAACTTCTTTTTGAAAGTTTCTATTTATTCCGATCAAG
TAGACTGCGGCCCACTATTTCAGCTCATATTGCTGATTTAAGATCAAACAT
CAGCTCGTGAGTTCTTTTGCAAGTGGTCTAGAAGAATTACCAGGACGTACT
TTCAATTCCATTGGACTGGATAAATTGATTTAAGGGAAAGGGTTAGATTG
GTTGTCTATGTTGCAAAACCCTTGATATTCATGAAGGGATTTAGATACTAT
AAGGTCCAATGTTTGAAAGCTTGGAACAAGTTTGGTTTATAT
>Phoenix dactylifera nuclear genomic sequence of SNP-03 of cultivar Halwai from
Pakistan (Accession # KT983268)
AGTAAAATGATTATAACTTCTTTTTGAAAGTTTCTATTTATTCCGATCAAG
TAGACTGCGGCCCACTATTTCAGCTCATATTGCTGATTTAAGATCAAACAT
CAGCTCGTGAGTTCTTTTGCAAGTGGTCTAGAAGAATTACCAGGACGTACT
TTCAATTCCATTGGACTGGATAAATTGATTTAAGGGAAAGGGTTAGATTG
GTTGTCTATGTTGCAAAACCCTTGATATTCATGAAGGGATTTAGATACTAT
AAGGTCCAATGTTTGAAAGCTTGGAACAAGTTTGGTTTATAT
>Phoenix dactylifera nuclear genomic sequence of SNP-03 of cultivar Qantar from
Pakistan (Accession # KT983269)
AGTAAAATGATTATAACTTCTTTTTGAAAGTTTCTATCTATTCCGATCAAG
TAGACTGCGGCCCACTATTTCAGCTCATATTGCTGATTTAAGATCAAACAT
CAGCTCGTGAGTTCTTTTGCAAGTGGTCTAGAAGAATTACCAGGACGTACT
TTCAATTCCATTGGACTGGATAAATTGATTTAAGGGAAAGGGTTAGATTG
GTTGTCTATTTTGCAAAACCCTTGATATTCATGAAGGGATTTAGATACTAT
AAGGTCCAATGTTTGAAAGCTTGGAACAAGTTTGGTTTATAT
133
>Phoenix dactylifera nuclear genomic sequence of SNP-03 of cultivar Haminwali
from Pakistan (Accession # KT983270)
AGTAAAATGATTATAACTTCTTTTTGAAAGTTTCTATTTATTCCGATCAAG
TAGACTGCGGCCCACTATTTCAGCTCATATTGCTGATTTAAGATCAAACAT
CAGCTCGTGAGTTCTTTTGCAAGTGGTCTAGAAGAATTACCAGGACGTACT
TTCAATTCCATTGGACTGGATAAATTGATTTAAGGGAAAGGGTTAGATTG
GTTGTCTATGTTGCAAAACCCTTGATATTCATGAAGGGATTTAGATACTAT
AAGGTCCAATGTTTGAAAGCTTGGAACAAGTTTGGTTTATAT
>Phoenix dactylifera nuclear genomic sequence of SNP-03 of cultivar Kupra from
Pakistan (Accession # KT983271)
AGTAAAATGATTATAACTTCTTTTTGAAAGTTTCTATCTATTCCGATCAAG
TAGACTGCGGCCCACTATTTCAGCTCATATTGCTGATTTAAGATCAAACAT
CAGCTCGTGAGTTCTTTTGCAAGTGGTCTAGAAGAATTACCAGGACGTACT
TTCAATTCCATTGGACTGGATAAATTGATTTAAGGGAAAGGGTTAGATTG
GTTGTCTATTTTGCAAAACCCTTGATATTCATGAAGGGATTTAGATACTAT
AAGGTCCAATGTTTGAAAGCTTGGAACAAGTTTGGTTTATAT
>Phoenix dactylifera nuclear genomic sequence of SNP-03 of cultivar Shakri from
Pakistan (Accession # KT983272)
AGTAAAATGATTATAACTTCTTTTTGAAAGTTTCTATCTATTCCGATCAAG
TAGACTGCGGCCCACTATTTCAGCTCATATTGCTGATTTAAGATCAAACAT
CAGCTCRTGAGTTCTTTTGCAAGTGGTCTAGAAGAATTACCAGGACGTACT
TTCAATTCCATTGGACTGGATAAATTGATTTAAGGGAAAGGGTTAGATTG
GTTGTCTATTTTGCAAAACCCTTGATATTCATGAAGGGATTTAGATACTAT
AAGGTCCAATGTTTGAAAGCTTGGAACAAGTTTGGTTTATAT
>Phoenix dactylifera nuclear genomic sequence of SNP-05 of cultivar Dhaki from
Pakistan (Accession # KT983273)
TTATTATAACTTGGCTTGATTTGGGCCTTACTCGACCCAGCTTAAACTGAT
ACACCTGGTTAAATAGACGTAATTGTGCTTGCTATCGAGCAGCAGGACTA
AAAATCGCTGTAATTAAGTACACAAAACAGTAGCTGACGCAAGAAAGAA
GATAGAACTGCAAACATATTAATTATTATGAAATCACGGGGCCTACACAA
GCATAGGAACAAAAAAGAGGATAAACTTAAACCACATCTTGGACCAGAT
CCAAAGTTAAACGACTGAACCTCAAAGTTTTGCATGACATGTTAAACAAG
AACAACGTAAGTAACATAAATGGAGGACATCCACGGCAAGCTATCGATA
GAAAGCTTTGTAACTGGCAATGTGGTCAGACCGGTCTACTGCCTCGAGCTT
CGCTCCTCTTGGA
>Phoenix dactylifera nuclear genomic sequence of SNP-05 of cultivar Aseel from
Pakistan (Accession #KT983274)
134
TTATTATAACTTGGCTTGATTTGGGCCTTACTCGACCCAGCTTAAACTGAT
ACACCTGGTTAAATAGACGTAATTGTGCTTGCTATCGAGCAGCAGGACTA
AAAATCGCTGTAATTAAGTACACAAAACAGTAGCTGACGCAAGAAAGAA
GATAGAACTGCAAACATATTAATTATTATGAAATCACGGGGCCTACACAA
GCATAGGAACAAAAAAGAGGATAAACTTAAACCACATCTTGGACCAGAT
CCAAAGTTAAACGACTGAACCTCAAAGTTTTGCATGACATGTTAAACAAG
AACAACGTAAGTAACATAAATGGAGGACATCCACGGCAAGCTATCGATA
GAAAGCTTTGTAACTGGCAATGTGGTCAGACCGGTCTACTGCCTCGAGCTT
CGCTCCTCTTGGA
>Phoenix dactylifera nuclear genomic sequence of SNP-05 of cultivar Halawi from
Pakistan (Accession # KT983275)
TTATTATAACTTGGCTTGATTTGGGCCTTACTCGACCCAGCTTAAACTGAT
ACACCTGGTTAAATAGACGTAATTGTGCTTGCTATCGAGCAGCAGGACTA
AAAATCGCTGTAATTAAGTACACAAAACAGTAGCTGACGCAAGAAAGAA
GATAGAACTGCAAACATATTAATTATTATGAAATCACGGGGCCTACACAA
GCATAGGAACAAAAAAGAGGATAAACTTAAACCACATCTTGGACCAGAT
CCAAAGTTAAACGACTGAACCTCAAAGTTTTGCATGACATGTTAAACAAG
AACAACGTAAGTAACATAAATGGAGGACATCCACGGCAAGCTATCGATA
GAAAGCTTTGTAACTGGCAATGTGGTCAGACCGGTCTACTGCCTCGAGCTT
CGCTCCTCTTGGA
>Phoenix dactylifera nuclear genomic sequence of SNP-05 of cultivar Qantar from
Pakistan (Accession # KT983276)
TTATTATAACTTGGCTTGATTTGGGCCTTACTCGACCCAGCTTAAACTGAT
ACACCTGGTTAAATAGACGTAATTGTGCTTGCTATCGAGCAGCAGGACTA
AAAATCGCTGTAATTAAGTACACAAAACAGTAGCTGACGCAAGAAAGAA
GATAGAACTGCAAACATATTAATTATTATGAAATCACGGGGCCTACACAA
GCATAGGAACAAAAAAGAGGATAAACTTAAACCACATCTTGGACCAGAT
CCAAAGTTAAACGACTGAACCTCAAAGTTTTGCATGACATGTTAAACAAG
AACAACGTAAGTAACATAAATGGAGGACATCCACGGCAAGCTATCGATA
GAAAGCTTTGTAACTGGCAATGTGGTCAGACCGGTCTACTGCCTCGAGCTT
CGCTCCTCTTGGA
>Phoenix dactylifera nuclear genomic sequence of SNP-05 of cultivar Haminwali
from Pakistan (Accession # KT983277)
TTATTATAACTTGGCTTGATTTGGGCCTTACTCGACCCAGCTTAAACTGAT
ACACCTGGTTAAATAGACGTAATTGTGCTTGCTATCGAGCAGCAGGACTA
AAAATCGCTGTAATTAAGTACACAAAACAGTAGCTGACGCAAGAAAGAA
GATAGAACTGCAAACATATTAATTATTATGAAATCACGGGGCCTACACAA
GCATAGGAACAAAAAAGAGGATAAACTTAAACCACATCTTGGACCAGAT
135
CCAAAGTTAAACGACTGAACCTCAAAGTTTTGCATGACATGTTAAACAAG
AACAACGTAAGTAACATAAATGGAGGACATCCACGGCAAGCTATCGATA
GAAAGCTTTGTAACTGGCAATGTGGTCAGACCGGTCTACTGCCTCGAGCTT
CGCTCCTCTTGGA
>Phoenix dactylifera nuclear genomic sequence of SNP-05 of cultivar Kupra from
Pakistan (Accession # KT983278)
TTATTATAACTTGGCTTGATTTGGGCCTTACTCGACCCAGCTTAAACTGAT
ACACCTGGTTAAATAGACGTAATTGTGCTTGCTATCGAGCAGCAGGACTA
AAAATCGCTGTAATTAAGTACACAAAACAGTAGCTGACGCAAGAAAGAA
GATAGAACTGCAAACATATTAATTATTATGAAATCACGGGGCCTACACAA
GCATAGGAACAAAAAAGAGGATAAACTTAAACCACATCTTGGACCAGAT
CCAAAGTTAAACGACTGAACCTCAAAGTTTTGCATGACATGTTAAACAAG
AACAACGTAAGTAACATAAATGGAGGACATCCACGGCAAGCTATCGATA
GAAAGCTTTGTAACTGGCAATGTGGTCAGACCGGTCTACTGCCTCGAGCTT
CGCTCCTCTTGGA
>Phoenix dactylifera nuclear genomic sequence of SNP-05 of cultivar Shakri from
Pakistan (Accession #KT983279)
