Professional Documents
Culture Documents
Agricultural Research
ISSN 2249-720X
Agric Res
DOI 10.1007/s40003-015-0165-7
1 23
1 23
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
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
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
123
Data Analysis
123
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.
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
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,
123
Primer name
No. of bands
1.
OPB-05
TGCGCCCTTC
13
2.
OPB-06
TGCTCTGCCC
3.
OPB-11
GTAGACCCGT
4.
OPB-12
5.
OPB-16
6.
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
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
123
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
Fig. 1 Amplification profile generated by RAPD primer OPN-5 and ISSR primer 16. M = 100 base pair (bp) DNA ladder
123
Reps
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
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
123
11.062
% Variation
37
63
100s
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
123
Among Pops
37%
Within Pops
63%
123
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