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Save Nature to Survive

9(2): 831-837, 2014 (Supplement on Genetics and Plant Breeding)
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ASSESSMENT OF MOLECULAR DIVERSITY IN WHEAT (TRITICUM

AESTIVUM L. AND TRITICUM DURUM L.) GENOTYPES
CULTIVATED IN SEMI-ARID REGION OF GUJARAT

HARSHVARDHAN ZALA1, TEJAS BOSAMIA1, KALYANI KULKARNI1* AND YOGESH SHUKLA2

1
Department of Agricultural Biotechnology, Anand Agricultural University, Anand - 388 110, Gujarat, INDIA
2
Department of Biochemistry, B. A. College of Agriculture, Anand Agricultural University,
Anand - 388 110, Gujarat, INDIA
e-mail: kalyaniaau@gmail.com

KEYWORDS ABSTRACT
Genetic diversity Molecular diversity was assessed among wheat genotypes cultivated in semi-arid region of Gujarat employing
RAPD molecular markers. In total, 18 RAPD and eight SSR markers amplified 5554 fragments with 66.83 % polymor-
SSR phism and 343 fragments with 90.32% polymorphism respectively. Unweighted pair group method with arith-
Rainfed metic mean (UPGMA) analysis generated by RAPD, SSR and combined RAPD and SSR based on genetic distance
Irrigated estimates displayed two major clusters consisting of irrigated aestivum (I), irrigated and rainfed durum (II) wheat
Wheat genotypes. A Jaccard’s similarity coefficient of all genotypes derived from RAPD data ranged from 0.65 to 0.90
Polymorphism and that of SSR ranged from 0.45 to 0.94. Marker data subjected to analysis of molecular variances revealed more
diversity within population. The PIC value for RAPD and SSR markers ranged from 0.91 to 0.96 and 0.57 to 0.76
Received on : respectively, with marker indexes of 11.35 and 5.35, demonstrating its utility in genetic diversity analysis. The
15.11.2013 results of PCoA analysis were comparable to dendrograms. Analysis of molecular variance (AMOVA) for com-
bined RAPD and SSR displayed more variance within and among population than RAPD and SSR. These
Accepted on : molecular markers can be put to use for characterization and selection of wheat genotypes suitable for cultivation
10.07.2014 in irrigated and rainfed conditions.

*Corresponding
author

INTRODUCTION semiarid regions has always been an important objective of

breeding programs (Khamssi et al., 2012). Molecular marker
Wheat (Triticum spp.), a self-pollinating annual plant, belong- technology offers such approaches for improvement with re-
ing to the family Poaceae (grasses), is globally one of the three spect to selection of desirable alleles. It is pre-requisite for
most important crops for human as well as livestock feed undertaking molecular breeding activities particularly identi-
(Shewry, 2009). It ranked third in the world with 86.9 million fying and localizing important genes controlling qualitatively
metric tons which is an increase over the past years (FAOSTAT, and quantitatively inherited traits (Varshney et al., 2006). They
2011). At least 60 million ha of wheat is grown in marginal are equally capable of tracking the introduced genomic re-
rainfed environments in developing countries (Monneveux gions in large numbers of lines for pre breeding (Allen et al.,
et al., 2012). Nevertheless, the crop productivity is under the 2011) and assessing the genetic diversity in the germplasm
constraints of drought stress which contributes nearly 17% of (Bahurupe et al., 2013). Various molecular markers have been
yield reductions (Boyer, 1982; Ashraf and Harris, 2005). In useful in breeding programs for assessment of genetic vari-
India, hexaploid (Triticum aestivum) and tetraploid (Triticum ability between genotypes such as restriction fragment length
durum and Triticum dicoccum) wheat spp. are cultivated polymorphisms (RFLPs), random amplification of polymor-
(Sramkova et al., 2009) and widely grown as rain-fed crop in phic DNAs (RAPD), amplified fragment length polymorphisms
semi-arid areas, where large fluctuations occur in the amount (AFLPs), inter simple sequence repeats (ISSRs), sequence char-
and frequency of rainfall events. Large part of the north-west acterized amplified region (SCAR), microsatellites or simple
India falls under arid and semi-region including the state of sequence repeats (SSR), single nucleotide polymorphisms
Gujarat (CFDA, 2007). Major efforts are underway worldwide (SNPs) etc. (Mallikarjuna et al., 2012). Among these markers,
to increase wheat production by extending genetic diversity RAPD was the first polymerase chain reaction (PCR) based
and analysing key traits (Brenchley et al., 2012). Understand- marker characterized by dominant nature and requiring no
ing the genotyping characteristics and relationships of the sequence information (Asif et al., 2005). There is a plethora of
germplasm is limited, mainly due to the polyploid nature of reports on genetic diversity studies employing RAPD markers
wheat (Haudry et al., 2007). Moreover, development of high- in wheat (Rahman et al., 2004; Asif et al., 2005; Iqbal et al.,
yielding wheat cultivars under drought conditions in arid and 2007; Rashed et al., 2008; Abd-El-Haleem et al., 2009; Patil

