GENETIC CHARACTERIZATION OF …prr.hec.gov.pk/jspui/bitstream/123456789/7097/1/Muhammad...Dr. Muhammad Akbar, Dr. Khurrum Shahzad, Imran Hussain Bhatti, Faizan Riaz Cheema Shahid Yar

GENETIC CHARACTERIZATION OF MITOCHONDRIAL

DNA IN MAKRANI AND KALASHI POPULATION FROM

PAKISTAN

BY

MUHAMMAD HASSAN SIDDIQI

DEPARTMENT OF ZOOLOGY

UNIVERSITY OF THE PUNJAB QUAID-I-AZAM CAMPUS

LAHORE, PAKISTAN (2014)

GENETIC CHARACTERIZATION OF

MITOCHONDRIAL DNA IN MAKRANI AND KALASHI

POPULATION FROM

PAKISTAN

A THESIS SUBMITTED TO

UNIVERSITY OF THE PUNJAB

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN

ZOOLOGY

BY

MUHAMMAD HASSAN SIDDIQI

SUPERVISOR

PROF. DR. TANVEER AKHTAR

DEPARTMENT OF ZOOLOGY

UNIVERSITY OF THE PUNJAB QUAID-I-AZAM CAMPUS

LAHORE, PAKISTAN

(2014)

IN THE NAME OF ALLAH, THE MOST BENEFICENT,

THE MOST MERCIFUL

AL QURAN

Translation: O ye, who believe, stand out firmly for

justice, as witnesses to Allah, even as against yourselves, or

your parents, or your kin, or whether it be (against) rich or

poor: for Allah can best protect both. Follow not the lusts

(of your hearts), lest ye swerve and if ye distort (justice) or

decline to do justice, verily Allah is well acquainted with

all that ye do (Quran 4:135).

CERTIFICATE

This is to certify that the research work described in this thesis is the original work of the

author Mr. Muhammad Hassan Siddiqi and has been carried out under my direct

supervision. I have personally gone through all the data/results/materials reported in the

manuscript and certify their correctness/authenticity. I further certify that the material

included in this thesis have not been used in part or full in a manuscript already submitted

or in the process of submission in partial/complete fulfillment of the award of any other

degree from any other institution. I also certify that the thesis has been prepared under my

supervision according to the prescribed format and I endorse its evaluation for the award

of Ph.D. degree through the official procedures of the University.

Prof. Dr. Tanveer Akhtar

Supervisor

Department of Zoology,

University of the Punjab, Lahore

DEDICATION

This work is dedicated to my Father, Mother (Late) and brother Muhammad

Aslam (Late) who have been the source of inspiration since my childhood

and who were always there to help me, gave me courage and strength to

accomplish all the goals of my life including this prestigious research

achievement. You are a part of every page, every thought and all the work.

CONTENTS

Title Page No.

SUMMARY ..................................................................................................................i

1. INTRODUCTION...................................................................................................1

2. LITERATURE REVIEW ......................................................................................6

2.1 Hypervariable Sites .....................................................................................10

2.2 Haplogroups ................................................................................................10

2.2.1 African Haplogroups ..........................................................................11

2.2.2 West Eurasian Haplogroups ...............................................................11

2.2.3 Southeast Asian Haplogroup..............................................................13

2.3 The Role of the mtDNA in Ancestry Studies ...............................................14

3. MATERIALS AND METHODS ..........................................................................16

3.1 Sample Collection Areas.............................................................................16

3.2 Makrani Population .....................................................................................17

3.2.1. Sample Collection .............................................................................17

3.3. Kalash Population ......................................................................................20

3.3.1. Sample Collectio ...............................................................................20

3.4 DNA Extraction and Quantification ...........................................................25

3.5 PCR Amplification......................................................................................25

3.5.1. Preparation of Agarose Gel ...............................................................27

3.6. Sequencing .................................................................................................27

3.7. Statistical Analysis .....................................................................................28

4. RESULTS ...............................................................................................................29

4.1. Sampled populations ..................................................................................29

4.2. Genomic DNA quality and PCR amplification of mtDNA control region 29

4.3. Sequencing the control region of mitochondrial DNA ..............................29

4.4. Reconstruction and alignment with rCRS..................................................39

4.5. Identification of haplotypes and assignment of haplogroups ....................45

4.6. Frequency of mtDNA haplogroups ............................................................55

4.7 The Haplogroups Diversity within Sub-ethnic group of Kalash Population 56

4.8 Frequency of mtDNA Haplogroups ............................................................58

4.9. The Construction of Median Joining (MJ) Networks ................................59

4.10 The Occurrence and Distribution of Nucleotide Variations in mtDNA

Control Region ..................................................................................................61

4.11. Heteroplasmy ...........................................................................................65

4.11.1. Point heteroplasmy..........................................................................65

4.11.2. Length heteroplasmy .......................................................................68

4.12. Comparison of haplogroup frequencies and continental origins in Sub-

populations of Pakistan .....................................................................................70

4.13. Comparative statistical analyses of different Pakistani subpopulations ..74

5. DISCUSSION ..........................................................................................................75

REFRENCES ..............................................................................................................86

APPENDIX

i

SUMMARY

Mitochondrial DNA (mtDNA) analysis has gained importance in forensic

investigations especially for cases where the genomic DNA found is highly degraded or

very less in quantity. Due to the high copy number of mtDNA in a cell increases the

possibility of some copies of mtDNA to be intact in such samples. The variations in

mitochondrial genome have been proven to be the most powerful genetic marker for

investigating gene pools and tracing maternal genetic relatedness of the suspect. The

control region of mtDNA including hypervariable segments (HVSI, HVSII and HVSIII)

has been considered the most important chunk of polymorphic DNA in the mitochondrial

genome.

This study reports the haplotype data of mtDNA control region (spanning

positions 16,024–16,569 and 1–576) including hypervariable segments (HVSI, HVSII

and HVSIII) for two-genetically distinct and isolated populations of Pakistan i.e. Makrani

& Kalashi. The genetic and forensic parameters were studied by sequencing the entire

mitochondrial DNA control region of 100 unrelated Makrani individuals (males, n = 96;

females, n = 4) and 111 Kalashi individuals (males, n = 63; females, n = 48).

A total of 149 polymorphic positions were detected in Makrani population. Based

on the entire profile of mutations along the mtDNA control region comparative to revised

Cambridge Reference Sequence (rCRS), seventy different haplotypes were observed in

the Makrani with 54 unique and 16 haplotypes shared by more than one individual in the

population. Point heteroplasmy was observed at 5 different positions in Makrani

accounting for 13% of the individuals. Only one individual presented more than one point

heteroplasmy in the Makranis. Median Joining Network analysis showed the substantial

divergence among the haplotypes in Makrani population. In Kalashi population, a total of

47 polymorphic positions were detected. After comparing with rCRS, 14 different

haplotypes were observed in the Kalashi population with 5 unique and 9 haplotypes

shared by more than one individual. Point heteroplasmy was observed at 6 different

positions accounting for 58.56% of the individuals. In this case, three individuals

presented more than one point heteroplasmy. Limited divergence among the haplotypes

has been observed in Kalashi population while plotting Median Joining Network.

ii

Based on identified haplotypes, the Makranis showed admixed mtDNA pool

consisting of African haplogroups (28%), West Eurasian haplogroups (26%), South

Asian haplogroups (24%), and East Asian haplogroups (2%), however, the origin of the

remaining individuals (20%) could not be confidently assigned in this population.

Moreover, two haplotypes observed in the Makranis, both carrying a characteristic

combination of two mutations in HVSII (154C and194T) could not be confidently

assigned to a known (sub) haplogroups, although the presence of both 16223T and 489C

indicate membership within macro-haplogroup M; this lineage was therefore tentatively

assigned to haplogroup named ‘‘M-154-194’’. Future studies performing complete

mitogenome sequencing, may elucidate the precise phylogenetic position of this lineage.

The high frequency of African mtDNA haplogroups in Makranis shows their origin with

major genetic contribution from Mozambique Bantu from southeastern Africa and Fulani

people of West-Central Africa as a result of African slave trade. In Kalashi population,

the dominating haplogroups were West Eurasians (98.2%) while a small proportion

(0.9%) of South Asians were also observed. However, one of the Kalashi sample could

not be assertively allocated with any of the known sub-haplogroups. The greater

frequency of West Eurasian haplogroups in Kalash might be the consequence of the Arab

and Muslim conquests, the rise of the British Indian Empire and invasion by the armies of

Alexander the Great.

The high genetic diversity (0.9688), consequently, a high power of discrimination

(0.9592) and low random match probability (0.048) reflects intense gene flow in the

Makrani population. In contrast, extremely low genetic diversity (0.8393), low power of

discrimination (0.832) and higher probability match between two random individuals

(0.168) in Kalashi population were observed. The low genetic diversity in Kalash may be

explained by genetic drift in the population due to either low population size or

endogamy. These data would be a valuable contribution to build a database of entire

mtDNA control-region sequences, which may significantly contribute for both the

populations to estimate the rarity of mtDNA profile under investigation in Pakistan.

ACKNOWLEDGEMENTS

All acclamations and appreciations are for ALIMIGHTY ALLAH, the

Omnipotent, the Omnipresent, the Compassionate, the Beneficent and the source of all

knowledge and wisdom, who bestowed upon me the intellectual ability, courage and

strength to complete this humble contribution towards knowledge. I am proud of being a

follower of the Holy Prophet Hazrat Muhammad (PBUH), the most perfect and exalted

among and of ever born on the surface of earth, which declared it to be an obligatory duty

of every man and woman to seek and acquire knowledge.

My wholehearted thanks goes to the worthy Chairman Department of Zoology,

Professor, Dr. Muhammad Akhtar for providing the established and an inspirational

environment and somewhat of a second home to me and other researchers. He has been

helpful in every facet of my graduate studies.

Furthermore, I feel highly privileged to take this opportunity to wish my profound

gratitude with a deep sense of obligation to my doctoral research supervisor,

Dr. Tanveer Akhtar, professor, Department of Zoology, for her personal interest,

inspiring guidance, helping attitude, and above all for providing necessary laboratory

facilities during the whole span of this research work.

I would like to thank all people who have helped and inspired me during my

doctoral study. My cordial thanks goes to Dr. Fazle Majid Khan, Dr. Allah Rakha,

Dr. Muhammad Akram Tariq and Dr. Muhammad Farooq Sabar, for their guidance

and cooperation whenever needed all the time.

I would like to thank, Sher Khan Kalash, Faizi Khan Kalash, Syed Said

Hussain Shah, Subhan Shah, Abid Naqvi, Dr. Jamil Ahmad, Akram Ali, Ghazanfar

Abbas, Sikandar Hayat, Dr. Muhammad Irfan, Syed Yasir Abbas Bokhari, Afia M

Akram, Sana Shahbaz, Naeem Haider, Ali Akhtar, Usman Akhtar, Farooq Akhtar,

Dr. Muhammad Akbar, Dr. Khurrum Shahzad, Imran Hussain Bhatti, Faizan Riaz

Cheema Shahid Yar Khan Khadija Fazal Karim who have helped and inspired me.

I am grateful to my senior friends Dr. Umar Farooq, Dr. Abdul Majid Khan,

Javed Akram, and Dr. Zafar Iqbal for helping me get through the difficult times and

for all the emotional support, camaraderie, entertainment, and caring they provided.

I wish to extend my thanks to members of Paleontology Lab., Physiology Lab.,

Cell and Molecular Biology Lab., Wild life and Environmental Health Lab.,

Biochemistry Lab., Microbiology Lab., Developmental Biology Lab., Entomology Lab.,

Fisheries Lab., all the scientific staff, especially Mr. Abbas Anjum, para scientific staff

especially Ashfaq Ahmad and Administrative staff of Department of Zoology, those had

been directly and indirectly instrumental in my research work.

My utmost gratitude goes to Mannis van Oven and Oscar Lao, Department of

Forensic Molecular Biology Erasmus MC, University Medical Center Rotterdam, The

Netherlands, for their helpful discussion.

No words can express and no deeds can return the love, affection, amiable

attitude, sacrifices, advices, unceasing prayers, support, and inspiration that my Father,

my brothers and my sister imparted in me during my whole academic career.

Muhammad Hassan Siddiqi

LIST OF TABLES

Table

No. Title

Page

No.

3.1 The detailed data of consent forms from Makrani population 17

3.2 The summarized information about sampling of Makrani

population from different cities of three provinces of Pakistan 20

3.3 The detailed data of consent form from Kalashi population 21

3.4 The summarized information about sampling from three different

valleys of Kalash population 25

3.5 List of oligonucleotides, along with melting temperatures (Tm),

concentrations and sequences used for amplification and

sequencing of the mtDNA control regions

26

4.1a The estimated haplotypes and haplogroups in Makrani population 46

4.1b The estimated haplotypes and haplogroups in Kalashi population 51

4.2a Differences observed in haplogroup estimation of Makrani

population either manually or by HaploGrep 55

4.2b Differences observed in haplogroup estimation of Kalashi

population either manually or by HaploGrep 56

4.3 The haplogroups diversity in each maternal sub-ethnic group of

Kalash 57

4.4a The occurrence and distribution of nucleotide variations in the

entire mtDNA control region of Makrani population 63

4.4b The occurrence and distribution of nucleotide variations in the

entire mtDNA control region of Kalashi population 64

4.5 Point heteroplasmy in the Makrani and the Kalashi populations 65

4.6 The length heteroplasmy distribution along the mtDNA control

region of the Makrani and Kalashi populations 69

4.7 The comparison of mtDNA haplogroups’ frequencies and their

continental origins among subpopulations of Pakistan 71

4.8 The comparison of diversity parameters estimated from the entire

mtDNA control region among subpopulations of Pakistan 74

LIST OF FIGURES

Fig. No. Title Page No.

2.1 Human mitochondrial DNA map showing CR (control region). 7

2.2 mtDNA control region schematic diagram 8

3.1 Map of Pakistan showing its administrative regions and

neighboring countries 16

4.1 Agarose gel electrophoretic analysis of genomic DNA extracted

from blood samples 30

4.2 Agarose gel electrophoretic analysis of the mtDNA control

region PCR products 30

4.3 (a) Chromatogram of Makrani individual (MKH080) for entire

mtDNA control region sequenced by forward primer (F15975) 32

4.3 (b) Chromatogram of Makrani individual (MKH080) for mtDNA

control region sequenced by reverse primer (R635) 34

4.4 (a) Chromatogram of Kalashi individual (KLH015) for the entire

mtDNA control region sequenced by forward primer (F15975) 36

4.4 (b) Chromatogram of Kalashi individual (KLH015) for the entire

mtDNA control region sequenced by reverse primer (R635) 38

4.5 (a) The haplotype of Makrani individual (MKH080) for entire

mtDNA control region 42

4.5 (b) The haplotype of Kalashi individual (KLH016) for entire

mtDNA control region 45

4.6 (a) Graphical illustration of frequencies of mtDNA based

haplogroups in Makrani population 58

4.6 (b) Graphical illustration of frequencies of mtDNA-based

haplogroups in Kalashi population 59

4.7 (a) Median-joining haplotype network of the Makrani population

(70 haplotypes). 60

4.7 (b) Median-joining haplotype network of the Kalashi population

(14 haplotypes). 61

4.8 Point heteroplasmy observed at different positions of mtDNA

control region in the Makrani population 66

4.9 Point heteroplasmies observed at different positions of mtDNA

control region in the Kalashi population 67

4.10 Chromatograms showing the homopolymeric patterns of length

heteroplasmy in the Makrani Population 69

ABBREVIATIONS

EDTA Ethylene diamine tetra acetic acid

KPK Khyber Pakhtunkhawa

nDNA Nuclear DNA

mtDNA Mitochondrial DNA

L-strand Light strand

H-strand Heavy Strand

D-loop Displacement loop

STR Short tandem repeat

SNP Single nucleotide polymorphism

RFLPs Restriction Fragment LengthPolymorphisms

PCR Polymerase chain reaction

HVR Hyper Variable region

pM Picomol

µl Micro liter

MgCl2 Magnesium Chloride

mM Milli mole

V Voltage

UV Ultraviolet

Rpm Revolution per minute

HG haplogroup

SA South Asian

WEA West Eurasian

SEA South East Asian

EEA East Eurasian

WA West Asian

SWA South West Asia

EA East Asia

AF Africa

KYA Thousand Years Ago

MCL Maximum Composite Likelihood

rCRS revised Cambridge Reference Sequence

HVSI Hypervariable Segment I

HVSII Hypervariable Segment II

HVSIII Hypervariable Segment III

1

1-INTRODUCTION

The genetic information is assembled within cells in the form of DNA sequences,

either 23 pairs of chromosomes in human cell nucleus or DNA molecules in

mitochondria. With the increase in the knowledge about the genetic differences in

humans, the analysis of silent biological witness, the DNA molecule from crime scene,

has become very important (Bandelt et al., 2012). There are two different kinds of DNA

makers being utilized for DNA studies such as autosomal markers and uniparental (Y-

chromosome, mitochondrial DNA) markers. The variations found within the autosomal

chromosomes are called as “autosomal DNA markers”, which provide high

discrimination power, and are considered powerful tool for human identifications. The Y

chromosomal markers as being uniparental markers have been used as valuable tool in

certain criminal investigations due to male specificity as males are usually culprits in

most sexual assault cases. Recently, the analyses of second type of uniparental markers

(mitochondrial DNA) in forensic investigations have gained remarkable importance

especially in cases where the DNA found is highly degraded (such as ancient samples) or

very less in quantity (such as stains, cigarette butts and fingernails etc.). The advantage of

using mtDNA is due to presence of 1000–2000 mitochondria per human cells (e.g. liver

cells) as well as five to ten copies of mtDNA per mitochondrion that increases the

possibility of obtaining some copies of mtDNA for analysis from such samples. Along

with copy number advantage of mtDNA, the clear-cut pattern of historical events can also

be judged by mtDNA studies (Legros et al., 2004; Chong et al., 2005; Nilsson et al.,

2008; Kavlick et al., 2011; Adachi et al., 2014).

Mitochondria are unique among cell organelles as they contain their own genome

and are quite distinctive from nuclear DNA. The human mtDNA is a circular double

stranded DNA molecule composed of ~16569 nucleotides (Taylor and Turnbull, 2005;

Lan, et al., 2008). The Cambridge Reference Sequence (CRS) of mtDNA published in

1981, has established the number of base pairs and functional genes in mtDNA

(Anderson et al., 1981). By re-sequencing the mtDNA, the CRS was revised and named

as revised Cambridge Reference Sequence (rCRS) that is used as standard for

comparisons (Andrews et al., 1999). The mtDNA consists of 37 genes, 28 genes are the

part of the H-strand while 9 are the part of the L-strand. Out of these, 13 genes encode

2

different types of proteins, which play different roles in respiration. The remaining 24

genes encode mature RNA products, out of these, 22 encode for mitochondrial tRNA

molecules and two encode for mitochondrial rRNA molecules (16 s rRNA and a 12 s

rRNA) (Andrews et al., 1999). Deletions or point mutations in mtDNA have been shown

to be involved in human genetic defects (Holt et al., 1988; Shoffner et al., 1989) and are

responsible for genetic differences between populations and advance the knowledge

about their phylogenetic relationships (Guha et al., 2013)

mtDNA has provided a wealth of interesting molecular enigmas since its

discovery and it is being utilized in different fields like evolution, anthropology, history,

inheritance and forensics (Brendan et al., 2013). The mtDNA and Y-chromosome

analyses are being utilized for assessing continental origin or ancestry. However, mtDNA

is advantageousin understandings about ancestry component and it provides valuable

information about the maternal inheritance as well as intercontinental movements of

humans. The continent specific polymorphisms in mtDNA are the key indicators to

determine historical human migration routes and assessing population affinities

(Chaitanya et al., 2014).

In mitochondrial genome, the control region is the most polymorphic region of

mtDNA, which is also called displacement loop (D- loop). This region is ~1122 bp in

size (spanning positions 16024-16569 and 1-576) and is hot spot for mtDNA alterations

(Michikawa et al., 1992; Tipirisetti et al., 2014). This region covers ~7% of the total

mitochondrial genome (Andrews et al., 1999) and contains three-hypervariable segments,

HVSI having fragment length of 342 bp (nps16024–16365), HVSII268 bp (nps73–340)

and HVSIII 137 bp (nps 438-576). Two hypervariable segments (HVSI and HVSII) are

the most polymorphic sites in mtDNA (Cano et al., 2014). Thus, knowledge of

polymorphisms harboring in control region of mtDNA and the classification of these

sequences in to haplogroups can be of great importance in the forensic cases of

identification, such as mass disaster and missing persons (Chong, et al., 2005; Nilsson, et

al., 2008). The mtDNA haplogroups have been considered as maternally derived

ancestral genomic markers (Ma et al., 2014). Analysis of mutational events along the

human mtDNA showed that individuals came from the same maternal lineage share the

same set of mutations (Senafi et al., 2014).

3

The length of mtDNA control region sequence varies among populations due to

the presence of indels and variable number of tandem repeats at the three-hypervariable

segments. The characteristic properties of mtDNA like; exclusively maternal inheritance

(Budowle et al., 2010), absence of recombination and high mutation rate (10-200 folds)

compared to nuclear DNA in the control region, laid the basis for high polymorphism in

mtDNA (Goncalves et al., 2011). This polymorphism in mtDNA is as a result of free

radicles production due to electron transport chain and limited DNA repair mechanism

(Larsen et al., 2005; Yu, 2011; Wallace, 2011). This feature has made mtDNA a useful

tool for exploring origin and migration in human populations and is widely applicable in

human ethnic group’s evolutionary relationships (Singh and Kulawiec, 2009).

