Mol Biol Rep (2014) 41:4329–4339
DOI 10.1007/s11033-014-3304-5
Population structure and genetic diversity analysis of Indian
and exotic rice (Oryza sativa L.) accessions using SSR markers
B. Kalyana Babu • Vimla Meena • Vasudha Agarwal
P. K. Agrawal
Received: 29 July 2013 / Accepted: 14 February 2014 / Published online: 2 March 2014
Ó Springer Science+Business Media Dordrecht 2014
Abstract In order to understand the population structure
and genetic diversity among a set of 82 rice genotypes
collected from different parts of the Asian countries
including India were characterized using 39 microsatellite
loci. The Population structure analysis suggested that the
optimum number of subpopulations was four (K = 4)
among the rice genotypes, whereas phylogenetic analysis
grouped them into three populations. The results obtained
from phylogenetic and STRUCTURE analysis proved to be
very powerful for the differentiation of rice genotypes
based on their place of origin. The genetic diversity ana-
lysis using 39 SSR loci yielded 183 scorable alleles, out of
which 182 alleles were observed to be polymorphic with an
average of 4.8 alleles per locus. The Polymorphism
Information Content (PIC) values for all the polymorphic
primers across 82 rice genotypes varied from 0.02 to 0.77,
with an average of 0.50. Gene diversity (He) was found to
be in the range of 0.02 (RM484) to 0.80 (OSR13) with an
average value of 0.55, while heterozygosity (Ho) was
observed with an average of 0.07, ranging from 0.01
(RM334) to 0.31 (RM316). The present study resulted in
identification of seven highly polymorphic SSR loci viz.,
OSR13, RM152, RM144, RM536, RM489, RM259 and
RM271 based on the parameters like PIC value (C0.70),
gene diversity (C0.71), and polymorphic alleles (C6).
These seven polymorphic primers can effectively be used
in further molecular breeding programs and QTL mapping
studies of rice since they exhibited very high polymor-
phism over other loci. SSR analysis resulted in a more
B. K. Babu
V. Meena
V. Agarwal
P. K. Agrawal (&)
Division of Crop Improvement, Vivekananda Institute of Hill
Agriculture VPKAS, Indian Council of Agricultural Research
(ICAR), Almora 263601, Uttarakhand, India
e-mail: pawankagrawal@hotmail.com
•
definitive separation of clustering of genotypes indicating a
higher level of efficiency of SSR markers for the accurate
determination of relationships between accessions.
Keywords Rice
Population structure
SSR
Gene
diversity
Polymorphism information content (PIC)
Introduction
Rice, one of the most widely cultivated crops, provides food
for one-half of the world population and is the second most
widely consumed cereal in the world, next to wheat. Rice
breeders are increasingly challenged to meet the rapidly
growing food demands for an increasing human population.
India occupies second place in the rice production, next to
China. In India, rice is cultivated for an area of 44 million
hectare area and contributes 140 million tonnes of grain
production, with a productivity of 3,124 kg/ha. The NW
Himalayan region of India have 0.63 million ha area under
rice cultivation producing about 1.26 million tonnes with a
productivity of 1,921 kg/ha [1]. Over two billion people in
Asia alone derive 80 % of their calorie needs from rice [2].
Rice protein, though small in amount, is of high nutritional
value [3]. Basmati rice makes a metallothionein-like protein,
rich in cystine that aids in iron absorption. Colored rice
(black and red) is rich in minerals (iron and zinc), polyphe-
nols and have anti-oxidant properties [4].
There is a requirement for increasing the production and
productivity of rice in order to meet the demand of ever-
increasing world population. One of the key factors for the
crop improvement efforts depend upon the amount and the
use of genetic variability in breeding programs. Charac-
terization and quantification of genetic diversity among
various germplasm is thus a major goal for effective
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genetic enhancement for crop productivity. Genetic diver-
sity based on the morphological characters possess several
limitations as they are influenced by environmental factors,
are limited in number and do not have the resolution power
for the differentiation between closely related genotypes
[5]. Advances in plant genetics and molecular biology have
lead to the development of molecular markers. DNA
markers have been used to evaluate genetic diversity in
different crop species [5]. Various molecular markers are
being used for fingerprinting and diversity analysis among
crop plants such as Restriction Fragment Length Poly-
morphism (RFLP) [6], Random Amplified Polymorphic
DNA (RAPD) [7], microsatellites or Simple Sequence
Repeats (SSRs) [8] and Amplified Fragment Length
Polymorphism (AFLP) [9]. Some of these techniques are
robust and reliable, e.g., RFLP and AFLP, while some are
quick, e.g., RAPD and some others are both quick and
reliable, e.g., SSRs. AFLP and RFLP are highly time
consuming and costly and therefore, their use is restricted.
PCR based techniques, i.e., SSRs and RAPD are fast and
cost effective, hence are widely used.