831
HARSHVARDHAN ZALA et al.,

et al., 2011; ElSayed and Rafudeen, 2012). Another widely μL), 0.5 dNTPs mix (10 mM each), 0.3 Taq DNA polymerase
utilized molecular marker is microsatellite or simple sequence (5 U/μL), 1.5 Template DNA (20 ng/μL) (Fermentas, India).
repeat marker (SSR). Microsatellite or SSRs are stretches of Amplification was carried in thermal Cycler (Whatman
DNA containing tandem repeating di-, tri-, or tetra- nucleotide Biometra T-Gradient, Germany) with the conditions as follows;
units distributed throughout the eukaryotic genomes (Pearson initial denaturation at 94 °C for four mins., 39 cycles of
and Sinden, 1998). These markers have gained considerable denaturation at 94 °C for 30 sec., annealing at 38 °C for 30
importance over other markers due to many desirable attributes sec., extension at 72 °C for one min. and final extension at 72
like hyper variability, multiallelic nature, co-dominant inherit- °C for seven mins. The amplified products of RAPD were
ance, reproducibility, relative abundance, extensive genome analysed on 1.8% agarose gel prepared in 1X TBE along with
coverage, chromosome specific location, amenability to au- 1 kb standard DNA ladder (Fermentas, India). The
tomation, high throughput genotyping and their ability to as- electrophoresis was conducted at 60 V current (constant) to
sociate with many phenotypes (Parida et al., 2009). SSRs have separate the amplified bands. The separated bands were
been widely applied to characterize the genetic diversity in visualized under UV transilluminator and photographed using
wheat and still continue to be the choice for genetic diversity BIORAD Gel Documentation system (BIORAD, USA).
analysis of trait of interest (Devos et al., 1995; Prasad et al., SSR markers specific for drought stress were selected from
2000; Huang et al., 2001; Quarrie et al., 2003; Somers et al., grain genes database based on their chromosomal position
2004; El-Maghraby et al., 2005; Eivazi et al., 2007; Prasad et (www.graingenes.org) and previous studies of Ciuca et al.
al., 2009; Bibi et al., 2010; Dodig et al., 2010; Achtar et al., (2009) (Table 3). Cocktail for PCR reaction was prepared by
2010; Hao et al., 2011; Kalia et al., 2011; El Siddig et al., adding Taq Buffer A (10X Tris with 15 mM MgCl2) followed by
2013). The molecular markers characterize genotypes accord- forward and reverse primers, dNTPs, Taq DNA polymerase
ing to a particular trait and thus molecular data is a must for (Fermentas, India) and template DNA. Cyclic amplification
executing improvement programmes in crops. Selection of were carried out in the Thermal Cycler (Whatman Biometra T-
drought tolerant genotypes adapted to the conditions of Gradient, Germany) by using following thermal amplification
Gujarat would preliminary require information on molecular conditions; initial denaturation at 94ºC for four mins., 39 cycles
data. Thus, molecular characterization of the local genotypes of denaturation at 94ºC for 30 sec., annealing temperature
which are released varieties or widely cultivated genotypes of ranged between 48-58ºC (5ºC less than the temperature
Gujarat would be useful for selection for molecular breeding melting of primers) for 45 sec., extension at 72ºC for one min.