Another source of polymorphism in the mtDNA is the occurrence of different

types of mtDNA or population of discrete mtDNA genomes in an individual, which is

also called as heteroplasmy (Melton et al., 2004). During heteroplasmy, there is

possibility of more than two types of mtDNA in an individual or in a single cell, or in a

single mitochondrion. Heteroplasmy is more frequent in the control region than in the

coding region of mtDNA (Santos et al., 2008; Li et al., 2010) and its level vary among

tissues (Irwin et al., 2009; He et al., 2010; Goto et al., 2011) and populations. Two

different types of heteroplasmies have been reported in mitochondrial genome including

sequence heteroplasmy and length heteroplasmy (Bendall et al., 1996; Melton et al.,

2004). The homopolymeric C-stretches at positions 16184–16193 (HVSI) and at

positions 303–310 (HVSII) are usually the source of length heteroplasmies (Stewart et

al., 2001). However, the occurrence of two nucleotides at one position in the mtDNA

shows the sequence heteroplasmy, which results in overlapping peaks in an

electropherogram. The mixture of wild type and variant mtDNA (heteroplasmic mtDNA)

has been reported in significant number of healthy individuals (25%) (Schonberg et al.,

2010). Moreover, homoplastic variation of mtDNA due to negligible or no recombination

at the population level has been very instrumental to determine global scale migrations of

populations (Torroni et al., 2006).

The mtDNA haplotypes have become popular tools for tracing maternal ancestry

(Ely et al., 2006). The haplotypes represent the entire profile of mutations along the

mtDNA molecule in comparison torCRS (Andrews et al., 1999). The similar haplotypes

4

which share a common ancestor with same single nucleotide polymorphism (SNP)

mutations form a haplogroup (Rosa and Brehm, 2011).There are seven macro-

haplogroups including L0’ L1’ L2’ L3’ L4’ L5’L6, which are African specific mtDNA

haplogroups, and M, N and R subgroups of macro-haplogroups are found in rest of the

world (Behar et al., 2008). The macrohaplogroups L have been predominantly reported in

western Africa (Barbieri et al., 2014).

It has been suggested in previous studies that the presence of African mtDNA

lineages in Makranis proves their recent origin as the Makrani haplotypes have also been

observed in modern sub-Saharan African populations fromMozambique (Salas et al.,

2002, Barbieri et al., 2014). Moreover, the lineages of Makranis including L1, L2, and L3

have been also found in Mozambique samples with the most frequent haplotypes

including L1a2, L2a1a, and L2a1b (Pereira et al., 2001; Salas et al., 2002). The previous

studies about Kalash suggested that highest contribution of western Eurasian haplogroups

in this population is due to their maternal lineages and no evidence of East or South Asian

lineages have been reported.The western Eurasian influence reached a frequency of 100%

in the Kalash population with U4 haplogroup (34%) being the most frequent mtDNA

haplogroup (Quintana-Murci et al.,2004). However, the molecular genetic studies of

mtDNA in the Makrani and Kalashi ethnic people have been relatively limited so far.

Present study reports the largest mtDNA survey so far of Makrani and Kalashi

peoples of Pakistan. The aim of this study wasto evaluate the genetic variability within

and between Makrani and Kalashi population using mtDNA control region.The entire

mtDNA control region (spanning positions 16,024–16,569 and 1–576) including

hypervariable segments (HVSI, HVSII and HVSIII) was sequenced for100 Makrani

individuals (males, n = 96; females, n = 4) and 111 Kalashi individuals (males, n = 63;

females, n = 48).The Makranis are the descendants of Bantu speaking, living in Turbat,

Panjgur, Awaran, Kharan, Nasirabad, Gwadar and Buleda cities of Baluchistan province,

Burewala city of Punjab Province and Karachi city of Sindh province of Pakistan. The

Kalasha or Kalash people are a group of Indo-European and Indo-Iranian speaking people

living in Bumburet, Birir and Rumbur valleys of Chitral district, Khyber-Pakhtunkhwa

province of Pakistan. The mitochondrial DNA variation in Makrani and Kalashi

populations from rCRS were utilized to infer mtDNA haplogroups. The haplogroups

5

profiles of Makrani and Kalashi individuals were compared with different populations,

which may provide an insight into the understanding of the history of their settlements in

Pakistan.

6

2-LITERATURE REVIEW

Pakistan is hypothesized to be one of the first regions where the modern humans

were settled (Qamar et al., 1999; Rakha et al., 2011) and is a South Asian association of

the four provinces i.e. Punjab, Sindh, Khyber Pakhtunkhwa (KPK), Baluchistan,

Islamabad Territory, Gilgit Baltistan formerly known as the Northern Areas and the Tribal

Areas in the northwest including the Frontier Regions, located within latitude and

longitude of 33.6667oN, 73.1667

oE and covering an area of 796,095 km

2. Pakistan is

considered the sixth most thickly populated country of the world with a population of

about 180 million people (Press Information Department, Pakistan 2009).

Pakistani population is usually divided into more than 18 ethnic and 60 linguistic

groups (Grimes, 1992). The major ethnic groups include the Punjabis, Pathans, Sindhi,

Saraiki, Muhajir, Balochi, Kalashi and Makrani (Rakha, et al., 2011). Makrani people

sometimes also called as “Negroid Makrani”, inhabit the Makran coast of Baluchistan

(Quintana-Murci et al., 2004). Baluchistan is the largest of Pakistani provinces with

respect to area and smallest in terms of population, with about 80% inter-mountainous

area, central Makran and Makran coast. Kalashi population is living in the Hindu Kush

Mountains of present day Pakistan which is divided into three remote mountain valleys at

the height of 1900M-2200M, exhibit the genes that certainly were originated in Europe

and may have been carried to East by the Alexander the Great The mitochondrial genome

analyses have been considered as a useful tool to study the ancestral relationship among

populations and their migratory routes on the globe (Cossins, 2014).

Mitochondria are unique among animal organelles which posses their own

genome and it is an extraordinarily distinctive from nuclear genome. The human mtDNA

is a closed circular double stranded DNA molecule composed of ~16569 building blocks

(Taylor and Turnbull, 2005; Lan, et al., 2008). The application of mitochondrial DNA

(mtDNA) analysis in forensic investigations has gained importance owing to the fact that

in the cases where the DNA found is highly degraded (such as stains, bones, saliva, and

fingernails etc) or very less in quantity, the mtDNA has proved very useful due to

presence of 1000–2000 mitochondria per human cell which increases the possibility of

obtaining some copies of mtDNA for analysis (Chong et al., 2005; Nilsson et al., 2008;

Kavlick et al., 2011; Adachi et al., 2014). In human cells, there are many mitochondria

7

found in each cell (50-100s) and each mitochondrion contains 5 to 10 copies of mtDNA

(Legros et al., 2004). The circular mtDNA is more resistant to the nucleases, which

usually easily can degrade the nuclear DNA. Its non-recombinational inheritance makes it

a valuable tool to track the human evolutionary history. Due to the presence of high copy

number of mtDNA in each cell, mtDNA analyses is more reliable than nuclear DNA in

the cases where the amount of starting material is low in quantity.

Figure 2.1: Human mitochondrial DNA map showing CR (control region).The hypervariable

segments (HVSI, HVSII and HVS III) in the control region that is important for forensic mtDNA

analyses are shown at the top.

The complete sequence of mitochondrial genome has revealed two different

strands of mtDNA including the purine-rich strand (heavy strand) and the pyrimidine-rich

strand (light strand) (Anderson et al., 1981). In mtDNA genome, the Nucleotide positions

have been numbered according to Anderson principle with small changes (Anderson et

al., 1981).

8

The each base on heavy strand has been numbered from base one to 16,569 base

pairs. The slight reliability of polymerase and the noticeable lack of repair mechanisms

are the major factors for high rates of mutations in mtDNA as compared to nuclear DNA.

The evolution rate of some regions in the mitochondrial genome has been reported about

ten times higher as compared to nuclear genes. These regions are being used for human

identity testing due to their hyper-variability. Mostly, the variations between the persons

are reported at two particular regions of the D-loop (Greenberg et al., 1983) known as the

hyper-variable segment I (HVSI) and the hyper-variable segment II (HVSII). Generally,

HVSI spans the region of 16,024 to 16,365 and HVSII from73 to 340 base pairs. Due to

small PCR product size of both regions, HVSI and HVSII are generally utilized for

forensic casework (Figure 2.1).

Figure 2.2: mtDNA control region schematic diagram (Courtesyhttp://forensic.yonsei.ac.kr/

protocols.html).

It has been found that mtDNA inherited maternally (Case and Wallace, 1981).

Without alteration, mtDNA sequences of all maternal relations are identical, due to which

mtDNA has become an effective tool in forensic investigations about missing persons

with the known maternal links. The source of evidence can be effectively analyzed with

reference to the maternal relatives that are several generations apart due to lack of

recombination. Hence nuclear DNA markers cannot present this characteristic (Ginther et

9

al.,1992). The presence of mtDNA in the hair shaft has made it an important tool for

forensic analysis besides bones and teeth. The mtDNA has also proved its importance

when it was used as a tool in the identification of persons from bones found from the

places of American war. In addition to this, mtDNA has been utilized to find evolutionary

links and anthropological studies as well as in the analyses of very old samples such as

old brain tissues (about 7000 years ago) (Yang et al., 2012).

The interpretations of mtDNA sequencing results have been simplified on account

of haploid and monoclonal characteristics of mtDNA. Although most of the individuals

are homoplasmic and heteroplasmy may be found at some sites (Bendall et al., 1997). If a

person carries more than one noticeable mtDNA types, he/she is considered to be

heteroplasmic. Due to its high copy number mtDNA molecule has become more valuable

as compared to nuclear DNA, for specific kinds of forensic analysis. However,

interpretational difficulties can be removed by keeping in mind the knowledge of

heteroplasmies about the populations when analyzing a sample under question(Alonso et

al., 2002). The associations of mtDNA haplogroups of different populations build the

base for the structures of genetic variations in human (Anderson et al., 1981; Andrew et

al., 1999). The variants sharing common ancestors cluster together with common

mutations forming a group called a haplogroup. In order to recognize the major human

haplogroups, phylogenetic methods have been used (Andrew et al., 1999).

Several methods are well characterized by the systematic community and used to

construct a phylogenetic tree of the mtDNA sequences, including neighbor-joining,

minimum spanning networks, maximum likelihood and maximum parsimony. The

phylogenetic method has also been used comprehensively to examine and describe The

variations in control region of mtDNA has been utilized comprehensively for

phylogenetic relationship and there is large amount data available for comparative studies

(Sobrino and Carracedo, 2005). The studies of mtDNA variation had been quite useful in

understanding the human origin and diffusion patterns in the last decade. The mtDNA

survey in the populations worldwide has shown the continent specific distributions of

mtDNA lineages (Mishmar et al., 2003).

10

2.1 Hypervariable Sites

The hypervariable sites have been identified in mtDNA during evolutionary

studies and these sites show variable nucleotides in different populations. The single

nucleotide polymorphisms have been utilized in many applications in the field of medical

genetics, human genetics and evolutionary genetics as well as in the field of forensics

(Quintana-Murci et al., 2004). The mtDNA polymorphisms have been reported as

precious for identity testing of degraded samples or samples with low quantity of starting

material (Quintana-Quintana-Murci et al., 2004). Due to lack of evidences, the

hypervariable sites were considered as mutational hotspots (Hagelberg et al., 1999).

However, a hypothesis was projected in recent times, which conveys that the shuffling of

earliest mtDNA mutations has occurred among different lineages via recombination,

which reflects the strength of hypervariable sites. Even though recent claim of

recombination in human mtDNA (Eyre-Walker et al., 1999) have not been verified in

few cases (Hagelberg et al., 2000) and remained notorious (Kumar et al., 2000; Parsons

and Irwin, 2000). The persistence of hypervariable sites in the non-coding mtDNA

control regions have been well recognized by means of different studies on mtDNA

variations (Hasegawa et al., 1993; Meyer et al., 1999).

2.2 Haplogroups

In the molecular progression, haplogroup is known as a group of related

haplotypes that contribute to a common ancestor that posse the same single nucleotide

polymorphism mutation in all haplotypes. Since a haplogroup posse’s similar haplotypes,

so it is possible to guess a haplogroup from the haplotypes. The evolution of the human

mitochondrial genome is characterized by the appearance of ethnically diverse

haplogroups. The haplogroups determined by utilizing the knowledge of mtDNA SNPs

become major clades of mtDNA tree (Torroni et al., 2000). Nine European, seven Asian

and three African mitochondrial DNA (mtDNA) haplogroups have been recognized

previously on the basis of the presence or absence of a comparatively small number of

restriction enzyme sites or on the basis of nucleotide sequences of the D-loop region. The

origin and distribution of some haplogroups is as follows.

11

2.2.1. African Haplogroups

Haplogroup L1 is known as African haplogroup and it is believed that this

haplogroup has appeared around 110,000 to 170,000 years before. It is normally used to

refer a family of lineages found in Africa and this haplogroup sometimes referred to as

haplogroup L1-6, which is known as the major haplogroup including mostly African

lineages with subclades L1, L2, L4, L5, L6 and also L3. It is also found mainly in Central

Africa and West Africa. The populations carrying the M1 haplogroup mostly favor Africa

as the place of origin instead of the Asia. It has also been observed that the population

distributions for both M1 and M1a make it obvious that majority M1 haplogroups emerge

across Sub- Saharan Africa, not in Asia and North Africa. Olivieri et al., 2006 claims

that M1haplogroup originated in Asia and also haplogroup represents a back-migration to

Ethiopia.

Haplogroup L2 is human mitochondrial DNA (mtDNA) haplogroup that is

normally found in Africa and its subclade L2a is a fairly common and prevalent in Africa,

as well as in the African diaspora Americans (Salas et al., 2002) and also L2a1b and

L2a1c appeared in Southeastern Africa. It is also believed that haplogroup L3 has

originated in East Africa (Gonder et al., 2006) and it is divided into numerous clades, two

of them are M and N haplogroups that become source of non-Africans haplogroups

(Wallace et al., 1999).

2.2.2. West Eurasian Haplogroups

About haplogroup HV it is believed that it is a west Eurasian haplogroup and is

normally found throughout western Asia, southern and Eastern Europe, especially Iran,

Anatolia and the Caucasus Mountains of southern Russia and the republic of Georgia.U7

has been reported as West Eurasian haplogroup with it origin from the Black Sea

(Metspalu et al., 2004). Presently, U7 haplogroup has been found in the western Siberian

tribes (Rudbeck et al., 2005), West Asia, Near East, in Iran (Metspalu et al., 2004) and

South Asia (Metspalu et al., 2004). It has been observed that the haplogroups U7, W and

R2 constitute about one third of the West Eurasian-specific haplogroups in India. It is

believed that haplogroup W6, collectively W3, W4 and W5, were descendants of a

woman from northwest India around 14,000 years ago. W6 haplogroup appeared in the

area between the Black and Caspian Sea and also in Georgia near about 10,000 years ago.

12

The J haplogroup has been found about 12% in native Europeans (Sykes, 2001).

Average frequency of the J was found with highest in the Near East followed by Europe,

Caucasus and North Africa. It has been observed that J1 haplogroup takes up four-fifths

of the total and is spread in the continent while J2 haplogroup is more localized around

the Mediterranean, Greece, Italy and Spain. However, this haplogroup has also been

found in Kalash (Quintana-Murci et al., 2004). It has been observed that haplogroup U4

has been originated near about 25,000 years ago in Upper Palaeolithic and has been

occupied since the time of settling of modern humans in Europe (Richards et al., 2000).

Haplogroup U4 is also found with highest frequency in Scandinavia as well as Baltic

states and is also linked to ancient European hunters conserved in Siberia (Sarkissian et

al., 2013).

Haplogroup R has extensive diversity having diverse ethnic position and different

families on language base in the South Asia. In western regions of India different castes

and tribes show higher haplogroup diversity than that of other regions which suggest their

autochthonous status (Maji, 2008). Haplogroup F is found in Asia and has appeared

throughout East Asia and Southeast Asia. It is a descendant haplogroup of haplogroup R.

Practically common in East Asia and Southeast Asia (David et al., 2004; Hill et al.,

2006). Its distribution extends at low frequency to the Tharu of southern Nepal and the

Bashkirs of the southern Urals (Fornarino et al., 2009). Haplogroup T is found in about

1% of native European population (Sykes, 2001). This haplogroup is rare in Africa and is

found to be absent in most of the populations. It has highest frequencies in the Amhara

and the Tigraipeople (Kivisild, 2004).

Haplogroup U2 has been found most common in South Asia (Metspalu et al.,

2004), with low frequency in Central Asia, West Asiaand Europe (U2e) (Maji et al.,

2008). R0 haplogroup is most derives than that of haplogroup R. Haplogroup R0 is found

frequently in the Arabian Plate with its highest frequency in Socotri (Cerny et al., 2009)

and has been also found in a high frequency in the Kalash in Pakistan (Quintana-Murci et

al., 2004). Haplogroup R2 have been reported with low frequencies in the Middle East

and India and is almost absent in another place. The haplogroup R2 have been only found

in few populations of the Volga in Europe. H2 haplogroup is fairly universal in Eastern

Europe and the Caucasus (Pereira et al., 2005). This is most widespread H subclades

13

among Central Asians and has also been observed in West Asia (Loogvali et al., 2004).

H2a5 haplogroup has been found in Spain (Alvarez-Iglesias et al., 2009) Norway, Ireland

and Slovakia (van Oven and Kayser, 2009).

2.2.3. Southeast Asian Haplogroup

It is thought that haplogroup Bhas been arisen in Asia near about 50,000 years

ago. Haplogroup R was its ancestral haplogroup. Its greater variety is found in China. It is

prominent with haplogroup B. haplogroup B is found frequently in southeastern Asia

(Yao et al., 2002). The haplogroup H is considered the descendant group of haplogroup

HV. Ina number of studies it has been concluded that perhaps haplogroup H has evolved

in West Asia 25,000 years ago. Then by migrations near about 20-25,000 years ago this

haplogroup was carried to Europe and spread with population of the southwest of the

continent (Richards et al., 2000; Pereira et al., 2005; Behar et al., 2012). Haplogroup HV

is derived form of the haplogroup R0. HV haplogroup is considered the ancestral

haplogroup of haplogroup H and haplogroup V while HV is a west Eurasian haplogroup

found throughout western Asia and southern & Eastern Europe, especially Iran, Anatolia

and the Caucasus Mountains of southern Russia and the republic of Georgia. It has also

been observed in some parts of the northeast Africa, in Arabs, while the Eurasian

frequency was found to be 22.5% (Afonso et al., 2008). Some other haplogroups and

their place of origin are as follows.

Haplogroup Place of origin Time of origin

(Years ago)

K South Asia or West Asia 40,000

T West Asia 30,000

J Middle East 30,000

R South Asia or Central Asia 28,000

E1b1b-M35 East Africa 26,000

I Balkans 25,000

R1a1 Southern Russia 21,000

R1b Around the Caspian Sea or Central Asia 20,000

E1b1b-M78 Egypt/Libya 18,000

G Between India and the Caucasus 17,000

14

I2 Balkans 17,000

J2 Northern Mesopotamia 15,000

I2b Central Europe 13,000

N1c1 Siberia 12,000

I2a Balkans 11,000

R1b1b2 North or south of the Caucasus 10,000

J1 Arabian peninsula 10,000

E1b1b-V13 Balkans 10,000

I2b1 Central Europe 9,000

I2a1 Pyrenees 8,000

I2a2 Dinaric Alps 7,500

E1b1b-M81 Maghreb 5,500

I1 Scandinavia or Central Europe 5,000

R1b-L21 Central or Eastern Europe 4,000

R1b-S21 Central Europe 3,000

I2b1a Britain < 3,000

2.3. The Role of the mtDNA in Ancestry Studies

The mtDNA follows maternal mode of inheritance strictly, the limited

recombination, high mutation rate and high level of population-specific polymorphisms.

Mutation accumulation in mtDNA is tenfold greater than in nuclear DNA. This feature

has created and characterized groups defined by having a maternal lineage legacy,

making mtDNA a useful tool for studying origin and migration in human populations; it

has been extensively used evolutionary associations studies among different ethnic

groups and their global migrations (Singh et al., 2009). The most variable segment of the

mtDNA is control region and the most polymorphic nucleotide sites are concentrated in

two hypervariable segments including HVS-I & HSV- II). Individuals’ geographical

origin has been identified by RFLP analysis (high-resolution) and HSVI sequencing

(Carracedo et al., 2000). mtDNA population databases serve as a mean to approximate

the expected frequency of haplotypes observed when an individual’s mtDNA sequence

matches that of a particular sample (Butler, 2009). Many researchers around the world

15

have spent a great amount of time and resources to compile mtDNA samples from

thousands of maternally unrelated samples to create databases. The databases must obtain

high-quality information so that potential random match probability may be estimated

reliably (Butler, 2009). The ability to accurately generate a frequency estimates for

random matches in a forensic setting is one of the most significance to forensic analysts

(Butler, 2009). Mitochondrial DNA forensic databases are drastically lacking in sample

size as well as population diversity.

This study reports the mtDNA survey of Makrani and Kalashi peoples of

Pakistan. The genetic variability within and between Makrani and Kalashi population

using mtDNA control region was evaluated. The entire mtDNA control region was

sequenced for 100 Makrani individuals and 111 Kalashi individuals. The mitochondrial

DNA variation in Makrani and Kalashi populations from rCRS were utilized to infer

mtDNA haplogroups. The haplogroups profiles of Makrani and Kalashi individuals were

compared with different populations to infer the ancestry of Kalashi and Makrani peoples

of Pakistan.

16

3-MATERIALS AND METHODS

3.1. Sample Collection Areas

Blood samples were collected from 211 maternally unrelated individuals from

two isolated populations of Pakistan, viz. Makrani and Kalashi. Sampling areas for both

populations examined during this study are shown in Fig.3.1. In this study, mtDNA

control region sequence data was generated for 211 individuals.

Figure 3.1: Map of Pakistan showing its administrative regions and neighboring

countries. Triangles represent sampling areas for Makrani and Kalashi populations.

