CHAPTER 4: RESULTS 4.1. Population genetic structure of …shodhganga.inflibnet.ac.in/bitstream/10603/43110/14/14_chapter 3.p… · five accessions from NP population exhibited the

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CHAPTER 4: RESULTS

4.1. Population genetic structure of turmeric (Curcuma longa) and its response to

Pythium aphanidermatum

4.1.1. Rhizome rot infection analysis

The statistical analysis on disease intensity showing the relative reaction of 62 turmeric

genotypes across 11 populations in response to Pythium aphanidermatum revealed large

and significant variation in the degree of disease incidence in the overall distribution of

the number of plants in all turmeric accessions within the 11 populations. However, no

significant difference was observed between isolates over the disease index scores

(Table 4.1). The two isolates were found equally aggressive or ineffective exhibiting

similar disease index (DI) score for any given isolate-accession combination. The

aggressiveness of each isolate was very much dependent on specific isolate accession

combinations.

Total 4.1: Analysis of variance for the distribution over percentage of infection, testing the

effect of turmeric genotypes across 11 populations and Pythiumapanidermatum isolates

Source df Sum of squares Mean squares F

Between isolates 1 108.98 108.98 0.0812

Between populations 10 52943.86 5294.39 3.947

Isolates x genotypes 102 1156.82 11.34 0.0008

Residual 10 13413.21 1341.32

Total 123 67622.87

The mean DI values ranged from 0.00% to 76.2% among the turmeric accessions. All

the six turmeric accessions from KR population had a disease index ≤ 10% and were

considered resistant. Similarly, six out of seven accessions from MK population and

five accessions from NP population exhibited the lowest disease index values. On the

other hand, turmeric accessions from CTK and CTN population represented the highest

DI values (58.35% to 76.2%). Turmeric accessions from KP, DK, KJ and MB

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populations also showed >20% DI and categorized as susceptible. Two turmeric lines

from Kandhamal (KM) population (KM2 and KM5) and one genotype from Nayagarh

(NG) population (NG4) revealed ≤ 20% DI although the rest of the turmeric lines were

found highly susceptible. The numbers of accessions from NG and KM populations

were also distributed across the DI classes. Of the 12 accession in the two populations, 1

accession was in first class, 2 accessions were in second class, 2 accessions were in

class III and 3 accessions were in class IV while rest of the four accessions were

grouped within class VI.

4.1.2. Overall genetic diversity in Curcuma longa

We surveyed 62 turmeric plants from 11 populations using a set of 29 molecular

markers. A total of 286 loci were presented from 29 primer (Table 4.2 to 4.5).

Combinations among 62 individuals of the 11 populations. Of these, 186 (65.03%) were

polymorphic The highest percentage of polymorphism was obtained with primer SSR1

(100%) followed by ISSR primer (GA)9T (90.9%). Similarly, RAPD primer OPN6

resulted in only three monomorphic bands (zero polymorphism) followed by OPA20

with only 16.6% polymorphic loci. Among the AFLP markers, the polymorphism

ranged from 52.9% in primer combination M-CAG x E-ACA to 55.5% in primer

combination M-CAT x E-ACA. At the population level, the percentage polymorphic

loci (PPL) ranged from 8.36% in the Dhenkanal (DK) population to 27.87% in

Mayurbhanj (MB) population with an average of 14.47%. All the populations had a PPL

<50%. Assuming Hardy-Weinberg equilibrium, the species wide diversity as measured

by Nei‟s unbiased gene diversity (He) across the total 62 sampled plants was

0.089±0.081. The average species wide observed heterozygosity (Ho) was 0.061±0.042

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Table 4.2: List of primers used for RAPD amplification, GC content, total number of loci, the level of polymorphism, resolving power and PIC

value.

Primer Primer sequence % GC content Annealing

Temperature

Total no. of

loci NPL (%) PPL

No. of fragments

amplified Rp PIC

A4 AATCGGGCTG 60 37 9 7 77.7 513 16.52 0.14

A11 CAATCGCCGT 60 37 10 7 70 499 16.08 0.29

A18 AGGTGACCGT 60 37 9 6 66.6 438 14.1 0.32

A3 AGTCAGCCAC 60 37 10 8 80 601 19.4 0.05

A20 GTTGCGATCC 60 37 6 1 16.6 362 11.68 0.05

D18 GAGAGCCAAC 60 37 7 3 42.8 428 13.78 0.04

D20 ACCCGGTCAC 70 37 7 5 71.4 325 10.46 0.37

N6 GAGACGCACA 60 37 3 0 0 186 6 0

N16 AAGCGACCTG 60 37 7 5 71.4 426 13.74 0.04

N4 GACCGACCCA 70 37 11 8 72.7 519 16.72 0.34

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Table 4.3: List of primers used for ISSR amplification, GC content, total number of loci, the level of polymorphism, resolving power and PIC

value.

ISSR Primer Primer sequence % GC

content

Annealing

Temperature

Total no.

of loci NPL (%) PPL

No. of fragments

amplified Rp PIC

(AGG)6 AGGAGGAGGAGGAGGAGG 66 55 6 5 83.3 354 11.42 0.09

(GA)9T GAGAGAGAGAGAGAGAGAT 42.8 51 11 10 90.9 686 22.12 0.13

(GAC)5 GACGACGACGACGAC 66.6 45 9 5 55.5 541 17.44 0.06

(GACA)4 GACAGACAGACAGACA 50 43 5 3 60 279 9.02 0.12

(GTG)5 GTGGTGGTGGTGGTG 66.6 45 7 5 71.4 414 13.34 0.09

(GTGC)4 GTGCGTGCGTGCGTGC 75 51 7 4 57.1 417 13.46 0.07

(CAA)5 CAACAACAACAACAA 33.3 35 3 2 66.6 176 5.68 0.1

(GGA)4 GGAGGAGGAGGA 66.6 35 7 4 57.1 413 13.32 0.09

(AG)8T AGAGAGAGAGAGAGAGT 47.1 45 11 7 63.6 621 20.02 0.15

(GT)8A GTGTGTGTGTGTGTGTA 47.1 45 8 5 62.5 463 14.92 0.12

T(GA)9 TGAGAGAGAGAGAGAGAGA 47.4 51 10 4 40 590 19.04 0.09

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Table 4.4: List of primers used for SSR amplification, GC content, total number of loci, the level of polymorphism, resolving power and PIC

value.