according to the drought tolerance characteristics. In the and final extension at 72ºC for seven mins. The amplified
present study, an attempt was made to characterize the irri- products of SSR were analyzed using 2.5% agarose gel
gated and rainfed wheat genotypes of Gujarat employing RAPD prepared in 1X TBE buffer (2.5 g agarose in 100mL 1X TBE
and SSR markers. and 3 μL ethidium bromide 10 mg/mL). The PCR amplified
products (7 μL and 2 μL loading dye) were loaded into the
MATERIALS AND METHODS wells along with 100 bp standard DNA ladder (marker). The
electrophoresis was conducted at 100 V current (constant) to
Plant material separate the amplified bands. The separated bands were
Total 22 irrigated and rainfed wheat genotypes (Table 1), visualized under UV transilluminator and photographed using
employed in the present study were procured from the BIORAD Gel Documentation system.
Regional Research Station, Arnej, Anand Agricultural University Coefficients of similarity were calculated by using Jaccard’s
and Main Wheat Research Station, Vijapur, Sardarkrishinagar similarity coefficient by SIMQUAL function and cluster analysis
Dantiwada Agricultural University. was performed by agglomerative technique using the UPGMA
DNA isolation (Un-weighted Pair Group Method with Arithmetic Mean)
Total DNA was extracted from the ten days old wheat seedling method by SAHN clustering function of NTSYS-pc (Rohlf,
of all 22 genotypes by cetyl trimethyl ammonium bromide 1994). Relationships between the wheat cultivars were
(CTAB) method as described by Zidani et al., 2005. The graphically represented in the form of dendrograms. The
isolated DNA was checked spectrophotometrically at 260/ polymorphism rates of RAPD and SSR markers were determined
280 and 260/230 ratio for quantity and quality (in terms of using polymorphism information content (PIC) values,
protein and RNA contamination) using software N.D. (V.3.3.0). calculated according to the formula: PIC = 1 “ ∑ P2i, where Pi
To check the form of DNA (linear or sheared) and RNA is the frequency of the ith allele (Anderson et al., 1993). The
contamination of isolated genomic DNA, electrophoresis was resolving power (Rp) of a primer was calculated according to
done using 0.8% agarose gel and quality was judged by Prevost and Wilkinson (1999) as Rp = ∑ IB where IB (band
viewing the image of single compact DNA band. The DNA informativeness) takes the value of: 1–[2 × (0.5–P)], P being
was diluted to a working concentration of 20 ng/μL and the proportion of the 22 genotypes containing the band. Nei’s
subsequently used for PCR amplification reactions through
genetic diversity (H) among wheat genotypes was calculated
RAPD and SSR markers.
by using POPGENE software (Nei, 1973). Data was obtained
RAPD and SSR analysis for observed number of alleles (Na), effective number of alleles
Random primers were selected based on the previous studies (Ne), Shannon’s information index (I), expected heterozygosity
of Pakniyat and Tavakol, 2007 and Gorji et al., 2010 (Table (He), unbiased expected heterozygosity (uHe), number of
2). Amplification reaction was performed with 25 μL volume polymorphic loci (NPL), number of band unique to a single
of 2.5 PCR buffer (10 X) with 15 mM MgCl2, primer (10 pmoles/ population (NUP), percentage polymorphic loci (PPL) across

