IJERD ± International Journal of Environmental and Rural Development (2018) 9-2

Review article
erd

Microsatellite Markers and their Application on Genetic
Diversity Studies of Rice Landraces (Oryza sativa L.) in
Myanmar - A Review
AYE AYE THANT
Czech University of Life Sciences, Prague, Czech Republic
Email: aye18988@gmail.com/thant@ftz.czu.cz

THAN MYINT HTUN
Yezin Agricultural University, Zeyar Thiri, Nay Pyi Taw, Myanmar

MARIE KALOUSOVÁ
Czech University of Life Sciences, Prague, Czech Republic

RAKESH KUMAR SINGHInternational Rice Research Institute, Los Baños, Laguna, Philippines

BOHDAN LOJKA*
Czech University of Life Sciences, Prague, Czech Republic
Email: lojka@ftz.czu.cz
Received 31 December 2017

Accepted 15 October 2018(*Corresponding Author)

Abstract Myanmar is one of the centers of genetic diversity of rice as it has heterogeneous
geographical and ecological conditions such as hills and mountains. Since 1988, owing tomarket oriented economic policy and international rice price, traditional varieties have been
replaced by improved varieties. Landraces are known for the excellent adaptation to the
local conditions; however, their productivity is very low. Increased productivity of thelandraces through high input application has not been possible because they are notresponsive to the inputs. However, these landraces and traditional varieties possess very
useful and important traits for further breeding and genetic improvement of rice. Thus, there
is an urgent need for germplasm collection, their conservation and characterization to study
the diversity of traditional rice varieties in Myanmar. In recent years, DNA sequencevariations have been used as markers for genome analysis since they are independent ofenvironment and growth stages of plant and more reliable than the other phenotypic orbiochemical markers. Of the different types of molecular markers, microsatellites have beenutilized most extensively, because they can be readily amplified by PCR, present large
amount of allelic variation at each locus, are highly informative, and require small amounts
of DNA. They have become the versatile molecular markers not only for germplasm
diversity studies but also for exploration of targeted gene. In Myanmar, some research on
rice landrace diversity by using microsatellites has been done. Therefore, this review aims
to take stock of the current status and summarize genetic diversity studies of traditional rice
varieties in Myanmar, through the use of microsatellite markers.
Keywords microsatellites, genetic diversity, rice landraces, Myanmar

INTRODUCTION
Myanmar is known as a rice-based agriculture country and large fraction of national economy relyon the surplus from rice export. From historical time to now, the country has a long history of riceproduction. Moreover, culture, religion and belief, politics and national economy were influenced
by rice and rice production. Therefore, civilization of Myanmar was established based on rice andⒸ ISERD

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IJERD ± International Journal of Environmental and Rural Development (2018) 9-2