TTATTATAACTTGGCTTGATTTGGGCCTTACTCGACCCAGCTTAAACTGAT
ACACCTGGTTAAATAGACGTAATTGTGCTTGCTATCGAGCAGCAGGACTA
AAAATCGCTGTAATTAAGTACACAAAACAGTAGCTGACGCAAGAAAGAA
GATAGAACTGCAAACATATTAATTATTATGAAATCACGGGGCCTACACAA
GCATAGGAACAAAAAAGAGGATAAACTTAAACCACATCTTGGACCAGAT
CCAAAGTTAAACGACTGAACCTCAAAGTTTTGCATGACATGTTAAACAAG
AACAACGTAAGTAACATAAATGGAGGACATCCACGGCAAGCTATCGATA
GAAAGCTTTGTAACTGGCAATGTGGTCAGACCGGTCTACTGCCTCGAGCTT
CGCTCCTCTTGGA
>Phoenix dactylifera nuclear genomic sequence of SNP-06 of cultivar Dhaki from
Pakistan (Accession # KT983280)
TTAGAGTTGATGCCATGCAACATGAGAACTCACTTATCCATTTAATAATCT
CCATAATCTACTCAGTGCACCATTAGTGAGAAAAGAAATCATATTGTATG
ACGCTACCATGTGATGAAAAGCACATGATTAAATTTGAGTGCAGATGTAT
ATGCATAGATCTACGATGTATACATTAAAAAGCAAAAGACATATTCTACA
AAATTCATAATAAGTGATCATTCTGCATTACTGATAGTAATAAAGTAATAC
ATAATAAACTTACCTGATTCAAGGCATTTTGCGGGGTCTCATACTCGGCCG
CTACAAAAACAAA
>Phoenix dactylifera nuclear genomic sequence of SNP-06 of cultivar Aseel from
Pakistan (Accession # KT983281)
TTAGAGTTGATGCCATGCAACATGAGAACTCACTTATCCATTTAATAATCT
CCATAATCTACTCAGTGCACCATTAGTGAGAAAAGAAATCATATTGTATG
136
ACGCTACCATGTGATGAAAAGCACATGATTAAATTTGAGTGCAGATGTAT
ATGCATAGATCTACGATGTATACATTAAAAAGCAAAAGACATATTCTACA
AAATTCATAATAAGTGATCATTCTGCATTACTGATAGTAATAAAGTAATAC
ATAATAAACTTACCTGATTCAAGGCATTTTGCGGGGTCTCATACTCGGCCG
CTACAAAAACAAA
>Phoenix dactylifera nuclear genomic sequence of SNP-06 of cultivar Halawi from
Pakistan (Accession # KT983282)
TTAGAGTTGATGCCATGCAACATGAGAACTCACTTATCCATTTAATAATCT
CCATAATCTACTCAGTGCACCATTAGTGAGAAAAGAAATCATATTGTATG
ACGCTACCATGTGATGAAAAGCACATGATTAAATTTGAGTGCAGATGTAT
ATGCATAGATCTACGATGTATACATTAAAAAGCAAAAGACATATTCTACA
AAATTCATAATAAGTGATCATTCTGCATTACTGATAGTAATAAAGTAATAC
ATAATAAACTTACCTGATTCAAGGCATTTTGCGGGGTCTCATACTCGGCCG
CTACAAAAACAAA
>Phoenix dactylifera nuclear genomic sequence of SNP-06 of cultivar Qantar from
Pakistan (Accession # KT983283)
TTAGAGTTGATGCCATGCAACATGAGAACTCACTTATCCATTTAATAATCT
CCATAATCTACTCAGTGCACCATTAGTGAGAAAAGAAATCATATTGTATG
ACGCTACCATGTGATGAAAAGCACATGATTAAATTTGAGTGCATATGTAT
ATGCATAGATCTACGATGTATACATTAAAAAGCAAAAGACATATTCTACA
AAATTCATAATAAGTGATCATTCTGCATTACTGATAGTAATAAAGTAATAC
ATAATAAACTTACCTGATTCAAGGCATTTTGCGGGGTCTCATACTCGGCCG
CTACAAAAACAAA
>Phoenix dactylifera nuclear genomic sequence of SNP-06 of cultivar Haminwali
from Pakistan (Accession # KT983284)
TTAGAGTTGATGCCATGCAACATGAGAACTCACTTATCCATTTAATAATCT
CCATAATCTACTCAGTGCACCATTAGTGAGAAAAGAAATCATATTGTATG
ACGCTACCATGTGATGAAAAGCACATGATTAAATTTGAGTGCAGATGTAT
ATGCATAGATCTACGATGTATACATTAAAAAGCAAAAGACATATTCTACA
AAATTCATAATAAGTGATCATTCTGCATTACTGATAGTAATAAAGTAATAC
ATAATAAACTTACCTGATTCAAGGCATTTTGCGGGGTCTCATACTCGGCCG
CTACAAAAACAAA
>Phoenix dactylifera nuclear genomic sequence of SNP-06 of cultivar Kupra from
Pakistan (Accession #KT983285)
TTAGAGTTGATGCCATGCAACATGAGAACTCACTTATCCATTTAATAATCT
CCATAATCTACTCAGTGCACCATTAGTGAGAAAAGAAATCATATTGTATG
ACGCTACCATGTGATGAAAAGCACATGATTAAATTTGAGTGCAGATGTAT
ATGCATAGATCTACGATGTATACATTAAAAAGCAAAAGACATATTCTACA
AAATTCATAATAAGTGATCATTCTGCATTACTGATAGTAATAAAGTAATAC
137
ATAATAAACTTACCTGATTCAAGGCATTTTGCGGGGTCTCATACTCGGCCG
CTACAAAAACAAA
>Phoenix dactylifera nuclear genomic sequence of SNP-06 of cultivar Shakri from
Pakistan (Accession # KT983286)
TTAGAGTTGATGCCATGCAACATGAGAACTCACTTATCCATTTAATAATCT
CCATAATCTACTCAGTGCACCATTAGTGAGAAAAGAAATCATATTGTATG
ACGCTACCATGTAATGAAAAGCACATGATTAAATTTGAGTGCAGATGTAT
ATGCATAGATCTACGATGTATACATTAAAAAGCAAAAGACATATTCTACA
AAATTCATAATAAGTGATCATTCTGCATTACTGATAGTAATAAAGTAATAC
ATAATAAACTTACCTGATTCAAGGCATTTTGCGGGGTCTCATACTCGGCCG
CTACAAAAACAAA
>Phoenix dactylifera nuclear genomic sequence of SNP-07 of cultivar Dhaki from
Pakistan (Accession # KT983287)
CTCGACCAACTGGACCTCAAAGACTCTCTTTGTAGTATTCTCATTCCATTTT
CTCTTCTCAGAGATAATCAGGTCACAAACTCTGCAGCCGGCCAAGTTCTCC
AGATCGATCATGGTAGGCCACAAGGCAAGCAGTCGCTCCGTCAGCCATCT
ATTATCCATCATGTTAATAGACAGACCATCTGATCCTAAGGAGTACCACC
AGTGCACAAGTACAGATCTTCCTCCATTTGAATGAACCGTGTTGCCCAGTA
TGGAAATCAAGGGCCGAGGTCTACGTGCCATTTTTGGCCCTCAGCAGGGT
GCTCCATAGGCTCTCAGGCTCAAGAAGGTACCTGAAAACGTATCTGGTAG
CAAGGAGCTCTCGCCTCACAAGTAAGGAGTGAATTCTCAACCCTCTACTG
CTAAGAGGCTGACAAACCACATCC
>Phoenix dactylifera nuclear genomic sequence of SNP-07 of cultivar Aseel from
Pakistan (Accession # KT983288)
CTCGACCAACTGGACCTCAAAGACTCTCTTTGTAGTATTCTCATTCCATTTT
CTCTTCTCAGAGATAATCAGGTCACAAACTCTGCAGCCGGCCAAGTTCTCC
AGATCGATCATGGTAGGCCACAAGGCAAGCAGTCGCTCCGTCAGCCATCT
ATTATCCATCATGTTAATAGACAGACCATCTGATCCTAAGGAGTACCACC
AGTGCACAAGTACAGATCTTCCTCCATTTGAATGAACCGTGTTGCCCAGTA
TGGAAATCAAGGGCCAAGGTCTACGTGCCATTTTTGGCCCTCAGCAGGGT
GCTCCATAGGCTCTCAGGCTCAAGAAGGTACCTGAAAACGTATCTGGTAG
CAAGGAGCTCTCGCCTCACAAGTAAGGAGTGAATTCTCAACCCTCTACTG
CTAAGAGGCTGACAAACCACATCC
>Phoenix dactylifera nuclear genomic sequence of SNP-07 of cultivar Halawi from
Pakistan (Accession # KT983289)
CTCGACCAACTGGACCTCAAAGACTCTCTTTGTAGTATTCTCATTCCATTTT
CTCTTCTCAGAGATAATCAGGTCACAAACTCTGCAGCCGGCCAAGTTCTCC
AGATCGATCATGGTAGGCCACAAGGCAAGCAGTCGCTCCGTCAGCCATCT
ATTATCCATCATGTTAATAGACAGACCATCTGATCCTAAGGAGTACCACC
138
AGTGCACAAGTACAGATCTTCCTCCATTTGAATGAACCGTGTTGCCCAGTA
TGGAAATCAAGGGCCAAGGTCTACGTGCCATTTTTGGCCCTCAGCAGGGT
GCTCCATAGGCTCTCAGGCTCAAGAAGGTACCTGAAAACGTATCTGGTAG
CAAGGAGCTCTCGCCTCACAAGTAAGGAGTGAATTCTCAACCCTCTACTG
CTAAGAGGCTGACAAACCACATCC
>Phoenix dactylifera nuclear genomic sequence of SNP-07 of cultivar Qantar from
Pakistan (Accession # KT983290)
CTCGACCAACTGGACCTCAAAGACTCTCTTTGTAGTATTCTCATTCCATTTT
CTCTTCTCCGAGATAATCAGGTCACAAACTCTGCAGCCGGCCAAATTCTCC
AGATCGATCATGGTAGGCCACAAGGCAAGCAGTCGCTCCGTCAGCCATCT
ATTATCCATCATGTTAATAGACAGACCATCTGATCCTAAGGAGTACCACC
AGTGCACAAGTACAGATCTTCCTCCATTTGAATGAACCGTGTTGCCCAGTA
TGGAAATCAAGGGCCAAGGTCTACGTGCCATTTTTGGCCCTCAGCAGGGT
GCTCCATAGGCTCTCAGGCTCAAGAAGGTACCTGAAAACGTATCTGGTAG
CAAGGAGCTCTCGCCTCACAAGTAAGGAGTGAATTCTCAACCCTCTACTG
CTAAGAGGCTGACAAACCACATCC