PAKISTAN

JA

MM

U &

KA

SH

MIR

C H I N A

DIS

PU

TE

D T

ER

RIT

OR

Y

GILGIT

I N

D

I

A

I R

A N

0 100 200 300 km

PUNJAB

SIND

BALOCHISTAN

A R A B I A N S E A

A F G

H A

N I

S T A

N

N

KH

YB

ER

PA

KH

TU

NK

HW

A

Burewala

Kharan

Panjgur

Turbat Awaran

Gwadar

Karachi(Lyari)

BuledaNasir-abad

BALTISTAN

AZA

D J

AM

MU

K

AS

HM

IR

Chitral

Burewala 14

Turbat 73

03Awaran

02Buleda

Kharan 01

02Panjgur

03Karachi (Lyari)

Gwadar 01

01Nasirabad

N

of sample NumberN

Sampling Area

BumburetRumbur

Birir

Chitral

24(i) Bumburet

(iii) Birir 42

45(ii) Rumbur

KALASHI POPULATION

111Total

MAKRANI POPULATION

Total 100

17

3.2. Makrani Population

3.2.1. Sample Collection

Blood samples (3-5 ml) were collected from 100 healthy, unrelated Makrani

individuals (males, n=96; females, n=4) from Pakistan, after obtaining oral and written

consent according to the declarations of Helsinki. Donor’s information was collected

individually according to the consent forms (Annexure 1 and 2). The detailed data of

consent forms from Makrani population is presented in Table 3.1.The summarized

information about sampling of Makrani population from different cities of three

provinces of Pakistan is presented in Table 3.2.

Table 3.1: The detailed data of consent forms from Makrani population.

Sr.

No.

Donor’s

ID Gender Age

(Yrs.)

Birth

Place

Ethnic

Group

Mother Father

Birth

Place

Ethnic

Group

Birth

Place

Ethnic

Group

1 MKH001 M 23 TRB MKB TRB MKB TRB MKB







8 MKH008 F 50 TRB MKB TRB MKB TRB MKB


19 MKH011 M 27 TRB MKB GWD MKB TRB MKB



13 MKH014 M 21 PJR MKB PJR MKB TRB MKB








21 MKH022 M 20 TRB MKB KRC MKB KRC MKB




18

Sr.

No.

Donor’s

ID Gender Age

(Yrs.)

Birth

Place

Ethnic

Group

Mother Father

Birth

Place

Ethnic

Group

Birth

Place

Ethnic

Group












36 MKH037 M 22 KRC MKB KRC MKB KRC MKB

37 MKH038 M 22 AWN MKB AWN MKB AWN MKB













50 MKH052 M 51 BRW MKB OKR MKB OKR MKB






56 MKH062 F 30 GWD MKB GWD MKB GWD MKB


58 MKH067 M 24 BRW MKB BRW MKB BRW MKB

59 MKH068 M 70 BRW MKB BRW MKB BRW MKB







19

Abbreviations: M, Male; F, Female; Yrs., Years; MKB, Makrani Baloch; TRB, Turbat; BLD,

Buleda; NSD, Nasirabad; KHN, Kharan; PJR, Panjgor; BRW, Burewala; GWD, Gawadar;

AWN, Awaran; KRC, Karachi, OKR, Okara.

Sr.

No.

Donor’s

ID Gender Age

(Yrs.)

Birth

Place

Ethnic

Group

Mother Father

Birth

Place

Ethnic

Group

Birth

Place

Ethnic

Group







72 MKH083 M 20 PJR MKB PJR MKB PJR MKB

73 MKH084 M 22 TRB MKB BLD MKB BLD MKB




77 MKH088 M 22 TRB MKB TRB MKB PJR MKB












89 MKH100 M 18 KHN MKB KHN MKB KHN MKB

90 MKH101 M 20 BLD MKB TRB MKB TRB MKB


92 MKH103 M 21 NSD MKB NSD MKB NSD MKB

93 MKH104 M 21 BLD MKB BLD MKB BLD MKB








20

Table 3.2: The summarized information about sampling of Makrani population from

different cities of three provinces of Pakistan

Province City Male

(n=96)

Female

(n=4)

Baluchistan

Turbat 70 3

Awaran 3 0

Buleda 2 0

Panjgur 2 0

Kharan 1 0

Gwadar 0 1

Nasirabad 1 0

Punjab Burewala 14 0

Sindh Karachi(Lyari) 3 0

3.3. Kalash Population

3.3.1. Sample Collection

Blood samples (3-5ml) were collected from 111 maternally unrelated healthy

Kalashi individuals (males=63 and females =48) from Pakistan after obtaining oral and

written consent according to the declarations of Helsinki. Donor’s information was

collected individually according to the consent forms (Annexure 1 and 2).The-sampling

areas for Kalashi population are shown in Fig.3.1 and detailed data of consent forms in

Table 3.3.For simplicity, the summarized data of samples from three different valleys of

Kalash are shown in Table 3.4.

21

Table 3.3: The detailed data of consent forms from Kalashi population.

Sr.

No

Donor’s

ID

Su

b-E

thn

ic

Gro

up

Eth

nic

Gro

up

Birth Place

Gen

der

Age(

Yea

rs)

Mother’s Father’s

Vil

lage

Vall

ey

Su

b-E

thn

ic

Gro

up

Bir

th P

lace

Su

b-E

thn

ic

Gro

up

Bir

th P

lace

1 KLH001 KLS KLS GBG BRR M 29 KLG BRR KLS BRR

2 KLH002 SKT KLS KRK BMT M 26 RJW BTK SKT KRK

3 KLH003 BBR KLS KRK BMT M 35 BLS BRN BBR KRK

4 KLH004 SKT KLS KRK BMT M 50 BZK BRN SKT KRK

5 KLH005 DHM KLS KRK RBR M 60 MTM RBR DHM KRK

6 KLH006 SKT KLS KRK BMT M 39 SHY DGU SKT KRK

7 KLH007 SKT KLS KRK BMT M 27 RJW BTK SKT KRK

8 KLH008 RJW KLS BTK BMT M 19 MHD BRR RJW BTK

9 KLH009 RJW KLS BTK BMT M 35 SHY DGU RJW BTK

10 KLH019 BMK KLS ANH BMT M 19 ASN ANH BMK ANH

11 KLH011 BZK KLS BRN BMT M 29 BGL RBR BZK BRN

12 KLH012 BZK KLS BRN BMT M 45 BRK ANH BZK BRN

13 KLH014 BD KLS KRK BMT M 35 BLS BRN BBR KRK

14 KLH015 BLS KLS BRN BMT M 50 SHY DGU BLS BRN

15 KLH016 BZK KLS BRN BMT M 28 BLO RBR BZK BRN

16 KLH018 BZK KLS BRN BMT M 18 SKT BRN BZK BRN

17 KLH019 BD KLS KRK BMT M 18 LGY AYN BBR KRK

18 KLH020 SKT KLS KRK BMT M 21 SKT KRK SKT KRK

19 KLH021 SKT KLS KRK BMT M 26 QRH SKH SKT KRK

20 KLH022 SKT KLS KRK BMT M 45 SHY DGU SKT KRK

21 KLH023 RJW KLS BTK BMT M 80 GSD BRR RJW BTK

22 KLH024 RJW KLS BTK BMT M 40 BLS BRN RJW BTK

23 KLH026 BLS KLS BRN BMT M 30 MTM RBR BLS BRN

24 KLH027 BLS KLS BRN BMT M 26 BZK BRN BLS BRN

25 KLH028 BBR KLS KRK BMT M 30 SKT KRK BBR KRK

26 KLH029 RJW KLS BTK BMT M 38 SKT KRK RJW BTK

27 KLH030 MTM KLS KTD RBR F 28 OMH MLD MTM KTD

28 KLH031 MTM KLS KTD RBR F 28 BBR KRK MTM KTD

29 KLH032 BLO KLS KLG RBR F 17 BLS BRN BLO KLG

30 KLH033 DHM KLS BGU RBR F 20 DHM BGU DHM BGU

31 KLH034 MTM KLS KTD RBR F 29 BBR KRK MTM BTT

32 KLH035 BLO KLS KLG RBR M 18 DHM BRR BLO KLG

22

Sr.

No

Donor’s

ID

Su

b-E

thn

ic G

rou

p

Eth

nic

Gro

up

Birth Place

Gen

der

Age

(Yea

rs)


Vil

lage

Vall

ey

Su

b-E

thn

ic

Gro

up

Bir

th P

lace

Su

b-E

thn

ic

Gro

up

Bir

th P

lace

33 KHL036 BLO KLS KLG RBR M 19 BZK BRN BLO KLG

34 KLH037 BLO KLS BGU RBR F 29 WKY BGU BLO GRM

35 KLH038 MTM KLS KLG RBR M 45 DHM BGU MTM KLG

36 KLH039 DHM KLS BGU RBR F 45 MTM KLG DHM BGU

37 KLH040 DHM KLS BGU RBR F 23 BLO GRM DHM BGU

38 KLH041 BGL KLS BGU RBR M 35 BLO GRM BGL BGU

39 KLH044 DHM KLS BGU RBR F 26 BGL BGU DHM BGU

40 LKH045 BLO KLS BTT RBR M 20 SKT KRK BLO BTT

41 KLH046 WKY KLS BGU RBR M 25 DHM BGU WKY BGU

42 KLH047 JRY KLS BGU RBR M 45 DHM BGU JRY BGU

43 KLH048 BGL KLS BGU RBR F 15 DHM MLD BGL BGU

44 KLH049 MTM KLS KTD RBR M 25 BLO KLS MTM KTD

45 KLH050 DHM KLS GRM RBR F 21 MTM BGU DHM GRM

46 KLH051 BLO KLS GRM RBR M 15 WKY BGU BLO GRM

47 KLH052 BLO KLS GRM RBR M 16 RJW BTT BLO GRM

48 KLH053 WKY KLS BGU RBR F 18 BLO KLS WKY BGU

49 KLH054 DHM KLS KLG RBR M 34 WKY KLS DHM KLG

50 KLH056 DHM KLS BGU RBR M 18 BGL BGU DHM BGU

51 KLH057 BGL KLS BGU RBR F 26 BLO KLS BGL BGU


53 KLH059 WKY KLS BGU RBR M 15 JRY BGU WKY BGU

54 KLH060 DHM KLS BGU RBR M 35 BLO GRM DHM BGU

55 KLH061 WKY KLS BGU RBR M 35 DHM BGU WKY BGU



58 KLH064 BGL KLS BGU RBR F 35 WKY BGU BGL BGU

59 KLH065 ZHO KLS BGU RBR F 18 BLO BTT ZHO BTT

60 KLH066 BGL KLS BGU RBR M 36 WKY BGU BGL BGU

61 KLH067 ZHO KLS BGU RBR M 30 BGL BGU ZHO MLD


63 KLH079 JRY KLS BGU RBR M 39 DHM BGU JRY BGU


65 KLH071 DHM KLS BGU RBR M 39 BGL BGU DHM BGU


23

Sr.

No.

Donor’s

ID

Su

b-E

thn

ic G

rou

p

Eth

nic

Gro

up

Birth Place

Gen

der

Age(

Yea

rs)


Vil

lage

Vall

ey

Su

b-E

thn

ic

Gro

up

Bir

th P

lace

Su

b-E

thn

ic

Gro

up

Bir

th P

lace

67 KLH073 JRY KLS BGU RBR F 39 DHM BGU JRY BGU


69 KLH075 DHM KLS GRM RBR F 25 BLO KLG DHM MLD

70 KLH076 MTM KLS KLG RBR F 28 BZK BRN MTM KLG

71 KLH077 ASN KLS GBG BRR M 45 BDI KDR ASN GMK

72 KLH078 LKD KLS NSP BRR M 19 LKD BPL RMI NSP

73 KLH079 AKW KLS ASP BRR F 19 MDI BWO AKW ASP

74 KLH080 TRK KLS GBG BRR F 35 LTK GBL TRK GBL

75 KLH081 TRK KLS GBG BRR F 32 LRH GBL TRK GBL

76 KLH082 LKD KLS Guru BRR F 40 LRK GBL TRK Guru

77 KLH083 LKD KLS NSB BRR F 35 BDI SWR LTK NSB

78 KLH084 AKW KLS ASP BRR M 19 LRK GBL AKW ASP

79 KLH085 GSD KLS ASP BRR M 20 DMD BSH GSD ASP

80 KLH086 TRK KLS GBG BRR M 18 GSD Guru TRK GBL

81 KLH087 LKD KLS Guru BRR M 18 RMI NSB LTK Guru

82 KLH088 CHS KLS GB BRR M 19 TRK ASP CHS GBL

83 KLH089 DRD KLS NSB BRR M 19 LRK GBL DRD NSB

84 KLH090 PNW KLS WDN BRR M 22 DMW BSH PNW WDN

85 KLH091 TRK KLS BRR BRR M 24 LTK RBR TRK Guru

86 KLH092 PNW KLS BRR BRR M 19 DMD BSH PNW WDN

87 KLH093 LKD KLS BRR BRR M 18 CHS BRR LTK GBL

88 KLH094 TRK KLS BRR BRR M 20 RKD DGU TRK GBL

89 KLH095 BRD KLS NSB BRR M 18 TRK Guru BDR GBL

90 KLH097 MHD KLS BWO BRR F 19 LTK GBL MHD BWO

91 KLH098 BP KLS BWO BRR F 19 AKW ASP AKW BWO

92 KLH099 TRK KLS Guru BRR F 20 LTK Guru TRK Guru

93 KLH100 AKW KLS ASP BRR F 19 LTK GBL AKW ASP

94 KLH102 RKD KLS NSB BRR F 18 CHS GBL LTK NSB

95 KLH103 LKD KLS GBG BRR F 19 LTK GBL AKW GBL

96 KLH04 CHS KLS GBG BRR F 20 LTK GBL CHS GBL

97 KLH105 BRD KLS BWO BRR F 20 TRK GBL LTK BWO

98 KLH106 TRK KLS GBG BRR F 24 BRD BWO TRK GBL

99 KLH107 TRK KLS GBG BRR F 19 AKW GBL AKW ASP

100 KLH109 PNY KLS WDN BRR F 24 RNW BSH AKW WDN

24

Sr.

No

Donor’s

ID

Su

b-E

thn

ic

Gro

up

Eth

nic

Gro

up

Birth Place

Gen

der

Age(

Yea

rs)


Vil

lage

Vall

ey

Su

b-E

thn

ic

Gro

up

Bir

thP

lace

Su

b-E

thn

ic

Gro

up

Bir

thP

lace

101 KLH110 AKW KLS ASP BRR F 19 TRK ASP AKW ASP

102 KLH111 PNY KLS WDN BRR F 19 ASN ABR PNW WDN

103 KLH112 DNW KLS BSH BRR F 20 AKW ASP DMW BSH

104 KLH113 LKD KLS Guru BRR F 19 TRK Guru LTK Guru

105 KLH114 LKD KLS GBG BRR F 20 RKD DGU LTK GBL

106 KLH115 TRK KLS GBG BRR F 19 LTK GBL TRK GBL

107 KLH116 MHD KLS BWO BRR F 20 MHD BWO PPW BWO

108 KLH117 TRK KLS GBG BRR M 21 TRK BWO TRK GBL

109 KLH118 TRK KLS BRR BRR M 19 RKD NSB TRK GBL

110 KLH119 LKD KLS PPG BRR M 19 TRK GBL LTK BPL

111 KLH121 AKW KLS ASP BRR M 19 TRK ASP AKW ASP

Abbreviations:BMT, Bumburete; BRR, Birir; RMB, Rumbur; KLS, Kalash; GBG, Grambetgol;

MTM, Mutimir; MHD, Mahadari; BGL, Bangalie; BRKBaramuk; SKH, Shahkhandeh; DGU,

Darasguru; RUR, Rumbur; MLD, Malidesh; BDI,Budadari; BMD,BumburDari;DHM, Dhramese;

LTK, L’atharuk; SKT, Sharakat; AKW,Al’ukshernawaw; RJW, Rajaway; GSD, Gil’asurdari;

CHS, Chagansey; DRD, Barburadari;RKD, Rashmukdari;DNW, DumuNawaw; MDI, Mahadari;

LRK, Latharuk; DMD, Damundari;RMI, Rashmukdari; TRK, Thararaik; DMW, Damunawawa;

RNW,RomaNawaw; SKT, Sharakat, , KLG, Kalashgram; DGU, Darasguru; BBR, Bumboor;

KDR,Kandisaar; BPL, phishpagole; WDN,Waridon; ANH, Anish. KRK, Krakal; ASP, Asper;

BLS; Bulasing BTK, Batrik; BRN, Broon; NSB, Nos’biaw; BSH, Bishal; PPG.Phishpagol; BWO,

Biawo; BZK, Bazik;BLO, Balo; KTD, Kotdish; BMK, Barmuk; SHY, Sharey; AYN,Ayun; QRH,

Quresh; WKY, Wakokay; ASN, Aspan’i; GRM, Groom; BTTBattet; JRY, Jaro’e’; LGY, L’agay;

ZHE, Zohe;NSP, Nosbiaw; PNY, Paney; OMH, Ohramsh; GMK, Gumbak;SWR, Saweri; PNW,

PanaiNawawo; PPW, Ponchapanaw

25

Table 3.4: The summarized information about sampling from three different valleys of

Kalash population

Valleys Villages Male

(n= 63)

Female

(n=48)

Bumburet

Anish 1 0

Batrik 5 0

Broon 7 0

Krakal 11 0

Rumbur

Battit 1 0

Groom 2 2

Kalash Gram 4 2

Kotdish 1 3

Rumbur 1 0

Balanguru 11 18

Birir

Asper 0 0

Biawo 0 4

Bishala 0 1

Grambetgol 8 0

Guru 2 3

Nosbia 2 2

Nospeawo 1 0

Pishpagol 1 0

Waridon 2 2

3.4. DNA Extraction and Quantification

DNA was extracted from blood samples using the QIAamp DNA Mini Kit

(Qiagen, Hilden, Germany) according to the manufacturer’s instructions.The extracted

DNA was incubated at 70C for 1 hour to avoid degradation by nucleases and then stored

at -40C .The quantity of the extracted DNA was determined by NanoDrop™ 1000

Spectrophotometer (Thermo Scientific, Wilmington, DE). The quality of the DNA was

determined by visualizing it using 0.8% agarose gel.

3.5. PCR Amplification

PCR was performed using Applied Biosystems thermal cycler (2720) in a 50 µl

reaction volume containing 1-2 ng of genomic DNA, 0.4 μM of each primer, and

AmpliTaq Gold®360 Master Mix (Applied Biosystems, Foster City, CA, USA) was used

according to the manufacturer’s instructions. The amplification program consisted of pre-

26

denaturation at 95C for 11 min, followed by 35 cycles consisting of denaturation step at

95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 90s, with a final

extension at 72°C for 7 mins. The primers listed in Table 3.5 were used for the

amplification and sequencing of the entire mtDNA control region in both Makrani and

Kalashi (http://forensic.yonsei.ac.kr/protocol/mtDNA-CR.pdf) populations.

Table 3.5: List of oligonucleotides, along with melting temperatures (Tm), concentrations and

sequences used for amplification and sequencing of the mtDNA control regions.

Sr.

No.

Primer name

(Control region) Primer Sequences (5→3)

Concentration

of each primer

(μM)

Melting

Temper-

ature

(°C)

1 Amplification and

sequencing primer-

F15975

CTC CAC CAT TAG CAC CCA AA 0.2 55.1

2 Sequencing primer-

F16327 CCG TAC ATA GCA CAT TAC AGT C 0.2 53.0


F155 TAT TTA TCG CAC CTA CGT TC 0.2 49.6


R16419m GAG GAT GGT GGT CAA GGG A 0.2 56.5


R042 AGA GCT CCC GTG AGT GGT TA 0.2 57.8

6

Amplification and

sequencing primer-

R635

GAT GTG AGC CCG TCT AAA CA 0.2 54.7


F403 CCG CTT CTG GCC ACA GCA CT 0.2 55.3


R389 CTG GTT AGG CTG GTG TTA GG 0.2 55.1


F16524 AAG CCT AAA TAG CCC ACA CG 0.2 55.1

27

3.5.1. Preparation of Agarose Gel

The amplified PCR products were electrophoresed in agarose gel (2%) stained

with ethidium bromide and were detected using UV transillumination (UVI doc gel

documentation systems UK). 2g of molecular grade agarose (molecular biology grade;

Sigma Chem. Co) was mixed in 100 ml of TAE electrophoresis buffer. The agarose was

melted in a microwave oven. When the agarose was dissolved completely, ethidium

bromide (Sigma-Aldrich, St. Louis, USA) was added and mixed thoroughly after

attaining mild temperature. A gel tray was sealed with rubber clamps and placed on a

level horizontal surface. The required combs were placed at appropriate positions (0.5-

1.0mm above the base of the gel). The gel was poured into the gel tray. After the gel

solidified, the combs and clamps were removed from the gel tray. The gel was placed in

an electrophoresis tank containing appropriate 1X TAE electrophoresis buffer.6X DNA

loading dye (Thermo Scientific USA) was added to each sample and the samples were

loaded on the gel. A 100 bp DNA ladder (Thermo Scientific Gene Ruler 100 bp Plus

DNA Ladder #SM0323) was loaded in the first well. Electrophoresis was carried out for

40 minutes at 100 volts using a Power Pac Basic, (B10-RAD). Photographs were taken

under UV transilluminator (PhotoDoc-It™ Imaging System, UK).

3.6. Sequencing

Unincorporated primers and dNTPs were removed from the amplified PCR

products by using ExoSAP-IT® (USB, Cleveland, OH, USA) according to

manufacturer’s instructions. Reactions were mixed briefly and incubated at 37°C for 90

min then 80°C for 20 min. An extended incubation at 37°C was implemented to ensure

digestion of all unincorporated PCR primers (Peter et al., 2004). Sequencing of the entire

mtDNA control region (spanning nucleotide positions 16024-16569 and 1-576) was done

using Big Dye Terminator Cycle Sequencing v3.1 Ready Reaction Kit (Applied

Biosystems; Carlsbad, CA, USA) according to the manufacturer’s instructions, as well as

commercial sequencing facilities from 1stBASE (http://www.base-asia.com) and the

National Center of Excellence in Molecular Biology (CEMB) Lahore, Pakistan, were

utilized for this research work.