Microsatellites are short tandem repeat motifs and offer
many advantages such as high level of polymorphism [10],
high accuracy and repeatability throughout the genome [11],
automated analysis [12], rapidity, technical simplicity, low
cost, and requirement of only a small quantity of DNA (few
nanograms). Thus, they are useful as genetic markers in many
plant species including Arabidopsis, rice, wheat, soybean and
sweet potato [13–15]. SSR markers are also widely used in
assessing genetic diversity in rice at both inter and intra-spe-
cific level [8, 16]. Structure [17] is the most extensively used
software applied to detect the population genetic structure. It
generates clusters based on both transient Hardy–Weinberg
disequilibrium and linkage disequilibrium caused by admix-
ture between populations. It works by clustering individuals in
groups, where linkage and Hardy–Weinberg disequilibrium
are both minimized, and therefore, the presence of linkage
disequilibrium in the data improves clustering results [18].
Herrera et al. [19] used 48 SSR markers to assess genetic
diversity among 11 Venezuelan rice cultivars. They observed
that the varieties were closely related and molecular identifi-
cation of seven of the cultivars could be done with nine primer
pairs producing 10 genotype specific alleles. Although, the
genetic diversity was observed to be low, SSRs proved to be an
efficient tool in assessing the genetic variability of rice
genotypes. However, no efforts have been made for the
assessment of genetic diversity of rice from North-West
Himalayan region of India and their comparison with other
rice cultivars from other parts of India and exotic genotypes.
The present study was undertaken with the objectives to assess
the population structural variation among the selected 82 rice
genotypes, and the level of genetic diversity among them
using SSR markers to compare and analysis of genetic
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variation of NW Himalayan genotypes with the other Indian
and exotic rice genotypes from different parts of the world,
and to better understand the association between genetic
diversity and geographic factors.
Materials and methods
Plant materials and DNA extraction
Seeds of 82 rice genotypes including of 27 varieties devel-
oped by Vivekananda Institute of Hill Agriculture, Almora
(Uttarakhand), for the NW Himalayan region of India; 23
genotypes from Northern and Southern parts of India, and 32
exotic rice accessions including two lines from IRRI, Phil-
ippines were taken for the present study. The details of the
rice genotypes along with their origin, and ecosystem are
given in Table 1. The genomic DNA was isolated from the
fresh young leaves following Agrawal and Katiyar [20].
Thirty nine SSR markers were used to detect polymorphism
among the 82 rice genotypes spread evenly over all the
chromosomes. The polymerase chain reactions and gel
documentation were carried out using standard procedures,
and the amplified products were resolved on 3.5 % agarose
gel [Super Fine Resolution (SFR) Agarose; Amresco, USA]
and scoring was carried out manually.
Data analysis
The SSR scores were used to create a data matrix to analyze
genetic relationships using the NTSYS-pc program version
2.11a [21]. The dendrogram was constructed based on Jac-
card’s similarity coefficient [22] using the marker data for all
the rice genotypes following unweighted pair group method
analysis (UPGMA) [23]. The Polymorphism Information
Content (PIC) was determined as described by Tang et al. [24],
by using the formula PIC = 1–Rfi2, where fi is the frequency
of the ith allele. For genotypes showing heterozygosity at a
specific SSR locus, the PIC values were calculated after
considering each allele as contributing one-half instead of one,
as suggested by Narvel et al. [25]. The heterozygosity, gene
diversity, allele frequency and inbreeding coefficients were
calculated using Power Marker V3.0 software [26].
Population structure analysis
The structure of the population was studied with the
structure version 2.3.4 software [27]. Clustering methods
with distinctive allele frequencies were used to identify the
optimum number of population (K) subgroups. Each indi-
vidual can be a member of multiple subpopulations with a