832
 ASSESSMENT OF MOLECULAR DIVERSITY IN WHEAT

Table 1: List of wheat genotypes

Triticum aestivum Triticum durum
Sr. No. Irrigated Sr. No. Rainfed Sr. No. Irrigated
1 LOK-1 8 A-206 17 GW-1139
2 GW-173 9 A-9-30-1 18 GW-1245
3 GW-273 10 AR-06-1 19 GW-1255
4 GW-322 11 AR-07-7 20 GW-1260
5 GW-366 12 AR-07-30 21 GW-1265
6 GW-496 13 AR-07-33 22 HI-8498
7 HW-2004 14 DR-08-6 - -
- - 15 DR-06-7 - -
- - 16 GW-1 - -

Table 2: List of primers used for RAPD amplification, GC content, total number of loci, the level of polymorphism, PIC value and resolving
power
Primer Primer sequence GC(%) Range of Total no Number of Percentage of Total number PICa RPb
band size of loci polymorphic polymorphic of fragment value
loci loci amplify
P-1 ACACAGAGGG 60 366-6114 31 20 64.52 503 0.96 10.82
P-2 CCTCTCGACA 60 397-6985 19 17 89.47 254 0.93 8.00
P-3 TCTCAGCTGG 60 258-3412 17 11 64.70 262 0.93 5.64
P-5 CCACGGGAAG 70 396-6407 23 12 52.17 388 0.95 6.36
P-7 CTGTATCGTG 50 501-6010 22 20 90.90 322 0.94 8.91
P-11 CCATTCCCCA 60 251-4448 22 15 69.18 345 0.95 9.73
P-12 GGTGAACGCT 60 218-4080 26 20 76.92 325 0.95 9.73
P-14 TTCCGGGTGA 60 315-4737 15 12 80.00 241 0.92 3.36
P-16 CCTGGGCTTC 70 305-4762 21 16 76.20 361 0.93 7.36
P-17 CCTGGGCTTG 70 235-3476 16 10 62.50 236 0.92 5.27
P-18 CCTGGGCCTA 70 406-4390 13 07 53.85 220 0.91 3.27
P-20 TGCCCCGAGC 80 326-4508 27 15 55.55 461 0.96 9.36
P-21 TTCCCCGACC 70 314-4256 16 11 68.75 256 0.93 8.00
P-24 GAGGTCCAGA 60 277-4501 18 10 55.55 323 0.94 3.18
P-25 GAGGTCCAGA 60 236-2046 20 15 75.00 294 0.94 6.91
P-26 ATCGGGTCCG 70 152-3137 15 08 53.33 248 0.92 4.00
P-27 CCGTGCAGTA 60 267-3216 18 16 88.88 266 0.93 6.73
P-30 TACGTGCCCG 70 226-2856 14 08 57.14 249 0.92 4.09
Total - - 353 243 66.83 5554 0.93 -
a
Polymorphic information content, bRP: Resolving power

22 wheat genotypes using GenAlEx 6.5. The RAPD and SSR RESULTS
data were subjected to a hierarchical analysis of molecular
variance (AMOVA) (Excoffier et al., 1992), using two RAPD polymorphism
hierarchical levels; individual and population. Principal In total, 5554 fragments were amplified using 18 RAPD primers
coordinates analysis (PCoA) that plots the relationship between with 353 loci, out of which 243 loci were polymorphic with
distance matrix elements based on their first two principal an average of 14 polymorphic loci per primer. The highest
coordinates (Peakall and Smouse, 2012) was calculated. (90.90%) polymorphism was exhibited by primer P-7, while
Diversity index (DI), effective multiplex ratio (EMR) and marker the lowest polymorphism (52.17%) was exhibited by primer
index (MI) were calculated according to Powell et al. (1996) P-5 and an average 68.83% polymorphism. The minimum
to determine the utility of each of the marker systems. MI is (152 bp) sized fragment was amplified by primer P-26, whereas
defined as the product of the average diversity index for maximum (6985 bp) sized fragment was amplified by primer
polymorphic bands in any assay and the EMR for that assay, P-2. The PIC value ranged from 0.91 (P-18) to 0.96 (P-1, P-20)
MI = DIavp × EMR. DI for genetic markers was calculated (Table 2) with an average of 0.93. The maximum number of
from the sum of the squares of allele frequencies: DIn = 1-∑ amplified fragments (503) were generated by primer P-1 with
pi2 (where ‘pi’ is the allele frequency of the ith allele). The resolving power of 10.82 and the minimum number of
arithmetic mean heterozygosity, Diav, was calculated for each amplified fragments (220) were generated by primer P-18 with
marker class: Diav = ∑ Din/n, (where ‘n’ is the number of a resolving power of 3.27. The respective values of Na, Ne, I,
markers (loci) analyzed). The DI for polymorphic markers is: He, uHe, NPL, NUP, PPL and MI across all the 22 genotypes
(Diav)p = ∑ Din/np (where ‘np’ is the number of polymorphic are presented in Table 4. Molecular variance within population
loci and n is the total number of loci). EMR (E) is the product of (64%) was more than among the population (36%).
the fraction of polymorphic loci and the number of Clustering pattern of dendrogram generated by RAPD data
polymorphic loci for an individual assay. EMR (E) = np (np/n). showed two major clusters I and II having similarity coefficient

