Indian J. Genet.

, 68(3): 255-260 (2008)

Assessment of genetic diversity among pigeonpea genotypes using

SSR markers
1

Shubhanjana Singh , K. N. Singh , Rama Kant , Sahil Mehfooz and S. Dutta

Department of Plant Molecular Biology and Genetic Engineering, N.D.U.A.&T., Kumarganj, Faizabad 224 229
Department of Genetics and Plant Breeding, N.D.U.A.&T., Kumarganj, Faizabad 224 229
3
Department of Biotechnology, IIPR, Kanpur 208 024
2

(Received: January 2008; Revised: June 2008; Accepted: June 2008)

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Abstract
Twenty two SSR markers of different crop species origin
were used to assess polymorphism through their SSR
fingerprinting of 16 cultivated pigeonpea genotypes. Four
hundred twenty five bands were amplified in all the sixteen
genotypes. A total of 46 SSR fragments were amplified.
Eight primers showed 100% polymorphism. Based on
dendrogram constructed using the similarity coefficient
values, 16 genotypes were grouped into two distinct
clusters. Cluster I comprises mostly late duration
genotypes while cluster II comprises medium duration
genotypes except CO-6 and Bahar. Both the clusters and
sub-cluster in the dendrogram were supported by high
bootstrap values, thus indicating that the SSR could be a
good choice to classify the genotypes. Genotypes with
high molecular diversity could be used in breeding
methodologies and development of gene pools with broad
genetic base. The genotype specific bands developed by
the SSR primers could also be used for cultivar
identification.

Key words:

Cajanus cajan, DNA isolation, fingerprinting, genetic diversity, SSR markers

Introduction
Pigeonpea [Cajanus cajan (L.) Millsp.] is an important
grain legume of the Indian sub-continent, Southern Asia,
Africa and central America. It is the most widely grown
legume after chickpea in India covering an area of 975
thousand hectares in the states of Maharashtra,
Karnataka, Andhra Pradesh, Uttar Pradesh, Madhya
Pradesh and Gujarat. In order to improve the production,
a large number of varieties have been released for
different agro-climatic zones. To identify unique primers
that can identify specific genotypes and determine the
genetic diversity has emerged as important aspect for
Corresponding authors e-mail: shubha_28097@yahoo.com

crop improvement programme. RAPD (Randomly

Amplified Polymorphic DNA), ISSR (Inter Simple
Sequence Repeats), microsatellites and AFLP
(Amplified Fragment Length Polymorphism) are the
commonly used molecular markers for characterization
of genotypes. The use of SSR markers, which are highly
polymorphic in nature, has been advocated for such
studies (Song et al., 1999). Further they are robust,
repeatable, co-dominant and follow mendelian
inheritance. SSR markers have been used in many
legumes like chickpea (Sharma et al., 1995; Sant et al.,
1999), alfalfa (Mengoni et al., 2000) and in soybean
(Doldi et al., 1997). The present study was undertaken
to investigate and quantify the magnitude of genetic
diversity at molecular level using SSR markers. The
results are communicated here under.
Materials and methods
A set of 16 genotypes of pigeonpea were obtained from
Pulse Section, Department of Genetics and Plant
Breeding, N.D.U.A.&T., Faizabad. These lines were
grown in the field during Kharif (2007-08). Fresh 30 days
old leaves were taken for DNA extraction. A modified
protocol without liquid nitrogen was used (Guillemant
and Laurence, 1992). Leaves from young seedlings were
pulled together and ground with grinding buffer until a
thick paste was formed. The composition of the grinding
buffer was as follows : 100 mM, sodium acetate. (pH
4.8) 500 mM NaCl, 50 mM EDTA; pH 8.0; 50 mM Tris,
pH 8.0; 2% PVP; 1.4% SDS. Purification of DNA was
done twice with extraction of phenol : chloroform :
Isoamyl alcohol (25:24:1). RNAse @ 40 l from 1 mg/
ml stock was applied in the supernatant to get rid of
RNA. DNA quality and quantity from all the individual

Shubhanjana Singh et al.

256

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genotypes were checked through 0.8% agarose

electrophoresis with standard DNA before PCR
amplification. A total of 22 SSR primers (Operon
technologies, Alameda, CA, USA) were selected for
PCR analysis based upon their performance and
reproducibility. Among them 14 primers have
given distinct polymorphism (Table 2) PCR mixture of
25 l contained 25 ng of genomic DNA template, 0.6
q of Tag DNA polymerase (Bangalore Genei,
Bangalore, India), 0.3 M of decamer primers, 2.5 l of
10 x PCR assay buffer (50 mM KCI, 10 mM Tris-Cl), 1.5
m MgCl2) and 0.25 l of pooled dNTPs (100 mM each
of dATP, dCTP, dGTP and DTTP from Fermentas Life
Sciences, USA). PCR condition used for SSR
amplification were follows. Initial denaturing step at 94C
for 3 minutes followed by 34 cycles of 94C for 1 minute,
55C for 1 minute and 72C for 2 minute. In the last
cycle, primer extension at 72C for 7 minutes. The
Table 1.