28

3.7. Statistical Analysis

All samples were sequenced bi-directionally and evaluated twice as recommended

(Parson and Bandelt, 2007) using the sequence analysis software Geneious (Version

7.0.3, Biomatters Ltd, New Zealand) (Drummond et al., 2009) as well as by two

independent researchers. MitoTool (Fan and Yao, 2011), mtDNA profiler (Yang et al.,

2013) and HaploGrep (Kloss-Brand et al., 2011), making use of the PhyloTree Build 16

(http://www.phylotree.org) (van Oven et al., 2008) as classification tree, were used to

assess the quality of mtDNA data (Fan and Yao, 2011, Yang et al., 2013). The Makrani

mtDNA sequences were assigned to haplogroups according to the published data

(Metspalu et al., 2004; van Oven et al., 2008; Behar, et al., 2008; van Oven et al., 2011;

Mostafa et al., 2013). The population statistical parameters such as haplotype diversity,

random match probability and power of discrimination were statistically calculated

according to the previous studies (Tajima, 1989; Prieto, 2011). The recommendations and

guidelines from the ISFG regarding the mtDNA population data reporting were followed

in this study (Parson and Bandelt, 2007). Median-joining haplotype networks (Bandelt et

al., 1999) were constructed using the software NETWORK (http://www.fluxus-

engineering.com/sharenet.htm).

29

4-RESULTS

4.1. Sampled Populations

During present study, maternal genetic ancestry of Makrani and Kalashi

populations living in Pakistan was characterized by analyzing mtDNA control region

(spanning positions 16,024–16,569 and 1–576) including hypervariable segments (HVSI,

HVSII& HVSIII). The sequences of mtDNA were obtained from 211healthy and

unrelated individuals belonging to the two ethnic populations.

4.2. Genomic DNA Quality and PCR Amplification of mtDNA Control

Region

Genomic DNA was extracted from blood samples of Makrani and Kalashi

individuals and quality of the DNA is shown in figure 4.1a & b. The mtDNA control

region was amplified for all samples and fragment size of the PCR products (~1122bp)

was determined using agarose gel electrophoresis. Due to indels that occurred in mtDNA

control region, the amplified PCR product was not always 1122 bp in length. The

fragment size and quality for PCR product of mtDNA control region for Makrani and

Kalashi populations are shown in figure 4.2a & b.

4.3. Sequencing the Control Region of Mitochondrial DNA

The amplified PCR products of mitochondrial DNA control region for all samples

were subjected to purification followed by Sanger sequencing using bidirectional

approach. Each sample was processed twice and chromatograms were counter checked

independently by laboratory fellows. For the confirmation of haplotype, an additional

sequencing for identification of relevant SNPs was carried out. Most of the questioned

haplogroups were assigned based on control region and relevant SNPs from coding

region. The bidirectional chromatograms of a Makrani (MKH080) and Kalashi

(KLH015) for entire mitochondrial DNA control region by using forward primer

(F15975) as well as reverse primer (R635) are shown as examples in figures 4.3 a-b and

4.4 a-b respectively.

30

(a) (b)

Figure 4.1: Agarose gel electrophoretic analysis of genomic DNA extracted from blood samples

(a) Makrani samples (1) MKH001, (2) MKH002, (3) MKH080, (4) Negative control, M=100bp

marker (ThermoTM SM # 0323) (b) Kalash samples (1) KLH001, (2) KLH002, (3) KLH003,

(4) KLH004, (5) KLH005, (6) Negative control, M=100bp DNA marker (Thermo TM

SM # 0323).

(a) (b)

Figure 4.2: Agarose gel electrophoretic analysis of the mtDNA control region PCR products

(a) Makrani samples (1) MKH001, (2) MKH002, (3) MKH003, (4) MKH004, (5) MKH005, (6)

MKH080, (7) Negative control, M=100bp DNA marker (Thermo TM

SM#0323)(b) Kalash

samples (1) KLH001 (2) KLH002 (3) KLH003 (4) KLH004, (5) KLH005, (6) KLH006,

(7) Negative control, M=100 bp DNA ladder (Thermo TM

SM # 0323).

31

32

Figure 4.3 (a): Chromatogram of Makrani individual (MKH080) for entire mtDNA control

region sequenced by forward primer (F15975)

33

34

Figure 4.3 (b): Chromatogram of Makrani individual (MKH080) for mtDNA control region

sequenced by reverse primer (R635).

35

36

Figure 4.4 (a): Chromatogram of Kalashi individual (KLH015) for the entire mtDNA control

region sequenced by forward primer (F15975).

37

38

Figure 4.4 (b): Chromatogram of Kalashi individual (KLH015) for the entire mtDNA control

region sequenced by reverse primer (R635)

39

4.4. Reconstruction and Alignment with rCRS

Expected sequence (~1122bp) for each sample was reconstructed from several

fragments sequences obtained from multiple amplification reactions by using “mtDNA

assembly tool” that is one of the tools from mtDNA profiler (Yang et al., 2013).By using

this tool, mtDNA sequences for Makrani as well as for Kalashi individuals were

reconstructed from different fragments sequences. The reconstructed sequences for all

samples of Makrani and Kalashi individuals were aligned to rCRS to identify the

differences from rCRS. The representative rCRS aligned sequences for one Makrani

(MKH080) and one Kalashi individual (KLH016) are shown in figure 4.5 a and b

respectively.

....16030.....16040.....16050.....16060.....16070.....16080

▼ ▼ ▼ ▼ ▼ ▼

rCRS TTCTTTCATGGGGAAGCAGATTTGGGTACCACCCAAGTATTGACTCACCCATCAACAACC

............................................................

MKH080 TTCTTTCATGGGGAAGCAGATTTGGGTACCACCCAAGTATTGACTCACCCATCAACAACC

....16090.....16100.....16110.....16120.....16130.....16140

▼ ▼ ▼ ▼ ▼ ▼

rCRS GCTATGTATTTCGTACATTACTGCCAGCCACCATGAATATTGTACGGTACCATAAATACT

............................................................

MKH080 GCTATGTATTTCGTACATTACTGCCAGCCACCATGAATATTGTACGGTACCATAAATACT

.....16150.....16160.....16170.....16180.....16190.....16200

▼ ▼ ▼ ▼ ▼ ▼

rCRS TGACCACCTGTAGTACATAAAAACCCAATCCACATCAAAACCCCCTCCCCATGCTTACAA

.S..............................S...........................

MKH080 TAACCACCTGTAGTACATAAAAACCCAATCCATATCAAAACCCCCTCCCCATGCTTACAA

.....16210.....16220.....16230.....16240.....16250.....16260

▼ ▼ ▼ ▼ ▼ ▼

rCRS GCAAGTACAGCAATCAACCCTCAACTATCACACATCAACTGCAACTCCAAAGCCACCCCT

...................S.....................................S..

MKH080 GCAAGTACAGCAATCAACCTTCAACTATCACACATCAACTGCAACTCCAAAGCCACCTCT

40

.....16270.....16280.....16290.....16300.....16310.....16320

▼ ▼ ▼ ▼ ▼ ▼

rCRS CACCCACTAGGATACCAACAAACCTACCCACCCTTAACAGTACATAGTACATAAAGCCAT

...............................................S............

MKH080 CACCCACTAGGATACCAACAAACCTACCCACCCTTAACAGTACATAGCACATAAAGCCAT

.....16330.....16340.....16350.....16360.....16370.....16380

▼ ▼ ▼ ▼ ▼ ▼

rCRS TTACCGTACATAGCACATTACAGTCAAATCCCTTCTCGTCCCCATGGATGACCCCCCTCA

............................................................

MKH080 TTACCGTACATAGCACATTACAGTCAAATCCCTTCTCGTCCCCATGGATGACCCCCCTCA

.....16390.....16400.....16410.....16420.....16430.....16440

▼ ▼ ▼ ▼ ▼ ▼

rCRS GATAGGGGTCCCTTGACCACCATCCTCCGTGAAATCAATATCCCGCACAAGAGTGCTACT

............................................................

MKH080 GATAGGGGTCCCTTGACCACCATCCTCCGTGAAATCAATATCCCGCACAAGAGTGCTACT

.....16450.....16460.....16470.....16480.....16490.....16500

▼ ▼ ▼ ▼ ▼ ▼

rCRS CTCCTCGCTCCGGGCCCATAACACTTGGGGGTAGCTAAAGTGAACTGTATCCGACATCTG

............................................................

MKH080 CTCCTCGCTCCGGGCCCATAACACTTGGGGGTAGCTAAAGTGAACTGTATCCGACATCTG

.....16510.....16520.....16530.....16540.....16550.....16560

▼ ▼ ▼ ▼ ▼ ▼

rCRS GTTCCTACTTCAGGGTCATAAAGCCTAAATAGCCCACACGTTCCCCTTAAATAAGACATC

...............S............................................

MKH080 GTTCCTACTTCAGGGCCATAAAGCCTAAATAGCCCACACGTTCCCCTTAAATAAGACATC

.....1........10........20........30........40........50...

▼ ▼ ▼ ▼ ▼ ▼

rCRS ACGATGGATCACAGGTCTATCACCCTATTAACCACTCACGGGAGCTCTCCATGCATTTGG

............................................................

MKH080 ACGATGGATCACAGGTCTATCACCCTATTAACCACTCACGGGAGCTCTCCATGCATTTGG

41

.....60........70........80........90........100.......110..

▼ ▼ ▼ ▼ ▼ ▼

rCRS TATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCC

..................S.........................................

MKH080 TATTTTCGTCTGGGGGGTGTGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCC

.....120.......130.......140.......150.......160.......170..

▼ ▼ ▼ ▼ ▼ ▼

rCRS TATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTATTATTTATCGCACCTACGTTC

............................................................

MKH080 TATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTATTATTTATCGCACCTACGTTC

.....180.......190.......200.......210.......220.......230..

▼ ▼ ▼ ▼ ▼ ▼

rCRS AATATTACAGGCGAACATACTTACTAAAGTGTGTTAATTAATTAATGCTTGTAGGACATA

............................................................

MKH080 AATATTACAGGCGAACATACTTACTAAAGTGTGTTAATTAATTAATGCTTGTAGGACATA

.....240.......250.......260.......270.......280.......290..

▼ ▼ ▼ ▼ ▼ ▼

rCRS ATAATAACAATTGAATGTCTGCACAGCCACTTTCCACACAGACATCATAACAAAAAATTT

............................S...............................

MKH080 ATAATAACAATTGAATGTCTGCACAGCCGCTTTCCACACAGACATCATAACAAAAAATTT

.....300.......310........320.......330.......340.......350.

▼ ▼ ▼ ▼ ▼ ▼

rCRS CCACCAAACCCCCCCTCCCCC─GCTTCTGGCCACAGCACTTAAACACATCTCTGCCAAAC

.....................I......................................

MKH080 CCACCAAACCCCCCCTCCCCCCGCTTCTGGCCACAGCACTTAAACACATCTCTGCCAAAC

......360.......370.......380.......390.......400.......410.

▼ ▼ ▼ ▼ ▼ ▼

rCRS CCCAAAAACAAAGAACCCTAACACCAGCCTAACCAGATTTCAAATTTTATCTTTTGGCGG

............................................................

MKH080 CCCAAAAACAAAGAACCCTAACACCAGCCTAACCAGATTTCAAATTTTATCTTTTGGCGG

42

......420.......430.......440.......450.......460.......470.

▼ ▼ ▼ ▼ ▼ ▼

rCRS TATGCACTTTTAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATAC

............................................................

MKH080 TATGCACTTTTAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATAC

......480.......490.......500.......510.......520.......530.

▼ ▼ ▼ ▼ ▼ ▼

rCRS TACTAATCTCATCAATACAACCCCCGCCCATCCTACCCAGCACACACACACCGCTGCTAA

...............S............................................

MKH080 TACTAATCTCATCAACACAACCCCCGCCCATCCTACCCAGCACACACACACCGCTGCTAA

......540.......550.......560.......570....

▼ ▼ ▼ ▼

rCRS CCCCATACCCCGAACCAACCAAACCCCAAAGACACCCCCCACA

...........................................

MKH080 CCCCATACCCCGAACCAACCAAACCCCAAAGACACCCCCCACA

Figure 4.5 (a): The haplotype of Makrani individual (MKH080) for entire mtDNA control region

The observed haplotype of MKH080after the alignment of reconstructed sequence with rCRS

(16145A, 16176T, 16223T, 16261T, 16311C, 16519C, 73G, 263G, 315.1C, 489C) I: Insertions,

S: Transitions, V: Transversions.

.....16030.....16040.....16050.....16060.....16070.....16080

▼ ▼ ▼ ▼ ▼ ▼

rCRS TTCTTTCATGGGGAAGCAGATTTGGGTACCACCCAAGTATTGACTCACCCATCAACAACC

...........................S................................

KLH016 TTCTTTCATGGGGAAGCAGATTTGGGTGCCACCCAAGTATTGACTCACCCATCAACAACC

.....16090.....16100.....16110.....16120.....16130.....16140

▼ ▼ ▼ ▼ ▼ ▼

rCRS GCTATGTATTTCGTACATTACTGCCAGCCACCATGAATATTGTACGGTACCATAAATACT

.............................................V..............

KLH016 GCTATGTATTTCGTACATTACTGCCAGCCACCATGAATATTGTACCGTACCATAAATACT

.....16150.....16160.....16170.....16180.....16190.....16200

▼ ▼ ▼ ▼ ▼ ▼

rCRS TGACCACCTGTAGTACATAAAAACCCAATCCACATCAAAACCCCCTCCCCATGCTTACAA

..........S.................................................

KLH016 TGACCACCTGCAGTACATAAAAACCCAATCCACATCAAAACCCCCTCCCCATGCTTACAA

43

.....16210.....16220.....16230.....16240.....16250.....16260

▼ ▼ ▼ ▼ ▼ ▼

rCRS GCAAGTACAGCAATCAACCCTCAACTATCACACATCAACTGCAACTCCAAAGCCACCCCT

............................................S...............

KLH016 GCAAGTACAGCAATCAACCCTCAACTATCACACATCAACTGCAATTCCAAAGCCACCCCT

.....16270.....16280.....16290.....16300.....16310.....16320

▼ ▼ ▼ ▼ ▼ ▼

rCRS CACCCACTAGGATACCAACAAACCTACCCACCCTTAACAGTACATAGTACATAAAGCCAT

............................................................

KLH016 CACCCACTAGGATACCAACAAACCTACCCACCCTTAACAGTACATAGTACATAAAGCCAT

.....16330.....16340.....16350.....16360.....16370.....16380

▼ ▼ ▼ ▼ ▼ ▼

rCRS TTACCGTACATAGCACATTACAGTCAAATCCCTTCTCGTCCCCATGGATGACCCCCCTCA

......................................S.....................

KLH016 TTACCGTACATAGCACATTACAGTCAAATCCCTTCTCGCCCCCATGGATGACCCCCCTCA

.....16390.....16400.....16410.....16420.....16430.....16440

▼ ▼ ▼ ▼ ▼ ▼

rCRS GATAGGGGTCCCTTGACCACCATCCTCCGTGAAATCAATATCCCGCACAAGAGTGCTACT

............................................................

KLH016 GATAGGGGTCCCTTGACCACCATCCTCCGTGAAATCAATATCCCGCACAAGAGTGCTACT

.....16450.....16460.....16470.....16480.....16490.....16500

▼ ▼ ▼ ▼ ▼ ▼

rCRS CTCCTCGCTCCGGGCCCATAACACTTGGGGGTAGCTAAAGTGAACTGTATCCGACATCTG

............................................................

KLH016 CTCCTCGCTCCGGGCCCATAACACTTGGGGGTAGCTAAAGTGAACTGTATCCGACATCTG

.....16510.....16520.....16530.....16540.....16550.....16560

▼ ▼ ▼ ▼ ▼ ▼

rCRS GTTCCTACTTCAGGGTCATAAAGCCTAAATAGCCCACACGTTCCCCTTAAATAAGACATC

...............S............................................

KLH016 GTTCCTACTTCAGGGCCATAAAGCCTAAATAGCCCACACGTTCCCCTTAAATAAGACATC

.....1........10........20........30........40........50...

▼ ▼ ▼ ▼ ▼ ▼

rCRS ACGATGGATCACAGGTCTATCACCCTATTAACCACTCACGGGAGCTCTCCATGCATTTGG

............................................................

KLH016 ACGATGGATCACAGGTCTATCACCCTATTAACCACTCACGGGAGCTCTCCATGCATTTGG

44

.....60........70........80........90........100.......110..

▼ ▼ ▼ ▼ ▼ ▼

rCRS TATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCC

..................S.........................................

KLH016 TATTTTCGTCTGGGGGGTGTGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCC

.....120.......130.......140.......150.......160.......170..

▼ ▼ ▼ ▼ ▼ ▼

rCRS TATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTATTATTTATCGCACCTACGTTC

.....................................S......................

KLH016 TATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCCATTATTTATCGCACCTACGTTC

.....180.......190.......200.......210.......220.......230..

▼ ▼ ▼ ▼ ▼ ▼

rCRS AATATTACAGGCGAACATACTTACTAAAGTGTGTTAATTAATTAATGCTTGTAGGACATA

..........................................S.................

KLH016 AATATTACAGGCGAACATACTTACTAAAGTGTGTTAATTAATCAATGCTTGTAGGACATA

.....240.......250.......260.......270.......280.......290..

▼ ▼ ▼ ▼ ▼ ▼

rCRS ATAATAACAATTGAATGTCTGCACAGCCACTTTCCACACAGACATCATAACAAAAAATTT

............................S...............................

KLH016 ATAATAACAATTGAATGTCTGCACAGCCGCTTTCCACACAGACATCATAACAAAAAATTT

.....300.......310........320.......330.......340.......350.

▼ ▼ ▼ ▼ ▼ ▼

rCRS CCACCAAACCCCCCCTCCCCC─GCTTCTGGCCACAGCACTTAAACACATCTCTGCCAAAC

.....................I........................S.............

KLH016 CCACCAAACCCCCCCTCCCCCCGCTTCTGGCCACAGCACTTAAACATATCTCTGCCAAAC

......360.......370.......380.......390.......400.......410.

▼ ▼ ▼ ▼ ▼ ▼

rCRS CCCAAAAACAAAGAACCCTAACACCAGCCTAACCAGATTTCAAATTTTATCTTTTGGCGG

............................................................

KLH016 CCCAAAAACAAAGAACCCTAACACCAGCCTAACCAGATTTCAAATTTTATCTTTTGGCGG

......420.......430.......440.......450.......460.......470.

▼ ▼ ▼ ▼ ▼ ▼

rCRS TATGCACTTTTAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATAC

............................................................

KLH016 TATGCACTTTTAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATAC

45

......480.......490.......500.......510.......520.......530.

▼ ▼ ▼ ▼ ▼ ▼

rCRS TACTAATCTCATCAATACAACCCCCGCCCATCCTACCCAGCACACACACACCGCTGCTAA

..................................S.........................

KLH016 TACTAATCTCATCAATACAACCCCCGCCCATCCTGCCCAGCACACACACACCGCTGCTAA

......540.......550.......560.......570....

▼ ▼ ▼ ▼

rCRS CCCCATACCCCGAACCAACCAAACCCCAAAGACACCCCCCACA

...........................................

KLH016 CCCCATACCCCGAACCAACCAAACCCCAAAGACACCCCCCACA

Figure 4.5 (b): The haplotype of Kalashi individual (KLH016) for entire mtDNA control region.

The observed haplotype of KLH016 after the alignment of reconstructed sequence with rCRS

(16051G 16129C 16154C 16248T 16362C, 16519C, 73G, 152C, 217C, 263G, 315.1C, 340T,

508G) I: Insertion, S: Transition, V: Transversions.

4.5. Identification of Haplotypes and Assignment of Haplogroups

Based on identified differences from rCRS in both Makrani and Kalashi

populations, 70 different haplotypes (of which 54 unique haplotypes) in former and 14

different haplotypes (of which 5 unique haplotypes) in later were observed. In Makrani

population, 16 haplotypes were shared by more than one individual. However, only5

haplotypes were shared by more than one individual in Kalashi population. Based on

haplotypes of mtDNA control region, haplogroups were assigned to each individual in

both populations, which are shown in (Table 4.1 a and b). The four variant positions in

mtDNA control region including 309 C insertions, 315C insertions, 524 indels and 16519

sites were disregarded during haplogroup assignment. The reason for disregarding these

positions is that these regions are length polymorphisms, which are often difficult to

determine precisely by Sanger sequencing. Therefore, it was safer to exclude these. The

16519 site is straight forward to determine by sequencing but it is a hypermutable

position (mutational hotspot; evidenced by the fact that it is extremely recurrent in the

mtDNA phylogeny) and therefore of very little phylogenetic value in haplogroup

assignment.

However, two haplotypes observed in the Makranis, both carrying a characteristic

combination of two mutations in HVS-II (154C and 194T) could not be confidently

assigned to any known (sub) haplogroup, although the presence of both 16223T and

46

489C indicate membership within Macrohaplogroup M; this lineage was therefore

tentatively assigned to haplogroup ‘‘M-154-194’’.

Table 4.1a: The estimated haplotypes and haplogroups in Makrani population

Sr.

No.