833
HARSHVARDHAN ZALA et al.,

Table 3: List of primers used for SSR amplification, sequence, GC content, total number of loci, the level of polymorphism, size range of
fragments and resolving power
Primer Chromosomal Primer sequence GC% Tm(p Total Number Percent Total PICa RPb
position C) no of of poly- of poly- number of value
loci morphic morphic fragment
loci loci amplify
Xbarc121 7A F-CTGATCAGCAATGTCAACTGAA 40.9 54 5 5 100% 41 0.76 3.18
R-CCGGTGTCTTTCCTAACGCTATG 52.2
Xwmc603 7A F-ACAAACGGTGACAATGCAAGGA 45.5 58 4 3 75% 45 0.58 0.64
R- CGCCTCTCTCGTAAGCCTCAAC 59.1
Xgwm332 7A F- AGCCAGCAAGTCACCAAAAC 50 56 4 4 100% 43 0.70 2.09
R- AGTGCTGGAAAGAGTAGTGAAGC 47.8
Xwmc233 5D F- GACGTCAAGAATCTTCGTCGGA 50 57 2 2 100% 28 0.57 0.72
R- ATCTGCTGAGCAGATCGTGGTT 50
Xgwm260 7A F- GCCCCCTTGCACAAATC 58.8 54 5 4 80% 70 0.69 1.09
R- CGCAGCTACAGGAGGCC 70.6
Xgwm276 7A F- ATTTGCCTGAAGAAAATATT 25 46 4 3 75% 43 0.63 1.72
R- AATTTCACTGCATACACAAG 35
Xwmc9 4A, 7A F- AACTAGTCAAATAGTCGTGTCCG 43.5 55 4 4 100% 40 0.65 2.00
R- GTCAAGTCATCTGACTTAACCCG 47.8
Xwmc695 3A, 3B, F- GAGGGCACCTCGTAAGTTGG 60 59 3 3 100% 33 0.61 1.72
4B, 7A
R- GGCAGGAGCCCCTACAAGAT 60
Total - - - - 31 28 90.32% 343 0.63 -
a
Polymorphic information content, bRP: Resolving power

LOK-1 LOK-1
Ia GW-173 GW-273
GW-273 GW-366
Ia
I GW-322 HW-2004
GW-366 I
GW-496
GW-496 IIb GW-173
Ib
HW2004 GW-322
A-206 A-206
AR-O6-1 A-9-30-1
IIa AR-07-33 AR-06-1
AR-07-7 IIa
AR-07-7
AR-07-30 AR-07-30
DR-08-06 DR-08-06
A-9-30-1 AR-07-33
DR-06-07 DR-06-07
II II
GW-1 GW-1
GW-1139 GW-1245
GW-1245 GW-1260
GW-1255 GW-1255
IIb
GW-1260 GW-1139
IIb
GW-1265 HI-8498
HI-8498 GW-1265

0.65 0.72 0.78 0.84 0.90 0.45 0.57 0.69 0.82 0.94
Co-efficient Co-efficient

Figure 1: Dendrogram showing clustering of 22 wheat genotypes Figure 2: Dendrogram showing clustering of 22 wheat genotypes
constructed using UPGMA based on Jacquard’s similarity coefficient constructed using UPGMA based on Jacquard’s similarity coefficient
obtained from RAPD analysis obtained from SSR analysis
of 0.65 to 0.90 (Fig. 1). Cluster I included T. aestivum genotypes primer. The average percent polymorphic loci observed were
and was further divided into two sub clusters Ia and Ib. Cluster 90.32 with an average of 3.5 polymorphic loci per primer.
II included T. durum genotypes which was further divided The PIC values ranged from 0.57 (Xwmc233) to 0.76
into two sub clusters IIa and IIb for rainfed and irrigated (Xbarc121) with an average of 0.63 and the resolving power
genotypes respectively. The results of PCoA analysis were ranged from 0.72 (Xwmc233) to 3.18 (Xbarc121) (Table 3).
comparable to the cluster analysis. The first and second The respective values for overall genetic variability for Na, Ne,
principal coordinates explained 46.22% and 65.52% of the
H, I, He, uHe, NPL, NUP, PPL, MI are presented in Table 4.
molecular variance, respectively.
The first and second principal coordinates explained 38.68%
SSR polymorphism and 56.79% of the molecular variance, respectively. Clustering
In total, 343 amplified fragments were generated using eight pattern of dendrogram generated by SSR data showed two
SSR primers with an average of 43 amplified fragments per major clusters I and II having similarity coefficient of 0.45 to

834
 ASSESSMENT OF MOLECULAR DIVERSITY IN WHEAT

No. of different alleles, bNo. of effective alleles = 1 / (p2 + q2), cNei’s gene diversity, dShannon’s information index = -1 × (p × Ln (p) + q × Ln(q)), eExpected heterozygosity = 2 × p × q, fUnbiased expected heterozygosity = (2N/ (2N-
0.94 (Fig. 2). Cluster I included irrigated T. aestivum genotypes
11.35

5.35
MI j
and cluster II included T. durum genotypes which was further

-
-
-
-

-
-
-

-
-
-
sub clustered into IIa and IIb comprising rainfed and irrigated
genotypes respectively.