rice cultivation. Since 1988, owing to market oriented economic policy and international rice price,
traditional varieties have been replaced by improved varieties. However, Myanmar traditional
landraces are still under cultivation by resource poor farmers who practice subsistence farming(Yamanaka et al., 2011) and the outcome is low yield. Although the output is lower, theselandraces have wide adaptation to local or harsh conditions and they are assumed as a harbor ofgreat genetic potential for rice improvement.
Landraces are also an integral part of cultural heritage due to their close link with the local
territory as well as the community with their associated traditional uses, knowledge and habits.
Landraces are, however, threatened and suffering from genetic erosion. Major contributing factors
include climate change, habitat loss, environmental degradation and changes in farming practices,leading to the introduction of new pests and diseases, a collapse of pollinators, and increased
drought and salinization. These precious and irreplaceable resources urgently need to be conserved
before they disappear. Therefore, in order to prevent the permanent loss of landraces, research
activities on genetic diversity, conservation and utilization become critical issues for the agriculture
sector in Myanmar.
Nowadays, several techniques are available for the detection of genetic diversity, i.e. for
identifying DNA polymorphism, including the microsatellite ones. This marker is easily amplified
by PCR, requires no large initial amounts of DNA samples and has high polymorphism due to alarge variation in the number of repetitions. Furthermore, the microsatellite loci have co-dominant
multi-allelic expression which permits the discrimination of homozygous and heterozygous
genotypes, facilitating the characterization of different populations by allele frequency analysis(Bruford, 1996). Therefore, genetic diversity data, based on microsatellites, can be used formonitoring the genetic variability of species and support management actions to prevent the loss ofgenetic diversity over time. Tens of thousands of potential SSRs have been identified in rice, andover 25,000 have been developed as molecular markers (Temnykh et al., 2000; McCouch et al.,
2002; IRGSP, 2005). These markers are currently being used to develop high density genetic maps,
genotype rice accessions, determine the genetic structure, optimize the assembly of core collections,and for marker-assisted breeding (McCouch et al., 2002; Yu et al., 2003; Garris et al., 2005). The
limiting feature of the application of these markers is the need for prior sequence information fordeveloping primers for locus-specific PCR amplification. This limitation is alleviated for the
economically important species and the ones closely related, since primer sequences of the SSR
DNA markers and the amplification conditions are available in the published reports and the riceannotation project database (rap-db). Generation of complex banding patterns for SSR loci could be
due to various reasons such as type of repeat, non-optimization of PCR conditions and the nature ofgenome (Mausa, 2014).
OBJECTIVE
This review aims to make an overall assessment of the current status and summarize genetic
diversity studies of traditional rice varieties in Myanmar, through the use of microsatellite markers.
RESULTS AND DISCUSSIONIn Myanmar, assessment of genetic diversity of rice germplasm have been conducted by using
various characters: viz: morphological, physiological, biochemical, and molecular characters.
Among them, molecular markers have been used for genome analysis since they are stable anddetectable in all tissues regardless of growth, differentiation, development, or defense status of thecell. Wunna et al., (2016) studied the genetic diversity of 175 rice accessions from Myanmar,
including landraces and improved types from upland and lowland ecosystems in five different areas.
They evaluated on the basis of polymorphism data for 65 microsatellite markers and confirmed
high genetic diversity with average polymorphism information content (PIC) value of 0.82 perlocus with the range of 0.519 to 0.919. The large range of PIC values for the respective accessions
provides greater confidence for the assessment of genetic diversity and relationships. ClusterⒸ ISERD

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IJERD ± International Journal of Environmental and Rural Development (2018) 9-2

analysis based on the polymorphism data from SSR markers clearly differentiated the riceaccessions into two groups. Group I, with 85 accessions, corresponded to Indica Group and was
dominant in all upland regions except for the Northeastern. Group II, with 90 accessions,corresponded to the Japonica Group and was dominant in the Southern (lowland) region (Table 1).
Table 1 Relationships among groups, areas, and rice cultivation ecosystems
Ecosystem
UplandLowland

OthersUplandLowlandTotal

Landrace or
improved

Area

Landrace

Northern
Western
Southeastern
Northeastern
Sum
Improved
Northeastern
Subtotal
Landrace
Southern
Improved
Southern
Subtotal
Improved
Landrace

Unknown
Unknown
Subtotal

No. of accessions (%)
Group I
16 ( 9.1)
11 ( 6.3)
29 (16.6)
12 ( 6.9)
68 (38.9)0 ( 0.0)68 (39.4)
3 ( 1.7)
12 ( 6.6)
15 ( 8.6)

Group II
2 ( 1.1)
1 ( 0.6)
4 ( 2.3)
18 (10.3)
25 (14.3)
7 ( 4.0)32 (18.3)
50 (28.6)
2 ( 1.1)
52 ( 29.7)

Total
18 ( 10.3)
12 ( 6.9)33 ( 18.9)30 ( 17.1)
93 ( 53.1)
7 ( 4.0)100 ( 57.7)
53 ( 30.3)
14 ( 8.0)
67 ( 38.3)