different coefficient, with the sum of all being equal to one
[28]. The number of subgroups (K) in the population was
Mol Biol Rep (2014) 41:4329–4339 4331

Table 1 Details of the 82 rice S. no. Genotype Origin (location) Ecosystem

genotypes used in the study
1. Kba-Sawrit B2 Exotic NA
2. Local Ahu Exotic NA
3. Milyang-15 Exotic Irrigated
4. Milyang-93 Exotic Irrigated
5. Mipun Exotic NA
6. Nanglwai Exotic NA
7. Naveen India Upland
8. Neela India Upland
9. Pusa Basmati 1 India Irrigated
10. Pusa Sugandha 2 India Irrigated
11. Pusa Sugandha 3 India Irrigated
12. Pusa Sugandha 5 India Irrigated
13. Rashi India Upland
14. Ratna India Irrigated
15. RP 2421 India Irrigated
16. Saket-4 Exotic Irrigated
17. Satabdi India Irrigated
18. Satari Exotic Irrigated
19. Satya India NA
20. Sinsatsu Exotic NA
21. Sneha India Irrigated
22. Stesiara-45 Exotic NA
23. Sulocon 235 Exotic NA
24. Suweon Exotic Irrigated
25. Swarna India Shallow rain-fed
26. Tai-Hikawi Exotic NA
27. Tangla Exotic NA
28. Tapaswini India Irrigated
29. Theberu Exotic NA
30. TN-1 Taiwan Irrigated
31. Vidhaya India NA
32. VL Dhan 82 VPKAS, Almora, India Irrigated
33. VL Dhan 30919 VPKAS, Almora, India Irrigated
34. VL Dhan 31330 VPKAS, Almora, India Irrigated
35. VL Dhan 31331 VPKAS, Almora, India Irrigated
36. VL Dhan 61 VPKAS, Almora, India Irrigated
37. VL Dhan 65 VPKAS, Almora, India Irrigated
38. VL Dhan 66 VPKAS, Almora, India Irrigated
39. VL Dhan 81 VPKAS, Almora, India Irrigated
40. VL Dhan 86 VPKAS, Almora, India Irrigated
41. VL Dhan 154 VPKAS, Almora, India Upland rain-fed
42. VL Dhan 163 VPKAS, Almora, India Upland
43. VL Dhan 206 VPKAS, Almora, India Upland
44. VL Dhan 209 VPKAS, Almora, India Upland
45. VL Dhan 221 VPKAS, Almora, India Upland
46. Yam-Nani Exotic NA
47. Yunlen-20 Exotic NA
48. Zenith Exotic NA
49. Abor-A Exotic NA

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Table 1 continued S. no. Genotype Origin (location) Ecosystem

50. Anjali India Upland

51. Barkot Exotic NA
52. Basmati 370 Land race (NW India) Irrigated
53. Boda-271 Exotic NA
54. BPT 5204 India Irrigated
55. Catrosa Exotic NA
56. Chandan India NA
57. Dhalahara India Upland
58. Dullo Exotic NA
59. Dullo-11 Exotic NA
60. Dullo-A Exotic NA
61. Geetanjali India Medium land and irrigated
62. GGAF-BYEO Exotic NA
63. Henjudo Exotic NA
64. IR-64 IRRI, Philippines Irrigated
65. IR 3941-34 IRRI, Philippines NA
66. Janam India NA
67. Jungurh Exotic NA
68. Kba-Thangmaw Exotic NA
69. Mujudo Exotic NA
70. Vandana India Upland
71. VL Dhan 7620 VPKAS, Almora, India Upland
72. VL Dhan 16 VPKAS, Almora, India NA
73. VL Dhan 85 VPKAS, Almora, India NA
74. VL Dhan 62 VPKAS, Almora, India Irrigated
75. VL Dhan 82 VPKAS, Almora, India Irrigated
76. S-1 NA NA
77. RP2421 NA NA
78. HPR-2143 NA Irrigated
79. VL Dhan207 VPKAS, Almora, India Upland
80. VL Dhan 208 VPKAS, Almora, India Upland
81. P1460 NA NA
82. P1121 NA Irrigated
NA not available

determined by running the program at different K values
with K carrying from 2 to 10. Five independent runs were
assessed for each K value. We used a burn-in period of
100,000 steps followed by 100,000 Monte Carlo Markov
Chain replicates, as suggested by Pritchard and Wen [29].
Results
Genetic variation of SSR loci among the rice genotypes
The genomic DNA of the 82 rice accessions were amplified
using 39 SSR markers and yielded 183 scorable alleles, out
of which 182 alleles were found to be polymorphic, while
one was monomorphic. All the 39 SSRs were spread across
all the chromosomes of rice evenly. Out of the 39 primers,
38 (98 %) loci were found to be polymorphic, while one
SSR locus, RM338 (2 %) was monomorphic. A total of 182
alleles were detected for the 38 polymorphic SSR markers
with an average of 4.8 alleles per locus, while it was 4.69
for all the 39 SSR loci including monomorphic marker
used in the study. The number of alleles generated with
polymorphic primers ranged from 2 to 9 among the rice
genotypes. The SSR locus RM144 was found to have
maximum number of allele (9) followed by the loci RM447
and RM316 (each seven allele) (Table 2). The banding

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Table 2 Parameters for genetic SSR loci PIC Heterozygosity Gene Allele Major allele Chromosome