44.19%

55.91%

42.36%
42.49%
43.63%
46.46%

61.29%
61.29%
45.16%

41.15%
45.05%
40.89%
RAPD + SSR combined data
PPLi

The average percent polymorphic for combined RAPD and

SSR data was 42.36%. The respective values for overall genetic
variability for Na, Ne, H, I, He, uHe, NPL, NUP, PPL, MI are
NUP h

presented in Table 4. Clustering pattern of dendrogram

10

23
10
7

6
0
1

3
generated by combined RAPD and SSR data displayed two
-

-
major clusters I and II having similarity coefficient of 0.64 to
0.90. Cluster I included irrigated T. aestivum genotypes and
NPLg

318
321
328

348
343
335 cluster II included T. durum genotypes which was further
27
22
22
-

- divided into two sub clusters IIa and IIb comprising rainfed
and irrigated durum genotypes respectively (Fig. 3). AMOVA
0.229(0.041)
0.244(0.041)
0.190(0.042)
0.161(0.011)
0.175(0.012)
0.187(0.012)

0.149(0.011)
0.180(0.011)
0.168(0.011)

for combined RAPD and SSR displayed more variance within

and among population than RAPD and SSR (Table 5). The first
0.174

0.221

0.166
uHe f

and second principal coordinates explained 46.09% and

65.40% of the molecular variance, respectively (Fig. 4).
0.212(0.038)
0.230(0.039)
0.175(0.039)
0.149(0.011)
0.165(0.011)
0.172(0.011)

0.138(0.010)
0.170(0.011)
0.154(0.010)

DISCUSSION
Table 4: Summary of genetic variation statistics for all loci of RAPD, SSR and RAPD + SSR among the populations

Evaluation of genetic divergence and relatedness among

0.162

0.206

0.154

breeding materials has significant implications for the

Hee

improvement of crop plants (Chandra et al., 2013). Molecular

analysis by RAPD displayed specific amplified products
0.317(0.053)
0.338(0.054)
0.256(0.055)
0.222(0.015)
0.243(0.016)
0.254(0.016)

0.207(0.014)
0.250(0.015)
0.227(0.015)

unique to irrigated aestivum and durum genotypes. These

1)) × He, gNumber of polymorphic loci, hNumber of band unique to a single population, iPercent polymorphic loci, jMarker index.

markers can be employed to screen the aestivum and durum

0.240

0.303

0.228

wheat genotypes as they follow distinct clusters. The cluster

analysis distinguishes genotypes on the basis of their diversity
Id

and could be used as basis of selection of genotypes for crop

0.212(0.210)
0.230(0.215)
0.174(0.214)
0.131(0.189)
0.165(0.206)
0.152(0.203)

0.138(0.191)
0.170(0.208)
0.154(0.204)

improvement (Bharose et al., 2014). These RAPD markers

can be used for determination of association with drought
0.241

0.287

0.245

tolerance which corroborates with the results reported by

ElSayed and Rafudeen, 2012 in wheat genotypes. Our results
Hc

are also in agreement with earlier results of Hashad et al.

1.374(0.073)
1.412(0.076)
1.313(0.073)
1.262(0.020)
1.293(0.021)
1.304(0.021)

1.239(0.018)
1.302(0.020)
1.275(0.020)

(2005); Fadly et al. (2007); Gorji et al. (2010). Pakniyat and

Tavakol (2007) observed specific bands present in drought
tolerant cultivars associated with drought tolerance in wheat
1.286

1.366

1.272
Neb

which corroborates with the present study. The SSR markers

were able to distinguish the aestivum and rainfed and irrigated
durum genotypes. The polymorphism exhibited among
1.326(0.034)
1.346(0.034)

1.484(0.130)
1.394(0.033)

1.323(0.163)
1.161(0.154)

1.318(0.032)
1.344(0.034)
1.281(0.035)

irrigated T. aestivum and rainfed and irrigated T. durum

consistent with the earlier findings by Younes (2009). SSR
1.355

1.323

1.314

markers were selected based on chromosomal position

Na a

associated with drought tolerance. Drought tolerance in wheat

is associated with chromosome 7A (Morgan and Tan, 1996;
Sample size

Galiba, 2002; Cattivelli et al., 2002). Membrane stability has

been suggested as a useful measure of drought tolerance in
wheat breeding programs. Morgan (1991) located a gene for
7
9
6

7
9
6

7
9
6

osmoregulation (‘or’) on chromosome 7A. Tian-Mei et al.