[Vol. 68, No. 3

amplified products as developed by the primers were

separated by agarose (1.5%) gel electrophoresis and
documented in gel documentation system (Bio Rad XR,
Biorad, USA). OGene Ruler 50 bp DNA Ladder plus
(ladder range 3000 bp to 100 bp from Fermentas Life
Sciences, USA) was used as molecular weight marker.
FNA bands were scored for its presence/absence (1/0)
for each primer genotypes combination. Software
NTSYS-pc, version 1.7 (Rohlf, 1992) was used for
estimation of genetic relatedness among the genotypes
using Jaccards similarity coefficient and clustering was
done with UPGMA (unweighted pair group method using
arithmetic averages). Strength of clusters was evaluated
by bootstrap analysis using win Boot software (Yap and
Nelson, 1995).
Results and discussion
Out of a total of 22 primers tested, 14 SSR primers

Details of pigeonpea genotypes used in the diversity assessment

S. No. Genotype

Pedigree

Area of adaptability

Special features

1.

CO-6

Mutant of SA1

Tamil Nadu

Medium duration, indeterminate

2.

BSMR-853

ICP7336/BDN-1//BDN-2

Western & Central

part of India

Spreading

3.

ICPL-87119

C11/ICPL-6

M.P., Gujarat,
Maharashtra, A.P.,
Karnataka, T.N.

Indeterminate, medium duration,

spreading, large brown seed, resistant
to wilt and sterility mosaic

4.

TAT-10

TT8/TT2

Maharashtra

Extra short duration

5.

GS-1

Selection from local

germplasm

Karnataka

Medium duration, white seeded

6.

LRG-38

Medium duration

7.

Bahar

Selection from land race of

Motihari

U.P., Bihar

Resistant to sterility mosaic, suitable for

pre-rabi sowing

8.

LRG-30

Local selection of Lam

Andhra Pradesh

White large seed, medium maturing

9.

MA-3

Selection from Malviya Arhar-2 Central zone

Spreading, constricted pods

10.

MA-6

MA-2/Bahar

Uttar Pradesh

Spreading

11.

MAL-13

MA-2/MA-166//Bahar

U.P., Bihar,
West Bengal

Spreading , resistant to sterility mosaic

and large seed size

12.

NDA-1

Selection from land race of

Faizabad (U.P.)

Uttar Pradesh

Resistant to sterility mosaic and tolerant

to wilt and phytophthora stem blight

13.

Pusa-9

UPAS-120/3673

U.P., Bihar,
West Bengal

Indeterminate, resistant to phytophthora

stem blight and sterility mosaic, suitable
for pre-rabi planting

14.

DA-11 (Sharad)

Bahar/NP (WR) 15

Bihar

Compact, resistant to phytophthora

blight and sterility mosaic, suitable for
pre-rabi sowing

15.

K91-25 (Azad)

Bahar/KPBR80-1

U.P., Bihar

Resistant to sterility mosaic

16.

KA32-1 (Amar)

Selection from Bahar

Uttar Pradesh

Compact, resistant to sterility mosaic

Source: Advances in pigeonpea research, 2005.

Genetic diversity among pigeonpea genotypes using SSR markers

August, 2008]

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Table 2.

Details of polymorphic primers used for diversity assessment in 16 genotypes of pigeonpea

S. No.

Primer code

Base sequence (5'3')

1.

PP4

2.

PP5

3.

PP7

4.

PP8

5.

PP9

6.

PV4

7.

PV13

8.

PV14

9.

PV16

10.

PV20

11.

PV21

12.

PV23

13.

L24

14.

L31

GGAGCTATGTTGGAGGATGA
CCTTTTTGCATGGGTTGTAT
GACAATTTTGCATGCATTGC
TTGCAAAAACACTTGGTTGG
CAACATTTGGACTAAAAACTG
ACTTGAGGCTGAATGGATTTG
TGCGTTTGTAAGCATTCTTCA
AGGTATCCAATATCCAACTTG
CACTTGGTTGGCTCAAGAAC
GCCAATGAAATCACATCCTTC
CTTCACCGATCTGACAGCAT
TTTCTCCACTGGAACACTCG
ACCTGGTCCCTAAAACCAAT
CAATGGAGCACCAAAGATCA
CCCCACCAACTCTTTCTTCC
TAGAATTGACTTGGCGAGAA
TGGTGAGAGAAGGACAATAGCA
GCCGCTTGTGACGTTTATTT
GGCTCCACCATCGACTACTG
GAATGAGGGCGCTAAGATC
GTCCGTTCATGGGTTTGACT
TCGAGATCTACGGAGGAGTTC
CATCAACAAGGACAGCCTCA
GCAGCTGGCGGGTAAAACAG
CTTGGAGCTGTTGGTC
GCCGCCTACATTATGG
TTACGAAAAAGGCAAACATA
ATCTTCTTCTTCTTCTTCTCA

Table 3.