>Phoenix dactylifera nuclear genomic sequence of SNP-07 of cultivar Haminwali
from Pakistan (Accession # KT983291)
CTCGACCAACTGGACCTCAAAGACTCTCTTTGTAGTATTCTCATTCCATTTT
CTCTTCTCAGAGATAATCAGGTCACAAACTCTGCAGCCGGCCAAGTTCTCC
AGATCGATCATGGTAGGCCACAAGGCAAGCAGTCGCTCCGTCAGCCATCT
ATTATCCATCATGTTAATAGACAGACCATCTGATCCTAAGGAGTACCACC
AGTGCACAAGTACAGATCTTCCTCCATTTGAATGAACCGTGTTGCCCAGTA
TGGAAATCAAGGGCCAAGGTCTACGTGCCATTTTTGGCCCTCAGCAGGGT
GCTCCATAGGCTCTCAGGCTCAAGAAGGTACCTGAAAACGTATCTGGTAG
CAAGGAGCTCTCGCCTCACAAGTAAGGAGTGAATTCTCAACCCTCTACTG
CTAAGAGGCTGACAAACCACATCC
>Phoenix dactylifera nuclear genomic sequence of SNP-07 of cultivar Kupra from
Pakistan (Accession # KT983292)
CTCGACCAACTGGACCTCAAAGACTCTCTTTGTAGTATTCTCATTCCATTTT
CTCTTCTCAGAGATAATCAGGTCACAAACTCTGCAGCCGGCCAAGTTCTCC
AGATCGATCATGGTAGGCCACAAGGCAAGCAGTCGCTCCGTCAGCCATCT
ATTATCCATCATGTTAATAGACAGACCATCTGATCCTAAGGAGTACCACC
AGTGCACAAGTACAGATCTTCCTCCATTTGAATGAACCGTGTTGCCCAGTA
TGGAAATCAAGGGCCAAGGTCTACGTGCCATTTTTGGCCCTCAGCAGGGT
GCTCCATAGGCTCTCAGGCTCAAGAAGGTACCTGAAAACGTATCTGGTAG
CAAGGAGCTCTCGCCTCACAAGTAAGGAGTGAATTCTCAACCCTCTACTG
CTAAGAGGCTGACAAACCACATCC
139
>Phoenix dactylifera nuclear genomic sequence of SNP-07 of cultivar Shakri from
Pakistan (Accession # KT983293)
CTCGACCAACTGGACCTCAAAGACTCTCTTTGTAGTATTCTCATTCCATTTT
CTCTTCTCCGAGATAATCAGGTCACAAACTCTGCAGCCGGCCAAATTCTCC
AGATCGATCATGGTAGGCCACAAGGCAAGCAGTCGCTCCGTCAGCCATCT
ATTATCCATCATGTTAATAGACAGACCATCTGATCCTAAGGAGTACCACC
AGTGCACAAGTACAGATCTTCCTCCATTTGAATGAACCGTGTTGCCCAGTA
TGGAAATCAAGGGCCAAGGTCTACGTGCCATTTTTGGCCCTCAGCAGGGT
GCTCCATAGGCTCTCAGGCTCAAGAAGGTACCTGAAAACGTATCTGGTAG
CAAGGAGCTCTCGCCTCACAAGTAAGGAGTGAATTCTCAACCCTCTACTG
CTAAGAGGCTGACAAACCACATCC
>Phoenix dactylifera nuclear genomic sequence of SNP-09 of cultivar Dhaki from
Pakistan (Accession # KT983294)
GCGGTGGCAGGGGCGAGAGGGGCCGTGGGTAGTGGTGGCCGCAAGCCAA
GGGGAGTCTCATTTTCTCCACCCCAGCTTGCTTGCTTGATTGCTTGGTCCCC
TACGCTCCTAGCTTTATTTTCGCTCATCCACCGCCGTAGCACTTGATACAC
AACGAACTGTAGGCGACCTGGGCCTGGTACTCCTAACTAGGTTACGGCTA
GGCTCGTCAATGGATCCCGTTTTGGCTCAAAATTGACCTAATTAGTAGGGT
TTGAAATTAACCTGACTCGATCAAAAAATATTTGTATTTCATGATAAAAAA
GGAAAAAAAACTTGAAG
>Phoenix dactylifera nuclear genomic sequence of SNP-09 of cultivar Aseel from
Pakistan (Accession # KT983295)
GCGGTGGCAGGGGCGAGAGGGGCCGTGGGTAGTGGTGGCCGCAAGCCAA
GGGGAGTCTCATTTTCTCCACCCCAGCTTGCTTGCTTGATTGCTTGGTCCCC
TACGCTCCTAGCTTTATTTTCGCTCATCCACCGCCGTAGCACTTGATGCAC
AACGAACTGTAGGCGACCTGGGCCTGGTACTCCTAACTAGGTTACGGCTA
GGCTCGTCAATGGATCCCGTTTTGGCTCAAAATTGACCTAATTAGTAGGGT
TTGAAATTAACCTGACTCGATCAAAAAATATTTGTATTTCATGATAAAAAA
GGAAAAAAAACTTGAAG
>Phoenix dactylifera nuclear genomic sequence of SNP-09 of cultivar Halawi from
Pakistan (Accession # KT983296)
GCGGTGGCAGGGGCGAGAGGGGCCGTGGGTGGTGGTGGCCGCAAGCCAA
GGGGAGTCTCATTTTCTCCACCCCAGCTTGCTTGCTTGATTGCTTGGTCCCC
TACGCTCCTAGCTTTATTTTCGGTCATCCACCGCCGTAGCACTTGATACAC
AACGAACTGTAGGCGACCTGGGCCTGGTACTCCTAACTAGGTTACGGCTA
GGCTCGTCAATGGATCCCGTTTTGGCTCAAAATTGACCTAATTAGTAGGGT
TTGAAATTAACCTGACTCGATCAAAAAATATTTGTATTTCATGATAAAAAA
GGAAAAAAAACTTGAAG
140
>Phoenix dactylifera nuclear genomic sequence of SNP-09 of cultivar Qantar from
Pakistan (Accession # KT983297)
GCGGTGGCAGGGGCGAGAGGGGCCGTGGGTAGTGGTGGCCGCAAGCCAA
GGGGAGTCTCATTTTCTCCACCCCAGCTTGCTTGCTTGATTGCTTGGTCCCC
TACGCTCCTAGCTTTATTTTCGCTCATCCACCGCCGTAGCACTTGATGCAC
AACGAACTGTAGGCGACCTGGGCCTGGTACTCCTAACTAGGTTACGGCTA
GGCTCGTCAATGGATCCCGTTTTGGCTCAAAATTGACCTAATTAGTAGGGT
TTGAAATTAACCTGACTCGATCAAAAAATATTTGTATTTCATGATAAAAAA
GGAAAAAAAACTTGAAG
>Phoenix dactylifera nuclear genomic sequence of SNP-09 of cultivar Haminwali
from Pakistan (Accession # KT983298)
GCGGTGGCAGGGGCGAGAGGGGCCGTGGGTAGTGGTGGCCGCAAGCCAA
GGGGAGTCTCATTTTCTCCACCCCAGCTTGCTTGCTTGATTGCTTGGTCCCC
TACGCTCCTAGCTTTATTTTCGCTCATCCACCGCCGTAGCACTTGATACAC
AACGAACTGTAGGCGACCTGGGCCTGGTACTCCTAACTAGGTTACGGCTA
GGCTCGTCAATGGATCCCGTTTTGGCTCAAAATTGACCTAATTAGTAGGGT
TTGAAATTAACCTGACTCGATCAAAAAATATTTGTATTTCATGATAAAAAA
GGAAAAAAAACTTGAAG
>Phoenix dactylifera nuclear genomic sequence of SNP-09 of cultivar Kupra from
Pakistan (Accession # KT983299)
GCGGTGGCAGGGGCGAGAGGGGCCGTGGGTAGTGGTGGCCGCAAGCCAA
GGGGAGTCTCATTTTCTCCACCCCAGCTTGCTTGCTTGATTGCTTGGTCCCC
TACGCTCCTAGCTTTATTTTCGCTCATCCACCGCCGTAGCACTTGATGCAC
AACGAACTGTAGGCGACCTGGGCCTGGTACTCCTAACTAGGTTACGGCTA
GGCTCGTCAATGGATCCCGTTTTGGCTCAAAATTGACCTAATTAGTAGGGT
TTGAAATTAACCTGACTCGATCAAAAAATATTTGTATTTCATGATAAAAAA
GGAAAAAAAACTTGAAG
>Phoenix dactylifera nuclear genomic sequence of SNP-09 of cultivar Shakri from
Pakistan (Accession # KT983300)
GCGGTGGCAGGGGCGAGAGGGGCCGTGGGTAGTGGTGGCCGCAAGCCAA
GGGGAGTCTCATTTTCTCCACCCCAGCTTGCTTGCTTGATTGCTTGGTCCCC
TACGCTCCTAGCTTTATTTTCGCTCATCCACCGCCGTAGCACTTGATACAC
AACGAACTGTAGGCGACCTGGGCCTGGTACTCCTAACTAGGTTACGGCTA
GGCTCGTCAATGGATCCCGTTTTGGCTCAAAATTGACCTAATTAGTAGGGT
TTGAAATTAACCTGACTCGATCAAAAAATATTTGTATTTCATGATAAAAAA
GGAAAAAAAACTTGAAG
>Phoenix dactylifera nuclear genomic sequence of SNP-10 of cultivar Dhaki from
Pakistan (Accession # KT983301)
141
ACATTCTCATTATTCCAGAAATGAGTCACCAGGTTTGAGAGATTTTTGGAA
CAACAGTATTAATCCTGATAAGCAAACATGGTTACTGCTAACCTTGAAAA
TCCACCAGAACCAGCTCCCACATTTTGGGCTATAATTCTAGAATCAATTCG
GACAACCTTTGAATCATCTTCTGGAATAGATATCTGGCGCCGGATAATTAA
CCCACCACCAATATCCCCTTCTAAGCAAAGTGATTCCTCTTCTCCTGATTG
TTCAAGATTTCTCCTACAAATACAATTTTTTGTTATATTTATTCATTATAAA
GGGAAACATCTATCATAACCAAAGATGCAGCAGTTATTTGAAATATT
>Phoenix dactylifera nuclear genomic sequence of SNP-10 of cultivar Aseel from
Pakistan (Accession # KT983302)
ACATTCTCATTATTCCAGAAATGAGTCACCAGGTTCGAGAGATTTTTGGAA
CAACAGTACTAATCCTGATAAGTAAACATGGTTACCGCTAACCTTGAAAA
TCCACCAGAACCAGCTCCCACATTTTGGGCTATAATTCTAGAATCAATTCG
GACAACCTTTGGATCATCTTCTGGAATAGATATCTGGCGCCGGATAATTAA
CCCACCACCAATATCCCCTTCTAAGCAAAGTGATTCCTCTTCTCCTGATTG
TTCAAGATTTCTCCTACAAATACATTTTTTTGTTATATTTATTCATTATAAA
GGGAAACATCTATCATAACCAAAGATGCAGCAGTTATTTGAAATATT