analysis of 39 SSR loci across diversity number frequency
the 82 rice accessions
RM338 0.00 0.00 0.00 1.00 1.00 3
RM484 0.02 0.00 0.02 2.00 0.99 10
RM283 0.11 0.00 0.12 2.00 0.94 1
RM452 0.28 0.25 0.31 4.00 0.82 2
RM125 0.31 0.07 0.33 4.00 0.81 7
RM215 0.31 0.01 0.38 3.00 0.75 9
RM454 0.32 0.00 0.39 3.00 0.74 6
RM277 0.32 0.01 0.37 3.00 0.76 12
RM133 0.34 0.00 0.41 3.00 0.73 6
RM284 0.36 0.00 0.40 3.00 0.75 8
RM312 0.37 0.21 0.41 3.00 0.75 1
RM433 0.39 0.22 0.42 4.00 0.75 8
RM510 0.41 0.01 0.50 3.00 0.64 6
RM44 0.45 0.06 0.53 3.00 0.60 –
RM287 0.46 0.03 0.51 4.00 0.66 11
RM161 0.46 0.02 0.56 3.00 0.53 5
RM118 0.47 0.00 0.53 3.00 0.62 7
RM25 0.49 0.16 0.54 5.00 0.63 8
RM413 0.57 0.03 0.60 7.00 0.59 5
RM316 0.57 0.31 0.60 8.00 0.61 9
RM431 0.60 0.02 0.65 5.00 0.52 1
RM154 0.60 0.02 0.66 5.00 0.43 2
RM105 0.60 0.00 0.67 4.00 0.41 9
RM334 0.61 0.01 0.64 5.00 0.54 5
RM237 0.61 0.29 0.67 5.00 0.44 1
RM474 0.61 0.05 0.64 6.00 0.57 10
RM408 0.61 0.03 0.67 6.00 0.44 8
RM55 0.62 0.00 0.67 6.00 0.47 3
RM514 0.63 0.02 0.68 4.00 0.44 3
RM447 0.66 0.15 0.69 8.00 0.51 8
RM271 0.66 0.08 0.71 6.00 0.38 10
RM259 0.68 0.01 0.72 6.00 0.41 1
RM5 0.68 0.01 0.72 5.00 0.35 –
RM552 0.68 0.24 0.72 5.00 0.41 11
RM489 0.71 0.15 0.75 6.00 0.32 3
RM536 0.73 0.00 0.76 7.00 0.38 11
RM144 0.75 0.14 0.78 9.00 0.34 11
RM152 0.76 0.07 0.79 7.00 0.32 8
OSR13 0.77 0.02 0.80 7.00 0.25 –
Mean 0.50 0.07 0.55 4.7 0.58

pattern of 82 rice genotypes with SSR loci RM17 was
shown in Fig. 1.
The PIC values for all the polymorphic primers across
82 rice genotypes varied from 0.02 to 0.77 with an average
value of 0.50. Out of the 39 primers, two primers (RM152
and OSR13) showed highest PIC values of 0.76 and 0.77
respectively, while three SSR loci, RM489, RM536 and
RM144 showed a PIC value of 0.71, 0.74 and 0.75
respectively. The lowest PIC values was observed in
primers RM484 (0.02), RM 283 (0.12) (Table 2). The
number of SSR loci based on PIC values with more than
the average was 21 in number. Among them, SSR loci
RM536, RM144, RM152 and OSR13 were noteworthy due
to their relatively higher level of polymorphism. A total of

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M 1 2 3 4 5 6
200bp
100bp
M 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
M 49 50 51 52 53 54
76 77 78 79 80 81 82
Fig. 1 The SSR banding profile of RM17 SSR loci among the 82 rice genotypes. Lane 1, M- Marker, Lane 1–82 rice genotypes (for labels please
refer to Table 1)
21 SSR loci came under the PIC range of 0.5–0.77 with an
average value of 0.65, while 18 loci came within the PIC
range of 0.00–0.49 with an average value of 0.33. The
SSRs which had PIC value more than 0.50 also generated
more number of loci with an average of 6.1, while it was
3.1 for the SSRs below the PIC value of 0.5 (Table 2).
Statistical analysis of genetic diversity
Gene diversity also known as ‘expected heterozygosity’ (He)
was in the range of 0.02 (RM484) to 0.80 (OSR13) with an
average value of 0.55. The heterozygosity, known as
‘observed heterozygosity’ (Ho) was observed with an aver-
age of 0.07 and range of 0.01 (RM334) to 0.31 (RM316).
Major alleles with highest frequency were observed for the
locus RM484 (80 bp allele) at 99 % followed by the locus
RM283 (150 bp) at 94 %. Based on the above SSR analysis
by considering the parameters of PIC value (C0.70), gene
diversity (C0.71), inbreeding coefficient (C0.62) and poly-
morphic alleles (C6), seven most highly polymorphic SSR
loci OSR13, RM152, RM144, RM536, RM489, RM259 and
RM271 were observed (Table 2).
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7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 7475
Associations among rice genotypes
Jaccard’s similarity coefficient between all the 82 rice
genotypes ranged from 22 % to 95 %. The maximum
similarity (88 %) was found between the genotype pairs
Geetanjali with Dullo-A and Chandan and Dullo-A (each
pair 88 %). The above results on similarity were also
substantiated by the dendrogram generated using the soft-
ware power marker (Fig. 2). The dendrogram was con-
structed by using both NTSYSpc2.11 and power marker
software (rooted and rectangular), and both were similar.
The similarity coefficients were used as input data for the
cluster analysis using NTSYSpc 2.11 program.