Sample

ID H

ap

loty

pe

ID

Differences to the rCRS (309ins, 315ins, 524ins and 16519 were disregarded)

Hap

logro

up

GenBank

Accession

Numbers

1 MKH001 h57

16071T, 16093C, 16129A, 16145A, 16187T,

16189C, 16213A, 16223T, 16234T, 16265C,

16278T, 16286G, 16294T, 16311C, 16360T,

16527T, 73G, 151T, 152C, 182T, 186A, 189C,

195C, 198T, 247A, 263G, 297G, 316A

L1c2a1a KM358171

2 MKH002 h31 16209C, 152C, 263G R30a1b KM358172

3 MKH003 h19 16129A, 16189C, 16223T, 16249C, 16311C,

16359C, 16519C, 73G, 195C, 263G, 489C M1a1 KM358173

4 MKH004 h25 16182C, 16183C, 16189C, 16223T, 16278T,

16290T, 16294T, 16309G, 16390A, 73G, 146C,

152C, 195C, 263G L2a1b1a KM358174

5 MKH005 h11 16124C, 16223T, 16319A, 73G, 150T, 152C,

263G L3d1a1a KM358175

6 MKH006 h36 16243C, 16519C, 153G, 263G ? KM358176

7 MKH007 h25 16182C, 16183C, 16189C, 16223T, 16278T,

16290T, 16294T, 16309G, 16390A, 73G, 146C,

152C, 195C, 263G L2a1b1a KM358177

8 MKH008 h24 16172C, 16189C, 16192T, 16223T, 16292T,

16325C, 16519C, 73G, 146C, 189G, 194T, 195C,

204C, 207A, 263G W6 KM358178

9 MKH009 h40 16519C, 199C, 263G ? KM358179

10 MKH011 h63 16069T, 16093C, 16126C, 16145A, 16172C,

16222T, 16259T, 16261T, 73G, 146C, 242T,

263G, 295T, 462T, 489C J1b1a1 KM358180

11 MKH012 h41 16519C, 263G ? KM358181 12 MKH013 h4 16093C, 16189C, 263G ? KM358182

13 MKH014 h17 16126C, 16163G, 16186T, 16189C, 16294T,

16325C, 16519C, 73G, 152C, 195C, 263G T1a8a KM358183

14 MKH015 h21

16129A, 16148T, 16168T, 16172C, 16187T,

16188G, 16189C, 16223T, 16230G, 16278T,

16293G, 16311C, 16320T, 93G, 95C, 185A,

189G, 236C, 247A, 263G

L0a1b KM358184

15 MKH016 h30 16182C, 16183C, 16189C, 16192T, 16223T,

16278T, 16290T, 16294T, 16309G, 16390A,

73G, 146C, 152C, 195C, 263G L2a1b1a KM358185

16 MKH017 h30 16182C, 16183C, 16189C, 16192T, 16223T,

16278T, 16290T, 16294T, 16309G, 16390A, L2a1b1a KM358186

47

Sr.

No.

Sample

ID

Hap

loty

pe

ID


Hap

logro

up

GenBank

Accession

Numbers

73G, 146C, 152C, 195C, 263G

17 MKH018 h18 16126C, 16163G, 16186T, 16189C, 16294T,

16519C, 73G, 152C, 195C, 263G, 372C T1a1'3 KM358187

18 MKH019 h34 16243C, 16311C, 16519C, 204C, 263G ? KM358188

19 MKH020 h30 16182C, 16183C, 16189C, 16192T, 16223T,

16278T, 16290T, 16294T, 16309G, 16390A,

73G, 146C, 152C, 195C, 263G L2a1b1a KM358189

20 MKH021 h53 16192T, 16270T, 73G, 150T, 152C, 263G, 264T,

275A U5b KM358190

21 MKH022 h35 16243C, 16519C, 153G, 200G, 263G ? KM358191

22 MKH023 h37 16309G, 16318T, 16519C, 73G, 151T, 152C,

263G U7a KM358192

23 MKH024 h31 16209C, 152C, 263G R30a1b KM358193

24 MKH025 h30 16182C, 16183C, 16189C, 16192T, 16223T,

16278T, 16290T, 16294T, 16309G, 16390A,

73G, 146C, 152C, 195C, 263G L2a1b1a KM358194

25 MKH026 h58 16071T, 16172C, 16223T, 16293T, 16311C,

16355T, 16362C, 16399G, 16519C, 73G, 189G,

244G, 263G L4b2a2 KM358195

26 MKH027 h14 16126C, 16223T, 16311C, 16519C, 73G, 204C,

217C, 263G, 482C, 489C M3a1 KM358196

27 MKH028 h9 16114A, 16129A, 16213A, 16223T, 16278T,

16355T, 16362C, 16390A, 73G, 150T, 152C,

182T, 195C, 198T, 204C, 263G, 418T L2b1a KM358197

28 MKH029 h68 16051G, 16145A, 16206C, 73G, 152C, 263G U2a KM358198

29 MKH030 h25 16182C, 16183C, 16189C, 16223T, 16278T,

16290T, 16294T, 16309G, 16390A, 73G, 146C,

152C, 195C, 263G L2a1b1a KM358199

30 MKH031 h10 16124C, 16223T, 16294T, 16319A, 73G, 150T,

152C, 263G L3d1a1a KM358200

31 MKH032 h54 16188T, 16223T, 16231C, 16264T, 16362C,

16519C, 73G, 146C, 263G, 461T, 489C M6a1b KM358201

32 MKH033 h33 16209C, 16223T, 16311C, 16519C, 73G, 150T,

189G, 200G, 263G L3f1b4a KM358202

33 MKH034 h50 16220C, 16265G, 16298C, 16362C, 73G, 150T,

152C, 249d, 263G F3b1 KM358203

34 MKH035 h16 16126C, 16163G, 16186T, 16189C, 16274A,

16294T, 16519C, 73G, 263G, 512G, 519G T1a7 KM358204

35 MKH036 h50 16220C, 16265G, 16298C, 16362C, 73G, 150T,

152C, 249d, 263G F3b1 KM358205

36 MKH037 h11 16124C, 16223T, 16319A, 73G, 150T, 152C,

263G L3d1a1a KM358206

37 MKH038 h62 16071T, 16111T, 16278T, 16311C, 16519C, R2 KM358207

48

Sr.

No.

Sample

ID

Hap

loty

pe

ID


Hap

logro

up

GenBank

Accession

Numbers

73G, 150T, 152C, 263G

38 MKH039 h47 16223T, 16278T, 16286T, 16294T, 16309G,

16390A, 16519C, 73G, 146C, 152C, 195C, 263G L2a1a2 KM358208

39 MKH040 h37 16309G, 16318T, 16519C, 73G, 151T, 152C,

263G U7a KM358209

40 MKH041 h45 16223T, 16297C, 16318T, 16519C, 73G, 93G,

194T, 246C, 263G, 489C M18a KM358210

41 MKH042 h2 16092C, 16311C, 16356C, 263G, 550G ? KM358211 42 MKH043 h32 16209C, 16311C, 73G, 152C, 263G R30a1b KM358212

43 MKH044 h49 16223T, 16234T, 16249C, 16278T, 16294T,

16295T, 16390A, 73G, 146C, 152C, 195C, 263G L2a1 KM358213

44 MKH045 h64 16069T, 16126C, 16145A, 16222T, 16261T,

16295T, 16301T, 16519C, 73G, 263G, 271T,

295T, 462T, 489C J1b1b KM358214

45 MKH046 h31 16209C, 152C, 263G R30a1b KM358215

46 MKH047 h67 16069T, 16114T, 16126C, 16193T, 16519C,

73G, 152C, 263G, 295T, 462T, 489C J1d KM358216

47 MKH048 h37 16309G, 16318T, 16519C, 73G, 151T, 152C,

263G U7a KM358217

48 MKH049 h46 16223T, 16519C, 73G, 154C, 194T, 263G, 489C

M-154-

194 KM358218

49 MKH050 h65 16069T, 16126C, 16239T, 16366T, 16399G,

73G, 150T, 195C, 263G, 295T, 489C, 573.1C,

573.2C J2a2 KM358219

50 MKH052 h17 16126C, 16163G, 16186T, 16189C, 16294T,

16325C, 16519C, 73G, 152C, 195C, 263G T1a8a KM358220

51 MKH055 h17 16126C, 16163G, 16186T, 16189C, 16294T,

16325C, 16519C, 73G, 152C, 195C, 263G T1a8a KM358221

52 MKH056 h15 16126C, 16223T, 16311C, 16519C, 73G, 263G,

482C, 489C M3 KM358222

53 MKH057 h69 16051G, 16172C, 73G, 263G U2b1 KM358223 54 MKH058 h35 16243C, 16519C, 153G, 200G, 263G ? KM358224

55 MKH061 h15 16126C, 16223T, 16311C, 16519C, 73G, 263G,

482C, 489C M3

56 MKH062 h15 16126C, 16223T, 16311C, 16519C, 73G, 263G,

482C, 489C M3 KM358225

57 MKH063 h17 16126C, 16163G, 16186T, 16189C, 16294T,

16325C, 16519C, 73G, 152C, 195C, 263G T1a8a KM358226

58 MKH067 h41 16519C, 263G ? KM358227 59 MKH068 h38 16311C, 16519C, 93G, 150T, 263G ? KM358228

60 MKH069 h7 16093C, 16189C, 16192T, 16223T, 16278T,

16284G, 16294T, 16309G, 16390A, 73G, 143A, L2a1 KM358229

49

Sr.

No.

Sample

ID

Hap

loty

pe

ID


Hap

logro

up

GenBank

Accession

Numbers

146C, 152C, 195C, 263G 61 MKH071 h69 16051G, 16172C, 73G, 263G U2b1 KM358230

62 MKH072 h17 16126C, 16163G, 16186T, 16189C, 16294T,

16325C, 16519C, 73G, 152C, 195C, 263G T1a8a KM358231

63 MKH073 h20 16129A, 16223T, 16519C, 73G, 154C, 194T,

263G, 489C M-154-

194 KM358232

64 MKH074 h26 16182C, 16183C, 16189C, 16223T, 16278T,

16290T, 16294T, 16309G, 16390A, 73G, 146C,

152C, 195C, 263G, 498.1C, 526.1G, 573.1C L2a1b1a KM358233

65 MKH075 h69 16051G, 16172C, 73G, 263G U2b1 KM358234

66 MKH077 h25 16182C, 16183C, 16189C, 16223T, 16278T,

16290T, 16294T, 16309G, 16390A, 73G, 146C,

152C, 195C, 263G L2a1b1a KM358235

67 MKH078 h23 16145A, 16176T, 16223T, 16261T, 16311C,

16519C, 73G, 263G, 489C M4 KM358236

68 MKH079 h51 16214T, 16217C, 16335G, 16519C, 73G, 152C,

246C, 263G HV2a KM358237

69 MKH080 h23 16145A, 16176T, 16223T, 16261T, 16311C,

16519C, 73G, 263G, 489C M4 KM358238

70 MKH081 h41 16519C, 263G ? KM358239

71 MKH082 h70

16051G, 16114T, 16189C, 16192.1T, 16223T,

16293T, 16311C, 16316G, 16355T, 16362C,

16399G, 16519C, 73G, 146C, 152C, 195C,

244G, 263G, 340T

L4b2b1 KM358240

72 MKH083 h6 16093C, 16182C, 16183C, 16189C, 16223T,

16278T, 16290T, 16294T, 16390A, 73G, 146C,

152C, 195C, 263G L2a1b1a KM358241

73 MKH084 h39 16355T, 73G, 195C, 263G, 343T, 499A, 573.1C,

573.2C U4'9 KM358242

74 MKH085 h52 16214T, 16217C, 16335G, 16519C, 73G, 152C,

246C, 263G, 573.1C, 573.2C HV2a KM358243

75 MKH086 h48 16223T, 16278T, 16294T, 16309G, 16390A,

16519C, 73G, 146C, 152C, 195C, 263G L2a1 KM358244

76 MKH087 h27 16182C, 16183C, 16189C, 16210C, 16223T,

16278T, 16290T, 16294T, 16309G, 16390A,

73G, 146C, 152C, 195C, 263G L2a1b1a KM358245

77 MKH088 h13 16126C, 16270T, 16278T, 16294T, 16357C,

16519C, 73G, 150T, 153G, 263G U5b KM358246

78 MKH089 h66 16069T, 16126C, 16193T, 16266T, 16519C,

73G, 152C, 263G, 295T, 324G, 376C, 462T,

489C J1d KM358247

79 MKH090 h3 16092C, 16207G, 16309G, 16318T, 16519C,

73G, 151T, 152C, 263G U7a KM358248

50

Sr.

No.

Sample

ID

Hap

loty

pe

ID


Hap

logro

up

GenBank

Accession

Numbers

80 MKH091 h1 16086C, 16148T, 16184T, 16223T, 16259T,

16278T, 16319A, 16399G, 16526A, 73G, 150T,

200G, 263G, 489C M32c KM358249

81 MKH092 h43 16256T, 263G ? KM358250

82 MKH093 h43 16256T, 263G ? KM358251

83 MKH094 h8 16093C, 16189C, 16192T, 16223T, 16278T,

16294T, 16309G, 16390A, 16519C, 73G, 146C,

152C, 195C, 263G L2a1 KM358252

84 MKH095 h12 16126C, 16294T, 16296T, 16519C, 73G, 217C,

263G, 490G T2 KM358253

85 MKH096 h22 16145A, 16223T, 16234T, 16261T, 16311C,

16519C, 73G, 263G, 489C M4 KM358254

86 MKH097 h56

16148T, 16172C, 16187T, 16188G, 16189C,

16223T, 16230G, 16242T, 16311C, 16320T,

16519C, 64T, 93G, 152C, 189G, 204C, 207A,

236C, 247A, 263G

L0a2a2 KM358255

87 MKH098 h42 263G ? KM358256

88 MKH099 h49 16223T, 16234T, 16249C, 16278T, 16294T,

16295T, 16390A, 73G, 146C, 152C, 195C, 263G L2a1 KM358257

89 MKH100 h60 16071T, 16519C, 73G, 152C, 263G R2 KM358258

90 MKH101 h29 16183C, 16189C, 16207G, 16309G, 16318C,

16519C, 73G, 151T, 152C, 263G, 573.1C,

573.2C U7a KM358259

91 MKH102 h61 16071T, 16519C, 73G, 263G R2 KM358260

92 MKH103 h5 16093C, 16189C, 93G, 263G ? KM358261

93 MKH104 h28 16189C, 16207C, 16309G, 16318C, 16519C,

73G, 151T, 152C, 263G, 573.1C, 573.2C U7a KM358262

94 MKH105 h35 16243C, 16519C, 153G, 200G, 263G ? KM358263

95 MKH106 h44 16234T, 152C, 263G ? KM358264

96 MKH107 h31 16209C, 152C, 263G R30a1b KM358265

97 MKH108 h60 16071T, 16519C, 73G, 152C, 263G R2 KM358266

98 MKH109 h59 16071T, 16519C, 73G, 152C, 185A, 263G R2 KM358267

99 MKH112 h61 16071T, 16519C, 73G, 263G R2 KM358268

100 MKH113 h55 16179T, 16356C, 16519C, 73G, 195C, 228A,

263G, 499A U4c1 KM358269

The mtDNA control-region sequences herein reported are available in GenBank under given

accession numbers. Polymorphic sites have been assigned with numbers in accordance with the

revised Cambridge Reference Sequence (rCRS) (Andrews et al., 1999). Haplotypes arranged

according to assigned haplogroups.

51

Table 4.1b: Kalashi samples with estimated haplotypes and haplogroups

Sr.

No.

Sample

ID

Hap

loty

pe

ID


Hap

logro

up

GenBank

Accession

Number

1 KLH001 h13 16051G, 16129C, 16154C, 16248T, 16362C, 16391A,

16519C, 73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358270

2 KLH002 h12 16069T, 16126C, 16193T, 16274A, 16278T, 73G, 150T,

152C, 263G, 295T, 489C J2b1a KM358271

3 KLH003 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358272 4 KLH004 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358273 5 KLH005 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358274 6 KLH006 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358275 7 KLH007 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358276

8 KLH008 h13 16051G, 16129C, 16154C, 16248T, 16362C, 16391A,

16519C, 73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358277

9 KLH009 h11 16071T, 16111T, 16147T, 16203G, 16311C, 16519C,

73G, 150T, 263G R2 KM358278

10 KLH010 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358279 11 KLH011 h4 16356C, 16519C, 73G, 195C, 198T, 263G, 499A U4 KM358280 12 KLH012 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358281

13 KLH014 h14 16051G, 16129C, 16154C, 16248T, 16362C, 16519C,

73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358282

14 KLH015 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358283

15 KLH016 h14 16051G, 16129C, 16154C, 16248T, 16362C, 16519C,

73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358284

16 KLH018 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358285 17 KLH019 h7 16223T, 16289G, 16519C, 73G, 263G, 489C, 511T M65a KM358286

18 KLH020 h14 16051G, 16129C, 16154C, 16248T, 16362C, 16519C,

73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358287

19 KLH021 h8 16223T, 55.1T, 57C, 59C, 62T, 73G, 146C, 152C, 195C,

263G, 489C ? KM358288

20 KLH022 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358289

21 KLH023 h13 16051G, 16129C, 16154C, 16248T, 16362C, 16391A,

16519C, 73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358290


23 KLH026 h12 16069T, 16126C, 16193T, 16274A, 16278T, 73G, 150T,

152C, 263G, 295T, 489C J2b1a KM358292


25 KLH028 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358294 26 KLH 029 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358295

27 KLH030 h12 16069T, 16126C, 16193T, 16274A, 16278T, 73G, 150T,

152C, 263G, 295T, 489C J2b1a KM358296

52

Sr.

No.

Sample

ID

Hap

loty

pe

ID


Hap

logro

up

GenBank

Accession

Number

28 KLH031 h14 16051G, 16129C, 16154C, 16248T, 16362C, 16519C,

73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358297


30 KLH033 h11 16071T, 16111T, 16147T, 16203G, 16311C, 16519C,

73G, 150T, 263G R2 KM358299

31 KLH034 h12 16069T, 16126C, 16193T, 16274A, 16278T, 73G, 150T,

152C, 263G, 295T, 489C J2b1a KM358300

32 KLH035 h6 16240G, 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358301

33 KLH036 h13 16051G, 16129C, 16154C, 16248T, 16362C, 16391A,

16519C, 73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358302

34 KLH037 h14 16051G, 16129C, 16154C, 16248T, 16362C, 16519C,

73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358303


36 KLH039 h12 16069T, 16126C, 16193T, 16274A, 16278T, 73G, 150T,

152C, 263G, 295T, 489C J2b1a KM358305

37 KLH040 h13 16051G, 16129C, 16154C, 16248T, 16362C, 16391A,

16519C, 73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358306

38 KLH041 h12 16069T, 16126C, 16193T, 16274A, 16278T, 73G, 150T,

152C, 263G, 295T, 489C J2b1a KM358307


40 KLH045 h12 16069T, 16126C, 16193T, 16274A, 16278T, 73G, 150T,

152C, 263G, 295T, 489C J2b1a KM358309

41 KLH046 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358310 42 KLH047 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358311

43 KLH048 h12 16069T, 16126C, 16193T, 16274A, 16278T, 73G, 150T,

152C, 263G, 295T, 489C J2b1a KM358312


45 KLH050 h11 16071T, 16111T, 16147T, 16203G, 16311C, 16519C,

73G, 150T, 263G R2 KM358314

46 KLH051 h4 16356C, 16519C, 73G, 195C, 198T, 263G, 499A U4 KM358315 47 KLH052 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358316 48 KLH053 h3 16354T, 263G H2a1 KM358317 49 KLH054 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358318

50 KLH056 h13 16051G, 16129C, 16154C, 16248T, 16362C, 16391A,

16519C, 73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358319


52 KLH058 h12 16069T, 16126C, 16193T, 16274A, 16278T, 73G, 150T,

152C, 263G, 295T, 489C J2b1a KM358321


54 KLH060 h12 16069T, 16126C, 16193T, 16274A, 16278T, 73G, 150T,

152C, 263G, 295T, 489C J2b1a KM358323

55 KLH061 h12 16069T, 16126C, 16193T, 16274A, 16278T, 73G, 150T,

152C, 263G, 295T, 489C J2b1a KM358324

53

Sr.

No.

Sample

ID

Hap

loty

pe

ID


Hap

logro

up

GenBank

Accession

Number

56 KLH062 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358325 57 KLH063 h4 16356C, 16519C, 73G, 195C, 198T, 263G, 499A U4 KM358326

58 KLH064 h12 16069T, 16126C, 16193T, 16274A, 16278T, 73G, 150T,

152C, 263G, 295T, 489C J2b1a KM358327

59 KLH065 h4 16356C, 16519C, 73G, 195C, 198T, 263G, 499A U4 KM358328

60 KLH066 h13 16051G, 16129C, 16154C, 16248T, 16362C, 16391A,

16519C, 73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358329

61 KLH067 h4 16356C, 16519C, 73G, 195C, 198T, 263G, 499A U4 KM358330

62 KLH068 h12 16069T, 16126C, 16193T, 16274A, 16278T, 73G, 150T,

152C, 263G, 295T, 489C J2b1a KM358331

63 KLH069 h4 16356C, 16519C, 73G, 195C, 198T, 263G, 499A U4 KM358332

64 KLH070 h12 16069T, 16126C, 16193T, 16274A, 16278T, 73G, 150T,

152C, 263G, 295T, 489C J2b1a KM358333

65 KLH071 h4 16356C, 16519C, 73G, 195C, 198T, 263G, 499A U4 KM358334

66 KLH072 h13 16051G, 16129C, 16154C, 16248T, 16362C, 16391A,

16519C, 73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358335


68 KLH074 h11 16071T, 16111T, 16147T, 16203G, 16311C, 16519C,

73G, 150T, 263G R2 KM358337

69 KLH075 h13 16051G, 16129C, 16154C, 16248T, 16362C, 16391A,

16519C, 73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358338

70 KLH076 h12 16069T, 16126C, 16193T, 16274A, 16278T, 73G, 150T,

152C, 263G, 295T, 489C J2b1a KM358339


72 KLH078 h13 16051G, 16129C, 16154C, 16248T, 16362C, 16391A,

16519C, 73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358341

73 KLH079 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358342 74 KLH080 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358343 75 KLH081 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358344 76 KLH082 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358345 77 KLH083 h1 16354T, 16519C, 199C, 263G H2a1 KM358346

78 KLH084 h13 16051G, 16129C, 16154C, 16248T, 16362C, 16391A,

16519C, 73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358347

79 KLH085 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358348 80 KLH086 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358349 81 KLH087 h2 16354T, 16519C, 263G H2a1 KM358350

82 KLH088 h13 16051G, 16129C, 16154C, 16248T, 16362C, 16391A,

16519C, 73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358351

83 KLH089 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358352 84 KLH090 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358353 85 KLH091 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358354 86 KLH092 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358355 87 KLH093 h12 16069T, 16126C, 16193T, 16274A, 16278T, 73G, 150T, J2b1a KM358356

54

Sr.