>Phoenix dactylifera nuclear genomic sequence of SNP-10 of cultivar Halawi from
Pakistan (Accession # KT983303)
ACATTCTCACTATTCCAGAAATGAGTCACCAGGTTCGAGAGATTTTTGGAA
CAACAGTACTAATCCTGATAAGCAAACATGGTTACTGCTAACCTTGAAAA
TCCACCAGAACCAGCTCCCACATTTTGGGCTATAATTCTAGAATCAATTCG
GACAACCTTTGGATCATCTTCTGGAATAGATATCTGGCGCCGGATAATTAA
CCCACCACCAATATCCCCTTCTAAGCAAAGTGATTCCTCTTCTCCTGATTG
TTCAAGATTTCTCCTACAAATACAATTTTTTGTTATATTTATTCATTATAAA
GGGAAACATCTATCATAACCAAAGATGCAGCAGTTATTTGAAATATT
>Phoenix dactylifera nuclear genomic sequence of SNP-10 of cultivar Qantar from
Pakistan (Accession # KT983304)
ACATTCTCATTATTCCAGAAATGAGTCACCAGGTTCGAGAGATTTTTGGAA
CAACAGTACTAATCCTGATAAGTAAACATGGTTACCGCTAACCTTGAAAA
TCCACCAGAACCAGCTCCCACATTTTGGGCTATAATTCTAGAATCAATTCG
GACAACCTTTGGATCATCTTCTGGAATAGATATCTGGCGCCGGATAATTAA
CCCACCACCAATATCCCCTTCTAAGCAAAGTGATTCCTCTTCTCCTGATTG
TTCAAGATTTCTCCTACAAATACATTTTTTTGTTATATTTATTCATTATAAA
GGGAAACATCTATCATAACCAAAGATGCAGCAGTTATTTGAAATATT
>Phoenix dactylifera nuclear genomic sequence of SNP-10 of cultivar Haminwali
from Pakistan (Accession # KT983305)
ACATTCTCACTATTCCAGAAATGAGTCACCAGGTTCGAGAGATTTTTGGAA
CAACAGTACTAATCCTGATAAGCAAACATGGTTACTGCTAACCTTGAAAA
TCCACCAGAACCAGCTCCCACATTTTGGGCTATAATTCTAGAATCAATTCG
142
GACAACCTTTGGATCATCTTCTGGAATAGATATCTGGCGCCGGATAATTAA
CCCACCACCAATATCCCCTTCTAAGCAAAGTGATTCCTCTTCTCCTGATTG
TTCAAGATTTCTCCTACAAATACAATTTTTTGTTATATTTATTCATTATAAA
GGGAAACATCTATCATAACCAAAGATGCAGCAGTTATTTGAAATATT
>Phoenix dactylifera nuclear genomic sequence of SNP-10 of cultivar Kupra from
Pakistan (Accession # KT983306)
ACATTCTCATTATTCCAGAAATGAGTCACCAGGTTCGAGAGATTTTTGGAA
CAACAGTACTAATCCTGATAAGCAAACATGGTTACCGCTAACCTTGAAAA
TCCACCAGAACCAGCTCCCACATTTTGGGCTATAATTCTAGAATCAATTCG
GACAACCTTTGGATCATCTTCTGGAATAGATATCTGGCGCCGGATAATTAA
CCCACCACCAATATCCCCTTCTAAGCAAAGTGATTCCTCTTCTCCTGATTG
TTCAAGATTTCTCCTACAAATACATTTTTTTGTTATATTTATTCATTATAAA
GGGAAACATCTATCATAACCAAAGATGCAGCAGTTATTTGAAATATT
>Phoenix dactylifera nuclear genomic sequence of SNP-10 of cultivar Shakri from
Pakistan (Accession # KT983307)
ACATTCTCACTATTCCAGAAATGAGTCACCAGGTTCGAGAGATTTTTGGAA
CAACAGTATTAATCCTGATAAGCAAACATGGTTACTGCTAACCTTGAAAA
TCCACCAGAACCAGCTCCCACATTTTGGGCTATAATTCTAGAATCAATTCG
GACAACCTTTGGATCATCTTCTGGAATAGATATCTGGCGCCGGATAATTAA
CCCACCACCAATATCCCCTTCTAAGCAAAGTGATTCCTCTTCTCCTGATTG
TTCAAGATTTCTCCTACAAATACAATTTTTTGTTATATTTATTCATTATAAA
GGGAAACATCTATCATAACCAAAGATGCAGCAGTTATTTGAAATATT
>Phoenix dactylifera nuclear genomic sequence of SNP-11 of cultivar Dhaki from
Pakistan (Accession # KT983308)
TAACATAAAAAATACACAAAAACATGTCCTGAAGTTTTATTATATTTATAA
AATAGAATAGAATATAGTCTAGTACATGCATCTACATGCCTCCCTTGTGCG
CCATAGCACCTAACAAACAAGTCCTCCTTTCCCCCTTTTCCTGAAACATAG
TTAAGCTGCTTTGTCATACTATAGACGGCCTTCACCAAAATTTACGTAGTG
ACCAATTCAACTACATTATATACAGGCAGCACCACAGCCTGATACAGAAG
AGCCAGAGAGAGGGAGGCCATCCACTGCAAACCTTCCATAAAGGCAGAT
AATGGAGATAAAGAGGTCCAGAAAGTAAAAAAGGTACTTCAGCCAGTAA
AGCACTACGAAATCCCACCACCGTCCAATTTTAAAAAGTTCAGAAAGTAT
TGGATCAGCTCAAAACATCGGTGCCTGCAGCTATCTACATGTTTCCCACCT
ACCTCTCGTCATCTCAGAATTAGTCAATTGCTTGTGTGACATCCTGCGCTG
CCAACCTCTCAGACACATTGGCCAAAGATTTGAGGTTGCACTTGATCAGG
GCCTCCACAAAGTAGCATGTCTCGTCCTTGGTGTTTCCATCAGGCACATCA
ACCACGAACGACTCGATCACCAGGGTCCCTGGTCTCCCATCAA
>Phoenix dactylifera nuclear genomic sequence of SNP-11 of cultivar Aseel from
Pakistan (Accession # KT983309)
143
TAACATAAAAAATACACAAAAACATGTCCTGAAGTTTTATTATATTTATAA
AATAGAATAGAATATAGTCTAGTACATGCATCTACATGCCTCCCTTGTGCG
CCATAGCACCTAACAAACAAGTCCTCCTTTCCCCCTTTTCCTGAAACATAG
TTAAGCTGCTTTGTCATACTATAGACGGCCTTCACCAAAATTTACGTAGTG
ACCAATTCAACTACATTATATACAGGCAGCACCACAGCCTGATACAGAAG
AGCCAGAGAGAGGGAGGCCATCCACTGCAAACCTTCCATAAAGGCAGAT
AATGGAGATAAAGAGGTCCAGAAAGTAAAAAAGGTACTTCAGCCAGTAA
AGCACTACGAAATCCCACCACCGTCCAATTTTAAAAAGTTCAGAAAGTAT
TGGATCAGCTCAAAACATCGGTGCCTGCAGCTATCTACATGTTTCCCACCT
ACCTCTCGTCATCTCAGAATTAGTCAATTGCTTGTGTGACATCCTGCGCTG
CCAACCTCTCAGACACATTGGCCAAAGATTTGAGGTTGCACTTGATCAGG
GCCTCCACAAAGTAGCATGTCTCGTCCTTGGTGTTTCCATCAGGCACATCA
ACCACGAACGACTCGATCACCAGGGTCCCTGGTCTCCCATCAA
>Phoenix dactylifera nuclear genomic sequence of SNP-11 of cultivar Halawi from
Pakistan (Accession # KT983310)
TAACATAAAAAATACACAAAAACATGTCCTGAAGTTTTATTATATTTATAA
AATAGAATAGAATATAGTCTAGTACATGCATCTACATGCCTCCCTTGTGCG
CCATAGCACCTAACAAACAAGTCCTCCTTTCCCCCTTTTCCTGAAACATAG
TTAAGCTGCTTTGTCATACTATAGACGGCCTTCACCAAAATTTACGTAGTG
ACCAATTCAACTACATTATATACAGGCAGCACCACAGCCTGATACAGAAG
AGCCAGAGAGAGGGAGGCCATCCACTGCAAACCTTCSATAAAGGCAGATA
ATGGAGATAAAGAGGTCCAGAAAGTAAAAAAGGTACTTCAGCCAGTAAA
GCACTACGAAATCCCACCACCGTCCAATTTTAAAAAGTTCAGAAAGTATT
GGATCAGCTCAAAACATCGGTGCCTGCAGCTATCTACATGTTTCCCACCTA
CCTCTCGTCATCTCAGAATTAGTCAATTGCTTGTGTGACATCCTGCGCTGC
CAACCTCTCAGACACATTGGCCAAAGATTTGAGGTTGCACTTGATCAGGG
CCTCCACAAAGTAGCATGTCTCGTCCTTGGTGTTTCCATCAGGCACATCAA
CCACGAACGACTCGATCACCAGGGTCCCTGGTCTCCCATCAA
>Phoenix dactylifera nuclear genomic sequence of SNP-11 of cultivar Qantar from
Pakistan (Accession # KT983311)
TAACATAAAAAATACACAAAAACATGTCCTGAAGTTTTATTATATTTATAA
AATAGAATAGAATATAGTCTAGTACATGCATCTACATGCCTCCCTTGTGCG
CCATAGCACCTAACAAACTAGTCCTCCTTTCCCCCTTTTCCTGAAACATAG
TTCAGCTGCTTTGTCATACTATAGACGGCCTTCACCAAAATTTACGTAGTG
ACCAATTCAACTACATTATATACAGGCAGCACCACAGCCTGATACAGAAG
AGCCAGAGAGAGGGAGGCCATCCACTGCAAACCTTCGATAAAGGCAGAT
AATGGAGATAAAGAGGTCCAGAAAGTAAAAAAGGTACTTCAGCCAGTAA
AGCACTACGAAATCCCACCACCGTCCAATTTTAAAAAGTTCAGAAAGTAT
TGGATCAGCTCAAAACATCGGTGCCTGCAGCTATCTACATGTTTCCCACCT
ACCTCTCGTCATCTCAGAATTAGTCAATTGCTTGTGTGACATCCTGCGCTG
CCAACCTCTCAGACACATTGGCCAAAGATTTGAGGTTGCACTTGATCAGG
144
GCCTCCACAAAGTAGCATGTCTCGTCCTTGGTGTTTCCATCAGGCACATCA
ACCACGAACGACTCGATCACCAGGGTCCCTGGTCTCCCATCAA
>Phoenix dactylifera nuclear genomic sequence of SNP-11 of cultivar Haminwali
from Pakistan (Accession # KT983312)
TAACATAAAAAATACACAAAAACATGTCCTGAAGTTTTATTATATTTATAA
AATAGAATAGAATATAGTCTAGTACATGCATCTACATGCCTCCCTTGTGCG