Primer Primer sequence % GC

content

Annealing

Temperature

Total no. of

loci NPL (%) PPL

No. of fragments

amplified Rp PIC

CBT2 F:TCCTCCCTCCCTTCGCCCACTG

R: GATGTTCGCCATGGCTGCTCC 65.9 55 6 4 66.6 346 11.18 0.13

CBT3 F:ATCAGCAGCCATGGCAGCGACC

R: AGGGGATCATGTGCCGAAGGCC 63.6 55 10 7 70 566 18.22 0.16

ClSSR1 F: ATGAACCCCTTCGGTCACC

R: TGTAACTCCTCTCGCCGTATG 55 60 7 7 100 373 12.04 0.26

ClSSR2 F: CCAAGTGCCCTATCCTAACA

R: CCTGGAAACCTGAATCCATTA 46.3 55 22 15 68.2 1300 41.9 0.05

ClSSR3 F: ACAGCACTCAACAACAGGAGA

R: CCTGGAAACCTGAATCCATTA 45.2 65 23 19 82.6 1349 43.52 0.09

Table 4.5: List of primers used for AFLP amplification, GC content, total number of loci, the level of polymorphism, resolving power and PIC

value.

Primer Primer sequence % GC

content

Annealing

Temperature

Total no.

of loci NPL

(%)

PPL

No. of fragments

amplified Rp PIC

MCAG-EACA GATGAGTCCTGAGTAACAG 50 56 17 9 52.9 1006 30.9 0.07

GACTGCGTACCAATTCACA

MCAT-EACA GATGAGTCCTGAGTAACAT 44.7 56 18 10 55.5 1084 34.98 0.06

GACTGCGTACCAATTCACA

MCAT-EAAC GATGAGTCCTGAGTAACAT 44.7 56 20 11 55 1202 38.78 0.06

GACTGCGTACCAATTCAAC

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(Table 4.6) The average population-level expected heterozygosity was 0.051±0.033

ranging from 0.027±0.019 in DK population to 0.114±0.109 in MB population.

Likewise, the population level observed heterozygosity ranged from 0.21±0.008 to

0.103±0.036 with an average of 0.045±0.022. Similar values were calculated for the

Shannon diversity index (I), which varied from 0.042±0.039 in population DK to

0.166±0.072 in population MB. The average Shannon diversity index (I) within

populations and species levels was

Table 4.6: Summary of genetic variation statistics for the combination of markers (RAPD,

ISSR, AFLP and SSR) among the genotypes of Curcuma longa with respect to their

distributions into 11 groups.

Population

ID

Sample

size

Na Ne He I Ho NPL PPL

CTK 5 1.125 (0.332) 1.064

(0.174)

0.042

(0.22)

0.065

(0.012)

0.037

(0.012) 36 12.54

CTN 5 1.087 (0.282) 1.043

(0.141)

0.028

(0.013)

0.044

(0.014)

0.023

(0.009) 25 8.71

KP 5 1.098 (0.297) 1.047

(0.147)

0.032

(0.027)

0.049

(0.051)

0.029

(0.009) 28 9.76

DK 5 1.084 (0.277) 1.039

(0.131)

0.027

(0.019)

0.042

(0.039)

0.021

(0.008) 24 8.36

NG 6 1.115 (0.320) 1.051

(0.150)

0.034

(0.018)

0.055

(0.032)

0.031

(0.010) 33 11.50

KM 6 1.174 (0.380) 1.068

(0.151)

0.049

(0.032)

0.079

(0.043)

0.042

(0.012) 50 17.42

KJ 5 1.157 (0.364) 1.126

(0.303)

0.069

(0.052)

0.098

(0.030)

0.056

(0.026) 45 15.68

MB 5 1.279(0.449) 1.202

(0.347)

0.114

(0.109)

0.166

(0.072)

0.103

(0.036) 80 27.87

KR 6 1.185 (0.389) 1.127

(0.280)

0.074

(0.059)

0.108

(0.063)

0.069

(0.025) 53 18.47

MK 7 1.139 (0.347) 1.066

(0.193)

0.042

(0.012)

0.066

(0.041)

0.034

(0.013) 40 13.94

NP 7 1.160 (0.367) 1.095

(0.233)

0.058

(0.037)

0.087

(0.048)

0.051

(0.019) 46 16.03

Mean 5.63 1.15 (0.363) 1.084

(0.312)

0.051

(0.033)

0.078

(0.043)

0.045

(0.022) 41.81 14.61

Total 62 1.173 (0.301) 1.119

(0.236)

0.089

(0.081)

0.132

(0.107)

0.061

(0.042)

Na= Observed number of alleles; Ne= Effective number of alleles; He= Nei‟s gene diversity; I=

Shannon‟s Information index; Ho= observed heterozygosity; NPL= Number of polymorphic

loci; PPL= Percentage of polymorphic loci

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0.078±0.043 and 0.132±0.107 respectively. The MB and DK populations yielded the

highest and lowest values respectively for all these gene diversity estimates (Table 4. 6).

The dice similarity coefficient among all analyzed turmeric genotypes from 11

populations varied from as close as 1.0 to as diverse as 0.71. Dendrogram based on

UPGMA analysis classified the 62 genotypes under two major clusters (Figure 4.1).

Cluster-A consisted of 50 accessions while cluster B grouped rest of the 12 turmeric

genotypes. Cluster-A included the genotypes representing the entire sampled plants

from Cuttack-2 (CTN), Dhenkanal (DK), Kandhamal (KM), Nayagarh (NG), Koraput

(KR), Malkangiri (MK) and Nabrangpur (NP) populations and all but one from Cuttack-

1 (CTK) and Kendrapada (KP) populations. On the other hand, Cluster-B included five

samples each from Keonjhar (KJ) and Mayurbhanj (MB) population. Interestingly,

CTK5 from Cuttack population and KP3 from Kendrapada population also grouped

under cluster B along with turmeric genotypes from Keonjhar population. Bootstrap

analysis higher support at lower branch points but represented a value less than 50 at

higher branch points in the dendrogram.