No.

Sample

ID

Hap

loty

pe

ID


Hap

logro

up

GenBank

Accession

Number

152C, 263G, 295T, 489C 88 KLH094 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358357 89 KLH095 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358358 90 KLH097 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358359 91 KLH098 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358360 92 KLH099 h2 16354T, 16519C, 263G H2a1 KM358361 93 KLH100 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358362 94 KLH102 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358363

95 KLH103 h14 16051G, 16129C, 16154C, 16248T, 16362C, 16519C,

73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358364

96 KLH104 h14 16051G, 16129C, 16154C, 16248T, 16362C, 16519C,

73G, 152C, 217C, 263G, 340T, 508G U2e1 KM358365

97 KLH105 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358366 98 KLH106 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358367 99 KLH107 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358368 100 KLH109 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358369 101 KLH110 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358370 102 KLH111 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358371

103 KLH112 h11 16071T, 16111T, 16147T, 16203G, 16311C, 16519C,

73G, 150T, 263G R2 KM358372

104 KLH113 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358373 105 KLH114 h9 16134T, 16356C, 16519C, 73G, 152C, 195C, 263G, 499A U4 KM358374 106 KLH115 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358375 107 KLH116 h10 16071T, 16519C, 16527T, 73G, 152C, 263G R2 KM358376 108 KLH117 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358377 109 KLH118 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358378 110 KLH119 h5 16362C, 16519C, 58C, 60.1T, 64T, 263G R0a'b KM358379 111 KLH121 h10 16071T, 16519C, 16527T, 73G, 152C, 263G R2 KM358380

The mtDNA control-region sequences herein reported are available in GenBank under given

accession numbers. Polymorphic sites have been assigned with numbers in accordance with the

revised Cambridge Reference Sequence (rCRS) (Andrews et al., 1999). Haplotypes were arranged

according to assigned haplogroups.

55

4.6 Differences Observed in Haplogroups Estimation Either Manually

(PhyloTreemt mtDNA tree Build 16) or by HaploGrep

During the haplogroup determination of Makrani samples, the discrepancies have

been observed between HaploGrep estimation and manual calls in 22 haplogroups out of

total 38 haplogroup found. Furthermore, 8 haplogroup calls by HaploGrep could not be

confirmed manually and remained unassigned as shown in (Table 4.2a). However, there

were total seven haplogroups found in the Kalash population and haplogroup estimation

was done manually, due to discrepancies in all haplogroup calls by HaploGrep as shown

in (Table 4.2b). Therefore, there may be a potential space to improve haplogroup

estimation by HaploGrep according to the observation of this study.

Table 4.2a: Differences observed in haplogroup estimation of Makrani population either

manually (PhyloTreemt mtDNA tree Build 16) or by HaploGrep

Sample ID

Haplogroup

Estimation by

HaploGrep

Manual

Haplogroup

Determination

MKH006, MKH022, MKH058, MKH105 H13a1b ?

MKH009, MKH012, MKH067, MKH081,

MKH098 H2a2a ?

MKH013, MKH103 H1f+16093 ?

MKH019 H1e1a4 ?

MKH027 M3a1+204 M3a1

MKH042 H1b ?

MKH044 L2a1+143+@.. L2a1

MKH049 D4b2b M-154-194

MKH057 U2 U2b1

MKH068 H1e1a1 ?

MKH069 L2a1b+143 L2a1

MKH071 U2 U2b1

MKH073 M5 M-154-194

MKH075 U2 U2b1

MKH084 U9b1 U4'9

MKH088 T U5b

MKH092, MKH093 H1e5 ?

MKH094 L2a1+16189 L2a1

MKH099 L2a1+143+@.. L2a1

MKH101 U8c U7a

MKH104 U8c U7a

MKH106 H13a1d ?

56

Table 4.2 b: Differences observed in haplogroup estimation of Kalashi population either

manually (PhyloTreemt mtDNA tree Build 16) or by HaploGrep

Samples IDs.

Haplogroup

estimation by

HaploGrep

Manual

Haplogroup

Determination

KLH001, KLH008, KLH014, KLH016, KLH020,



KLH078, KLH084, KLH088, KLH103, KLH104

U2e1h U2e1






KLH118, KLH119

H57 R0a'b

KLH019 M65a+@16311 M65a

KLH021 M24 ?

KLH022, KLH 029, KLH032, KLH046,




KLH106, KLH107, KLH109, KLH113, KLH114

U4a1 U4

KLH024, KLH038, KLH052, KLH062, R0a+60.1T R0a'b

KLH035 H1j8 R0a'b

4.7 The Haplogroups Diversity within Sub-ethnic group of Kalash

Population

The Kalash maternal sub-ethnicity and their relevant haplogroups were sorted out

to see the haplogroup diversity in each maternal sub-ethnic group of Kalash as shown in

(Table 4.3).

57

Table 4.3: The haplogroup diversity in each maternal sub-ethnic group of Kalash

Maternal Sub-ethnicity

Haplogroups

Total number

of samples/

sub-ethnic

group

Al’ukSher R2, U4 2

Al’ukShernawaw R0a'b 1

Aspan’i R0a'b 2

Babura dari U4, H2a1 4

Bagalie J2b1a, R0a'b, U4 7

Bal’o’e R0a'b, H2a1, U4, U2e1 9

Baramuk R0a'b 1

Bazik J2b1a, U2e1, R0a'b 4

Budadari R0a'b 1

Bulasing U4, U2e1, R0a'b 4

Bumboor J2b1a, U2e1 2

Chagans’eynawaw J2b1a 1

Chagansey U4 1

Damunawaw U4 2

Damudari U4 1

Dhrames J2b1a, U4, R0a'b, R2 9

Gil’asur R0a'b, U2e1 2

Jaro’e’ R0a'b, U2e1 2

L’agay M65a 1

L’atharuk U2e1, H2a1, R0a'b, U4 13

Mahadari R2, U4, U2e1 3

Mutimir R2, J2b1a, R0a'b 4

Ohramsh J2b1a 1

Quresh ? 1

Raja way R0a'b, J2b1a, U2e1 4

Rashmukdari R0a'b, U4, H2a1 4

RumoNowaw U4 1

Sharakat J2b1a, U4, U2e1, R0a'b 5

Sharey U4, R2, R0a'b 4

Tharariek U2e1, U4, R0a'b, R2 8

Wakoke U2e1, U4, R0a'b, J2b1a, R2 7

58

4.8. Frequency of mtDNA Haplogroups

The frequencies of mtDNA haplogroups in Makrani and Kalashi populations were

calculated. The most frequent haplogroup observed in Makrani population was L2a1b1a

(a southeastern African haplogroup found mostly in Mozambique) carried by 11% of the

samples and R0a'b haplogroup was carried by 28.8% of Kalashi was found to be the most

frequent haplogroup. In Makrani population, 17% of mtDNA profiles could not be

confidently assigned to any known haplogroup. However, only 1% of the mtDNA profile

remained unassigned to any haplogroup in Kalashi population. The frequencies of

mtDNA haplogroups for both Makrani and Kalashi populations are summarized in pie

charts (Fig.4.6 a and b) respectively.

Figure 4.6 (a): Graphical illustration of frequencies of mtDNA based haplogroups in Makrani population.

L2a1b1a

11%

L2a1

5%

L2b1a

1%

L3d1a1a

3%

L3f1b4a

1%

L2a1a2

1%

L0a2a2

1%

L1c2a1a

1% L0a1b

1%

L1c2a1a

1%

L4b2b1

1%

M1a1

1%

R2

6%

R30a1b

5%

M3

3%

M3a1

1%

M4

3%

M6a1b

1% M18a

1% U2a

1%

U2b1

3%

U7a

6% U5b

2%

U4c1

1%

U4'9

1%

T1a8a

5% T1a7

1%

T2

1%

T1a1'3

1%

J1b1b

1%

J2a2

1%

J1b1a1

1%

J1d

2%

HV2a

2%

W6

1%

F3b1

2%

M-154-194

2%

?

17%

M32c

1%

59

Figure 4.6 (b): Graphical illustration of frequencies of mtDNA-based haplogroups in Kalashi population.

4.9. The Construction of Median Joining (MJ) Networks

The median joining (MJ) networks was plotted from all control region haplotypes

to reveal the possible relationships among haplotypes in Makrani and Kalashi

populations, which are shown in figure 4.7 a & b respectively. The haplotypes of

Makranis including (h43, h41, h40, h36, h35, h32, h5 and h4) clustered well with rCRS

haplotype in the middle of network with highest frequency of h44 haplotype. The

substantial divergence was observed among haplotypes in the population with several

independent branches including in major haplotypes, h25 and h30, in the first branch,

major haplotypes, h15 and h28, in the second branch and major haplotypes, 2 and h17, in

the third branch. The median joining network analyses of Kalash population has shown a

considerable divergence between haplotypes. This network shows a large number of

independent branches giving rise to many sub-branches that are separated by several

mutations.

60

Figure 4.7 (a): Median-joining haplotype network of the Makrani population (70

haplotypes). Mutations 309.1C, 309.1CC, 315.1C, 16182C, 16183C, 16519C, as well

as length variation in the AC stretch spanning pos. 515-524, were ignored for network

construction. The sizes of circlesare proportional to the number of respective

haplotypes and branch lengths are proportional to nucleotide changes.

61

Figure 4.7 (b): Median-joining haplotype network of the Kalashi population (14 haplotypes) Mutations 309.1C, 309.1CC, 315.1C, 16182C, 16183C, 16519C, as well as length

variation in the AC stretch spanning pos. 515-524, were ignored for network

construction. Circle sizes are proportional to the number of mtDNAs with that haplotype

and branch lengths are proportional to nucleotide changes.

4.10. The Occurrence and Distribution of Nucleotide Variations in

mtDNA Control Region

After comparing sequence profile of Makrani individuals with rCRS, 149 variable

sites were observed in the entire mtDNA control region. The distribution of polymorphic

sites across the mtDNA control region of Makrani samples clearly shows that control

region of the human mitochondrial DNA is a highly polymorphic region. Three

hypervariable segments have been described, HVSI (np16024– 16365), HVSII (np73–

340) and HVSIII (np438-576). The highest density of polymorphic sites was obtained for

HVSI, which contains 86 variable positions in total length of 342 bp (25.14 %), HVSII

displays 39 mutable sites in 268 bp (14.55%), and HVSIII exhibits a comparatively lower

variability with 13 polymorphic sites in 137 bp (9.48%). In contrast, the segment located

between HVSI &HVSII (np16366–16569, 1–72) shows 7 polymorphic sites in 275 bp

(2.54%) and the other segment located between HVSII & HVSIII (np341–437) deviate

62

from rCRS at 4 positions in 98 bp (4.08%) (Table 4.4a).

A comparison of sequence data of Makrani population with the rCRS in the

region studied here revealed nucleotide substitutions; insertions or deletions had taken

place. Sequence deviations caused by nucleotide substitutions predominate over

insertions and deletions. Transitions make up the majority of the nucleotide substitutions.

Transversions insertions and deletions were observed with significantly lower frequency.

This excessive amount of transitions may indicate that mispairing during replication is

the major source of spontaneous mutations in mitochondrial DNA. Among the

transitions, pyrimidine substitutions are predominantly found and T–C transitions occur

with particular high frequency.

After comparing sequence profiles of Kalashi individuals with rCRS, 47 variable

sites were observed in the entire mtDNA control region. The distribution of polymorphic

sites across the mtDNA control region of Kalashi individuals clearly shows that control

region of the human mitochondrial DNA is a highly polymorphic region. Three

hypervariable segments have been described, HVSI (np16024– 16365), HVSII (np73–

340) and HVSIII (np438-576). The highest density of polymorphic sites was obtained for

HVSI, which contains 21variable positions in total length of 342 bp (6.1%), HVSII

displays 11 mutable sites in 268 bp (4.1%), and HVSIII exhibits a slightly lower

variability with 4 polymorphic sites in 137 bp (2.91%). In contrast, the segments located

between HVSI &HVSII (np16366–16569, 1–72) show 11 polymorphic sites in 275 bp (4

%) and the other segment located between HVSII & HVSIII (98 bp; np 341–437) did not

show any deviation from rCRS (Table 4.4b).

A comparison of sequence data of Kalashi population with the rCRS in the region

studied here revealed nucleotide substitutions; insertions or deletions had taken place.

Sequence deviations caused by nucleotide substitutions predominate over insertions and

transversions. Furthermore no deletion was observed. Transitions make up the majority

of the nucleotide substitutions. Transversions and insertions were observed with

significantly lower frequency. This excessive amount of transitions may indicate that

mispairing during replication is the major source of spontaneous mutations in

mitochondrial DNA. Among the transitions, pyrimidine substitutions were predominant

and C–T transitions occurred with particular high frequency.

63

Table 4.4a: The occurrence and distribution of nucleotide variations in the entire mtDNA

control region of Makrani population

Mutation

type

HVSI

(np16024-

16365)

Segment

between HVS1

& HVSII

(np16366-

16569, 1-72)

HVSII (np73-340)

Segment

between

HVSII&

HVSIII

(np341-437)

HVSIII (np438-574)

Nu

mb

er o

f

po

siti

on

s

To

tal

nu

mb

er

of

mu

tati

on

s

Nu

mb

er o

f

po

siti

on

s

To

tal

nu

mb

er

of

mu

tati

on

s

Nu

mb

er o

f

po

siti

on

s

To

tal

nu

mb

er

of

mu

tati

on

s

Nu

mb

er o

f

po

siti

on

s

To

tal

nu

mb

er

of

mu

tati

on

s

Nu

mb

er o

f

po

siti

on

s

To

tal

nu

mb

er

of

mu

tati

on

s

Substitutions

Transitions Py-Py

C–T 37 186 3 3 10 37 2 2 2 5

T–C 20 124 1 57 9 120 1 1 1 4

Pu-Pu

A–G 10 43 1 4 8 200 0 0 3 3

G–A 5 19 2 19 7 11 0 0 1 2

Total 72 372 7 83 34 368 3 3 7 14

Transversions Pu-Py

A–C 8 31 0 0 2 2 1 1 1 18

G–C 0 0 0 0 0 0 0 0 0 0

A–T 2 7 0 0 0 0 0 0 0 0

G–T 0 0 0 0 0 0 0 0 0 0

Py-Pu

C–A 1 1 0 0 1 1 0 0 0 0

C–G 2 3 0 0 1 1 0 0 1 1

T–A 0 0 0 0 0 0 0 0 0 0

T–G 0 0 0 0 0 0 0 0 0 0

Total 13 42 0 0 4 4 1 1 2 19

Insertions

C 0 0 0 0 0 0 0 0 3 12

G 0 0 0 0 0 0 0 0 1 1

T 1 1 0 0 0 0 0 0 0 0

AC 0 0 0 0 0 0 0 0 0 0

CA 0 0 0 0 0 0 0 0 0 0

Total 1 1 0 0 0 0 0 0 4 13

Deletions

-G 0 0 0 0 0 0 0 0 0 0

-A 0 0 0 0 1 2 0 0 0 0

-C 0 0 0 0 0 0 0 0 0 0

-T 0 0 0 0 0 0 0 0 0 0

Total 0 0 0 0 1 2 0 0 0 0

Abbreviations: HVSI: Hypervariable Segment I, HVSII: Hypervariable Segment II, HVSIII:

Hypervariable Segment 3, Py: pyrimidine-base,Pu: purine-base, A: adenine, C: cytosine, G: guanine, T:

thymin

64

Table 4.4b: The occurrence and distribution of nucleotide variations in the entire

mtDNA control region of Kalashi population

Mutation

type

HVSI

(16024bp-

16365bp)

Segment

between HVSI

& HVSII

(16366bp-72bp)

HVSII

(73bp-340bp)

Segment

between

HVSII&

HVSIII

(341bp-437bp)

HVSIII

(438-574)

Nu

mb

er o

f

po

siti

on

s

To

tal

nu

mb

er o

f

mu

tati

on

s

Nu

mb

er o

f

po

siti

on

s

To

tal

nu

mb

er o

f

mu

tati

on

s

Nu

mb

er o

f

po

siti

on

s

To

tal

nu

mb

er o

f

mu

tati

on

s

To

tal

nu

mb

er o

f

mu

tati

on

s

Nu

mb

er o

f

po

siti

on

s

Nu

mb

er o

f

po

siti

on

s

To

tal

nu

mb

er o

f

mu

tati

on

s

Substitutions

TransitionsPy-Py

C–T 10 114 2 34 4 61 0 0 1 1

T–C 5 122 5 128 5 115 0 0 1 18

Pu-Pu

A–G 4 26 2 196 0 0 1 19

G–A 1 16 1 12 0 0 0 0 1 31

Total 20 278 8 174 11 372 0 0 4 69

TransversionsPu-Py

A–C 0 0 0 0 0 0 0 0 0 0

G–C 1 19 0 0 0 0 0 0 0 0

A–T 0 0 0 0 0 0 0 0 0 0

G–T 0 0 1 1 0 0 0 0 0 0

Py-Pu

C–A 0 0 0 0 0 0 0 0 0 0

C–G 0 0 0 0 0 0 0 0 0 0

T–A 0 0 0 0 0 0 0 0 0 0

T–G 0 0 0 0 0 0 0 0 0 0

Total 1 19 1 1 0 0 0 0 0 0

Insertions

C 0 0 0 0 0 0 0 0 0 0

2C 0 0 0 0 0 0 0 0 0 0

3C 0 0 0 0 0 0 0 0 0 0

T 0 0 2 33 0 0 0 0 0 0

AC 0 0 0 0 0 0 0 0 0 0

CA 0 0 0 0 0 0 0 0 0 0

2CA 0 0 0 0 0 0 0 0

Total 0 0 2 33 0 0 0 0 0 0

-G 0 0 0 0 0 0 0 0 0 0

-A 0 0 0 0 0 0 0 0 0 0

-C 0 0 0 0 0 0 0 0 0 0

-CA 0 0 0 0 0 0 0 0 0 0

Total 0 0 0 0 0 0 0 0 0 0

Abbreviations: HVSI: Hypervariable Segment 1, HVSII:Hypervariable Segment 2, HVSIII: Hypervariable

Segment 3, Py: pyrimidine-base,Pu: purine-base,A: adenine, C: cytosine, G: guanine, T: thymine

65

4.11. Heteroplasmy

4.11.1. Point Heteroplasmy

Point heteroplasmy was observed at 5 different positions (16497A/G, 199C/T,

16168C/A, 16173C/A, 16249T/A) accounting 13% of the individuals in the Makrani

population (Table 4.5). The point heteroplasmy observed at the different positions in

Makrani population is shown in figure 4.8 (MKH009, MKH010, MKH029, MKH026).

Moreover, only one individual (MKH026) presented more than one point heteroplasmy at

positions 16168bp & 16173bp in HVSI of control region in this population Fig. 4.8.

In Kalashi population, a significant number of samples showed the sequence

heteroplasmy at 6 different positions (199C/T, 16168C/A, 16169C/A, 297A/C, 412G/A,

16295C/T) accounting for 58.56% of the individuals (Table 4.5). The point heteroplasmy

observed at the different positions in Kalashi population are shown in figure 4.8 fig. 4.9

KLH 025, KLH121, KLH007, KLH112, KLH052, KLH119, KLH066 & KLH087. In

this population, three individuals (KLH007, KLH011, KLH081) presented more than one

point heteroplasmy in HVSI, HVSII segments of control region as shown in fig. 4.9.

Table 4.5: Point heteroplasmy in the Makrani and the Kalashi populations

Sr. No. Heteroplasmic

positions Symbol

No. of samples

Kalashi Makrani

1 16497 A/G R 0 1

2 199 C/T Y 2 2

3 16168 C/A M 58 8

4 16173 C/A M 0 1

5 16169 C/A M 3 0

6 297 A/C M 3 0

7 412 G/A R 1 0

8 16295 C/T Y 1 0

9 16249T/A W 0 1

66

Figure 4.8: Point heteroplasmy observed at different positions of mtDNA control region in the

Makrani population. rCRS; revised Cambridge Reference Sequence

67

Figure 4.9: Point heteroplasmies observed at different positions of mtDNA control region in the

Kalashi population. rCRS; revised Cambridge Reference Sequence.

68

4.11.2. Length Heteroplasmy

In Makrani population, length heteroplasmy was observed in all samples as shown

in (Table 4.6). In this case, one length heteroplasmy was observed in each individual (100

individuals), and two of them possessed more than one length heteroplasmy. The

segments of mtDNA control region depicting length heteroplasmy are shown in (Table

4.6). The length heteroplasmy were observed in the common poly-C tracts with highest

frequency (100%) in HVS II (between positions 303–315) of the control region. The

length heteroplasmy in the common poly-C tracts in HVS II are shown in two Makrani

individuals as an example in fig. 4.10. Moreover, two individuals presented length

heteroplasmy in the common poly-C tracts in different segments of mtDNA control

region (HVSI: np16184-16193 & HVSII: np303-315). The length heteroplasmy in the

common poly-C tracts of a Makrani individual found in two different regions such as

HVSI and HVSII is shown as an example in fig. 4.10.

