Analysis of Genetic Diversity and

Population Structure in Horsegram

(Macrotyloma uniflorum) Using RAPD
and ISSR Markers
Vikas Sharma, Tilak Raj Sharma, Jai
Chand Rana & Rakesh Kumar Chahota

Agricultural Research
ISSN 2249-720X
Agric Res
DOI 10.1007/s40003-015-0165-7

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Agric Res
DOI 10.1007/s40003-015-0165-7

FULL-LENGTH RESEARCH ARTICLE

Analysis of Genetic Diversity and Population Structure

in Horsegram (Macrotyloma uniflorum) Using RAPD and ISSR
Markers
Vikas Sharma1 Tilak Raj Sharma1 Jai Chand Rana2 Rakesh Kumar Chahota1

Received: 1 December 2014 / Accepted: 2 March 2015

NAAS (National Academy of Agricultural Sciences) 2015

Abstract Macrotyloma uniflorum (horsegram) is an underutilized warm season pulse crop used as food and fodder in
many semi arid regions of the world. In the present study, genetic structure and diversity of M. uniflorum was analysed
using RAPD and ISSR markers. Two other species of the genus Macrotyloma namely M. axillare and M. sar-gharwalensis
were also included to assess genetic inter-relationships. In total, 25 polymorphic primers amplified 156 fragments ranging
in size from 300 to 3000 bp. Primer wise fragments ranged from 2 (OPR-20) to 13 (OPB-5, OPB-12 and OPS-14) with an
average of 6.24 fragments per primer. Highest PIC value of 0.499 was recorded for primer OPR-20 and lowest (0.013) for
primer OPR-2 with an average of 0.344. STRUCTURE analysis clustered accessions on the basis of their geographic origin
and showed the presence of two distinct gene pools. Dendrogram based on Jaccards similarity coefficient also grouped the
accessions into two groups and revealed that M. sar-gharwalensis is more distantly related to the cultivated than M.
axillare. PCA analysis also confirmed clustering results shown by STRUCTURE. Partitioning of genetic variation using
AMOVA revealed 63 % within population variation with only 37 % genetic variation among populations. Few horsegram
accessions showed high levels of genetic diversity which can be exploited in crop breeding programmes for its genetic
improvement.
Keywords

Horsegram  Genetic diversity  Genetic structure  RAPD  ISSR

Introduction
Macrotyloma uniflorum (Lam.) Verdcourt, commonly
known as horsegram, is an underutilized warm season food
legume with little genetic and genomic information available. Horsegram is mainly cultivated as pulse crop in India
and as a forage crop in many other semi arid regions of the

Electronic supplementary material The online version of this

article (doi:10.1007/s40003-015-0165-7) contains supplementary
material, which is available to authorized users.
& Rakesh Kumar Chahota
rkchahota@yahoo.com
1

Department of Agricultural Biotechnology, CSKHP

Agricultural University, Palapmur 176062, HP, India

National Bureau of Plant Genetic Resources, Regional

Station, Phagli, Shimla, HP, India

world [3, 17]. The wild members of this plant species are
found both in South Africa and India, however, India is
considered to be the centre of origin of horsegram [3, 30,
37, 40, 41, 45]. Horsegram has huge potential as future
pulse crop due to its therapeutic values and agronomically
important characteristics. It possesses many desirable traits
such as tolerance to salinity, heavy metals and drought [33,
38]. Moreover, the species exhibits many medicinal properties such as antioxidant activity, antimicrobial properties
and is also shown to be effective in dissolution and dislocation of kidney stones [4, 20, 31, 32]. Extracts from
horsegram seeds have shown significant activity against
many bacteria [15]. It is also shown to be effective against
type 2 diabetes and seeds of horsegram contains insoluble
dietary fibre which are required in normal functioning of
lower intestine [1, 18, 19]. As a fodder crop, horsegram has
shown better performance than other forage crops such as
Stylosanthes hamata, Vigna unguiculata and Crotalaria

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juncea [24]. Despite all these properties, its importance has

still not been realized and the species remained neglected
in terms of practical breeding and basic research. The
production and area under horsegram cultivation in India
has been gradually declining due to plant types unsuitable
for intensive agriculture. In order to meet this challenge,
introgression of desirable traits to well adapted genotypes
from cultivated and wild species is required. However,
before initiating systematic horsegram improvement programme, it is imperative to understand the extent of genetic
diversity present in the available germplasm. In horsegram,
few studies on phenotypic and biochemical diversity have
been reported [22, 23, 28], however there is no report on
diversity based on molecular markers. Therefore, the study
was conducted to assess the genetic diversity present in
available horsegram germplasm using randomly amplified
polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers.