The dendrogram generated through UPGMA analysis
grouped all the 82 rice genotypes into three major groups
A, B, and C. The clustering of the rice genotypes was
largely based on their centre of development. The cluster C
comprised of all the exotic genotypes with five Indian rice
accessions viz, Vandana, Satya, VL Dhan 221, Swarna and
Geetanjali. All the exotic lines were grouped under the
cluster C with few exceptions like Jungurh, Abor-A, Dullo,
Barkot and Catrosa which were placed in the cluster A. The
Mol Biol Rep (2014) 41:4329–4339
Fig. 2 The phylogram of 82 rice genotypes as obtained from POWER MARKER software based on UPGMA analysis
major cluster A comprised of 40 genotypes, while the
cluster B and C comprised of 14 and 28 genotypes
respectively. The genotypes from the NW Himalayan
region were grouped in the cluster A and B. Cluster ‘A’
comprised of genotypes from different centers that includes
exotic and Indian genotypes, from different centers of
development like CRRI, Cuttack; IARI, New Delhi;
VPKAS, Almora and IRRI, Philippines. The cluster B
consisted of most of the VPKAS genotypes from VPKAS
along with the genotypes from IARI, New Delhi. The
genotypes BPT 5204 and Swarna were from Bapatla,
4335
Andhra Pradesh, but were scattered into two different
clusters A and D respectively. The genotypes from CRRI,
Cuttack were spread randomly in the cluster A.
Population structure analysis
The rice genotypes consisted of popular varieties of India
and exotic accessions from different regions were evalu-
ated for estimation of population structure using a panel of
39 SSR loci spread across all the 12 chromosomes. For
estimation of the exact population structure (K), Ks from 1
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Fig. 3 The population structure of 82 rice genotypes as obtained from structure 2.3.4 software

to 10 (with ten iterations) were ran and the LnP(D) value Table 3 Allele frequencies, [divergence among pops (net nucleotide
was used to group all the genotypes. The maximum DK distance)], computed using point estimates of P
value was observed for K = 4. The inferred ancestry at Clusters 1 2 3 4
K = 4 suggested that the rice genotypes were grouped into
four subpopulations (Fig. 3). However power marker 1 – 0.5327 0.2665 0.3109
software was able to differentiate them into three main 2 0.5327 – 0.5359 0.5175
clusters. The subpopulation (G1) consisted of exotic rice 3 0.2665 0.5359 – 0.1936
genotypes with few Indian genotypes viz., Basmati 370, 4 0.3109 0.5175 0.1936 –
Geetanjali, VL Dhan 7620 and Anjali. This subpopulation
had admixture of Anjali, Dullo and Dhalaheera. The sub-
population (G2) consisted of all the Indian accessions, estimates of P] was 0.5327 between clusters 1 and 2,
where as G3 consisted of all the exotic germplasm with 0.2665 between clusters 1 and 3, and 0.3109 between
Satya and Swarna rice varieties of India. The subpopula- clusters 1 and 4 (Table 3).
tions G2 and G3 did not have any admixture populations.
The subpopulation (G4) consisted of mostly Indian geno-
types with few exotic germplasm. Similar grouping pattern Discussion
(except, exotics were under same cluster) was also
observed in the case of power marker, however with the Microsatellite (SSR) analysis
help of a structure bar plot, depicting the estimated mem-
bership of each variety in each of the populations Cultivated varieties of rice were the result of several
(K = 1–10), the admixtures could easily be identified, thousands of years of human selection from the available
which can explain the grouping pattern better than the genetic diversity in various environments and human cul-
dendrogram. The mean value of alpha was 0.0284, and the tures. Modern breeding in the last two century has done
ancestry-inferred cluster proportions of the membership of little more than to control the process of hybridization and
the samples were 0.265, 0.135, 0.256 and 0.345. The selection in a more efficient way and the result has been the
average distances (expected heterozygosity) between indi- development of varieties adapted to better controlled
viduals in the same cluster were 0.1728, 0.3374, 0.1387 environments. Cultivation of few genotypes in any crop
and 0.1834 and the allele frequency [divergence among including rice leads to narrow genetic base. The crop is
pops (Net nucleotide distance), computed using point prone to many biotic and abiotic stresses. Improvement of

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rice breeding requires identification of highly diverse
germplasm and highly polymorphic molecular markers and
their effective utilization in mapping and breeding pro-
grammes. In the present study, 39 primers yielded 183
scorable alleles, out of which 182 were found to be poly-
morphic with an average of 4.69 alleles per locus with a
range of 2–9 alleles per locus. Michael et al. [30] also
found similar results where they used 30 SSR loci across
the 183 Indonesian rice landraces on the islands of Borneo.