Similarly, length heteroplasmy of poly-C tracts in HVS II (between positions

303–315) of the control region were observed in all Kalashi individuals (n=111). Out of

111 individuals, only 16 individuals showed heteroplasmy with poly-C in HVSI (16184-

16193) and 23 individuals with poly-AC in HVSIII (np514-525) in addition to poly-C

tract of HVSII (np303-315) (Table 4.6).

During the sequencing of mtDNA control region, it was observed that the

homopolymeric patterns of length heteroplasmy (poly-C types) affected the quality of

sequencing profiles. In sequencing results, different kinds of homopolymeric C patterns

like 7+6C (i.e. 7+6 C means that C repeated seven times interrupted by one T and then 6

more Cs), 8+6C and 9+6C were observed. All the samples with 7+6 C produced good

quality sequencing chromatograms. However, homopolymer 8+6 C and 9+6 C produced

noise after poly-C stretch as shown in figure 4.10. The quality of sequencing for such

homopolymeric patterns was improved by using additional sequencing primers close to

the poly-C stretches

69

Table 4.6: The length heteroplasmy distribution along the mtDNA control region of the

Makrani and Kalashi populations

Numbers of heteroplasmy and frequency

Makrani Kalashi

HVSI poly-C (16184–16193)

02 16

HVSII poly-C (303–315) 100 111

HVSIII

Poly-AC (514–525)

Poly-C (568–573)

00

00

23

00

Figure 4.10: Chromatograms showing the homopolymeric patterns of length heteroplasmy in the

Makrani Population

70

4.12. Comparison of Haplogroup Frequencies and Continental Origins

in Subpopulations of Pakistan

The haplogroup frequencies and continental origins observed in Makranis and

Kalashi during this study was compared with the other previously studied subpopulations

of Pakistan. The comparative analysis revealed that the most frequent haplogroup

observed in Makrani population is L2a1b1a (a southeastern African haplogroup found

mostly in Mozambique) showing a high degree of genetic association with southeast

Africans. In this study, African haplogroups (28%) including L2a1b1a, L2a1, L3d1a1a,

L2ba1, L3f1b4a, L4b2a2, M1a1, L0a1b, L1c2a1a, L4b2b1, L2a1a2 and L0a2a2; West

Eurasian haplogroups (26%) including U7a, T1a8a, U5b, J1d, HV2a, T1a7, U4c1, U4‘9,

J1b1b, W6, T2, T1a103, J2a2 and J1b1a1; South Asian haplogroups (24%) including R2,

R30a1b, M3, M4, U2b1, M3a1, M6a1b, M18a and U2a; and East Asian haplogroup,

F3b1 (1%), were observed. The remaining 20% mtDNA of the sampled individuals could

not be confidently assigned to a continental origin.

On the other hand, Western Eurasian mtDNA haplogroups (U4, U2e1, R0a’b, R2, H2a1

and J2b1a) were found to be most prominent in Kalash. Only one haplogroup (M65a)

found in Kalashi belongs to South Asian origin and one sample could not be assertively

assigned with any of the known sub-haplogroup and its origin. The haplogroups,

R0a’b&U4, were observed to be the most frequent haplogroups (28.8%, 27.9%

respectively) in Kalash, only U4 haplogroup, the least frequent in Pathans (0.9%) and

none of the individual belongs to R0a’b&U4 haplogroups in Makranis and Saraiki of

Pakistan. L2a1b1a is the most frequent (11%) haplogroup in Makranis and was not found

in Kalashi, Saraiki and Pathans (Table 4.7).

71

Table 4.7: The comparison of mtDNA haplogroups’ frequencies and their continental

origins among subpopulations of Pakistan

Haplogroups Haplogroup

Origin

Kalash

N=111 (Present study)

Makrani

N=100 (Present study)

Saraiki

N=85 (Hayatet al.,

2014)

Pathan

N=230

Rakha et

al., 2011)

n % n % n % n %

B4b1 0 0 0 0 0 0 1 0.4

C4a1 0 0 0 0 0 0 2 0.9

D4 0 0 0 0 0 0 4 1.7

D4a EA 0 0 0 0 1 1.1 0 0 D4j1a EA 0 0 0 0 1 2.3 0 0 D4j1b2 SEA 0 0 0 0 1 1.1 0 0

D4q 0 0 0 0 0 0 1 0.4

F3b1 EA 0 0 2 2 0 0 0 0 H WE 0 0 0 0 0 0 9 3.9

H1 0 0 0 0 0 0 2 0.9

H14a 0 0 0 0 0 0 1 0.4

H2a+152,16311 SWA 0 0 0 0 1 1.1 0 0 H2a1 WEA 4 3.6 0 0 0 0 1 0.4 H2a2a SWA 0 0 0 0 3 1.1 0 0

H2b 0 0 0 0 0 0 6 2.6

H5 0 0 0 0 0 0 2 0.9

H6 0 0 0 0 0 0 1 0.4

H7a 0 0 0 0 0 0 1 0.4

HV WEA 0 0 0 0 0 0 24 10.4 HV0 WE 0 0 0 0 0 0 1 0.4 HV2 WE 0 0 0 0 0 0 2 0.9 HV2a WEA 0 0 2 2 1 1.1 0 0 I WA 0 0 0 0 1 1.1 1 0.4

I1 0 0 0 0 0 0 4 1.7

J1 0 0 0 0 0 0 1 0.4

J1b 0 0 0 0 0 0 2 0.9

J1b1a 0 0 0 0 0 0 1 0.4

J1b1a1 WEA 0 0 1 1 0 0 0 0 J1b1b WEA 0 0 1 1 1 1.1 0 0 J1d WEA 0 0 2 2 0 0 0 0 J2a2 WEA 0 0 1 1 0 0 0 0

J2b 0 0 0 0 0 0 1 0.4

J2b1a WEA 16 14.4 0 0 0 0 0 0

K1a 0 0 0 0 0 0 4 1.7

K1a11 0 0 0 0 0 0 1 0.4

K2a5 0 0 0 0 0 0 1 0.4

L0a1b AF 0 0 1 1 0 0 0 0 L0a2a2 AF 0 0 1 1 0 0 0 0 L1c2a1a AF 0 0 1 1 0 0 0 0

72

L2a1 AF 0 0 5 5 0 0 0 0 L2a1a2 AF 0 0 1 1 0 0 0 0 L2a1b1a AF 0 0 11 11 0 0 0 0 L2b1a AF 0 0 1 1 0 0 0 0

L3d1a1a AF 0 0 3 3 0 0 0 0

L3e'i'k'x EEA/SA 0 0 0 0 3 2.3 0 0 L3f1b4a AF 0 0 1 1 0 0 0 0 L4b2b1 AF 0 0 1 1 0 0 0 0 M EE 0 0 0 0 0 0 5 2.2

M-154-194 0 0 2 2 0 0 0 0

M12a 0 0 0 0 0 0 2 0.9

M18 0 0 0 0 0 0 1 0.4

M18a SA 0 0 1 1 2 2.3 0 0 M1a1 AF 0 0 1 1 0 0 0 0

M25 0 0 0 0 0 0 1 0.4

M2a1a SA 0 0 0 0 1 1.1 0 0

M2a1a2 0 0 0 0 0 0 1 0.4

M3 SA 0 0 3 3 0 0 20 8.7 M30 SA 0 0 0 0 1 1.1 7 3 M30+16234 SA 0 0 0 0 1 1.1 0 0

M30b 0 0 0 0 0 0 1 0.4

M30c1 0 0 0 0 0 0 2 0.9

M32c 0 0 1 1 0 0 0 0

M37e 0 0 0 0 0 0 1 0.4

M3a1 SA 0 0 1 1 0 0 0 0 M3a1+204 SA/AF 0 0 0 0 1 1.1 0 0

M3c1 0 0 0 0 0 0 2 0.9

M4 SA 0 0 3 3 1 1.1 0 0

M4a 0 0 0 0 0 0 2 0.9

M4b1 0 0 0 0 0 0 3 1.3

M4b2 0 0 0 0 0 0 1 0.4

M5 EEA/SA 0 0 0 0 2 2.3 5 2.2

M5a1 0 0 0 0 0 0 4 1.7

M5a2a1a EEA/SA 0 0 0 0 1 3.5 0 0 M5b2 EEA/SA 0 0 0 0 1 1.1 0 0 M5c1 EEA/SA 0 0 0 0 10 11.7 0 0 M65a SA 1 0.9 0 0 0 0 0 0 M6a1b SA 0 0 1 1 0 0 0 0

M7b 0 0 0 0 0 0 1 0.4

N 0 0 0 0 0 0 1 0.4

N10a SEA 0 0 0 0 1 1.1 0 0

N1b 0 0 0 0 0 0 1 0.4

N5 0 0 0 0 0 0 1 0.5

Pre-M30e 0 0 0 0 0 0 4 1.7

Pre-R0a2, 3 0 0 0 0 0 0 4 1.7

R0a’b WEA 32 28.8 0 0 0 0 0 0 R2 WEA 7 6.3 6 6 2 2.3 4 1.7

73

R30 0 0 0 0 0 0 1 0.4

R30a 0 0 0 0 0 0 3 1.3

R30a1b SA 0 0 5 5 0 0 0 0 R31 SA 0 0 0 0 1 1.1 0 0

R5a1 0 0 0 0 0 0 1 0.4

R5a2 0 0 0 0 0 0 2 0.9

R5a2b 0 0 0 0 0 0 1 0.4

R6 0 0 0 0 0 0 2 0.9

R9 SA 0 0 0 0 1 1.1 0 0

T1 0 0 0 0 0 0 3 1.3

T1a 0 0 0 0 0 0 6 2.6

T1a1’3 WEA 0 0 1 1 0 0 0 0 T1a7 WEA 0 0 1 1 0 0 0 0 T2 WEA 0 0 1 1 0 0 3 1.3

T2b 0 0 0 0 0 0 3 1.3

T2c 0 0 0 0 0 0 2 0.9

T2c1b 0 0 0 0 0 0 1 0.4

U1a 0 0 0 0 0 0 1 0.4

U2 WEA/SA 0 0 0 0 1 1.1 1 0.4 U2+152 SA 0 0 0 0 2 1.1 0 0 U2a WEA 0 0 1 1 0 0 1 0.4 U2a1a WEA/SA 0 0 0 0 3 3.5 0 0 U2b WE 0 0 0 0 0 0 2 0.9 U2b1 WEA 0 0 3 3 0 0 0 0 U2b2 SA 0 0 0 0 8 9.4 5 2.2

U2c 0 0 0 0 0 0 5 2.2

U2c'd SA 0 0 0 0 2 2.3 0 0 U2e1 WEA 19 17.1 0 0 0 0 0 0 U4 WEA 31 27.9 0 0 0 0 2 0.9 U4’9 WEA 0 0 1 1 0 0 0 0

U4a2 0 0 0 0 0 0 1 0.4

U4a2a SA 0 0 0 0 2 1.1 0 0 U4c1 WEA 0 0 1 1 0 0 0 0 U5b WEA 0 0 2 2 0 0 1 0.4 U7 WEA/SA 0 0 0 0 6 7 26 11.3 U7a WEA 0 0 6 6 7 8.2 0 0

U8c SA 0 0 0 0 1 2.3 0 0

W 0 0 0 0 0 0 3 1.3

W3, 5 0 0 0 0 0 0 3 1.3

W5a 0 0 0 0 0 0 1 0.4

W6 WEA 0 0 1 1 11 12.9 3 1.3 X2 WA 0 0 0 0 1 1.1 0 0 X2d WA 0 0 0 0 1 1.1 0 0 Z EE 0 0 0 0 0 0 1 0.4

? 1 0.9 17 17 0 0 0 0

N, total number of individuals for each population; n, number of times haplogroup observed,

Major haplogroups and their frequencies are represented in bold italics.

74

4.13. Comparative Statistical Analyses of Different Pakistani

Subpopulations

The statistical parameters observed in Makrani and Kalashi population during this

study were compared with the other previously studied subpopulations of Pakistan. The

comparative analysis revealed that the Pathans population showed the highest power of

discrimination (0.9978), genetic diversity (0.993) and lowest random match probability

(0.0065) among the subpopulations of Pakistan such as Makrani, Kalashi and Saraiki.

However, Kalashi population observed to be the least diverse population, having genetic

diversity (0.8393), power of discrimination (0.832) and random match probability

(0.16824) among the present and previously studied subpopulations of Pakistan (Table

4.8).

Table 4.8: The comparison of diversity parameters estimated from the entire mtDNA

control region among subpopulations of Pakistan

Makrani

(Present study)

Kalashi (Present study)

Saraiki (Hayat et al., 2014)

Pathans (Rakha et al., 2011)

Sample size 100 111 85 230

Number of different

haplotypes

70 (of which

54 unique)

14 (of which 5

unique)

63 (of which 58

unique)

193 (of which 171

unique)

Polymorphic

Positions 149 47 140 215

Random Match

Probability 0.0408 0.1682 0.0542 0.0065

Power of

Discrimination 0.9592 0.832 0.9458 0.9978

Genetic Diversity 0.9688 0.8393 0.957 0.993

75

5-DISCUSSION

DNA serves as a molecular passport that tells us the story of early human’s

intercontinental movements. In addition to nuclear genome humans have a second

genome, the mitochondrial DNA (mtDNA) that follows a maternal mode of inheritance.

Random mutations add up to form familial signature after every few generations. So a

comparison of two samples of mtDNA with respect to mutations reveals ancestral origin

and degree of relationship (Radford, 2011; Hellenthal et al., 2014).

The present study reports the haplotype data of mtDNA control region (spanning

positions 16,024–16,569 and 1–576) including hypervariable segments (HVSI, HVSII

and HVSIII) for two isolated populations of Pakistan i.e. Makrani & Kalashi. The entire

mitochondrial DNA control region of 100 unrelated Makrani and 111 Kalashi individuals

were sequenced. In the Makrani data set, 149 polymorphic positions were observed

(Siddiqi et al., 2014) while 47 polymorphic positions were detected in Kalashi population

(Table 4.8).

Overall, the entire mtDNA control region sequence analyses revealed an

extremely high level of genetic diversity in the Makrani population (0.9688) with a high

number of unique haplotypes (54) (Table 4.8). These results are consistent with two other

studies involving Saraiki ethnic group (Hayat et al., 2014) and Pakistani-Karachi

(Quintana-Murci et al., 2004). However, the highest number of unique haplotypes has

been reported previously in Pathans but this was based on a larger sample size (n = 230).

Furthermore, the high number of unique haplotypes in the Pathans population is also

reflected in high genetic diversity (Rakha et al., 2011) among different ethnic groups of

Pakistan, closely followed by Hazara, Sindhi and Pakistani-Karachi (Quintana-Murci, et

al., 2004). The high genetic diversity in the Makrani population (0.9688) is comparable

to the other regional ethnic groups such as Iran such as Qashqais (0.996) Persians (0.999)

and Azeris (1.00) (Derenko et al., 2013). Similarly higher genetic diversities were also

observed in Tajiks from Tajikistan (Ovchinnikov et al., 2014) and Mansi populations

(Pimenoff et al., 2008).Moreover, Median Joining Network analysis showed the

substantial divergence among the haplotypes in Makrani population (Fig.4.7).

However, in the case of Kalash, low genetic diversity (0.875)was observed which

might be due to either low number of haplotypes (14: only 5 are unique) or due to

76

presence of shared haplotypes. For example; only two shared haplotypes including h5

(16362C, 16519C, T58C, 60.1T, 64T, 263G) and h9 (16134T, 16356C, 16519C, 73G,

152C, 195C, 263G, 499A) represent >50% of Kalash population. Furthermore, high

frequency of few haplogroups in Kalash further provided the evidence for low genetic

diversity. For example, two haplogroups including R0a’b and U4 constituted ~60% of

Kalash population which may be called as Kalash specific haplogroups (Fig. 4.6 b).The

extremely low genetic diversity has been reported in the Waorani population as a

consequence of genetic drift based on low population size (Beckerman et al., 2009;

Cardoso et al., 2012).Moreover, the least genetic diversity (0.00) by analyzing

hypervariable segment I (HVSI) of mtDNA has also been reported in Malbari population

from Thailand (Oota et al., 2005). Out of total population of 300 individuals of Malbari

population, only one haplotype have been found in analyzing HVSI of mtDNA of 58

individuals. The authors suggested exceptional display of poor gene pool of this

population might be the result of founder event or bottleneck. Therefore, low genetic

diversity in Kalash may be explained by genetic drift in the population due to either low

population size (Oota et al., 2005) orendogamy. Furthermore, Median Joining Network

analysis showed limited divergence among the haplotypes of Kalash (Fig. 4.7 b).

Furthermore, out of total seven different haplogroups found in Kalash, the diverse

haplogroup distribution was observed within each maternal sub-ethnic group (1-5

haplogroups/maternal sub-ethnic group) despite Kalash being isolated population (Table

4.3). However, few haplotypes were reported in all analyzed samples from two isolated

populations including Waorani (three haplotypes) and Malbari (one haplotype) in contrast

to our study (Beckerman et al., 2009; Cardoso et al., 2012). Therefore, the diversity

within the sub ethnic group of Kalash may be the indicator of their strict custom about

prohibition of marriages within their own sub-ethnic groups.

The present study revealed a strongly admixed mtDNA pool composed 28% of

African haplogroups (L2a1b1a, L2a1, L3d1a1a, L2ba, L3f1b4a, L4b2a2, M1a1, L0a1b,

L1c2a1a, L4b2b1, L2a1a2, L0a2a2), 26% of West Eurasian haplogroups (U7a, T1a8a,

U5b, J1d, HV2a, T1a7, U4c1, U4‘9, J1b1b, W6, T2, T1a103, J2a2, J1b1a1),24% of

South Asian haplogroups (R2, R30a1, M3, M4, U2b1, M3a1, M6a1b, M18a, U2a.The

remaining 20% mtDNA of the sampled individuals could not be confidently assigned a

77

continental origin (Table 4.1a).

Among the African haplogroups, L2 lineage was observed as dominant with

eighteen mtDNA sequences (18%) in the Makranis with no traces of this lineage in other

sub populations of Pakistan such as Pathans (Rakha et al., 2011), Saraiki (Hayat et al.,

2014) and Kalash (Present study). Among the L2 sub-clades, L2a1b1a was found to be

highly frequent (11%), L2a1 as a moderately frequent (5%), while remaining sub-clades

such as L2a1a2 and L2ba1 were found to be 1% each in the Makranis. In some earlier

investigations haplogroup L2a has been reported as the most frequent and widespread

mtDNA cluster (reaching over 40%) in different ethnic groups such as Tuareg, Mali, Fali,

Western Pygmies and Mozambique Bantu from Africa (Watson et al., 1997; Pereira et

al., 2001; Salas et al., 2002; Quintana-Murci et al., 2004; Coia et al., 2005). During

present study the haplotype (16189 16192 16223 16278 16294 16309 16390) of L2a1

haplogroup was observed in Makranis and this haplotype has been considered as most

extensive haplotype in pan-African individuals especially people belonging to Niger-

Congo family from West Africa, Afro-Asiatic family including the Hausa from North

Africa and the Niger-Congo family including the Bamileke from Central Africa (Ely et

al., 2006). However, very low frequency of L2a haplogroup has been reported in Persians

(1.11%) indicating least gene flow from Africa to Persia in contrast to Makranis. Thus,

identical mitochondrial haplotypes are often shared among ethnic groups with common

origin as seen in the case of Makranis and African populations (Derenko et al., 2013).

L3 lineage constituted a frequency of 4% in the Makranis with two sub-clades

including L3d1a1a (3%) and L3f1b4a (1%) and no evidence of this lineage was reported

in other subpopulations of Pakistan (Rakha et al., 2011; Hayat et al., 2014 Kalash:

Present study). L3 is more related to Eurasian haplogroups than to the most divergent

African clusters L1 and L2 (Meyer et al., 2001). L3 is the haplogroup from which all

modern humans outside of Africa were originated. In a recent study, the L3d1a1a has

been predominantly seen in Fulani people of West-Central Africa (John et al., 2014).

Another evidence of L3d was also found in Damara people of Africa but L3f was entirely

absent (Barbieri et al., 2014) in contrast to the Makranis which may suggest this genetic

contribution from southwestern African and some population of Cameron as frequency of

L3f has been reported >20% in both southwestern Africans and Cameroonians (Cerny et

78

al., 2009; Cerezo et al., 2011). The sub-clade L3f1b4a has also been reported in Himba

and Herero populations as well as several other Bantu-speaking populations from

Namibia and Angola of Africa (Barbieri et al., 2014). L3f1b6, found at 1% in Asturias

Spain, which diverged from African L3 lineages at least 10,000 years ago (Pardinas et al.,

2014). L3d has also been found(1.79%)in Qashqais people of Iran living in nearby areas

of Baluchistan (Makran Coast) which may further strengthen the suggestion of common

African genetic influence in both Makranis and Qashqais people as result of slave trade

from Africa (Derenko et al., 2013).

L0 lineage was observed in Makranis with two sub-clades including L0a1b (1%),

and L0a2a2 (1%) and was not observed in other subpopulations of Pakistan (Rakha et al.,

2011; Hayat et al., 2014; Kalash: Present study). L0 lineage has been predominantly

found in Khoisan mtDNA gene pool, which is more than 60% with either L0d or L0k

lineages in sub-Saharan Africa (Vigilant et al., 1991; Chen et al., 2000; Knight et al.,

2003; Tishkoff et al., 2007). However, Haplogroup L0a is the most prevalent in

Southeast African populations (25% in Mozambique) (Rosa et al., 2004). Moreover, L0a

has been reported with low frequency in some other ethnic groups including Balanta

(5%) and Guineans (1%) (Ridl et al., 2009). Particularly, L0a2a1 sub-clade is the most

prevalent sub-clade in central/West Africa and L0a2a2a, is mostly associated with Bantu-

speaking populations (Rito et al., 2013).