9.0, 50 mM KCl, 0.01 % Geletin, 1.5 mM MgCl2), 0.2 ll

dNTPs (25 mM), 1.0 ll MgCl2 (25 mM), 1.0 ll primer
(2 lM/ll), 0.1 ll Taq DNA polymerase (5 U/ll, Himedia
Pvt. Ltd. Bombay, India) and rest deionised water to make
a final volume of 25 ll. The PCR cycles were one denaturation cycle at 94 C for 5 min, followed by 45 cycles
at 94 C for 1 min, annealing at 37 C for 1 min and extension at 72 C for 2 min. The final extension cycle was
carried out at 72 C for 7 min. For ISSR primers, reaction
mixture and PCR cycles remained same as for RAPD except annealing temperatures, which were 54 C for ISSR
11, 55 C for ISSR 17, ISSR 18, ISSR 23, ISSR 24 and
57 C for ISSR 16. All PCR reactions were carried in
I-Cycler (ABI, USA). PCR products were run on 1.5 %
agarose gel and size of each fragment was estimated using
100 bp plus ladder (MBI Fermentas, Lithuania). For band
visualization ethidium bromide was used and permanent
photographs of gels were taken in gel documentation system (Bio-Rad laboratories-segrate, Milan, Italy).

Materials and Methods

Data Analysis

Plant Material and DNA Extraction

All RAPD and ISSR fragments were scored manually and

converted into binary data i.e. 1 for the presence of band
and 0 for the absence of band. Polymorphism information
content (PIC) was calculated using formula given by Roldan-Ruiz et al. [36] as below:

Fifty-one horsegram accessions were used in this study, of

which forty-nine accessions belonged to M. uniflorum and
one accession each to Macrotyloma axillare and
Macrotyloma gharwalensis (Table 1). Of the forty-nine M.
uniflorum accessions, twelve were mutants or hybrids
generated by our group previously [5]. Seeds of remaining
thirty-seven accessions were collected from different
horsegram producing regions of the country. Seeds of M.
axillare and M. sar-gharwalensis accessions were procured
from Australia and Uttarakhand, respectively. Seeds were
grown in experimental fields and fresh young leaves were
taken for isolation of DNA following CTAB method [7].
DNA stocks were prepared in Tris (10 mm) EDTA (1 mm)
buffer and quantification of DNA was done in 0.8 %
agarose gel by comparing it with uncut lambda DNA
(Fermentas, Lithuania). Working stock of each sample was
prepared by diluting them to make a concentration of
13 ng/ll. These dilutions were further checked on 0.8 %
agarose before using in PCR reactions.
RAPD and ISSR Fingerprinting
RAPD technique [42] was followed for fingerprinting the
DNA of accessions. Initially, 45 RAPD primers and 30
ISSR primers were screened and out of these, 19 polymorphic RAPD primers and 6 polymorphic ISSR primers
(Table 2) were selected for genotyping of 51 horsegram
accessions. RAPD reaction mixture consisted of 1.5 ll
DNA (13 ng/ll), 2.5 ll 109 PCR buffer (10 mM Tris, pH