CCATAGCACCTAACAAACAAGTCCTCCTTTCCCCCTTTTCCTGAAACATAG
TTAAGCTGCTTTGTCATACTATAGACGGCCTTCACCAAAATTTACGTAGTG
ACCAATTCAACTACATTATATACAGGCAGCACCACAGCCTGATACAGAAG
AGCCAGAGAGAGGGAGGCCATCCACTGCAAACCTTCCATAAAGGCAGAT
AATGGAGATAAAGAGGTCCAGAAAGTAAAAAAGGTACTTCAGCCAGTAA
AGCACTACGAAATCCCACCACCGTCCAATTTTAAAAAGTTCAGAAAGTAT
TGGATCAGCTCAAAACATCGGTGCCTGCAGCTATCTACATGTTTCCCACCT
ACCTCTCGTCATCTCAGAATTAGTCAATTGCTTGTGTGACATCCTGCGCTG
CCAACCTCTCAGACACATTGGCCAAAGATTTGAGGTTGCACTTGATCAGG
GCCTCCACAAAGTAGCATGTCTCGTCCTTGGTGTTTCCATCAGGCACATCA
ACCACGAACGACTCGATCACCAGGGTCCCTGGTCTCCCATCAA
>Phoenix dactylifera nuclear genomic sequence of SNP-11 of cultivar Kupra from
Pakistan (Accession #KT983313)
TAACATAAAAAATACACAAAAACATGTCCTGAAGTTTTATTATATTTATAA
AATAGAATAGAATATAGTCTAGTACATGCATCTACATGCCTCCCTTGTGCG
CCATAGCACCTAACAAACAAGTCCTCCTTTCCCCCTTTTCCTGAAACATAG
TTAAGCTGCTTTGTCATACTATAGACGGCCTTCACCAAAATTTACGTAGTG
ACCAATTCAACTACATTATATACAGGCAGCACCACAGCCTGATACAGAAG
AGCCAGAGAGAGGGAGGCCATCCACTGCAAACCTTCCATAAAGGCAGAT
AATGGAGATAAAGAGGTCCAGAAAGTAAAAAAGGTACTTCAGCCAGTAA
AGCACTACGAAATCCCACCACCGTCCAATTTTAAAAAGTTCAGAAAGTAT
TGGATCAGCTCAAAACATCGGTGCCTGCAGCTATCTACATGTTTCCCACCT
ACCTCTCGTCATCTCAGAATTAGTCAATTGCTTGTGTGACATCCTGCGCTG
CCAACCTCTCAGACACATTGGCCAAAGATTTGAGGTTGCACTTGATCAGG
GCCTCCACAAAGTAGCATGTCTCGTCCTTGGTGTTTCCATCAGGCACATCA
ACCACGAACGACTCGATCACCAGGGTCCCTGGTCTCCCATCAA
>Phoenix dactylifera nuclear genomic sequence of SNP-11 of cultivar Shakri from
Pakistan (Accession #KT983314)
TAACATAAAAAATACACAAAAACATGTCCTGAAGTTTTATTATATTTATAA
AATAGAATAGAATATAGTCTAGTACATGCATCTACATGCCTCCCTTGTGCG
CCATAGCACCTAACAAACAAGTCCTCCTTTCCCCCTTTTCCTGAAACATAG
TTAAGCTGCTTTGTCATACTATAGACGGCCTTCACCAAAATTTACGTAGTG
ACCAATTCAACTACATTATATACAGGCAGCACCACAGCCTGATACAGAAG
AGCCAGAGAGAGGGAGGCCATCCACTGCAAACCTTCCATAAAGGCAGAT
145
AATGGAGATAAAGAGGTCCAGAAAGTAAAAAAGGTACTTCAGCCAGTAA
AGCACTACGAAATCCCACCACCGTCCAATTTTAAAAAGTTCAGAAAGTAT
TGGATCAGCTCAAAACATCGGTGCCTGCAGCTATCTACATGTTTCCCACCT
ACCTCTCGTCATCTCAGAATTAGTCAATTGCTTGTGTGACATCCTGCGCTG
CCAACCTCTCAGACACATTGGCCAAAGATTTGAGGTTGCACTTGATCAGG
GCCTCCACAAAGTAGCATGTCTCGTCCTTGGTGTTTCCATCAGGCACATCA
ACCACGAACGACTCGATCACCAGGGTCCCTGGTCTCCCATCAA
>Phoenix dactylifera nuclear genomic sequence of SNP-12 of cultivar Dhaki from
Pakistan (Accession # KT983315)
TAATAACAAAAAATAAAAACTAAAAACACTTTCTGAAGCTTAATTCAGTC
TTTTTGGTGTAGTGTATTATAGTTAATATTAGAATTCATTCTTTGAGTTGTC
TCTAGGCTTTGTCTGAGTCAACCCATGCCCAGCTTGAATCAAATTCCGCCC
AGATTATGGAGTTTGGCAAGGGCCTAAAAACTGATTTTCTGAACTCAAAC
TAACGCATGAAGCTCGCCTAGATCAGTGGTGCTCTCTCTGGTTTCGTCCAT
TTCCAGCCTGAAATG
>Phoenix dactylifera nuclear genomic sequence of SNP-12 of cultivar Aseel from
Pakistan (Accession # KT983316)
TAATAACAAAAAATAAAAACTAAAAACACTTTCTGAAGCTTAATTCAGTC
TTTTTGGTGTAGTGTATTATAGTTAATATTAGAATTCATTCTTTGAGTTGTC
TCTAGGCTTTGTCTGAGTCAACCCATGCCCAGCTTGAATCAAATTCCGCCC
AGATTATGGAGTTTGGCAAGGGCCTAAAAACTGATTTTCTGAACTCAAAC
TAACGCATGAAGCTCGCCTAGATCAGTGGTGCTCTCTCTGGTTTCGTCCAT
TTCCAGCCTGAAATG
>Phoenix dactylifera nuclear genomic sequence of SNP-12 of cultivar Halawi from
Pakistan (Accession # KT983317)
TAATAACAAAAAATAAAAACTAAAAACACTTTCTGAAGCTTAATTCAGTC
TTTTTGGTGTAGTGTATTATAGTTAATATTAGAATTCATTCTTTGAGTTGTC
TCTAGGCTTTGTCTGAGTCAACCCATGCCCAGCTTGAATCAAATTCCGCCC
AGATTATGGAGTTTGGCAAGGGCCTAAAAACTGATTTTCTGAACTCAAAC
TAACGCATGAAGCTCGCCTAGATCAGTGGTGCTCTCTCTGGTTTCGTCCAT
TTCCAGCCTGAAATG
>Phoenix dactylifera nuclear genomic sequence of SNP-12 of cultivar Qantar from
Pakistan (Accession # KT983318)
TAATAACAAAAAATAAAAACTAAAAACACTTTCTGAAGCTTAATTCAGTC
TTTTTGGTGTAGTGTATTATAGTTAATATTAGAATTCATTCTTTGAGTTGTC
TCTAGGCTTTGTCTGAGTCAACCCATGCCCAGCTTGAATCAAATTCCGCCC
AGATTATGGAGTTTGGCAAGGGCCTAAAAACTGATTTTCTGAACTCAAAC
TAACGCATGAAGCTCGCCTAGATCAGTGGTGCTCTCTCTGGTTTCGTCCAT
TTCCAGCCTGAAATG
146
>Phoenix dactylifera nuclear genomic sequence of SNP-12 of cultivar Haminwali
from Pakistan (Accession # KT983319)
TAATAACAAAAAATAAAAACTAAAAACACTTTCTGAAGCTTAATTCAGTC
TTTTTGGTGTAGTGTATTATAGTTAATATTAGAATTCATTCTTTGAGTTGTC
TCTAGGCTTTGTCTGAGTCAACCCATGCCCAGCTTGAATCAAATTCCGCCC
AGATTATGGAGTTTGGCAAGGGCCTAAAAACTGATTTTCTGAACTCAAAC
TAACGCATGAAGCTCGCCTAGATCAGTGGTGCTCTCTCTGGTTTCGTCCAT
TTCCAGCCTGAAATG
>Phoenix dactylifera nuclear genomic sequence of SNP-12 of cultivar Kupra from
Pakistan (Accession # KT983320)
TAATAACAAAAAATAAAAACTAAAAACACTTTCTGAAGCTTAATTCAGTC
TTTTTGGTGTAGTGTATTATAGTTAATATTAGAATTCATTCTTTGAATTGTC
TCTAGGCTTTGTCTGAGTCAACCCATGCCCAGCTTGAATCAAATTCCGCCC
AGATTATGGAGTTTGGCAAGGGCCTAAAAACTGATTTTCTGAACTCAAAC
TAACGCATGAAGCTCGCCTAGATCAGTGGTGCTCTCTCTGGTTTTGTCCAT
TTCCAGCCTGAAATG
>Phoenix dactylifera nuclear genomic sequence of SNP-12 of cultivar Shakri from
Pakistan (Accession # KT983321)
TAATAACAAAAAATAAAAACTAAAAACACTTTCTGAAGCTTAATTCAGTC
TTTTTGGTGTAGTGTATTATAGTTAATATTAGAATTCATTCTTTGAGTTGTC
TCTAGGCTTTGTCTGAGTCAACCCATGCCCAGCTTGAATCAAATTCCGCCC
AGATTATGGAGTTTGGCAAGGGCCTAAAAACTGATTTTCTGAACTCAAAC
TAACGCATGAAGCTCGCCTAGATCAGTGGTGCTCTCTCTGGTTTCGTCCAT
TTCCAGCCTGAAATG
>Phoenix dactylifera nuclear genomic sequence of SNP-14 of cultivar Dhaki from
Pakistan (Accession #KT983322)
AAGAAAAGCAGAGATAGCAGTAACAAGAATGGCAGGCATTAATGGAGGA
GAAGATTTCTAACGTTCATCCATGTACCAGTCGCTCTTCACCTTTGGTCCC
GTAGGGCCTTCCTCTCCTAGCACTTCATCAGGGAGGCTCTCCACCAGATGC
TTGAACTCAGTCTTGAGATTCCCAGGCAGGAGCTTCACCGCCTTATCGATC
TCCTTCGCGATGTCGTAGGTTATCTTCGGCCCCCATTCCCTCTCGTAGTTCA
GCCATGACGGCTCGACGACGGCGGAGCCCAAGTACTCGGCCGC
>Phoenix dactylifera nuclear genomic sequence of SNP-14 of cultivar Aseel from
Pakistan (Accession #KT983323)
AAGAAAAGCAGAGATAGCAGTAACAAGAATGGCAGGCATTAATGGAGGA
GAAGATTTCTAACGTTCATCCATGTACCAGTCGCTCTTCACCTTTGGTCCC
GTAGGGCCTTCCTCTCCTAGCACTTCATCAGGGAGGCTCTCCACCAGATGC
TTGAACTCAGTCTTGAGATTCCCAGGCAGGAGCTTCACCGCCTTATCGATC
147
TCCTTCGCGATGTCGTAGGTTATCTTCGGCCCCCATTCCCTCTCGTAGTTCA
GCCATGACGGCTCGACGACGGCGGAGCCCAAGTACTCGGCCGC