As far as L4 lineage is concerned it was observed in Makranis in the form of two

sub-clades including L4b2a2 (1%) and L4b2b1 (1%) and this lineage was not detected in

other subpopulations of Pakistan (Rakha et al., 2011; Hayat et al., 2014, Kalash: Present

study). The haplogroup L4 is a sister clade of L3, typically found in East and Northeast

Africa, with low frequencies (Watson et al., 1997; Krings et al., 1999; Kivisild et al.,

2004; Tishkoff et al., 2007; Castri et al., 2009; Rito et al., 2013). In some earlier studies,

the L4a motif has been found in Sudan (Salas et al., 2002) and is also frequent in

Tanzania, Amhara and Gurages from Ethiopia (Salas et al., 2002; Kivisild et al., 2004;

Gonder et al., 2006). Furthermore this haplogroup is most concentrated in the southern

tip of the Arabian Peninsula. Consistent with present study there is also evidence of low

frequency (1.8%) for this haplogroup in the Rio de Janeiro (Brazil) (Bernardo et al.,

2014).

79

Haplogroup L1c reaches its highest frequencies in West and Central Africa,

notably among the Mbenga Pygmy peoples (Quintana-Murci et al., 2008). Other

populations in which L1c is particularly prevalent include the Tikar (100%), Baka people

from Gabon (97%) and Cameroon (90%).Furthermore, it is also common in São Tomé

(20%) and Angola (16-24%), however, L1c2a1athe sub-clade of L1, was found in

Makranis with very low frequency (1%) during the present investigation.

The macro-haplogroup M, like its sibling N, is a descendant of haplogroup

L3.The geographical distribution of M and N are as a result of out of Africa migrations

and the subsequent colonization of the rest of the world. The highest frequencies of

macrohaplogroup M worldwide have been observed in Asia, specifically in Bangladesh,

India, Japan, Nepal, and Tibet, where it ranges from 60%-80% (Maruyama et al., 2003;

Rajkumar et al., 2005; Thangaraj et al., 2006).The sub clade haplogroup M1, which is the

only variant of macrohaplogroup M, has been reported in Africa (Metspalu et al., 2004).

Furthermore, India has shown several M lineages that may have appeared directly from

the root of haplogroup M. The percentage of individuals carrying this haplogroup in this

study was observed between 13.0to 55.4% for M1 and 11.0 to 68.0% for M1a1

(Rajkumar et al., 2005; winters, 2010). In the present study, very low frequency (1%) of

haplogroup M1a1 was observed in Makranis in contrast to some previous studies where

no evidence for this haplogroup was found in other subpopulations of Pakistan (Rakha et

al., 2011; Hayat et al., 2014, Kalash: Present study).

The Western Eurasian component was represented by haplogroups J, T, and U of

the macrohaplogroup R (van Oven and Kayser, 2008).Among the West Eurasian

haplogroups observed in Makranis, U haplogroup was predominant with 10 mtDNA

sequences (10%). Among the U sub-clades, U7a displays the highest percentage (6%) in

Makranis. A few sequences were assigned to other branches of U such as U5b (2%),

U4c1 (1%), and U4‘9 (1%) in Makranis.U5b has also been reported with very low

frequency (0.4%) in Pathans (Rakha et al., 2011). The sub-clade U5b2a has been

characterized by its presence in Poland, Slovakia and the Czech Republic showing its

Central European origin (Malyarchuk et al., 2010). The sub-clade U7a (having highest

frequency in U sub-clade) has also been found in Saraikis (8.7%), another ethnic group of

Pakistan (Hayat et al., 2014) and was not observed in Pathans (Rakha et al., 2011) and

80

Kalash (present study). The highest frequencies (up to 10%) of the haplogroup U7a have

been also registered in some Iranian populations and in Gujarat (over 12%), the

westernmost state of India (Quintana-Murci et al., 2004, Metspalu et al., 2004; Terreros

et al., 2011; Derenko et al., 2013). Haplogroup U7 has been reported in three

phylogenetic clusters based on its distribution either in Southwest Asian/Indian such as

U7a and U7c or European such as U7b. Haplogroup U7a has been reported in Tajiks with

frequency of 4.4% (Ovchinnikov et al., 2014), which is consistent with Makranis (Siddiqi

et al., 2014).

T lineage was observed in Makranis in the form of four sub-clades including

T1a8a (5%), T1a1'3 (1%), T1a7 (1%), and T2 (1%). This lineage was not found in

Saraikis (Hayat et al., 2014) and Kalash (Present study) of Pakistan. However, only T2

sub-clade of T lineage was observed in Pathans (1.3%) of Pakistan (Rakha et al., 2011),

in Indo-European-speaking Persians (7.18) and Azeris (18.2%) living in Iran (Derenko et

al., 2013). This lineage has been detected in Amhara and the Tigrai of Ethopia suggesting

its contribution may due to bidirectional migrations from Europe to Africa (Kivisild,

2004). The presence of T lineage in Makranis may support the idea of slave trade from

Africa to South Asia (India) as suggested previously (Kivisild, 2004). Two sub-clades of

T linage (8.8%), T1a1’3 and T2b, have been found in Tajiks with equal frequencies in

recent study (Ovchinnikov et al., 2014). Herein this study, the sequence similarity cannot

be ignored between the rare T1a1'3 (16126C, 16163G, 16186T, 16189C, 16294T,

16519C, 73G, 152C, 195C, 263G, 309.1C, 315.1C, 372C) in Makranis sample

(MKH018) and a sample from Region of Republican Subordination, (16126, 16163,

16186, 16189, 16294, 16519, 73, 152, 195, 263, 309.1C, 315.1C) of Tajikistan

(Ovchinnikov et al., 2014)that may suggest their common genetic origin.

The J lineages were found with moderate frequency (5%) in Makranis, having

sub-cladesJ1b1b (1%), J2a2 (1%), J1b1a1 (1%) and J1d (2%). The two sub-clades J1b1a

and J2a1a have been considered as indicators for the Near East towards Europe

expansions in the Late Glacial period. Furthermore, J2b1a has been suggested as an

exclusive mtDNA marker for Europeans (Pala et al., 2012). In a recent study,

haplogroups J1 has been found (2.2%) in Tajiks (Ovchinnikov et al., 2014). As a result of

northwest European expansion, J1b1a1a sub-clade has been found in Georgians and

81

Russians (Forster et al., 2004) similar to Makranis that may suggest their common

genetic origin. The present study reports HV2a (2%), which has been found in Saraikis

(1.1%) (Hayat et al., 2014) but not observed in Pathans (Rakha et al., 2011) and Kalash

(present study).

The south Asian component was comprised of twenty-four mtDNA sequences in

the Makranis and it has been suggested that the macrohaplogroups M and Rare South

Asian specific lineages (van Oven and Kayser, 2008). Macrohaplogroup R was

represented by eleven mtDNA sequences (11%) belonging to R2 (6%) and R30a1b (5 %)

in Makranis. The haplogroup R2 was found 6.3% in Kalash (present study), 2.3% in

Saraiki population (Hayat et al., 2014) and 1.7% in Pathans (Rakha et al., 2011).

Furthermore, R2 haplogroup has been reported 3.31% in Persians and 2.68% in Azeris

(Derenko et al., 2013). Haplogroup R2, which is concentrated in southern Pakistan and in

India, and is present at low frequencies in the most of adjacent regions, including the

Near East, the Caucasus, the Iranian Plateau, the Arabian Peninsula, and Central Asia

(Quintana-Murci et al., 2004; Al-Abri et al., 2012). The extensive sequencing of

complete mtDNAs from a large part of the Iranian Plateau led to the identification of

several highly divergent Qashqai lineages within the entire haplogroup R2 and revealed a

new Persian-specific sub-clade within haplogroup R2a (Derenko et al., 2013).

Furthermore, the subline ages of a south Asian autochthonous subhaplogroup of the

macrohaplogroup R including U2b1 (3%) and U2a (1%) was found in Makranis. The

other lineage of South Asian component, M was found 9% including M3 (3%), M4 (3%),

M3a1(1%) M6a1b (1%) and M18a (1%). The sublineageM18a (2.3%) and M4 (1.1%)

have been found in Sarakis (Hayat et al., 2014). Moreover, M3 (8.7%) has been reported

in Pathans (Rakha et al., 2011). Here in this study, the similarity of haplogroup deciding

nucleotides cannot be ignored between the rare lineage M3a1 (16126C, 16223T, 16311C,

16519C, 73G, 204C, 217C, 263G, 482C, 489C) in Makrani sample (MKH027) from

Turbat and a sample, (16126, 16223, 16519, 73, 204, 263, 315.1C, 482, 489) from Pamir,

Tajikistan (Ovchinnikov et al., 2014). Moreover, the presence of M haplogroup in Saudi

Arab gene pool also suggested gene flow from South Asia (Abu-Amero et al., 2007).

Two haplotypes observed in the Makrani, both carrying a characteristic

combination of two mutations in HVS-II (154C and 194T) could not be confidently

82

assigned to a known (sub) haplogroup, although the presence of both 16223T and 489C

indicate membership within macrohaplogroup M; this lineage was therefore tentatively

assigned to haplogroup ‘‘M-154-194’’. Future studies, performing complete mitogenome

sequencing, will be needed to elucidate the precise phylogenetic position of this lineage.

Another isolated population, Kalash, living in the three valleys namely,

Bumburet, Birrir and Rumbur of Chitral district was also analyzed for mtDNA-based

haplogrouping to determine their origin. The Kalash showed greater affinity with West

Eurasia comprising mainly of West Eurasian haplogroups (98.2%) includingR0a’b, U4,

U2e1, J2b1a, R2 and H2a1 (Table 4.1b).

Among West Eurasian haplogroups, highest frequency was observed for

haplogroup R0a’b (28.8%) in Kalash population (present study) and no evidence was

found in Makranis (Siddiqi et al., 2014), Pathans (Rakha et al., 2011) and Saraikis for

this haplogroup (Hayat et al., 2014). Similarly, its highest frequency (23%) was observed

in a previous study on Kalash population (Quintana-Murci, et al., 2004).In South Asia,

haplogroup R has been reportedin western and southern India (Maji et al., 2008).

Haplogroup R0 has been frequently reported in the Arabian Plate with its highest

frequency in Socotri (Yemen) i.e. 38% (Torroni et al., 2006, Cerny et al., 2009) and least

frequently observed in North Africa, the Horn of Africa, Anatolia, Iranian Plateau and

Dalmatia (Achilli et al., 2011). The R0a’b, node of R, has been observed in Italian

individuals. However, the greater frequency of macro-haplogroup R in the Arabian Plate

suggests that the origin of R0a is Arabian Peninsula (Abu-Amero et al., 2008).

MtDNA haplogroup U4 was found to be second most frequent haplogroup

(27.9%) in Kalash population and the haplogroup has not been observed in Makranis

(Siddiqi et al., 2014) Pathans (Rakha et al., 2011) and Saraikis (Hayat et al., 2014).

Similarly, U4 haplogroup was observed with the highest frequency of 34% in a previous

study on Kalashi population (Quintana-Murci et al., 2004). The association of U4

haplogroup has been observed with the remnants of ancient European hunting-gatherers

preserved in the indigenous populations of Siberia (Sarkissian et al., 2013) and with

Europeans having highest concentrations in Scandinavia and the Baltic states. The

isolated populations such as Mansi (16.3%) an endangered people of Russia (Derbeneva

et al., 2002) and the Ket people (28.9%) of the Yenisey River (born in the heart of

83

Mongolia and flows north, through Siberia, and on to the Arctic Ocean) (Derbeneva et

al., 2002) also showed high frequency of U4 haplogroup, which is consistent with present

study. U4 is an ancient mitochondrial haplogroup and is relatively rare in modern

populations (Malyarchuk et al., 2010) indicating ancient origin of Kalashi population.

Further evidence of U4 as Western Eurasian haplogroup has been found in the form of

relatively high frequencies (up to 18%) in Altai-Sayan region in Altaians and

Khakassians (Derenko et al., 2013). Therefore, the evidence of highest frequency of U4

haplogroup in the Kalash may suggest Western Eurasian influence in the population

(Quintana-Murci, et al., 2004).

Another West Eurasian haplogroup, U2e1, was found 17.1% in the Kalash

population but no evidence of this haplogroup, has been found in the Makranis (Siddiqi et

al., 2014) Pathans (Rakha et al., 2011) and Saraikis (Hayat et al., 2014) of Pakistan.

U2Haplogroup is most common in South Asia (Metspalu et al., 2004) but it is also found

in low frequency in Central and West Asia, as well as in Europe (Maji et al., 2008). The

U2i, the sub clade of U2, largely account for overall higher frequency in South Asians

(Indians) whereas sub cladeU2e, common in Europe, is entirely absent in the region.

These lineages diverged approximately 50,000-years-ago and very low maternal-line

gene flow has been observed between South Asia and Europe throughout this period

(Metspalu et al., 2004). In Siberia, the frequency of U2e had been reported as the average

frequency of about 0.9%, but in some Altaian populations (such as Altaian-Kizhi, Teleuts

and Telenghits) this haplogroup reached higher than the average frequencies (up to 3%)

(Duliket al., 2012). The higher frequency of haplogroupU2e1 found in Kalash population

may suggest its West Eurasian origin with contribution from Altaian population.

The haplogroup J2b1a (14.4 %) was observed in the Kalash population and this

haplogroup was not found in Makranis, Pathans (Rakha et al., 2011) and Saraikis (Hayat

et al., 2014) of Pakistan. The J haplogroup has been observed to be the most frequent in

haplogroup in the Near East (12%) followed by Europe (11%), Caucasus (8%) and North

Africa (6%). J1, a subgroup of J, is spread on the European continent accounting up to

four-fifths of the total while J2 is found more localized around the Mediterranean,

Greece, Italy/Sardinia and Spain. J1 haplogroup has been also reported in Tajiks (West

Eurasians) with a frequency of 2.2% (Ovchinnikov et al., 2014). J2 has been reported

84

with significant frequency (9%) in the Kalashi during the previous investigations

(Quintana-Murci, et al., 2004), which is consistent with the findings of this study. The

presence of significantly high frequency (14.4 %) of Western Eurasian lineage (J2b1a) in

Kalash supports the idea of genetic contribution of West Eurasians.

The moderate frequency (6.3%) for haplogroup R2 in Kalash people was

observed during present study. R2 haplogroup is an example of West Eurasian

haplogroup that has been found in other ethnic groups of Pakistan such as Makranis,

(6%) Pathans (1.7%) (Rakha et al., 2011) and Saraiki (2.3%) (Hayat et al., 2014). The

haplogroup R2 in Europe has been observed in few populations of Volga basin, Russia,

low frequencies in near and Middle East and India and is virtually absent elsewhere. The

phylogeographic analysis of R2 haplogroup has pointed out its presence in southern

Arabia, and Near East has been considered as a possible place of origin for R2 (Al-Abri

et al., 2012). High concentration of haplogroup R2has been reported in southern Pakistan

and in India, while low frequencies were observed in adjacent areas; the Near East, the

Caucasus, the Iranian Plateau, the Arabian Peninsula, and Central Asia. Another study of

Persian Peninsula provided the evidence for its possible origin from Iran as its sub clade

has been found mostly in southern Arabians and Persians. R2 haplogroup belongs to

Persian Peninsula as evidenced in previous studies that may possibly have Western

Eurasian influence due to conquest of Persia by Alexander the Great. Hence, the presence

of R2 haplogroup in Kalash may support the idea of greater genetic contribution of West

Eurasians in Kalash (Hellenthal et al., 2014).

Low frequency (3.6%) for haplogroup H2a1 in Kalash people was observed

during this study. H2a1 haplogroup is another example of West Eurasian haplogroup that

is absent in Makranis, and Saraiki (Hayat et al., 2014) of Pakistan. However, H2a1 has

been reported in Pathans although in very low frequency (0.7%) (Rakha et al., 2011). It

has been reported in previous studies that the likely origin of haplogroup H is in

Southwest Asia (Achilli et al., 2004). In a recent study on Tajiks, haplogroup H was

reported as predominant haplogroup with a frequency of 23.1%. Furthermore, H2 a sub-

clades of haplogroup H displayed the highest percentage (4.4%) among Tajiks

(Ovchinnikov et al., 2014) consistent with the findings of this study. According to

(Ovchinnikov et al., 2014) the majority of Minoans (Greece) were classified in

85

haplogroup H (43.2%). Moreover none of the Minoans carried the African haplogroup

such as L haplogroup (Jeffery et al., 2013), which is consistent with the population data

of Kalash.

CONCLUSION

1) Makrani population

The high frequency of African mtDNA haplogroups such as L2, L1, L3, and L0 in

Makrani population shows their origin with major genetic contribution from

Mozambique Bantu from southeastern Africa and Fulani people of West-Central

Africa. This may be the result of strong slave-trade connection between the

Makran ports of Gwadar, the principal ports of Oman during the Omani Empire.

The African component in the Makrani community may therefore represent the

genetic gift of this slave trade.

The presence of West Eurasian lineages such as U7a, T and J in the Makranis

similar to Irani, Tajiks and west most Indians may suggest their common genetic

contribution and may indicate their likely origin to be within West Eurasia.

Another major South Asian contribution including M and R lineages were

observed in Makranis, which may support the previous suggestions of mating

between African women and autochthonous males and it, resulted in to genetic

admixture.

2) Kalash population

The mtDNA genetic analysis of Kalashi population revealed that the frequency of

West Eurasian haplogroups including R0a’b, U4, U2e1, J2b1a, R2 and H2a1

reaches to 98.2%.

Only one South Asian haplogroup (M65a) was found in the population with least

frequency (0.9%). African haplogroup L and East Asian haplogroup were not

observed in Kalash at all.

Hence, greater frequency of West Eurasian haplogroups in Kalash may suggest

their West Eurasians origin that might be the consequence of the major

historical movements during the Arab and Muslim conquests, the rise of the

British Indian Empire and invasion by the armies of Alexander the Great.

86

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Characterization of Genetic Markers in Pakistani Population

CONSENT FORM

Donor’s Name:_______________________________ Donor’s ID:___________________________________

Gender:_________________________________________Age:_________________________________________

Place of Birth:__________________________________Ethnic Group:______________________________

Education:______________________________________Religion:____________________________________

Mother’s Place of Birth: _____________________Mother’s Ethnic Group:____________________

Grand Mother’s Place of Birth:_______________Grand Mother’s Ethnic Group:_____________

Father’s Place of Birth:________________________Father’s Ethnic Group:_____________________

Grand Father’s Place of Birth:________________Grand Father’s Ethnic Group:_____________

Mother Language:_____________________________Other Languages:___________________________

Contact Number:_____________________________________________________________________________

Consanguinity of Parents: (Father married to):

First Cousin Khalazad Mamonzad Chachazad Phupizad

Second Cousin Khalazad Mamonzad Chachazad Phupizad

Any Other Within Caste (sub-caste) Outside Caste (Sub-caste)

I am a Ph.D Scholar at Department of Zoology, University of the Punjab, Lahore,

working on the research project “Characterization of Genetic Markers in Pakistani

Population”. I assure you that the samples collected over here during this study will

be utilized solely for research purposes.

________________________________

Muhammad Hassan Siddiqi

Ph.D Research Scholar

For Donor:

I voluntarily agree to take part in this research project. My blood sample may be

collected and used in population genetic studies as defined in this consent form.A

person, well versed in my native language, has conveyed to me the aim of this study.

_______________________________________ _______________________ ______________________

Native Person ‘s Name & Signature Donor’s Signature Date

PUBLICATIONS

(A) From the Thesis

1) Siddiqi, M. H., Akhtar, T., Rakha, A., Abbas, G., Ali, A., Haider, N., Ali, A., Hayat,

S., Masooma, S., Ahmad, J., Tariq, M. A. and Khan, F. M., 2014. Genetic

characterization of the Makrani people of Pakistan from mitochondrial DNA control

region data. J. Leg. Med., [In press]

http://dx.doi.org/10.1016/j.legalmed.2014.09.007

(B) Other than the Thesis

1) Hayat, S., Akhtar, T., Siddiqi, M. H., Rakha, A., Haider, N., Tayyab, M., Abbas, G.,

Ali, A., Yassir, S. A. B., Tariq, M. A. and Khan, F. M., 2014. Mitochondrial DNA

Control Region Sequences Study in Saraiki Population from Pakistan. J. Legal

Med., [In press].

2) Jahan, S., Khaliq, S., Siddiqi, M. H., Ijaz, B., Ahmad, W., Ashfaq, U. A. and

Hassan, S., 2011. Anti-apoptotic effect of HCV Core gene of Genotype 3a in Huh-7

cell line. Virology Journal.

3) Jahan, S., Samreen, B., Khaliq, S., Ijaz, B., Khan, M., Siddiqi, M. H., Ahmad, W.,

Hassan, S., 2011. HCV entry receptor as potential targets for siRNA based

inhibition of HCV. Journal Genetic Vaccines and Therapy.

4) Rakha, A., Kyoung-Jin S., Yoon, J. A., Kim, Na, Y., Siddiqi, M. H., Yang, S., Yang,

W. L. and Lee, H. Y., 2010. Forensic and genetic characterization of mtDNA from

Pathans of Pakistan. Int. J. Legal Med.

5) Malik, F., Kayani, M. A., Ansar, M., Obaid-ullah, M. S., Chohan, S., Abbas, Y.,

Shahzad, S., Raza, A., Rehman, R., Qurat-ul-ain, Siddiqi, M. H., Rakha, A., Zia ur

Rehman, Ahmad, Z., 2008. Development of 19-plex YSTR system and

polymorphism studies in Pakistani population. J ACAD J XJTU.

http://dx.doi.org/10.1016/j.legalmed.2014.09.007

Documents

GENETIC CHARACTERIZATION OF …prr.hec.gov.pk/jspui/bitstream/123456789/7097/1/Muhammad...Dr. Muhammad Akbar, Dr. Khurrum Shahzad, Imran Hussain Bhatti, Faizan Riaz Cheema Shahid Yar