They obtained 166 alleles with a range of 2–15. This high
number of alleles may be due to that fact that they used
land races from different parts of Indonesia, where land-
races are expected to exhibit higher diversity. However, the
number of alleles obtained during the present study is also
quite high that could differentiate the rice genotypes
matching to their centers of development largely. This
showed that these markers are highly useful and can be
effectively useful for genetic diversity and fingerprinting
studies. Similarly, Xinghua et al. [31] used SSRs for esti-
mating the temporal changes from 1950s to 1990s across
310 major Chinese rice varieties and obtained 221 alleles
with an average of 5.7 alleles per locus. However, Cha-
kravarthi and Rambabu [32] found 2–7 alleles per locus
with 30 SSR loci on 15 elite rice genotypes which con-
tained Indian and exotic lines from IRRI, Philippines. Out
of the total 39 primers used, all the primers amplified
among all the genotypes. However, 38 primers (98 %)
were found to be polymorphic and one SSR was mono-
morphic. This high level of polymorphism existed among
the selected genotypes has great potential for their use in
breeding programs. In the present study, close propor-
tionate relationship between the number of alleles and the
PIC values of SSR loci was observed. PIC demonstrates the
informativeness of the SSR loci and their potential to detect
differences among the genotypes based on their genetic
relationships. The SSR loci, RM144, RM152 and OSR13
having high number of alleles and highest PIC values, will
be very useful for further genetic and mapping studies. A
conclusion may be derived that the loci with more number
of alleles can be highly useful for the assessment of genetic
diversity. Similar results were also observed by Ravi et al.
[33] where it was observed that the SSR loci RM202 pro-
duced 11 alleles with highest PIC of 0.89. The PIC values
of all the polymorphic primers across 82 rice genotypes in
the present study were in the range of 0.02–0.77 with an
average value of 0.50. The number of SSR loci with higher
than average PIC values was found to be 21. The average
PIC value determined during the present investigation
agreed well with the earlier findings reported based on SSR
marker in rice genotypes [30, 31, 33]. Michael et al. [30]
reported an average PIC value of 0.45 which was lower
than the PIC value observed during the present study. This
showed the polymorphic ability of the SSR loci reported
4337
and their wide applicability for genetic analysis of rice
accessions. The high level of polymorphism was due to
diverse germplasm and highly polymorphic SSR loci used
in the present study. Among the SSRs, RM536, RM144,
RM152 and OSR13 were noteworthy due to their relatively
higher polymorphism, high PIC values and more number of
polymorphic alleles per locus and they can effectively be
used in the genetic diversity studies.
The observed and expected heterozygosity within the
genotypes showed obvious deviations from Hardy–Wein-
berg expectations. Gene diversity (He) was in the range of
0.02–0.80 with an average value of 0.55 which was cor-
related with the earlier findings. Xinghau et al. [31]
observed gene diversity to be 0.62 among the 310 major
Chinese landraces which was slightly more than the He
value observed during the present study. The heterozy-
gosity, was observed with an average of 0.07 and ranged
from 0.01 (RM334) to 0.31 (RM316) which is higher than
the findings observed by Michael et al. [30] where they
observed an average value of 0.03 (Table 2) emphasizing
that the selection practiced during the development of
hybrids, could have led to the deficit of heterozygous
individuals. Based on the above SSR analysis by consid-
ering the parameters of PIC value (C0.70), gene diversity
(C0.71), inbreeding coefficient (C0.62) and polymorphic
alleles (C6), seven most highly polymorphic SSR loci
observed were OSR13, RM152, RM144, RM536, RM489,
RM259 and RM271 (Table 2). These primers can effec-
tively be used in molecular breeding programs and QTL
mapping studies since they exhibited very high level of
polymorphism over other loci.
Genetic diversity among the rice genotypes
The ability to provide distance measures between the
genotypes that reflect pedigree relatedness ensures a more
stringent evaluation of the adequacy of a marker profile
data. The fact that minimum genetic distance revealed
during the study is a good indication confirming the power
of SSR markers to distinguish between geographically
similar genotypes and closely related genotypes. The
average gene diversity existing among all the genotypes
were relatively high (55 %), indicating existence of high
levels of polymorphisms among the inbreds. These results
are in close agreement with the findings reported among
the Chinese rice accessions using the SSR marker system
[31].
The dendrogram constructed using the UPGMA clus-
tering algorithm grouped the rice accessions into three
clusters. These groupings, in most instances, revealed
evidence of associations related to their centre of devel-
opment i.e., geographic origin. However, Michael et al.
[30] did not get such type of correlation which may be
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because of less number of polymorphic primers used by
them. Alternatively, the 82 rice genotypes were broadly
grouped into exotic rice accessions, accessions from
VPKAS, Almora with few exceptions. For example, VL
Dhan 163 grouped with BPT 5204 and Pusa Basmati 1
which were from Bapatla, Andhra Pradesh and IARI, New
Delhi respectively. It showed that during selection/evolu-
tion allele introgression has occurred from several sources.
The cluster ‘A’ was comprised of mixed genotypes from
different origins. This is also an indication that modern
breeding programmes involved diverse genotypes in
developing highly suitable genotypes adapted to their local
environments by crossings from the popular varieties of
different origins for important agronomic traits. The cluster
B mostly composed of genotypes developed from VPKAS,
Almora with exceptions like Pusa Sugandha accessions.
The interesting point in our study was that all the exotic
lines with some exceptions were grouped under the cluster
C with Indian genotypes like Vandana, Satya, VL Dhan
221, Swarna and Geetanjali, which may be due to that
exotic pedigree involved in breeding these five genotypes.