4.1.3. Molecular variance and gene flow

Analysis of Molecular Variance (AMOVA) based on the two gene clusters showed that

majority of genetic variation occurred within populations (60.68%), while the variation

between the clusters was 39.31% (Table 4.7). AMOVA analysis based on individual

marker data also revealed similar results with negligible differences. However, the

genetic differentiation among 11 populations of C. longa was high and significant with

Fst = 0.382, P<0.001 and Gst = 0.486.

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Figure4.1 UPGMA dendrogram of 62 Curcuma longa individuals generated by the NJOIN module of NTSYSpc based on DICE genetic

distance from molecular data. Major clusters are indicated by alphabets. Bootstrap values >50 are given at the branch points.

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Table 4.7: Summary of analysis of molecular variance (AMOVA) based on (a) RAPD, (b)

ISSR, (c) AFLP and (d) SSR profiling markers among the genotypes of Curcuma longa.

Levels of significance are based on 1000 iteration steps. Source of

variation

Degree of

freedom

Variance

component Percentage of variation P-value

RAPD

Among populations 10 5.932 33.82 < 0.001

Among genotypes 61 9.161 67.22 < 0.001

ISSR


Among genotypes 61 27.361 64.37 < 0.001

SSR


Among genotypes 61 36.083 60.99 < 0.001

AFLP


Among genotypes 61 27.036 68.54 < 0.001

COMBINATION


Among genotypes 61 9.019 60.68 < 0.001

The estimated gene flow, Nm (Nm = 0.5(1-Gst)/Gst) was found to be 0.529. This

suggest that the gene flow among turmeric populations throughout the entire

distribution region is highly restricted and the genetic differentiation is highly

significant.

4.1.4. Population structure in Curcuma longa

A Bayesian model based clustering method was implemented to further test the

population structure with regards to turmeric. Under an admixed model without prior

information, STRUCTURE calculated that the log likelihood of the data (LnP(D)) was

found to be greatest at K=2 (Figure 4.2A). For K>2, LnP(D) meagrely increased but

mostly represented a plateaued plot. ∆K reached its maximum value when K=2 (Figure

4.2 B) suggesting that all populations fell into one of the two clusters. The geographic

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distribution of turmeric populations corresponded well with the two genetically distinct

clusters. The red cluster included all the genotypes form Kendrapada (KP), Nayagarh

(NG), Cuttack (CTK & CTN), Malkangiri (MK), Nabrangpur (NP), Koraput (KP),

Dhenkanal (DK) and Kandhamal (KM) while the remaining genotypes from Keonjhar

(KJ) and Mayurbhanj (MJ) were grouped in the green cluster (Figure 4.2C).

Furthermore, one individual each from Cuttack (CTK) and Kendrapada (KP) population

grouped within the green cluster. This result shows identical splitting with that of

UPGMA tree. The two dimensional principal coordinate analysis (PCoA) plot showed

that the first principal coordinate accounts for 24.8% of total variation and separates the

turmeric accessions from the Keonjhar (KJ) and Mayurbhanj (MJ) populations from rest

of the other populations analyzed in this study (Figure 4.3). The second principal

coordinate with 16.3% of total variation separated majority of turmeric accessions of

CTK, CTN, DK and KP population from that of KR, MK and NP populations. PCoA

analysis revealed broad overlap amongst the individuals of KM and NG populations.

However, in accordance with STRUCTURE, KJ and MJ populations were separated

from all other populations.

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Figure4.2: STRUCTURE analysis of Curcuma longa populations based on molecular data. A)

The relationship between the number of cluster (K) and the estimated likelihood of data

(LnP(D)). A model based clustering of 62 genotypes using STRUCTURE without prior

knowledge about the populations and under an admixed model calculated that LnP (D) was

greatest when K = 2. (B) The relationship between K and ΔK., i.e. ΔK reaches its maximum

when K = 2, suggesting that all genotypes fall into one of the 2 clusters. (C) Grouping of

genotypes when K = 2. The genotypes were more likely clustered with respect to one of the 2

clusters. Genotypes from different clusters are represented with different colours: cluster 1(red)

and cluster 2 (blue).

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Figure4.3: PCA-Principal coordinate analysis of the eleven sampled populations based

on genetic distances amongst all sampled individuals.

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4.2. Development of sequence tagged diagnostic markers (SCAR/STS) linked to

rhizome rot resistance

4.2.1. Identification of ISSR marker linked with rhizome rot resistance

The rhizome rot resistant and susceptible genotypes of Curcuma longa were screened in

a bulked segregant analysis approach to identify ISSR markers associated with the

resistance. Forty ISSR primers were used to identify polymorphisms between the

susceptible bulks and six susceptible individuals and the resistant bulk and five

resistantindividuals. In total, 38 out of the 40 primers yielded 319 reproducible ISSR

(Table 4.8) bands ranging from 200bp to 3500bp. The number of ISSR bands per primer

varied from 4 to 12 with an average of 8 bands per primer. Majority of the ISSR bands

were identical among the genotypes while 98 bands were found to be polymorphic.

Although each primer generated multiple polymorphic bands, only ISSR2 could amplify

a differential band linked to resistance locus among the turmeric genotypes (Figure 4.4).

ISSR2 with the primer sequence 5‟-GAGAGAGAGAGAGAGAGAT-3‟ [(GA)9T]

resulted in a clear band of ≈1200bp (ClRSM- Curcuma longa resistance specific marker)

found in resistant bulk and all the five resistant individual while absent in the six

susceptible plants of C. longa.

4.2.2. Development of STS marker linked with rhizome rot resistance

The resistant specific fragment corresponding to ISSR marker (GA)9T amplified in the

five resistant individuals were cloned into pTZ57R/T vector and sequenced. Alignment

of the ClRSM sequences from the five resistant individuals of C. longa revealed high

homology. The five fragments have the length of 1203bp and showed variation at only

five nucleotide positions (Figure 4.5). Comparison of deduced ClRSM sequences

against the nucleotide and EST databases at the NCBI homepage showed no significant

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homology between ClRSM and coding sequences of any structural genes. Further, the

sequences

Table 4.8: Primers used for ISSR analysis in Curcuma longa L.