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PICi 2f i 1  f i ;
where PICi is the polymorphic information content of
marker I, fi is the frequency of the marker bands present
and (1-fi) is the frequency of marker bands absent. Marker
index (MI) was calculated by applying the formula given
by Varshney et al. [39]. Other genetic diversity estimates
such as Neis gene diversity (h), number of effective alleles
(Ne) and Shannon information index (I) were calculated
with the help of POPGENE version 1.32 [43]. Distancebased cluster analysis was performed and dendrogram
based on the unweighted pair-group method of arithmetic
mean (UPGMA) was constructed using Jaccards similarity
coefficient with the help of NTSYSpc 2.0 [35]. Neighbourjoining (N-J) tree was constructed using Dice coefficient
with the help of DARwin [27]. Bayesian model-based
clustering method implemented in STRUCTURE software,
version: 2.3.3 [13, 29] was used to assess the genetic
structure at population level as well as to detect gene pools
contributing to this germplasm collection. Ancestry model
with admixture and correlated allele frequency model was
set to get the estimates of posterior probability of data. Ten
independent runs were given setting the value of K from 1
to 5 with three iterations for each value of K. Both, length
of burn-in period and number of Markov Chain Monte
Carlo (MCMC), repeat after burn-in was set at 100,000.

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Table 1 List of horsegram accessions used in present study

Table 1 continued

S. no.

Genotype

Location

S. no.

Genotype

Location

1.

HPKC-114

Kangra

49.

IC-544828

Kinnaur

2.

IC-56142

Andhra Pradesh

50.

IC-547543

Kullu

3.

HPKM-151

Kangra

51.

IC-544834

Kangra

4.

IC-94591

India

5.

IC-43510

Karnataka

6.

HPKC-113

Kangra

7.

IC-120821

India

8.

HPKM-317

Kangra

9.

HPKC-132

Kangra

10.

HPKM-1000

Kangra

11.
12.

HPKM-193
HPKM-319

Kangra
Kangra

13.

HPK-4

Kangra

14.

VLG-1

Uttarakhand

15.

HPKC-2

Kangra

16.

HPKM-201

Kangra

17.

HPKC-119

Kangra

18.

HPKM-150

Kangra

19.

M.gharwalensis

Uttarakhand

20.

HPKM-249

Kangra

21.

IC-139356

Rajasthan

22.

IC-22766

Madhya Pradesh

23.

IC-139329

Maharashtra

24.

IC-139449

Bihar

25.

Himganga

Kullu

26.

IC-139444

Rajasthan

27.
28.

M.axillare
IC-107337

Australia
Chamoli

29.

IC-107344

Pithoragarh

30.

IC-94636

Uttarkashi

31.

IC-94637

Uttarkashi

32.

IC-107446

Pithoragarh

33.

IC-106912

Doda

34.

IC-106914

Doda

35.

IC-108079

Chamba

36.

IC-107188

Kangra

37.

IC-139555

Mandi

38.

IC-278827

Sirmour

39.

IC-280031

Chamba

40.

IC-278826

Sirmour

41.

IC-313262

Chamba

42.
43.

IC-469259
IC-469266

Shimla
Kangra

44.

IC-469271

Chamba

45.

IC-469272

Kangra

46.

IC-469273

Kangra

47.

IC-544826

Shimla

48.

IC-544827

Shimla

Evannos method [12]-based programme STRUCTURE

HARVESTER developed by Earl and Vonholdt [10] was
used to determine the value of estimated Ln probability of
data- LnP(K) and to get the best-fit value of K for the data.
Genetic differentiation (Fst) estimates among inferred
clusters were also measured by STRUCTURE software.
Genetic relationships among the genotypes were also
analysed by principal coordinate analysis (PCA) using the
NTSYSpc 2.0 [35]. Analysis of molecular variance
(AMOVA) was performed with the help of GenAlex version 6.41 [26].