This showed that these selected markers were not only able
to differentiate based on geographic origin but also based
on the pedigree of the genotypes. The accession VL Dhan
221 was derived from the cross between IR 2053-521-1-1-
1/CH 1039, however Swarna was derived from Vasista and
Mahsuri, and Mahsuri was derived from Taichung
65/ZxMoyzng EB05080. Likewise Sri Satya was derived
from RGL1232/Phalguna/RGL1231/IR36, Vandana from
C-22 and Kalakeri. Hence there was introgression of alleles
from the exotic lines into these genotypes while crossings
for the development of superior hybrids for this region. The
rice variety Swarna is highly popular in the most parts of
eastern India due to its good taste, yield, and adaptability.
Population structure analysis
Since some geographical grouping was observed in the
phylogenetic analysis, we evaluated them to understand
whether alternative clustering methods such as model-
based clustering would resolve the geographic patterning.
From the phylogenetic analysis, it was observed that there
were at least three population subgroups, largely corre-
sponding to Indian, exotic and Indian with few exotic rice
genotypes. However, by the structure analysis these exotic
accessions were further divided into two different sub-
groups. The phylogenetic analysis also provided some
evidence for gene flow between the subpopulations. There
was good correspondence between the geographic pattern
observed in the dendrogram and the population structure
identified using structure. Although the population sub-
groups corresponded largely to geographic regions, there
were some notable exceptions. In the subpopulation (G4),
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Mol Biol Rep (2014) 41:4329–4339
genotypes VL Dhan 221, VL Dhan 206 and VL Dhan 209
carried more than 35 % of the exotic germplasm alleles.
For example, the accession VL Dhan 221 was derived from
the cross between IR 2053-521-1-1-1/CH 1039. The exotic
rice variety TN-1 grouped with Indian genotypes, which
showed that 70 % of alleles were common with Indian
germplasm. This showed that the exotic variety TN-1 was
very highly used in rice breeding programmes during 1960s
for developing high yielding rice varieties. Evidence of
admixture was also found in exotic population where Dullo
and Dhalahara accessions carried 8–10 % alleles from
Indian germplasm. Thus, the present study showed that
genotyping combined with phylogenetic and population
structure analysis is a powerful method for characterizing
germplasm. The present study was able to identify highly
polymorphic SSR loci which can be effectively used in
genetic analysis studies and molecular breeding pro-
grammes. Our results also showed that the polymorphic
SSR loci used in the study were able to differentiate the
Indian and exotic rice genotypes based on their geo-
graphical regions. There is also a great scope to derive
further improved materials by hybridizing with distant
lines identified during the present study and for further
selection of the desired line to derive more agronomically
useful lines for their commercial production.
References
1. ICAR (2011-12) Annual report. Vivekananda Parvatiya Krishi
Anusanthan Sansthan, Almora
2. Juliano BO (1985) Rice chemistry and technology. American
Association of Cereal Chemists, Minnesota, p 757
3. Chaudhary RC, Tran DV (2001) Speciality rices of the world-a
prologue. In: Chaudhary RC, Tran DV (eds) Speciality rices of
the world: breeding, production and marketing. FAO, Rome;
Oxford and IBH Publishing, New Delhi, pp 3–14
4. Zhang MW, Guo BJ, Chi JW, Wei ZC, Xu ZH, Zhang Y, Zhang
RF (2005) Antioxidants and their correlation with total flavonoid
and anthocyanin contents in different black rice varieties. Sci
Agric Sin 38(7):1324–1331
5. Cooke RJ (1995) Variety identification of crop plants. In: Skerrit
JH, Appels R (eds) New diagnostics in crop science. Biotech-
nology in Agriculture no. 13. CAB International, Wallingford,
pp 33–63
6. Sun CQ, Wang XK, Li ZC, Yoshimura A, Iwata N (2001)
Comparison of the genetic diversity of common wild rice (Oryza
rufipogon Griff.) and cultivated rice (O. sativa L.) using RFLP
markers. Theor Appl Genet 102(1):157–162
7. Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV
(1991) DNA polymorphisms amplified by arbitrary primers are
useful as genetic markers. Nucl Acids Res 18:6531–6535
8. Gao LZ, Zhang CH, Chang LP, Jia JZ, Qiu ZE, Dong YS (2005)
Microsatellite diversity of Oryza sativa with emphasis on indica-
japonica divergence. Genet Res 85:1–14
9. Aggarwal RK, Brar DS, Nandi S, Huang N, Khush GS (1999)