Primer code Oligonucleotide

name Sequence (5‟-3‟)

Annealing

temp (0C)

No. of

bands

amplified

ISSR1 T(GA)9 TGAGAGAGAGAGAGAGAGA 51 9

ISSR2 (GA)9T GAGAGAGAGAGAGAGAGAT 51 9

ISSR3 (GGA)4 GGAGGAGGAGGA 35 8

ISSR4 (GTG)5 GTGGTGGTGGTGGTG 45 8

ISSR5 (GAC)5 GACGACGACGACGAC 45 7

ISSR6 (CAA)5 CAACAACAACAACAA 35 Smear

ISSR7 (AGG)5 AGGAGGAGGAGGAGG 55 9

ISSR8 (GACA)4 GACAGACAGACAGACA 43 6

ISSR9 (GTGC)4 GTGCGTGCGTGCGTGC 51 12

ISSR10 (AT)8T ATATATATATATATATT 34 4

ISSR11 (AT)8G ATATATATATATATATG 36 7

ISSR12 (AT)8C ATATATATATATATATC 36 8

ISSR13 (TA)8A TATATATATATATATAA 34 Smear

ISSR14 (TA)8C TATATATATATATATAC 36 9

ISSR15 (TA)8G TATATATATATATATAG 36 9

ISSR16 (AG)8T AGAGAGAGAGAGAGAGT 50 4

ISSR17 (GA)8T GAGAGAGAGAGAGAGAT 50 7

ISSR18 (GT)8A GTGTGTGTGTGTGTGTA 50 7

ISSR19 (GT)8T GTGTGTGTGTGTGTGTT 50 8

ISSR20 (AG)8C AGAGAGAGAGAGAGAGC 47 11

ISSR21 (AG)8G AGAGAGAGAGAGAGAGG 47 12

ISSR22 (GA)8C GAGAGAGAGAGAGAGAC 47 9

ISSR23 (GA)8A GAGAGAGAGAGAGAGAA 45 8

ISSR24 (CT)8T CTCTCTCTCTCTCTCTT 45 8

ISSR25 (CT)8A CTCTCTCTCTCTCTCTA 45 4

ISSR26 (CT)8G CTCTCTCTCTCTCTCTG 47 9

ISSR27 (CA)8T CACACACACACACACAT 45 6

ISSR28 (CA)8A CACACACACACACACAA 45 12

ISSR29 (CA)8G CACACACACACACACAG 47 10

ISSR30 (TG)8A TGTGTGTGTGTGTGTGA 45 6

ISSR31 (TG)8C TGTGTGTGTGTGTGTGC 47 4

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ISSR31 (TG)8G TGTGTGTGTGTGTGTGG 47 9

ISSR33 (AC)8T ACACACACACACACACT 45 9

ISSR34 (GT)8YC GTGTGTGTGTGTGTGTYC 51 13

ISSR35 (GA)8YC GAGAGAGAGAGAGAGAYC 51 10

ISSR36 (GT)8YA GTGTGTGTGTGTGTGTYA 53 6

ISSR37 (GT)8YG GTGTGTGTGTGTGTGTYG 51 12

ISSR38 (GA)8YT GAGAGAGAGAGAGAGAYT 49 10

ISSR39 (AG)8YT AGAGAGAGAGAGAGAGAYT 55 11

ISSR40 (AG)8YC AGAGAGAGAGAGAGAGAYC 53 9

319

Figure 4.4: ISSR amplification patterns of five resistance and six susceptible

germplasm of turmeric obtained with the primer ISSR2 [(GA)9T]. The arrow

represents the putative fragment of 1200bp present in the resistant bulk (RB)

as well as five resistance individuals. M, 100bp ladder plus; RB, resistance

bulk; SB, Susceptible bulk; R1-R5 represents the resistant individuals CBT-

Cl23, CBT-Cl63, CBT-Cl87, CBT-Cl114 and CBT-Cl136 respectively; S1-

S6 represents the susceptible genotypes CBT-Cl09, CBT-Cl26, CBT-Cl35,

CBT-Cl57, CBT-Cl73 and CBT-Cl121 respectively.

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CBT-Cl136 GAGAGAGAGAGAGAGAGATCCCCCCTTTTTCCGAACACCTCCTTCCTCCCTTTATACATCAAGAGCTTCCTTGAAAGTATGACCAGTTCTGCCAAAACATGGTCTTGAGAAGACAACCAA 120





************************************************************************************************************************

CBT-Cl136 TCACATCCGCATTTAATGCTTGGGTAGGTTATTCATTGGAGAGAGAGAGATCGTCTTGACAAGTGACCTCTCCCATTAATGCTTGACACAGTTGTTTAGTAGAGAGAGAAGATCATTTCT 240





************************************************************************************************************************

CBT-Cl136 GACAAGTCACATCCCAAGTGATGTTTGATTATTAGAGAGAGATCATTTATGATGGGACTGTTGAGCCGAGTGGGCTTGGCAGAATTGAGCTAAGTGTTGAGTGGCCTGGGTGGAGGTGTC 360





************************************************************************************************************************

CBT-Cl136 GGATGGCAGAGCGGAGCTGCCGGATGGCAGTGCTGAGCTGCCGGGTAGCAGTGCTGAGCTGTCGGATAGCAGTGCTGAGCTGCCGGGTAGCAGTGCTGAGCTGCCGGATAGCAGTGCTGA 480





************************************************************************************************************************

CBT-Cl136 GCTGCCGAATAGCAGTGCTGAGCTGCCGGGTCGAATGAGCCAAGTCCTAGAGGGGGGCTATAACACCCATGTGGCCGAATGGGCGATTCGGAGACGCGACCGAAGAGATAACTTGACTTC 600





************************************************************************************************************************

CBT-Cl136 TTTAGGCCTTTAGGGATAGACCGTGACGGGAGCCGACCGAACCCATGGATGAGGCTCGAATTAGACTCGAGTCAAGTCTGGGTCCTCCAAAATACTCACCGGAACAGTTATAATTATTTT 720





************************************************************************************************************************

(Figure 4.5 )