257

Forward/ Reverse

Source

F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R

C. cajan
C. cajan
C. cajan
C. cajan
C. cajan
P. vulgaris
P. vulgaris
P. vulgaris
P. vulgaris
P. vulgaris
P. vulgaris
P. vulgaris
L. culinaris
L. culinaris

Amplified product and per cent polymorphism obtained by using SSR markers in 16 genotypes of pigeonpea

S. No.

Primer

No. of
amplified bands
(y)

No. of
polymorphic bands
(x)

Size
range (bp)

Polymorphism
(%) x x 100
y

Unique
bands

PIC
value

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Total

PP1
PP2
PP3
PP4
PP5
PP6
PP7
PP8
PP9
PP10
PV4
PV13
PV14
PV16
PV20
PV21
PV22
PV23
PV24
L14
L24
L31
46

1
2
1
2
3
1
1
3
5
1
2
2
5
1
2
1
1
2
1
1
5
3
32

0
0
0
2
3
0
1
3
5
0
2
1
4
1
1
1
0
1
0
0
5
2
-

200
180, 200
70
250, 270
200-215
225
165
80-120
150-470
70
375, 480
150, 325
140-600
135
70, 150
200
80
260, 410
60
100
150-900
180-490
69.5

0
0
0
100
100
0
100
100
100
0
100
50
80
100
50
100
0
50
0
0
100
66.6
-

120 bp (Pusa-9)
600 bp (Co-6)
-

0.01
0.07
0.13
0.67
0.58
0.07
0.01
0.68
0.78
0.54
0.39
0.48
0.54
0.08
0.25
0.36
0.32
0.03
0.10
0.17
0.78
0.63

Shubhanjana Singh et al.

258

Fig. 1b. SSR profile of pigeonpea genotypes with primer

PP-5. Genotypes (1-16): MA-3, MA-6, MAL-13,
NDA-1, Pusa-9, DA-11, Azad, Amar, CO-6, BSMR853, ICP-87119, TAT-10, GS-1, LRG-38, Bahar and
LRG-30

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Fig. 1a. SSR profile of pigeonpea genotypes with primer

PP-9. Genotypes (1-16): MA-3, MA-6, MAL-13, NDA1, Pusa-9, DA-11, Azad, Amar, CO-6, BSMR-853,
ICP-87119, TAT-10, GS-1, LRG-38, Bahar and LRG30

[Vol. 68, No. 3

Fig. 2a: Amplification with primer PV-14

Table 4.

Fig. 2b: Amplification with primer PP-8

Jaccards similarity coefficients based on SSR markers in 16 cultivated pigeonpea genotypes

MA-3

MA-6

Mal-13 NDA-1

Pusa-9 DA-11

Azad

Amar

CO-6

BSMR-853

ICP-87119 TAT-10

GS-1 LRG-38 Bahar

1.

1.00

2.

0.69

1.00

3.

0.63

0.63

1.00

4.

0.40

0.60

0.63

1.00

5.

0.32

0.48

0.50

0.80

6.

0.42

0.63

0.65

0.88

0.77

1.00

7.

0.63

0.82

0.76

0.63

0.50

0.65

8.

0.38

0.58

0.48

0.77

0.68

0.80

0.61

1.00

9.

0.26

0.36

0.43

0.68

0.71

0.65

0.43

0.67

1.00

10.

0.32

0.42

0.43

0.45

0.45

0.46

0.50

0.60

0.64

1.00

11.

0.28

0.48

0.38

0.50

0.55

0.52

0.50

0.59

0.69

0.70

1.00

12.

0.42

0.40

0.50

0.33

0.27

0.35

0.50

0.42

0.43

0.67

0.57

1.00

13.

0.37

0.48

0.30

0.44

0.41

0.41

0.43

0.48

0.43

0.38

0.64

0.42

1.00

14.

0.40

0.50

0.39

0.52

0.47

0.48

0.52

0.44

0.50

0.46

0.73

0.53

0.68

15.