Results
RAPD and ISSR Polymorphism
Only unambiguous and reliable fragments amplified by 19
RAPD and 6 ISSR primers were scored. In total, 25 primers
amplified 156 polymorphic fragments ranging from 2 to 13
with an average of 6.2 fragments per primer. Size range of
amplified fragments varied from 300 to 3000 bp. Primer
OPR-20 amplified minimum two fragments while three
primers namely OPB-5, OPB-12 and OPS-14 amplified a
maximum of 13 fragments. A representative amplification
profile of a RAPD and an ISSR is shown in Fig. 1. PIC
value ranged from 0.013 for primer OPR-2 to 0.499 for
primer OPR-20 with an average of 0.344 (Table 2). MI was
highest (6.22) for primer OPB-12 and lowest (0.02) for
OPR-8 with a mean value of 2.14. Highest (0.48) and
lowest (0.03) Neis gene diversity (h) values were obtained
with primers OPS-04 and OPB-16, respectively. A highest
(0.68) Shannons Information index (I) value was obtained
with primer OPS-04 and lowest (0.07) with OPB-16 primer, with an average of 0.45 as shown in Table 3.
Bayesian Genetic Structure
Bayesian clustering methods are powerful computational
tools meant for estimation of various features of population. STRUCTURE assigns the individuals to different
populations and hybrid zones on the basis of allele frequencies of genotypes. The method assumes K (unknown)
populations for the given data set and the value of K can be
estimated by posterior probability of the data for a given K,

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Table 2 Characteristics of 19 RAPD and 6 ISSR primers used in present study
S. no.

Primer name

Primer sequence (50 30 )

No. of bands

1.

OPB-05

TGCGCCCTTC

13

2.

OPB-06

TGCTCTGCCC

3.

OPB-11

GTAGACCCGT

4.

OPB-12

5.

OPB-16

6.

Size range (bp)

PIC

MI

5102200

0.295

3.83

8001900

0.448

2.24

3501900

0.490

3.92

CCTTGACGCA

13

4002500

0.479

6.22

TTTGCCCGGA

550950

0.413

2.47

OPC-08

TGGACCGGTG

11

6502400

0.499

5.48

7.

OPC-14

TGCGTGCTTG

4501900

0.496

1.48

8.

OPN-02

ACCAGGGGCA

5001500

0.466

2.79

9.

OPN-03

GGTACTCCCC

4201300

0.359

2.15

10.

OPN-05

ACTGAACGCC

4501650

0.424

3.39

11.
12.

OPO-06
OPR-02

TCGGCGGTTC
CACAGCTGCC

3
11

3501100
3501520

0.424
0.013

1.27
0.14

13.

OPR-04

CCCGTAGCAC

4001750

0.389

1.16

14.

OPR-08

CCCGTTGCCT

8201000

0.020

0.06

15.

OPR-20

ACGGCAAGGA

600650

0.499

0.99

16.

OPS-04

CACCCCCTTG

4001600

0.299

1.19

17.

OPS-07

TCCGATGCTG

4501520

0.427

2.13

18.

OPS-09

TCCTGGTCCC

10

3503000

0.039

0.39

19.

OPS-14

AAAGGGGTCC

13

3001900

0.478

6.21

20.

ISSR-11

(AG)10C

5001250

0.348

1.74

21.

ISSR-16

(CTC)6G

11501200

0.464

0.92

22.

ISSR-17

(CTC)6T

11503000

0.413

1.65

23.

ISSR-18

(CTC)6A

6501200

0.020

0.10

24.

ISSR-23

(CAC)6T

8801110

0.039

0.11

25.

ISSR-24

(CAC)6A

6201150

0.359

1.43

Total

25

0.344

2.14

Mean

156
6.24

PIC polymorphism information content, bp base pair, MI marker index

Pr (X|K) [29]. Delta K, which is used to determine the bestfit value of K, was computed by STRUCTURE HARVESTER for the given range of 15 and highest value was
shown at K = 2 (Table 4). Therefore, STRUCTURE analysis was conducted for K = 2. Admixture model with
correlated allele frequency was used to infer genetic
structure. One of these clusters (Cluster-II in Fig. 2) was
containing the accessions of Himalayan region. While
other genetic cluster (Cluster-I) contained accessions from
remaining parts of the country with the exception of two
accessions namely Himganga and VLG-1 from Himalayan
region. Both clusters showed very low extent of admixture
within accessions. Percentages of pure accessions in
Cluster-I and II were 92 % and 84 %, respectively.
Cluster and Diversity Analyses
Dendrogram based on Jaccards similarity coefficient and
UPGMA method showed major two groups as shown in
Fig. 3. Group-I represented accessions from different