>Phoenix dactylifera nuclear genomic sequence of SNP-14 of cultivar Halawi from
Pakistan (Accession #KT983324)
AAGAAAAGCAGAGATAGCAGTAACAAGAATGGCAGGCATTAATGGAGGA
GAAGATTTCTAACGTTCATCCATGTACCAGTCGCTCTTCACCTTTGGTCCC
GTAGGGCCTTCCTCTCCTAGCACTTCATCAGGGAGGCTCTCCACCAGATGC
TTGAACTCAGTCTTGAGATTCCCAGGCAGGAGCTTCACCGCCTTATCGATC
TCCTTCGCGATGTCGTAGGTTATCTTCGGCCCCCATTCCCTCTCGTAGTTCA
GCCATGACGGCTCGACGACGGCGGAGCCCAAGTACTCGGCCGC
>Phoenix dactylifera nuclear genomic sequence of SNP-14 of cultivar Qantar from
Pakistan (Accession #KT983325)
AAGAAAAGCAGAGATAGCAGTAACAAGAATGGCAGGCATTAATGGAGGA
GAAGATTTCTAACGTTCATCCATGTACCAGTCGCTCTTCACCTTTGGTCCC
GTAGGGCCTTCCTCTCCTAGCACTTCATCAGGGAGGCTCTCCACCAGATGC
TTGAACTCAGTCTTGAGATTCCCAGGCAGGAGCTTCACCGCCTTATCGATC
TCCTTCGCGATGTCGTAGGTTATCTTCGGCCCCCATTCCCTCTCGTAGTTCA
GCCATGACGGCTCGACGACGGCGGAGCCCAAGTACTCGGCCGC
>Phoenix dactylifera nuclear genomic sequence of SNP-14 of cultivar Haminwali
from Pakistan (Accession #KT983326)
AAGAAAAGCAGAGATAGCAGTAACAAGAATGGCAGGCATTAATGGAGGA
GAAGATTTCTAACGTTCATCCATGTACCAGTCGCTCTTCACCTTTGGTCCC
GTAGGGCCTTCCTCTCCTAGCACTTCATCAGGGAGGCTCTCCACCAGATGC
TTGAACTCAGTCTTGAGATTCCCAGGCAGGAGCTTCACCGCCTTATCGATC
TCCTTCGCGATGTCGTAGGTTATCTTCGGCCCCCATTCCCTCTCGTAGTTCA
GCCATGACGGCTCGACGACGGCGGAGCCCAAGTACTCGGCCGC
>Phoenix dactylifera nuclear genomic sequence of SNP-14 of cultivar Kupra from
Pakistan (Accession #KT983327)
AAGAAAAGCAGAGATAGCAGTAACAAGAATGGCAGACATTAATGGAGGA
GAAGATTTCTAACGTTCATCCATGTACCAGTCGCTCTTCACCTTTGGTCCC
GTAGGGCCTTCCTCTCCTAGCACTTCATCAGGGAGGCTCTCCACCAGATGC
TTGAACTCAGTCTTGAGATTCCCAGGTAGGAGCTTCGCCGCCTTATCGATC
TCCTTCGCGATGTCGTAGGTTATCTTCGGCCCCCATTCCCTCTCGTAGTTCA
GCCATGACGGCTCGACGACGGCGGAGCCCAAGTACTCGGCCGC
>Phoenix dactylifera nuclear genomic sequence of SNP-14 of cultivar Shakri from
Pakistan (Accession #KT983328)
148
AAGAAAAGCAGAGATAGCAGTAACAAGAATGGCAGACATTAATGGAGGA
GAAGATTTCTAACGTTCATCCATGTACCAGTCGCTCTTCACCTTTGGTCCC
GTAGGGCCTTCCTCTCCTAGCACTTCATCAGGGAGGCTCTCCACCAGATGC
TTGAACTCAGTCTTGAGATTCCCAGGTAGGAGCTTCGCCGCCTTATCGATC
TCCTTCGCGATGTCGTAGGTTATCTTCGGCCCCCATTCCCTCTCGTAGTTCA
GCCATGACGGCTCGACGACGGCGGAGCCCAAGTACTCGGCCGC
>Phoenix dactylifera nuclear genomic sequence of SNP-17 of cultivar Dhaki from
Pakistan (Accession # KT983329)
AATTTAGTCTGCATCACACCCTCTGCCTCACCATGTTATCTTGTGACCTTTG
GATGAGCCCAAGTCCATAGAAGAGAAGAAGGTGATCGATGATCAGATGA
CTGGGATGAGAATGAGGAGGAGAGGGGCACATCAGCAGCTGGTCACAGC
TAGCAGCAAAGAGGGAGGAAGCAAGTATCAATGTGTGGCATGTGCAAGA
AGAATGACGAAGCTAAGAATGAAGAAGTAGAAGGGATATTCATGGACCT
AGGCCAATTAAAAGGTCAACAAGTCGAA
>Phoenix dactylifera nuclear genomic sequence of SNP-17 of cultivar Aseel from
Pakistan (Accession #KT983330)
AATTTAGTCTGCATCACACCCTCTGCCTCACCATGCTATCTTGTGACCTTTG
GATGAGCCCAAGTCCATAGAAGAGAAGAAGGTGATCGATGATCAGATGA
CTGGGATGAGAATGAGGAGGAGAAGGGCACATCAGCAGCTGGTCACAGC
TAGCAGCAAAGAGGGAGGAAGCAAGTATCAATGTGTGGCATGTGCAAGA
AGAATGACGAAGCTAAGAATGAAGAAGTAGAAGGGATATTCATGGACCT
AGGCCAATTAAAAGGTCAACAAGTCGAA
>Phoenix dactylifera nuclear genomic sequence of SNP-17 of cultivar Halawi from
Pakistan (Accession # KT983331)
AATTTAGTCTGCATCACACCCTCTGCCTCACCATGCTATCTTGTGACCTTTG
GATGAGCCCAAGTCCATAGAAGAGAAGAAGGTGATCGACGATCAGATGA
CTGGGATGAGAATGAGGAGGAGAAGGGCACATCAGCAGCTGGTCACAGC
TAGCAGCAAAGAGGGAGGAAGCAAGTACCAATGTGTGGCATGTGCAAGA
AGAACGACGAAGCTAAGAATGAAGAAGTAGAAGGGATATTCATGGACCT
AGGCCAATTAAAAGGTCAACAAGTCGAA
>Phoenix dactylifera nuclear genomic sequence of SNP-17 of cultivar Qantar from
Pakistan (Accession #KT983332)
AATTTAGTCTGCATCACACCCTCTGCCTCACCATGCTATCTTGTGACCTTTG
GATGAGCCCAAGTCCATAGAAGAGAAGAAGGTGATCGATGATCAGATGA
CTGGGATGAGAATGAGGAGGAGAAGGGCACATCAGCAGCTGGTCACAGC
TAGCAGCAAAGAGGGAGGAAGCAAGTATCAATGTGTGGCATGTGCAAGA
AGAATGACGAAGCTAAGAATGAAGAAGTAGAAGGGATATTCATGGACCT
AGGCCAATTAAAAGGTCAACAAGTCGAA
149
>Phoenix dactylifera nuclear genomic sequence of SNP-17 of cultivar Haminwali
from Pakistan (Accession #KT983333)
AATTTAGTCTGCATCACACCCTCTGCCTCACCATGCTATCTTGTGACCTTTG
GATGAGCCCAAGTCCATAGAAGAGAAGAAGGTGATCGATGATCAGATGA
CTGGGATGAGAATGAGGAGGAGAAGGGCACATCAGCAGCTGGTCACAGC
TAGCAGCAAAGAGGGAGGAAGCAAGTATCAATGTGTGGCATGTGCAAGA
AGAATGACGAAGCTAAGAATGAAGAAGTAGAAGGGATATTCATGGACCT
AGGCCAATTAAAAGGTCAACAAGTCGAA
>Phoenix dactylifera nuclear genomic sequence of SNP-17 of cultivar Kupra from
Pakistan (Accession #KT983334)
AATTTAGTCTGCATCACACCCTCTGCCTCACCATGCTATCTTGTGACCTTTG
GATGAGCCCAAGTCCATAGAAGAGAAGAAGGTGATCGATGATCAGATGA
CTGGGATGAGAATGAGGAGGAGAAGGGCACATCAGCAGCTGGTCACAGC
TAGCAGCAAAGAGGGAGGAAGCAAGTATCAATGTGTGGCATGTGCAAGA
AGAATGACGAAGCTAAGAATGAAGAAGTAGAAGGGATATTCATGGACCT
AGGCCAATTAAAAGGTCAACAAGTCGAA
>Phoenix dactylifera nuclear genomic sequence of SNP-17 of cultivar Shakri from
Pakistan (Accession #KT983335)
AATTTAGTCTGCATCACACCCTCTGCCTCACCATGCTATCTTGTGACCTTTG
GATGAGCCCAAGTCCATAGAAGAGAAGAAGGTGATCGATGATCAGATGA
CTGGGATGAGAATGAGGAGGAGAAGGGCACATCAGCAGCTGGTCACAGC
TAGCAGCAAAGAGGGAGGAAGCAAGTATCAATGTGTGGCATGTGCAAGA
AGAATGACGAAGCTAAGAATGAAGAAGTAGAAGGGATATTCATGGACCT
AGGCCAATTAAAAGGTCAACAAGTCGAA
>Phoenix dactylifera nuclear genomic sequence of SNP-20 of cultivar Dhaki from
Pakistan(Accession #KT983336)
CTTCTCATAGGCTAATCTGCAAAGGGCCAAATGATCCTACCTTTATATAGG
AACACACTCAAAAATGAGTGCCAATATCCTGTTCTTCCAATGAAAATTTGC
CTTCAAAGGTTAGACACTCCATTATCGATCCTCTTTGCCTGATAACCCAAA
TCAATGTTACTAAATGTATCCAATTTCATTACTCATTTTGATTTAACGAAC
AACAATTTCAGTCTTGCACCTTACAAAGATAATAATAAAGCCAAAAAA
>Phoenix dactylifera nuclear genomic sequence of SNP-20 of cultivar Aseel from
Pakistan(Accession #KT983337)
CTTCTCATAGGCTAATCTGCAAAGGGCCAAATGATCCTACCTTTATATAGG
AACACACTCAAAAATGAGTGCCAATATCCTGTTCTTCCAATGAAAATTTGC
CTTCAAAGGTTAGACACTCCATTATCGATCCTCTTTGCCTGATAACCCAAA
TCAATGTTACTAAATGTATCCAATTTCATTACTCATTTTGATTTAACGAAC
AACAATTTCAGTCTTGCACCTTACAAAGATAATAATAAAGCCAAAAAA
150
>Phoenix dactyliferanuclear genomic sequence of SNP-20 of cultivar Halawi from
Pakistan(Accession #KT983338)