0.33

0.38

0.33

0.42

0.34

0.38

0.40

0.40

0.37

0.41

0.48

0.47

0.47

0.67

1.00

16.

0.35

0.50

0.45

0.57

0.56

0.59

0.59

0.56

0.66

0.58

0.78

0.52

0.52

0.76

0.50

LRG-30

1.00
1.00

1.00
1.00

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August, 2008]

Genetic diversity among pigeonpea genotypes using SSR markers

showing polymor phism and providing proper

amplification pattern were selected. Out of these, two
primers showed uniqueness by producing two genotype
specific bands in two genotypes (Table 2). A total of 46
amplified products were obtained with an average of
2.1 amplicons/primers, out of which 32 (78%) bands
were polymorphic. DNA amplification pattern as detected
by two of the SSR primers used in the present study
(PV-13 and PV-23) has been presented in Fig. 1a and
1b. Molecular diversity analysis vis-a-vis identification
of cultivars is important for variety registration, protection
of plant breeders right and identification of quite
similar/duplicate genotypes. Moreover, alongwith the
morphological data, DNA data is well accepted in
defining DUS (Distinctiveness, Uniformity and Stability).
The study identified two primers (PP8 and PV14) giving
unique bands (Fig. 2 a&b) in genotypes Pusa 9 and CO
6. These infor mative pr imers either singly or in
combination may be of great potential use in the
establishment of identities of unknown genotypes and
to monitor the designed crosses involving these
genotypes. Genotype specific DNA amplification
products could be used as reference for fingerprinting
and in due course these bands could be converted into
Sequence Characterized Amplified Regions (SCARs)
or Cleaved Amplified Polymorphic Sequence (CAPS)
marker for varietal conformity tests.
In order to quantify the level of polymorphism
detected through Jaccards estimate of similarity
coefficients based on the probability that an amplified

259

fragment from one plant will also be found in another

was used to generate a similarity matrix (Table 4). All
genotypes fall in the range of 0.26 to 0.88 (shown by
bold figure in Table 4) with the average being 0.57. Earlier
Ratnaparkhe et al. (1995) detected pigeonpea
genotypes with RAPD markers for their identification and
genetic divergence. They got little polymor phism
(similarity matrix ranges from 0.7 to 0.9) in 10 pigeonpea
genotypes and suggested the use of microsatellites to
study the genetic diversity among the genotypes.
The similarity coefficients values were highest
between the genotypes NDA-1 and DA-11 (0.88),
followed by MA-6 and Azad (0.82), NDA-1 and Pusa-9
(0.80) and DA-11 and Amar (0.80), ICP-87119 and LRG30 (0.78), Pusa-9 and DA-11 (0.77), NDA-1 and Amar
(0.77), MAL-13 and Azad (0.76) and LRG-38 and LRG30 (0.76). These genotypes showed maximum degree
of similarity (> 75%) in their genetic makeup. However,
the minimum values of similarity coefficients were
observed between MA-3 and CO-6 (0.26) followed by
Pusa-9 and TAT-10 (0.27) and MA-3 and ICP-87119
(0.28). Thus these genotypes were highly diverse. The
crossing among these genotypes would likely to produce
highly heterotic individuals and could be used in
heterosis breeding.
The dendrogram based on UPGMA clearly
separate late and medium varieties into two distinct
clusters (Fig. 3) with the exception of CO6 and Bahar
(shown by astrick in figure 2). The grouping of CO6 with

Fig. 3. UPGMA dendrogram based on 45 SSR alleles in markers in 16 cultivated pigeonpea genotypes

Shubhanjana Singh et al.

260

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late genotypes can be attributed to the fact that CO6 is

a result of mutation in SA-1 causing an abrupt change
in the nucleotide sequence of DNA. Thus many cryptic
changes in the DNA base composition may cause
micromutations in the polygenic or quantitative traits.
These two clusters further divided into two subclusters
of each. Subcluster IA comprises of varieties, MA3, MA6,
Azad and MAL 13 while, subcluster IB consisted 5
genotypes, namely, NDA 1, DA 11, Amar, Pusa-9 and
CO-6. The genotypes BSMR 853 and TAT 10 were
presented in the IIA and the genotypes ICPL 87119,
LRG 30, LRG 38, GS 1 and Bahar in the subcluster IIB.
In cluster I, NDA I and DA 11 showed maximum similarity
(high bootstrap values) which is due to the fact that both
genotypes belong to nearly similar geographical regions.
The similarity is closely followed by MA3 and Azad which
is due to one of their common parents.
SSRs are relatively quick and easy to use, but
refractory to many environmental influences. However,
they can be generated in large numbers and can
complement traditional character that may be limited in
availability. SSRs also provide a valuable new resource
for phylogenetic studies.
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