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regions while Group-II included accessions of western

Himalayan region only. The maximum genetic similarity
value was 0.968 between IC94591 and IC43510 showing
them the most similar accessions, whereas HPKM193 and
IC139329 were found to be genetically most dissimilar
with the similarity value of 0.421. These four accessions
belonged to M. uniflorum. When related species of M.
gharwaleis and M. axillare were taken into consideration
for similarity estimates, M. gharwaleis showed maximum
dissimilarity of 0.934 with IC544826 (M uniflorum). Unrooted N-J tree based on Dice coefficient also separated M
uniflorum accessions on the basis of different geographical
origins (Fig. 4). N-J tree also showed that divergence of
accessions was attributed to their cultivated and wild nature. Two-dimensional graphical view of genetic diversity
in 51 accessions was represented in PCA analysis (Fig. 5)
which clearly showed the two groups of M. uniflorum and
two distantly related species. Clustering patterns in PCA
supported clustering of both dendrogram and STRUCTURE. Further, AMOVA analysis (Table 5; Fig. 6) in the

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Table 3 Various diversity estimates of each primer used in present study
Primer

Ne

OPB-05

1.2737 (0.1239)

0.2075 (0.0917)

0.3513 (0.1441)

OPB-06

1.6000 (0.3544)

0.3558 (0.1308)

0.5355 (0.1442)

OPB-11

1.7244 (0.2725)

0.4073 (0.1014)

0.5936 (0.1115)

OPB-12

1.3079 (0.3220)

0.1957 (0.1941)

0.3073 (0.2757)

OPB-16

1.0448 (0.0776)

0.0395 (0.0684)

0.0786 (0.1361)

OPC-08

1.6659 (0.4418)

0.3558 (0.2094)

0.5136 (0.2748)

OPC-14

1.6680 (0.4434)

0.3785 (0.1652)

0.5598 (0.1821)

OPN-02

1.3622 (0.3871)

0.2138 (0.2206)

0.3211 (0.3117)

OPN-03

1.6506 (0.2471)

0.3873 (0.0917)

0.5739 (0.0999)

OPN-05

1.3501 (0.1537)

0.2507 (0.0914)

0.4112 (01204)

OPO-06
OPR-02

1.7796 (0.0779)
1.3508 (0.1623)

0.4375 (0.0246)
0.2499 (0.0981)

0.6292 (0.0257)
0.4090 (0.1314)

OPR-04

1.7630 (0.1242)

0.4308 (0.0418)

0.6218 (0.0443)

OPR-08

1.1622 (0.2293)

0.1224 (0.1732)

0.2051 (0.2900)

OPR-20

1.6897 (0.0000)

0.4082 (0.0000)

0.5983 (0.0000)

OPS-04

1.9600 (0.0000)

0.4898 (0.0000)

0.6829 (0.0000)

OPS-07

1.7027 (0.3887)

0.3763 (0.2002)

0.5377 (0.2696)

OPS-09

1.3243 (0.0000)

0.2449 (0.0000)

0.4101 (0.0000)

OPS-14

1.5288 (0.3362)

0.3188 (0.1455)

0.4879 (0.1720)

ISSR-11

1.3243 (0.0000)

0.2449 (0.0000)

0.4101 (0.0000)

ISSR-16

1.7569 (0.2434)

0.4253 (0.0796)

0.6152 (0.0841)

ISSR-17

1.3823 (0.2565)

0.2587 (0.1450)

0.4155 (0.1887)

ISSR-18

1.1622 (0.2293)

0.1224 (0.1732)

0.2051 (0.2900)

ISSR-23

1.3243 (0.0000)

0.2449 (0.0000)

0.4101 (0.0000)

ISSR-24

1.5585 (0.3311)

0.3435 (0.1395)

0.5221 (0.1584)

Mean

1.4966

0.3004

0.4562

Value within bracket shows standard deviation

Ne number of effective alleles, h Nies gene diversity, I Shannon information index

Fig. 1 Amplification profile generated by RAPD primer OPN-5 and ISSR primer 16. M = 100 base pair (bp) DNA ladder