90

CBT-Cl136 GTATGGTGTGATCTGAGCGAAAGAGCGACTAGCTTTTCGATACGTTGAAGAAAAATGATTTTGGCACCGACAGAAATAAGCTCCAAGAAGAAGAGGAAGGAAATTTATACATGCTGCCCG 840





************************************************************************************************************************

CBT-Cl136 ATGTGGCAGATCTTGACTGGTCTGACGTGGTCAAAAAAGATGAACCTTTCTTCTAATTGACTCAGATTTATGCCTCATCCAAAAGGATAAGTACGACGACCAAGTTTCCCAAGTTGACCC 960

CBT-Cl114 ATGTGGCAGATCTTGACTGGTCTGACGTGGTCAAAAAAGATGAACCTTTCTTCTAATTGACTCAGATTTATGCCTCATCCAAAAGGATAAGTACGACGACCAAGTTTCCAAAGTTGACCC 960



CBT-Cl63 ATGTGGCAGATCTTGACTGGTCTGACGTGGTCAAAAAAGATGAACCTTTCTTCTAATGGACTCAGATTTATGCCTCATCCAAAAGGATAAGTACGACGACCAAGTTTCCAAAGTTGACCC 960

*********************************************************.***************************************************.**********

CBT-Cl136 CCTAAAATCCTGCATACTAATGCATGAAACTGTCAAAATGAAGATCGGCAAATCTAAACCCTTTGGATTTTCGAAAACTCTCCAAAGATTATCCTTACCCACTTAATAACCGCTCCAGAA 1080

CBT-Cl114 CCTAAAATCCTGCATACTAATGCATGAAACTGTCAAAATGAAGATCGGCTAATCTAAACCCTTTGGATTTTCGAAAACTCTCCAAAGATTATCCTTACCCACTTAATAACCGCTCCAGAA 1080

CBT-Cl87 CCTAAAATCCTGCATACTAATGCATGAAACTGTCAAAATGAAGATCGGCAAATCTAAGCCCTTTGGATTTTCGAAAACTCTCCAAAGATTATCCTTACCCACTTAATAACCGCTCCAGAA 1080

CBT-Cl23 CCTAAAATCCTGCATACTAATGCATGAAACTGTCAAAATGAAGATCGGCAAATCTAAACCCTTAGGATTTTCGAAAACTCTCCAAAGATTATCCTTACCCACTTAATAACCGCTCCAGAA 1080

CBT-Cl63 CCTAAAATCCTGCATACTAATGCATGAAACTGTCAAAATGAAGATCGGCAAATCTAAACCCTTTGGATTTTCGAAAACTCTCCAAAGATTATCCTTACCCACTTAATAACCGCTCCAGAA 1080

*************************************************:*******.*****:********************************************************

CBT-Cl136 GTCAATTACTTGGCCTGAATTCCCCGAAAAAAACGCTTTGCCCGGACCCAATTTAAAGCCCCCCCGGGTTAAAAAAAAAGAAAGGGGAAGCGAAGGCTTCCTTTATCTCTCTCTCTCTCTCTC 1203

CBT-Cl114 GTCAATTACTTGGCCTGAATTCCCCGAAAAAAACGCTTTGCCCGGACCCAATTTAAACCCCCCCCGGGTTAAAAAAAAAGAAAGGGGAAGCGAAGGCTTCCTTTATCTCTCTCTCTCTCTCTC 1203




***************************************************************************************************************************

Figure 4.5: Multiple sequence alignment of the ClRSM sequences isolated from five turmeric germplasm resistant to

Pythiumaphanidermatum. The shaded regions indicate the nucleotide base variation among the five sequences.

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also revealed no open reading frame and multiple stop codons existed in all the three

reading structures.

Based on the complete sequence data of ClRSM, a pair of STS primers (ClSTS-F and

ClSTS-R) was designed (Figure 4.6). The primer pair was used to amplify the five

resistant and four susceptible genotypes of turmeric to confirm their validity. PCR with

ClSTS-F (5‟-CCTTTTTCCGAACACCTCCT-3‟) forward sequence and reverse is

CISTS–R. (5‟CTTTCGCTCAGATCACACCA-3‟) resulted in the amplification of a

720bp fragment (ClSTS) only in the resistant and not in the susceptible individuals of

turmeric (Figure 4.8A). Accurate amplification of a 720bp fragment in the five resistant

genotypes proved the stability of the designed SCAR marker and suggests that ClSTS

amplification during ISSR-PCR is independent to the base changes in the primer

binding sites. Further, 720bp fragment of ClSTS was also obtained with wild Curcuma

zedoaria genotype Cze512-11 resistant to P. aphanidermatum while no amplification

was found in two susceptible wild samples- Curcuma zedoaria Cze102-03 and

Curcuma amada Cam1703 genotypes. This suggests that the specific DNA region of

ClSTS is conserved across wild and cultivated Curcuma species that confers resistant to

P. aphanidermatum. Southern hybridization was performed to determine the nature of

the STS sequence within the turmeric genome. Genomic DNA of the resistant turmeric

line CBT-Cl23 and susceptible line CBT-Cl09 digested with EcoRI, HindIII, XbaI or

SacI was subjected to southern blotting using a digoxigenin labeled ClSTS fragment as

probe. DNA digested with different enzymes revealed single band only with the

resistant genotype CBT-Cl23 in the southern blots (Figure 4.7). This specifies that

ClSTS is represented by a single copy locus in the turmeric genome.

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Figure 4.6: DNA sequence of the Curcuma longa resistance specific marker ClRSM.

The underlined region represents the annealing position of ISSR2 [(GA)9T]. The arrow

with the shaded region indicates the position of primers ClSTS-F and ClSTS-R for

amplifying the sequenced tagged sites marker ClSTS.

93

Figure 4.7: Southern blot analysis of STS marker ClSTS. Restriction enzymes

used are EcoRI, HinDIII, XbaI and SacI. M, Molecular weight marker; R,

resistant genotype CBT-Cl23; S, susceptible genotype CBT-Cl09.

Hybridization patterns of the genomic DNA digested with all the four

enzymes revealed single band only in the resistant genotype.