-

(2010) established association of SSR markers located on the

5D and 3B with drought tolerance in wheat cultivars. The
Marker and Population

T. aestivum (Irrigated)

T. aestivum (Irrigated)
T. aestivum(Irrigated)
T. Durum (Irrigated)

markers selected from these chromosomes showed PIC value

T. durum (Irrigated)

T. durum (irrigated)
T. durum (Rainfed)

T. durum (Rainfed)

T. durum (rainfed)

more than 0.5, pointing out its utility in distinguishing the

genotypes according to their response to water deficit
RAPD + SSR

conditions (Sonmezoglu et al., 2012). Recent study by Siddig

et al., 2013 showed that SSR markers were associated with
RAPD

Mean

Mean

Mean

drought tolerance in wheat. Dendrograms generated through

SSR

RAPD, SSR and combined RAPD and SSR indicated major

a

835
HARSHVARDHAN ZALA et al.,

Table 5: Analysis of molecular variance (AMOVA) based on RAPD and SSR individually and in combination, among the populations of wheat.
Levels of significance are based on 1000 iteration steps
Among population Within population
d.f. 2 19
Marker RAPD SSR RAPD + SSR RAPD SSR RAPD + SSR
S.S.D. 304.716 36.420 374.159 568.921 65.262 601.159
Variance component 16.938 2.044 21.507 29.943 3.435 31.640
Percentage 36% 37% 40% 64% 63% 60%
P-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

LOK-1
GW-273
GW-322
Ia
GW-366
I GW-496
A-206
GW-173 A-9-30-1
IIb HW-2004 AR-07-7

IIa A-206 AR-06-1

A-9-30-1
AR-07-30
AR-06-1 HW-2004

AR-07-7 AR-07-33

II AR-07-33
DR-08-6
T. aestivum
(irrigated)
AR-07-30 GW-322 T. durum
GW-173 GW-366
DR-08-06 (Rainfed)
GW-496 GW-273 T. durum
DR-06-7 LOK-1 (irrigated)
DR-06-7
IIb GW-1
GW-1139
GW-1139
GW-1245 GW-1
GW-1255 HI-8498
GW-1260
GW-1260 GW-1255 GW-1265
GW-1265
HI-8498
0.45 0.57 0.69 0.82 0.94
Co-efficient
Figure 3: Dendrogram showing clustering of 22 wheat genotypes Figure 4: Two-dimensional plot of principal component analysis
constructed using UPGMA based on Jacquard’s similarity coefficient (PCoA) of 22 wheat genotypes using RAPD + SSR analysis
obtained from RAPD+SSR analysis

three clusters consisting irrigated aestivum, rainfed durum and rainfed and irrigated conditions of Gujarat. The information
irrigated durum genotypes. The rainfed durum genotypes, DR- provided by the molecular markers in wheat would be
06-7 and GW1 clustered with irrigated durum genotypes which beneficial for breeding for drought tolerance and selection of
can be ascribed to the common parentage of this genotype genotypes for cultivation in the rainfed areas of Gujarat.
with the irrigated durum genotypes. The similarity coefficient
for RAPD (0.65 to 0.90), SSR (0.45 to 0.94) and combined ACKNOWLEDGEMENT
RAPD and SSR (0.64 to 0.90) indicated more diversity for SSR
markers. Our results are in agreement with earlier studies of The authors are grateful to Dr. G. C. Jadeja, Department of
Ciuca and Petcu (2009); Dreisigacker et al. (2004) in assessing Agricultural Botany and Dr. R. S. Fougat, Department of
wheat genetic diversity. The mean values of Na, Ne, H, I, He, Agricultural Biotechnology, Anand Agricultural University,
uHe, NPL, NUP were found to be higher for SSR than RAPD. Anand for providing research facilities.
The molecular markers employed in the present study
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