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Table 4 The Evanno table output showing maximum value of delta k at K= 2
K

Reps

Mean LnP (K)

Stdev LnP (K)

Ln0 (K)

|Ln

00

-2831.166667

0.680686

-2213.066667

0.568624

618.100000

477.100000

839.042919

-2072.066667

0.550757

141.000000

2.466667

4.478684

-1933.533333

17.724653

138.533333

76.833333

4.334829

-1871.833333

8.140229

61.700000

(K)|

Delta K

Fig. 2 Genetic structure of 49

horsegram accessions as
inferred by STRUCTURE
v2.3.3 with 19 RAPD and 6
ISSR markers data set. Single
vertical line represents an
individual genotype and
different colours represent
genetic stocks/gene pools.
Segments of each vertical line
show extent of admixture in an
individual

Table 5 Summary of AMOVA

Source

df

SS

MS

Est. Var.

Among pops

106.504

106.504

4.063

Within pops

47

328.925

6.998

6.998

Total

48

435.429

Fig. 3 Dendrogram of 51
horsegram genotypes based on
156 RAPD and ISSR fragments
and constructed using Jaccards
similarity coefficient with
UPGMA method. All the
genotypes were grouped into
two main groups. Two wild
species namely M. axillare and
M. sar-gharwalensis out
grouped showing their distant
relation with M. uniflorum

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11.062

% Variation
37
63
100s

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Fig. 4 Unrooted Neighbourjoining tree of 51 horsegram
genotypes representing three
species. Two major clusters
representing M. uniflorum
accessions were shown. Red
coloured cluster (cluster-I)
represents accessions from
Himalayan region while green
coloured cluster (cluster-II)
represent mixed accessions.
(Color figure online)

present study revealed that the major portion of genetic

variation resided within different populations of horsegram
(63 %) rather than among populations (37 %).

Discussion
Diversity of horsegram remained unexplored except few
studies in which morphological and biochemical traits were
analysed [22, 28]. The lack of genetic and genomic information and resources have been major limiting factor for
genetic improvement of horsegram. This study is the first
attempt wherein, molecular markers have been used for
assessing genetic diversity and analysing structure of
horsegram gene pool. While both RAPD and ISSR marker
techniques revealed polymorphism in analysed germplasm

lines, RAPD has proven to be a more effective technique to

explore the genetic diversity of horsegram. The amplification of 6.2 fragments per primer revealed that considerable level of polymorphism was detected, which is in
accordance with earlier studies in horsegram by Durga [8,
22] who studied phenotypic characteristics and on the basis
of Mahalanobis D2 analysis concluded that high level of
diversity is present in horsegram. Morris [23] also reported
greater variability for seed number and weight in horsegram. Average PIC (0.34) in current study is comparable to
barley, cowpea and pigeon pea [9, 16, 21] where authors
reported average PIC of 0.34, 0.34 and 0.38 on the basis of
SNP and SSR markers, respectively. This PIC value was
greater than obtained in Trifolium species and lentil [14,
34]. Similarly, mean Neis gene diversity (h), MI and
Shannons Information index (I) were found higher than

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Fig. 5 Principal component
analysis (PCA) of 51
horsegram genotypes based on
156 RAPD and ISSR
fragments. Numbering of
accessions is in correspondence
to numbering of Table 1

Among Pops
37%
Within Pops
63%

Fig. 6 Graphical representation of Percentages of variations shown

by AMOVA within and among populations

reported in other legume species [6, 11, 25]. High values of

the diversity indices showed that horsegram accessions
revealed high genetic variability which can be exploited in
breeding programmes for development of desired cultivars.
Further, Bayesian method-based clustering by STRUCTURE indicated two gene pools for horsegram accessions
included in the present study. One of the clusters (Cluster-II)
represented accessions from hill regions (North western
Himalayan region) while another cluster (Cluster-I) represented mixed accessions from central and southern part of
India, and two accessions from North western hill region. As
very low admixture was recorded and accessions in two
different gene pools represented purity of 92 % and 84 % for
Cluster-I and Cluster-II, respectively. This genetic structuring indicated two centres of diversity of horsegram, one
pointing towards region of north western Himalayas and
other in southern parts of the country. Although STRUCTURE results showed low admixture in accessions of both