94

4.2.3. Application of ClSTS marker to diagnose rhizome rot resistance in turmeric

Fifteen turmeric accessions collected from different agroclimatic zones of Odisha, India

with unknown response to rhizome rot disease were tested for the presence or absence

of the STS marker ClSTS. PCR amplification of the isolated DNA from the 15

individuals with STS primer resulted in single expected DNA band of 720bp in ten

accessions Cl13-07, Cl23-11, Cl27-03, Cl51-06, Cl63-07, Cl96-03, Cl126-17, Cl69-01,

Cl87-19 and Cl29-17. The rest of the five accessions Cl75-15, Cl102-13, Cl42-12,

Cl33-09 and Cl134-05 did not result in the expected PCR product (Fig 4.8 B). Thus, the

amplification pattern of ClSTS suggested that ten out of the 15 unknown turmeric

accessions exhibit resistant response against rhizome rot. An inoculation experiment

was carried out with the fifteen turmeric accessions to determine the associations of

rhizome rot susceptibility with the absence of ClSTS locus. Pseudostems of the fifteen

turmeric accessions were inoculated with fungal zoospore suspension, kept under green

house conditions and observed for disease symptoms (Figure 4.9). Ten turmeric

accessions with positive amplification of ClSTS showed no symptoms of infection,

either foliar or rhizomic. In contrast to them, the rest of the five genotypes without

ClSTS locus showed significant levels of foliar decline and rhizome rot score. The

pseudostems of the five genotypes were completely wilted and their rhizomes were

decayed within four week of inoculation. These results indicate that the five turmeric

accessions are indeed susceptible to P. aphanidermatum infection.

95

Figure 4.8: (A) Analysis of STS marker ClSTS in turmeric genotypes either

resistant or susceptible to rhizome rot. ClSTS amplified a single 720bp band in

five resistant Curcuma longa genotypes CBT-Cl23, CBT-Cl63, CBT-Cl87,

CBT-Cl114, CBT-Cl136 and one resistant Curcuma zedoaria genotype Cze512-

11. No amplification was found with four susceptible C. longa genotypes CBT-

Cl09, CBT-cl26, CBT-cl57, CBT-cl121, one susceptible C zedoaria genotype

Cze102-03 and one susceptible C amada genotype Cam17-03. (B) STS analysis

carried out using ClSTS marker in 15 unknown turmeric accessions listed in

table 1. Sample that amplified a single 720bp band with ClSTS was identified as

resistant while the samples with no amplification were detected as susceptible to

rhizome rot. M, 100bp ladder; R, resistant genotype CBT-Cl23; S, susceptible

genotype CBT-Cl09; 1, Cl13-07;2, Cl23-11; 3, Cl27-03; 4, Cl29-17; 5, Cl33-09;

6, Cl42-12; 7, Cl51-06; 8, Cl63-07; 9, Cl69-01; 10, Cl75-15;11, Cl87-19;12,

Cl96-03; 13, Cl102-13; 14, Cl126-17; 15, Cl134-05.

96

Figure 4.9: Rhizome rot inoculation experiment with resistant genotype CBT-Cl23 and

the identified turmeric accession Cl75-15 (Plant no 10) susceptible to P.

aphanidermatum. A) CBT-Cl23 plant inoculated with P. aphanidermatum 40 days PI.

B) Disease symptoms of Cl75-15 (Plant no 10) inoculated with P. aphanidermatum 40

days PI. C) Rhizome of rot resistant CBT-Cl23 genotype inoculated with P.

aphanidermatum 40 days PI. D) Decayed rhizome of Cl75-15 (Plant no 10) inoculated

with P. aphanidermatum 40 days PI.

97

4.3. Cloning and characterization of resistance related candidate gene sequences

from wild Curcuma spp.

4.3.1. Amplification and cloning of RGCs of the NBS-type from wild Curcuma spp.

Using the three R-gene specific degenerate primers, a ≈530bp fragment was amplified

between the P-loop and GLPLA motifs of the NBS region of the NBS-LRR class of R-

genes from the three wild turmeric genotypes. An additional band of 100/250/300bp

was also observed in a few samples. Different PCR conditions, including the

concentrations of template DNA, Mg2+ and Taq DNA polymerase, were tested to

further confirm the PCR amplification. Although the 530-bp band could be reproduced

under all conditions tested, the DNA bands of other sizes could not. Therefore, the 530-

bp band was cloned and sequenced. Nine amplicons of 530-bp in size (Figure 4.10)

were cloned and 38 clones were isolated that comprised of 10, 17 and 11 clones

respectively from Curcuma aromatica, Curcuma angustifolia and Curcuma zedoaria.

BLASTx search revealed 21 (nine, five and seven from Curcuma aromatica, Curcuma

angustifolia and Curcuma zedoaria respectively) out of 38 clones (Table 4.9). having

high homology to the NBS domain of known R-genes or RGCs from other plant

species. These sequences have been submitted in the Gene Bank database

(http://www.ncbi.nlm.nih.gov) with accession number from JN426969 to JN426989.

The rest of the 17 clones showed homology to putative polyprotein and hypothetical

leucine-rich repeats and were excluded from further analysis.

4.3.2. Sequence analysis of RGCs from wild Curcuma spp.

The 21 nucleotide sequences were translated using ExPaSy translate tool and the

deduced amino acid sequences that showed a high level of sequence similarity with

more than 100 amino acids to the NBS regions of disease resistance R-genes were

published in the GenBank database. The level of identity of these sequences

98

Figure 4.10: Amplification of 530 bp fragment using a degenerate primer of

wild relatives of Curcuma.