Phylogenetic relationships among Oryza species revealed by
AFLP markers. Theor Appl Genet 98:1320–1328
Mol Biol Rep (2014) 41:4329–4339
10. Smith JSC, Chin ECL, Shu H, Smith OS, Well SJ, Senior ML,
Mitchell SE, Kresovich S, Ziegle J (1997) An evaluation of the
utility of SSR loci as molecular markers in maize (Zea mays L.):
comparisons with data from RFLPs and pedigree. Theor Appl
Genet 95:163–173
11. Heckenberger M, Bohn M, Ziegle JS, Joe LK, Hauser JD, Hutton
M, Melchinger AE (2002) Variation of DNA fingerprints among
accessions within maize inbred lines and implications for iden-
tification of essentially derived varieties. Mol Breed 10:181–191
12. Sharon EM, Kresovich S, Jester CA, Hernandez CJ, Szewc-
McFadden AK (1997) Application of multiplex PCR and fluo-
rescence-based, semi-automated allele sizing technology for
genotyping plant genetic resources. Crop Sci 37:617–624
13. Depeige A, Goubely C, Lenoir A, Cocherel S, Picard G, Raynal
M, Grellet F, Delseny M (1995) Identification of the most rep-
resented repeated motifs in Arabidopsis thaliana microsatellite
loci. Theor Appl Genet 91:160–168
14. Roder MS, Plaschke J, Konig SU, Borner A, Sorrels ME,
Tanksley SD, Ganal MW (1995) Abundance, variability and
chromosomal location of microsatellites in wheat. Mol Gen
Genet 246:327–333
15. Senior ML, Heun M (1993) Mapping maize microsatellites and
polymerase chain reaction confirmation of the targeted repeats
using a CT primer. Genome 36:884–889
16. Olufowote JO, Xu Y, Chen X, Park WD, Beachel HM, Dilday
RH, Goto M, McCouch SR (1997) Comparative evaluation of
within-cultivar variation of rice (Oryza sativa L.) using micro-
satellite and RFLP markers. Genome 40:370–378
17. Pritchard JK, Stephens M, Donnelly P (2000) Inference of pop-
ulation structure using multilocus genotype data. Genetics
155:945–959
18. Falush D, Stephens M, Pritchard JK (2003) Inference of popu-
lation structure using multilocus genotype data: linked loci and
correlated allele frequencies. Genetics 164:1567–1587
19. Herrera TG, Duque DP, Almeida IP, Núñez GT, Pieters AJ,
Martinez CP, Tohme JM (2008) Assessment of genetic diversity
in Venezuelan rice cultivars using simple sequence repeats
markers. Electron J Biotechnol 11(5)
20. Agrawal PK, Katiyar AK (2008) Validation of chickpea-STMS
markers and DNA fingerprinting in lentil (Lens culinaris subsp.
culinaris) cultivars of India. Indian J Genet 68(2):149–156
4339
21. Rohlf FJ (1997) NTSYS-pc. Numerical taxonomy and multivar-
iate analysis system, version 2.0. State University of New York,
New York
22. Jaccard P (1908) Nouvelles recherches sur la distribuition florale.
Bull Soc Vaud Nat 44:223–270
23. Gawel NJ, Jarret RL (1991) A modified CTAB DNA extraction
procedure for Musa and Ipomoea plant. Mol Biol Rep 9:262–266
24. Tang S, Zhang Y, Zeng L, Luo L, Zhong Y, Geng Y (2010)
Assessment of genetic diversity and relationships of upland rice
accessions from southwest China using microsatellite markers.
Plant Biosyst 144(1):85–92
25. Narvel JM, Fehr WR, Chu WC, Grant D, Shoemaker RC (2000)
Simple sequence repeat diversity among soybean plant intro-
ductions and elite genotypes. Crop Sci 40:1452–1458
26. Liu K, Muse SV (2005) Powermarker: integrated analysis envi-
ronment for genetic marker data. Bioinformatics 21(9):2128–2129
27. Siwach P, Jain S, Saini N, Chowdhury VK, Jain RK (2004)
Allelic diversity among Basmati and non-Basmati long-grain
indica rice varieties using microsatellite markers. J Plant Bio-
chem Biotechnol 13:25–32
28. Aranzana MJ, Abbasi E, Howard W, Arus P (2010) Genetic
variation, population structure and linkage disequilibrium in
peach commercial varieties. BMC Genet 11:69
29. Pritchard JK, Wen W (2003) Documentation for the structure
software, version 2. Department of Human Genetics, University
of Chicago, Chicago. http://pritch.bsd.uchicago.edu/software
30. Michael JT, Nicholas RP, Joko PKR, Trijatmiko, Tiur SS,
McCouch SusanR (2009) Genetic diversity of isolated popula-
tions of Indonesian landraces of Rice (Oryza sativa L.) collected
in east Kalimantan on the Island of Borneo. Rice 2:80–92
31. Xinghua W, Yuan X, Yu H, Wang Y, Qun Xu, Tang S (2009)
Temporal changes in SSR allelic diversity of major rice cultivars
in China. J Genet Genom 36:363–370
32. Chakravarthi B, Rambabu N (2006) SSR marker based DNA
fingerprinting and diversity study in rice (Oryza sativa. L). Afr J
Biotechol 5(9):684–688
33. Ravi M, Geethanjalil S, Sameeyafarheen F, Maheswaran M
(2003) Molecular marker based genetic diversity analysis in rice
(Oryza sativa L.) using RAPD and SSR markers. Euphytica
133:243–252
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