123

clusters, cluster-II corresponding to hilly regions, was

showing greater extent of admixture in its accessions. This
can be attributed to cultivation and consumption patterns of
horsegram within country. Indigenous varieties of horsegram are grown in hill regions of north western Himalayas but
horsegram production does not fulfil the demand of this region and most of the horsegram produce in market comes
from central and southern regions of country where it is
cultivated in plenty. Therefore in addition to consumption,
the cultivation of lines coming from central and southern
parts of the country cannot be denied. Thus, admixture
shown by STRUCTURE may be due to the mixing of accessions across regions. Structuring of accessions into two
clusters with low admixture also points towards isolated and
conserved genetic background of the crop species. Although
STRUCTURE assigned most of the accessions into two
clusters according to their geographical origin, the clustering
of two hilly region accessions (Himganga and VLG-1)
against their parent cluster (Cluster-II) is exceptional which
needs further validation.
Dendrogram based on Jaccards similarity coefficient and
UPGMA method showed two major groups as shown in
Fig. 3. Group-I consisted of mixed accessions representing
almost all geographical locations of country while group-II
consisted of accessions from Himalayan regions. In addition,
each accession of M. axillare and M. sar-gharwalensis appeared as out groups. Group-I was having mixed accessions
but majority of them were from central and southern India
indicating their distinct genetic background. In this group
three accessions namely HPKC-4, IC-56142 and HPKM151 were more diverse as compared to others. Group-II was

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Agric Res

representing accessions from hill regions of north western

Himalayas. Accessions in this group consisted from Doda
district of Jammu and Kashmir and from different hilly regions of Himachal Pradesh and Uttarakhand. HPKM-193
was the most diverse accession in M. uniflorum germplasm
analysed in this study and grouped distantly in dendrogram.
Except few accessions, grouping was in correspondence to
geographical origin of germplasm lines i.e. each belonging
to Himalayan region and southern parts of country as reported earlier [2, 44]. Jaccards similarity matrix showed
highest genetic similarity of 0.96 between IC94591 and
IC43510 showing them the most adjoining accessions,
whereas HPKM193 and IC139329 were found to be genetically most diverse accessions with the similarity value of
0.421. At interspecific level M. gharwaleis and IC544826
were most distant with a dissimilarity of 0.934. N-J tree
based on Dice coefficient also separated accessions on the
basis of different locations (Fig. 4). N-J tree showed M. sargharwalensis more distant relative to cultivated germplasm
than M. axillare. It also showed HPKM-193 as most diverse
among M. uniflorum accessions. The N-J tree also showed
divergence of accessions attributed to their cultivated and
wild nature. All the clustering methods complemented each
other and pointed towards occurrence of two primary gene
pools in horsegram. Two-dimensional representations of
data by PCA also showed two major groups with HPKM-193
as the most distant accession (Fig. 5). Clustering patterns of
PCA complemented clustering of both dendrogram and
STRUCTURE. AMOVA partitioned 63 % of genetic variation within populations while only 37 % genetic variation
among populations. AMOVA results showed that major part
(63 %) of genetic variation is residing within population
while only 37 % genetic variation is residing among
populations. This also indicates that there is frequent flow of
alleles within populations as compared to among
populations.
In conclusion, this is the first report of genetic diversity
study using DNA markers in horsegram and we found that
high level of genetic diversity is prevailing in this crop.
Genetic structure showed the presence of two gene pools
and low level of natural admixture within studied accessions, hence showed a strong genetic structuring of
horsegram germplasm. Genetically diverse accessions detected in the present study can be useful in future horsegram improvement programmes using conventional as well
as molecular breeding tools. The novel information on
diversity provided in this work can be beneficial for genetic
diversity analysis in larger sets, population genetic studies
and in genetic mapping studies.
Acknowledgments We gratefully acknowledge Department of
Science and Technology (DST), Ministry of Science and Technology,
Government of India, New Delhi for providing financial support.

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