99

Table 4.9: Results of the search for similarity between wild turmeric NBS-

sequences and GenBank accessions carried out using the BLASTX algorithm

Curcuma

NBS

clone

GenBank protein

accession from

Zingiberaceae

showing the

highest similarity

Amino

acid

identity

(%)

E

value

Other GenBank

protein accession

showing the

highest similarity

Amin

o acid

identit

y (%)

E

value

Cap11 Kaempferia galanga

NBS-LRR protein

(ADZ73428)

98% 2e-

123

Disease resistance

like protein, Coffea

arabica

(CAC82610)

51% 4e-35


NBS-LRR protein

(ADZ73428)

98% 5e-

122

Disease resistance

like protein, Coffea

arabica

(CAC82610)

53% 3e-36


NBS-LRR protein

(ADZ73428)

99% 2e-

123

Resistance like

protein, Vitis davidii

(ABS50350)

52% 2e-35


NBS-LRR protein

(ADZ73431)

98% 5e-

124

Resistance like

protein, Vitis davidii

(ABS50350)

52% 7e-39


NBS-LRR protein

(ADZ73429)

98% 1e-

126

NBS containg R-

gene [Musa

acuminata subsp.

malaccensis]

(ABY75804)

55% 1e-38


NBS-LRR protein

(ADZ73430)

98% 3e-

129

NBS containg R-

gene [Musa

acuminata subsp.

malaccensis]

(ABY75804)

60% 3e-37


NBS-LRR protein

(ADZ73428)

96% 3e-

120

NBS-LRR type

resistance protein

[Cynodon x

magennisii]

(ADC54190)

60% 1e-37


NBS-LRR protein

(ADZ73430)

97% 1e-

121

NBS-LRR type

resistance protein

[Cynodon x

magennisii]

(ADC54190)

59% 1e-36


NBS-LRR protein

(ADZ73431)

89% 2e-

126

NBS-LRR type

resistance protein

[Cynodon x

magennisii]

(ADC54190)

56% 2e-36

Can11 Kaempferia galanga

NBS-LRR protein

ADZ73429

89% 2e-

112

NBS containg R-

gene [Musa

acuminata subsp.

malaccensis]

(ABY75804)

56% 4e-35


NBS-LRR protein

87% 5e-

107

NBS containg R-

gene [Musa

56% 5e-32

100

(ADZ73428) acuminata subsp.

malaccensis]

(ABY75804)


NBS-LRR protein

(ADZ73428)

86% 2e-

104

NBS containg R-

gene [Musa

acuminata subsp.

malaccensis]

(ABY75804)

55% 8e-34


NBS-LRR protein

(ADZ73429)

88% 1e-

108

NBS-LRR type

resistance protein

[Cynodon x

magennisii]

(ADC54190)

56% 2e-32


NBS-LRR protein

(ADZ73429)

88% 2e-

107

NBS-LRR type

resistance protein

[Cynodon x

magennisii]

(ADC54190)

54% 3e-30

Czp11 Kaempferia galanga

NBS-LRR protein

(ADZ73429)

87% 4e-

109

NBS containg R-

gene [Musa

acuminata subsp.

malaccensis]

(ABY75804)

57% 3e-36


NBS-LRR protein

(ADZ73431)

86% 9e-

106

NBS containg R-

gene [Musa

acuminata subsp.

malaccensis]

(ABY75804)

57% 5e-36


NBS-LRR protein

(ADZ73428)

85% 3e-

103

NBS containg R-

gene [Musa

acuminata subsp.

malaccensis]

(ABY75804)

63% 1e-35


NBS-LRR protein

(ADZ73429)

93% 2e-

123

NBS containg R-

gene [Musa

acuminata subsp.

malaccensis]

(ABY75804)

58% 2e-39


NBS-LRR protein

ADZ73429

89% 2e-

112

NBS containg R-

gene [Musa

acuminata subsp.

malaccensis]

(ABY75804)

59% 5e-39


NBS-LRR protein

(ADZ73430)

87% 3e-

109

NBS containg R-

gene [Musa

acuminata subsp.

malaccensis]

(ABY75804)

59% 1e-38


NBS-LRR protein

(ADZ73428)

87% 3e-

104

NBS containg R-

gene [Musa

acuminata subsp.

malaccensis]

(ABY75804)

59% 3e-36

101

to RGCs isolated from other plant species ranged from 51% between Cap11 and NBS-

LRR resistance like protein from Coffea arabica (e-value: 4e-35) to 63% between

Czp21 and NBS-containing disease resistance-like protein from Musa acuminata subsp.

malaccensis (e-value: 1e-35). The level of sequence identity of isolated sequences with

RGCs isolated from other zingiberaceous species ranged from 85% between Czp21 and

resistance like protein KRGC1 from Kaempferia galanga (e-value: 3e-103) to 99%

between Cap21 and Kaempferia galanga resistance like protein KRGC4 (e-value: 1e-

04). BLASTP searches of deduced amino acid sequences of the 21 clones revealed the

presence of NB-ARC (nucleotide binding and similarity to Apaf-1, R genes and Ced-4)

domain and significant homology to well characterized RGCs and R-genes from

angiosperms. Analysis of the sequences using the ORF finder at the NCBI server

(http://www.ncbi.nlm.nih.gov/projects/gorf) revealed that all the 21 sequences could be

translated into open reading frame (ORF) with more than 100 amino acids. Southern

blot analysis of selected resistance gene candidates resulted in hybridization pattern

consisting of multiple bands for each of the three RGC tested (Figure 4.11) suggesting

that RGCs occurs as multiple copies in the turmeric genome. The RGCs Cap12, Czp11

and Can12 showed the hybridization pattern representing seven, six and five copies in

the genome of Curcuma aromatica, Curcuma zedoaria and Curcuma angustifolia

respectively. Such types of RGCs attribute to be a member of the multi resistance gene

family.

Analysis of the putative amino acid sequences of the wild Curcuma RGCs using

CLUSTALX program revealed the presence of appropriately located consensus P-

loop/Kinase-1a (GGVGKTT), kinase-2 (LLVFDDVW), RNBS-B (GSRVIVTTRI) and

GLPL motifs characteristic of the NBS domain of R-genes (Figure 4.12). The non-TIR

102

Figure 4.11: Southern hybridization of three selected resistance gene candidates

Cap12, Czp11 and Can12 of Curcuma aromatica, Curcuma angustifolia and

Curcuma zedoaria plants respectively. The restriction enzymes used are (1)

EcoRI and (2) HindIII. Molecular weight marker values are indicated in the left

side of each blot. Hybridization pattern consisting of multiple bands was realized

for each of the three RGC tested with both EcoRI and HindIII suggesting that

the RGCs belongs to a multi-copy gene family in the turmeric genome.