CTTCTCATAGGCTAATCTGCAAAGGGCCAAATGATCCTACCTTTATATAGG
AACACACTCAAAAATGAGTGCCAATATCCTGTTCTTCCAATGAAAATTTGC
CTTCAAAGGTTAGACACTCCATTATCGATCCTCTTTGCCTGATAACCCAAA
TCAATGTTACTAAATGTATCCAATTTCATTACTCATTTTGATTTAACGAAC
AACAATTTCAGTCTTGCACCTTACAAAGATAATAATAAAGCCAAAAAA
>Phoenix dactylifera nuclear genomic sequence of SNP-20 of cultivar Qantar from
Pakistan(Accession # KT983339)
CTTCTCATAGGCTAATCTGCAAAGGGCCAAATGATCCTACCTTTATATAGG
AACACACTCAAAAATGAGTGCCAATATCCTGTTCTTCCAATGAAAATTTGC
CTTCAAAGGTTAGACACTCCATTATCGATCCTCTTTGCCTGATAACCCAAA
TCAATGTTACTAAATGTATCCAATTTCATTACTCATTTTGATTTAACGAAC
AACAATTTCAGTCTTGCACCTTACAAAGATAATAATAAAGCCAAAAAA
>Phoenix dactylifera nuclear genomic sequence of SNP-20 of cultivar Haminwali
from Pakistan (Accession #KT983340)
CTTCTCATAGGCTAATCTGCAAAGGGCCAAATGATCCTACCTTTATATAGG
AACACACTCAAAAATGAGTGCCAATATCCTGTTCTTCCAATGAAAATTTGC
CTTCAAAGGTTAGACACTCCATTATCGATCCTCTTTGCCTGATAACCCAAA
TCAATGTTACTAAATGTATCCAATTTCATTACTCATTTTGATTTAACGAAC
AACAATTTCAGTCATGCACCTTACAAAGATAATAATAAAGCCAAAAAA
>Phoenix dactylifera nuclear genomic sequence of SNP-20 of cultivar Kupra from
Pakistan(Accession #KT983341)
CTTCTCATAGGCTAATCTGCAAAGGGCCAAATGATCCTACCTTTATATAGG
AACACACTCAAAAATGAGTGCCAATATCCTGTTCTTCCAATGAAAATTTGC
CTTCAAAGGTTAGACACTCCATTATCGATCCTCTTTGCCTGATAACCCAAA
TCAATGTTACTAAATGTATCCAATTTCATTACTCATTTTGATTTAACGAAC
AACAATTTCAGTCTTGCACCTTACAAAGATAATAATAAAGCCAAAAAA
>Phoenix dactylifera nuclear genomic sequence of SNP-20 of cultivar Shakri from
Pakistan (Accession #KT983342)
CTTCTCATAGGCTAATCTGCAAAGGGCCAAATGATCCTACCTTTATATAGG
AACACACTCAAAAATGAGTGCCAATATCCTGTTCTTCCAATGAAAATTTGC
CTTCAAAGGTTAGACACTCCATTATCGATCCTCTTTGCCTGATAACCCAAA
TCAATGTTACTAAATGTATCCAATTTCATTACTCATTTTGATTTAACGAAC
AACAATTTCAGTCTTGCACCTTACAAAGATAATAATAAAGCCAAAAAA
>Phoenix dactylifera nuclear genomic sequence of SNP-21 of cultivar Dhaki from
Pakistan(Accession #KT983343)
151
CAACAACTCCCCGTTTCACTTCCTTTCCGATTGATAGAAAGATACGAAGAA
ACCATGGCTGCTAAGTTCCGACCTGTTCTCGCTGCTCTCTGCCTCATTTCA
GCCCTCCTCGGGATCGCTGATACCACCCCAACTCCAACCTTTCACGTTCGC
GGTCGAGTTTACTGCGACACCTGCCGTGCTGGCTTCGTTCATGAATACACC
GAATACCTCGAAGGTACATCAATACTTGCATCTTGCCTAAGCACAGCCAG
TTTAGCTATCAGTTTTGGTTGTTGAGGTC
>Phoenix dactylifera nuclear genomic sequence of SNP-21 of cultivar Aseel from
Pakistan(Accession #KT983344)
CAACAACCCCCCGTTTCACTTCCTTTCCGATTGATAGAAAGATACGAAGA
AACCATGGCTGCTAAGTTCCGACCTGTTCTCGCTGCTCTCTGCCTCATTTC
AGCCCTCCTCGGGATCGCTGATACCACCCCAACTCCAACCTTTCACGTTCG
CGGTCGAGTTTACTGCGACACCTGCCGTGCTGGCTTCGTTCATGAATACAC
CGAATACCTCGAAGGTACATCAATACTTGCATCTTGCCTAAGCACAGCCA
GTTTAGCTATCAGTTTTGGTTGCTGAGGTC
>Phoenix dactylifera nuclear genomic sequence of SNP-21 of cultivar Halawi from
Pakistan (Accession #KT983345)
CAACAACCCCCCGTTTCACTTCCTTTCCGATTGATAGAAAGATACGAAGA
AACCATGGCTGCTAAGTTCCGACCTGTTCTCGCTGCTCTCTGCCTCATTTC
AGCCCTCCTCGGGATCGCTGATACCACCCCAACTCCAACCTTTCACGTTCG
CGGTCGAGTTTACTGCGACACCTGCCGTGCTGGCTTCGTTCATGAATACAC
CGAATACCTCGAAGGTACATCAATACTTGCATCTTGCCTAAGCACAGCCA
GTTTAGCTATCAGTTTTGGTTGCTGAGGTC
>Phoenix dactylifera nuclear genomic sequence of SNP-21 of cultivar Qantar from
Pakistan (Accession #KT983346)
CAACAACTCCCCGTTTCACTTCCTTTCCGATTGATAGAAAGATACGAAGAA
ACCATGGCTGCTAAGTTCCGACCTGTTCTCGCTGCTCTCTGCCTCATTTCA
GCCCTCCTCGGGATCGCTGATACCACCCCAACTCCAACCTTTCACGTTCGC
GGTCGAGTTTACTGCGACACCTGCCGTGCTGGCTTCGTTCATGAATACACC
GAATACCTCGAAGGTACATCAATACTTGCATCTTGCCTAAGCACAGCCAG
TTTAGCTATCAGTTTTGGTTGTTGAGGTC
>Phoenix dactylifera nuclear genomic sequence of SNP-21of cultivar Haminwali
from Pakistan (Accession # KT983347)
CAACAACCCCCCGTTTCACTTCCTTTCCGATTGATAGAAAGATACGAAGA
AACCATGGCTGCTAAGTTCCGACCTGTTCTCGCTGCTCTCTGCCTCATTTC
AGCCCTCCTCGGGATCGCTGATACCACCCCAACTCCAACCTTTCACGTTCG
CGGTCGAGTTTACTGCGACACCTGCCGTGCTGGCTTCGTTCATGAATACAC
CGAATACCTCGAAGGTACATCAATACTTGCATCTTGCCTAAGCACAGCCA
GTTTAGCTATCAGTTTTGGTTGTTGAGGTC
152
>Phoenix dactylifera nuclear genomic sequence of SNP-21of cultivar Kuprafrom
Pakistan (Accession #KT983348)
CAACAACCCCCCGTTTCACTTCCTTTCCGATTGATAGAAAGATACGAAGA
AACCATGGCTGCTAAGTTCCGACCTGTTCTCGCTGCTCTCTGCCTCATTTC
GGCCCTCCTCGGGATCGCTGATACCACCCCAACTCCAACCTTTCACGTTCG
CGGTCGAGTTTACTGCGACACCTGCCGTGCTGGCTTCGTTCATGAATACAC
CGAATACCTCGAAGGTACATCAATACTTGCATCTTGCCTAAGCACAGCCA
GTTTAGCTATCAGTTTTGGTTGTTGAGGTC
>Phoenixdactylifera nuclear genomic sequence of SNP-21 of cultivar Shakri from
Pakistan (Accession #KT983349)
CAACAACCCCCCGTTTCACTTCCTTTCCGATTGATAGAAAGATACGAAGA
AACCATGGCTGCTAAGTTCCGACCTGTTCTCGCTGCTCTCTGCCTCATTTC
AGCCCTCCTCGGGATCGCTGATACCACCCCAACTCCAACCTTTCACGTTCG
CGGTCGAGTTTACTGCGACACCTGCCGTGCTGGCTTCGTTCATGAATACAC
CGAATACCTCGAAGGTACATCAATACTTGCATCTTGCCTAAGCACAGCCA
GTTTAGCTATCAGTTTTGGTTGTTGAGGTC
>Phoenix dactylifera nuclear genomic sequence of SNP-23 of cultivar Dhaki from
Pakistan (Accession #KT983350)
TCTCCTTGATCACGCTAAGCTTGAACATATTGGTCCAGCCATCTACGTCAG
ATGAGTGCCTCGATCTCGTCCTTAAGCTAGAGGCATTCCTCATTATCATGA
CCGTAGTCTCGATGGAAGCAGTAGTACTTCTCTTTTTTCGTTTGGCTGTCG
ATGCCTTTATTCGACGTAGAGGGCACTAGTAGCCTCGGCTTTCGATCTCTA
TGAGTATCTGTGTTCTTGGGGCTATGAGAGTGGTGTACCTTTCAAACTTCT
GAGGAGAGCTCTTGGGCCATC
>Phoenix dactylifera nuclear genomic sequence of SNP-23 of cultivar Aseel from
Pakistan (Accession #KT983351)
TCTCCTTGATCACGCTAAGCTTGAACATATTGGTCCAGCCATCTACGTCAG
ATGAGTGCCTCGATCTCGTCCTTAAGCTAGAGGCATTCCTCATTATCATGA
CCATAGTCTCGATGGAAGCAGTAGTACTTCTCTTTTTTCGTTTGGCTGTCG
ATGCCTTTATTCGACGTAGAGGGCACTAGTAGCCTCGGCTTTCGATCTCTA
TGAGTATCTGTGTTCTTGGGGCTATGAGAGTGGTGTACCTTTCAAACTTCT
GAGGAGAGCTCTTGGGCCATC
>Phoenix dactylifera nuclear genomic sequence of SNP-23 of cultivar Halawi from
Pakistan (Accession #KT983352)
TCTCCTTGATCACGCTAAGCTTGAACATATTGGTCCAGCCATCTACGTCAG
ATGAGTGCCTCGATCTCGTCCTTAAGCTAGAGGCATTCCTCATTATCATGA
CCATAGTCTCGATGGAAGCAGTAGTACTTCTCTTTTTTCGTTTGGCTGTCG
ATGCCTTTATTCGACGTAGAGGGCACTAGTAGCCTCGGCTTTCGATCTCTA
153
TGAGTATCTGTGTTCTTGGGGCTATGAGAGTGGTGTACCTTTCAAACTTCT
GAGGAGAGCTCTTGGGCCATC