103

Figure 4.12: CLUSTAL_X multiple alignment of deduced amino acid sequences of

the NBS region of wild Curcuma sequences and analogous domains in known R-

genes. RPS2 (A54809) confers resistance to Pseudomonas syringae in Arabidopsis

thaliana, N (U15605) confers resistance to Tobacco mosaic virus in Nicotiana

tabaccum, L6 (U27081) confers resistance to Melampsora lini in Linum usitatissimum

and Pi-ta (AAK00132) confers resistance to Magnaporthe grisea in rice. Conserved

motifs are shaded and indicated above the alignment. The arrows indicate the position

and orientation of degenerate primers.

104

motif (WVxxIRELAYDIEDIVDxY) associated with non-TIR subclass of NBS

sequences was also present in the N-terminal region of all the 21 wild Curcuma

sequences. In addition, the analysis showed a tryptophan (W) residue at the end of

kinase-2 motif, which is also a characteristic feature of non-TIR subclass of NBS-LRR

R-genes. Sequence comparisons of the wild Curcuma RGCs (using the region spanning

the P-loop and GLPLA motifs of the NBS domain) revealed a high amino acid identity

among them. The amino acid identity ranged from 85% (between Czp11 and Cap12,

Cap21) to 99% (between Cap21 and Cap11; between Cap22 and Cap23, Cap24, Cap31

and Cap32; between Can11 and Can12, Can21; between Can22 and Can31; between

Czp11 and Czp12, Czp21). Similarly, the amino acid identity between wild Curcuma

sequences and known R-genes varied from 41% between Can11, Can12, Can21, Can31

and Linium L6 (U27081) to 53% between Cap23 and Arabidopsis RPS2 (A54809).

Likewise, the amino acid identity among R-genes utilized in the study ranged from 42%

(between RPS2, N and L6) to 53% (between L6 and N).

4.3.3. Phylogenetic analysis of wild turmeric RGCs

A phylogenetic tree was constructed using the Neighbor Joining algorithm implemented

in the Molecular Evolutionary Genetics Analysis (MEGA) software package version 2.1

to examine the relationships of wild Curcuma RGCs among themselves and to the R-

genes and RGCs from other plant species. The analysis was performed using only the

NBS domain (P-loop to GLPLA) as it contains numerous conserved motifs that

facilitate proper alignment. The resulting tree consisted of two major branches- TIR and

non-TIR NBS-LRR disease resistance proteins (Figure 4.13). The TIR branch consisted

of seven known TIR-NBS-LRR genes namely L (AAD25976), L6 (U27081), M

(P93244), P2 (AAK28806), N (U15605), RPP4 (NM117790) and RPP1 (NM114316).

105

Figure 4.13: Neighbour joining tree based on amino acid sequence alignment of resistance gene candidates isolated from wild Curcuma

spp., other genotypes of Zingiberaceae and NBS sequences of R-genes from other plant species. Numbers on the branches indicate

the percentage of 1000 bootstrap replications supporting the particular nodes.The bold branches indicate the TIR-NBS cluster.

106

All the wild Curcuma RGCs clustered with the non-TIR subclass of NBS sequences,

which supports our earlier results that the turmeric RGCs lacks a TIR domain at the N-

terminal region. The wild Curcuma RGCs were distributed into four classes. 12 out of

21 RGCs were in class A and 3 RGCs each in class B, C and D. The distribution of wild

Curcuma RGCs in different branches of the non-TIR-NBS cluster reflects a high level

of sequence divergence for these sequences. However, in all the subtrees, the Curcuma

RGCs were clustered with RGCs of known function from other members of the family

Zingiberaceae such as Zingiber officinale and Kaempferia galanga.Thus, the 21

sequences identified from three taxa can be considered as resistance gene candidates

based on their high level of sequence identity to R-genes/RGCs and presence of true

open reading frames with desired conserved motifs.

4.3.4. Transcript expression analysis of the wild turmeric RGCs

Total RNA was isolated from the healthy plants of three wild turmeric genotypes either

resistant or susceptible to Pythium aphanidermatum and subjected to RT-PCR to study

the transcript expression profiles. Cap12 and Can12 resulted in constitutive

amplification of PCR product in both resistant and susceptible accession of Curcuma

aromatica and Curcuma angustifolia. However, in case of Curcuma zedoaria, the

mRNA encoding Czp11 expressed in the plant resistant to Pythium aphanidermatum but

not in plants susceptible to the fungal pathogen (Figure 4.14). Similar results were

alsoobtained with cultivated turmeric (Curcuma longa L.); Czp11 was detected in as

many as seven resistant accessions (CBT no. C106, C109, C110, C122, C123, C134,

C137) but not in susceptible lines (data not shown). This suggests a transcriptional

variance of Czp11 might account for resistance against Pythium aphanidermatum.

Control reactions were set up to ensure that the PCR products were not amplified from

107

Figure 4.14: RT-PCR analysis of three selected resistance gene candidates Cap12,

Czp11 and Can12 in root tissue of Curcuma aromatica, Curcuma zedoaria and

Curcuma angustifolia plants resistant (R1, R2, R3) or susceptible (S1, S2, S3)

respectively to Pythium aphanidermatum. Cap12 and Can12 showed a constitutive

expression in both resistant (R1, R3) and susceptible (S1, S3) plants of Curcuma

aromatica and Curcuma angustifolia while Czp11 expression was realized only in

resistant line of Curcuma zedoaria (R2) but not in susceptible line (S2). C+ denotes a

positive control and show the expected ≈300bp fragment amplified from turmeric actin

1 cDNA.C- denotes the negative control which show no amplification of turmeric actin

1 amplification due to lack of reverse transcription. M denotes the 1kb ladder plus

molecular weight marker.

108

genomic DNA. PCR was done carrying a positive control with actin-specific primers

and negative control with actin primers but with RNA instead of cDNA as the template.

Amplification of the expected cDNA fragment (≈300bp) in the positive control for Actin

1 gene and no amplification in the negative control confirmed that the mRNA samples

were free of genomic DNA contamination.

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CHAPTER 4: RESULTS 4.1. Population genetic structure of …shodhganga.inflibnet.ac.in/bitstream/10603/43110/14/14_chapter 3.p… · five accessions from NP population exhibited the