>Phoenix dactylifera nuclear genomic sequence of SNP-23 of cultivar Qantar from
Pakistan (Accession #KT983353)
TCTCCTTGATCACGCTAAGCTTGAACATATTGGTCCAGCCATCTACGTCAG
ATGAGTGCCTCGATCTCGTCCTTAAGCTAGAGGCATTCCTCATTATCATGA
CCGTAGTCTCGATGGAAGCAGTAGTACTTCTCTTTTTTCGTTTGGCTGTCG
ATGCCTTTATTCGACGTAGAGGGCACTAGTAGCCTCGGCTTTCGATCTCTA
TGAGTATCTGTGTTCTTGGGGCTATGAGAGTGGTGTACCTTTCAAACTTCT
GAGGAGAGCTCTTGGGCCATC
>Phoenix dactylifera nuclear genomic sequence of SNP-23 of cultivar Haminwali
from Pakistan (Accession #KT983354)
TCTCCTTGATCACGCTAAGCTTGAACATATTGGTCCAGCCATCTACGTCAG
ATGAGTGCCTCGATCTCGTCCTTAAGCTAGAGGCATTCCTCATTATCATGA
CCATAGTCTCGATGGAAGCAGTAGTACTTCTCTTTTTTCGTTTGGCTGTCG
ATGCCTTTATTCGACGTAGAGGGCACTAGTAGCCTCGGCTTTCGATCTCTA
TGAGTATCTGTGTTCTTGGGGCTATGAGAGTGGTGTACCTTTCAAACTTCT
GAGGAGAGCTCTTGGGCCATC
>Phoenix dactylifera nuclear genomic sequence of SNP-23 of cultivar Kupra from
Pakistan (Accession #KT983355)
TCTCCTTGATCACGCTAAGCTTGAACATATTGGTCCAGCCATCTACGTCAG
ATGAGTGCCTCGATCTCGTCCTTAAGCTAGAGGCATTCCTCATTATCATGA
CCRTAGTCTCGATGGAAGCAGTAGTACTTCTCTTTTTTCGTTTGGCTGTCG
ATGCCTTTATTCGACGTAGAGGGCACTAGTAGCCTCGGCTTTCGATCTCTA
TGAGTATCTGTGTTCTTGGGGCTATGAGAGTGGTGTACCTTTCAAACTTCT
GAGGAGAGCTCTTGGGCCATC
>Phoenix dactylifera nuclear genomic sequence of SNP-23 of cultivar Shakri from
Pakistan (Accession #KT983356)
TCTCCTTGATCACGCTAAGCTTGAACATATTGGTCCAGCCATCTACGTCAG
ATGAGTGCCTCGATCTCGTCCTTAAGCTAGGGGCATTCCTCATTATCATGA
CCGTAGTCTCGATGGAAGCAGTAGTACTTCTCTTTTTTCGTTTGGCTGTCG
ATGCCTTTATTCGACGTAGAGGGCACTAGTAGCCTCGGCTTTCGATCTCTA
TGAGTATCTGTGTTCTTGGGGCTATGAGAGTGGTGTACCTTTCAAACTTCT
GAGGAGAGCTCTTGGGCCATC
>Phoenix dactylifera nuclear genomic sequence of SNP-32 of cultivar Dhaki from
Pakistan (Accession #KT983357)
154
CCAGTGGATGAAAGCTTGGGGATAGTCATGGGCCTCGTGGCACAAGGGAA
GAGTGGAAAATAAATCATACATCCTTACATGCAGCAATATTCCTAAAATA
GCTTAATCTAAAGGTGACAGAAATAGTGAAATAGCATACCTCATTCAATT
TACATTGCATATAACTTGGTTGTGTCCTGTTCTTGTATACTTATCCATTTCA
TTCTTTGTATGACATTGTACATGGGATGTACATGATAGCCCATCATTGTTT
T
>Phoenix dactylifera nuclear genomic sequence of SNP-32 of cultivar Aseel from
Pakistan (Accession #KT983358)
CCAGTGGATGAAAGCTTAGGAATAGTCATGGGCCTCGTGGCGCAAGGGAA
GGGTGGAAAATAAATCATACATCCTTACCTGCAGCAATATTCCTAAAATA
GCTTAATCTAAAGGTGATAGAAATAGTGAAATAGCATACCTCATTCAATT
TACATTGCATATAACTTGGTTGTGTCCTGTTCTTGTATACTTATCCATTTCA
TTCTTTGTATGAGATTGTACATGGGATGTACATAATAGCCCATCATTGTTT
T
>Phoenix dactylifera nuclear genomic sequence of SNP-32 of cultivar Halawi from
Pakistan (Accession #KT983359)
CCAGTGGATGAAAGCTTAGGAATAGTCATGGGCCTCGTGGCGCAAGGGAA
GGGTGGAAAATAAATCATACATCCTTACCTGCAGCAATATTCCTAAAATA
GCTTAATCTAAAGGTGATAGAAATAGTGAAATAGCATACCTCATTCAATT
TACATTGCATATAACTTGGTTGTGTCCTGTTCTTGTATACTTATCCATTTCA
TTCTTTGTATGAGATTGTACATGGGATGTACATAATAGCCCATCATTGTTT
T
>Phoenix dactylifera nuclear genomic sequence of SNP-32 of cultivar Qantar from
Pakistan (Accession #KT983360)
CCAGTGGATGAAAGCTTGGGGATAGTCATGGGCCTCGTGGCACAAGGGAA
GGGTGGAAAATAAATCATACATCCTTACATGCAGCAATATTCCTAAAATA
GCTTAATCTAAAGGTGACAGAAATAGTGAAATAGCATACCTCATTCAATT
TACATTGCATATAACTTGGTTGTGTCCTGTTCTTGTATACTTATCCATTTCA
TTCTTTGTATGAGATTGTACATGGGATGTACATGATAGCCCATCATTGTTT
T
>Phoenix dactylifera nuclear genomic sequence of SNP-32 of cultivar Haminwali
from Pakistan (Accession #KT983361)
CCAGTGGATGAAAGCTTGGGGATAGTCATGGGCCTCGTGGCGCAAGGGAA
GGGTGGAAAATAAATCATACATCCTTACATGCAGCAATATTCCTAAAATA
GCTTAATCTAAAGGTGACAGAAATAGTGAAATAGCATACCTCATTCAATT
TACATTGCATATAACTTGGTTGTGTCCTGTTCTTGTATACTTATCCATTTCA
TTCTTTGTATGAGATTGTACATGGGATGTACATGATAGCCCATCATTGTTT
T
155
>Phoenix dactylifera nuclear genomic sequence of SNP-32 of cultivar Kupra from
Pakistan (Accession #KT983362)
CCAGTGGATGAAAGCTTAGGAATAGTCATGGGCCTCGTGGCGCAAGGGAA
GGGTGGAAAATAAATCATACATCCTTACCTGCAGCAATATTCCTAAAATA
GCTTAATCTAAAGGTGATAGAAATAGTGAAATAGCATACCTCATTCAATT
TACATTGCATATAACTTGGTTGTGTCCTGTTCTTGTATACTTATCCATTTCA
TTCTTTGTATGAGATTGTACATGGGATGTACATAATAGCCCATCATTGTTT
T
>Phoenix dactylifera nuclear genomic sequence of SNP-32 of cultivar Shakri from
Pakistan (Accession #KT983363)
CCAGTGGATGAAAGCTTGGGGATAGTCATGGGCCTCGTGGCGCAAGGGAA
GGGTGGAAAATAAATCATACATCCTTACATGCAGCAATATTCCTAAAATA
GCTTAATCTAAAGGTGACAGAAATAGTGAAATAGCATACCTCATTCAATT
TACATTGCATATAACTTGGTTGTGTCCTGTTCTTGTATACTTATCCATTTCA
TTCTTTGTATGAGATTGTACATGGGATGTACATGATAGCCCATCATTGTTT
T
Appendix XIV: Accession numbers of sequences of different genes/fragments of date
palm submitted to Genbank
Genes/ Genome fragment Dhaki Aseel Halawi Qantar Haminwali Kupra Shakri
matK KT803890 KT803889 KT803891 KT803892 KT803893 KT803894 KT803895
GGR KT983259 KT983260 KT983261 KT983262 KT983263 KT983264 KT983265
RBCL KT803883 KT803882 KT803884 KT803885 KT803886 KT803887 KT803888
atpB KT781683 KT781682 KT781684 KT781685 KT781686 KT781687 KT781688
16s rRNA KT983365 KT983364 KT983366 KT983367 KT983368 KT983369 KT983370
SNP03 KT983266 KT983267 KT983268 KT983269 KT983270 KT983271 KT983272
SNP05 KT983273 KT983274 KT983275 KT983276 KT983277 KT983278 KT983279
SNP06 KT983280 KT983281 KT983282 KT983283 KT983284 KT983285 KT983286
SNP07 KT983287 KT983288 KT983289 KT983290 KT983291 KT983292 KT983293
SNP09 KT983294 KT983295 KT983296 KT983297 KT983298 KT983299 KT983300
SNP10 KT983301 KT983302 KT983303 KT983304 KT983305 KT983306 KT983307
SNP11 KT983308 KT983309 KT983310 KT983311 KT983312 KT983313 KT983314
SNP12 KT983315 KT983316 KT983317 KT983318 KT983319 KT983320 KT983321
SNP14 KT983322 KT983323 KT983324 KT983325 KT983326 KT983327 KT983328
SNP17 KT983329 KT983330 KT983331 KT983332 KT983333 KT983334 KT983335
SNP20 KT983336 KT983337 KT983338 KT983339 KT983340 KT983341 KT983342
SNP21 KT983343 KT983344 KT983345 KT983346 KT983347 KT983348 KT983349
SNP23 KT983350 KT983351 KT983352 KT983353 KT983354 KT983355 KT983356
SNP32 KT983357 KT983358 KT983359 KT983360 KT983361 KT983362 KT983363