1 ( 0.6)
1 ( 0.6)
2 ( 0.11)
85 (48.6)0 ( 0.0)
6 ( 3.4)
6 ( 3.4)
90 (51.4)

1 ( 0.6)
7 ( 4.0)
8 ( 4.6)
175 (100.0)

Pawsan rice cultivars, quality rice, seems to be originated in Myanmar and due to theirdelightful aroma and good eating quality, they are recognized as premium rice cultivars in
Myanmar. Min Soe Thein et al., (2014) collected 38 Pawsan cultivars mainly from Ayeyarwaddy
region and documented the variation of genetic structure of these collected Pawsan cultivars inorder to fingerprint, conserve and exploit potential cultivars for breeding program. Twenty two
SSR loci of Pawsan cultivars with two controls generated 112 alleles with an average of 5.09, andPIC ranged from 0.22 to 0.80 with an average of 0.54. SSR genotyping revealed that pair-wise
genetic similarity of Pawsan cultivars ranged from 14% to 84% and averaged of 49%, and Pawsan
group was separated from two controls (IR36 and Koshihikari). However, genetic similarity of
Pawsan group was closer to Koshihikari (japonica). Among the local varieties in Myanmar,
Meedon rice, which is one of the five rice varietal groups in Myanmar, is important for local
adaptability, grain quality, premium price and market availability. Minn San Thein et al., (2012)carried out a study to assess genetic diversity and to analyze population structure of Meedon rice
germplasm conserved in Myanmar Seed Bank using SSR markers. 154 accessions of Meedon rice
germplasm were analyzed with nine SSR markers. A total of 86 alleles were detected with anaverage of 9.6 alleles per locus. All the loci were found to be polymorphic, and there wereconsiderable genetic variation among accessions with mean values of expected heterozygosity(HE) = 0.5774 and polymorphic information content (PIC) = 0.5496. High frequency of rare alleles
was identified, among which 35 unique (accession-specific) alleles were observed. Based on cluster
analysis, the three population groups consist of 105 accessions, 43 accessions and 6 accessions
indicating the inclusion of non-Meedon rice accessions, which was about one-third of the
germplasm, and it was a large proportion. This study revealed that the small number of SSR
markers is possible for discriminating Meedon and non-Meedon rice. Yamanaka et al., (2011)
conducted a study on genetic variation in Myanmar by using 12 SSR markers. 41 out of 83 strains
were obtained from the Myanmar seedbank. The other 42 strains were directly sampled fromagricultural fields in the areas where the seed-bank materials were originally derived. The geneⒸ ISERD

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diversity were 0.809 in seed-bank and 0.826 in on-farm. This suggested that on-farm material wascomparatively more diverse than the seed-bank samples.
Isozyme analyses classified majority of the rice cultivars into six groups; group-I corresponds
to the indica and group-VI refers to both temperate and tropical japonica. Groups II, III, IV and V
were classified as indicas conventionally (Glaszmann, 1987). Recently, a diversity analysis was
conducted using DNA markers on Myanmar rice varieties and found that some varieties fromisozyme group-V appeared to be japonica types. Thawda et al., (2005) analyzed 31 rice varieties
from Myanmar and they reported that the rice varieties, in isozyme group-V (Singh, 2000; Khushet al., 2003) could also be considered as japonica. Therefore, Moe Moe Oo et al., (2009) conducteda research to obtain more information on Myanmar rice varieties from isozyme group-V for proper
genetic resource conservation and management. A total of 43 microsatellite primers were utilized
and 41 primers showed polymorphism among the 52 accessions. The number of alleles per locus
detected by microsatellite primers varied from 2 to 15 with an average of 5.63 alleles per primer.
The highest polymorphism was observed at RM334 on chromosome no.5 which showed 15 alleles.
According to the cluster analyses, most of the Myanmar rice varieties from the isozyme group-V
are genetically closer to japonica and a few of them are found closer to indica. The dendrogram
reveals two main groups with the genetic similarity of 0.476. The group-I consisted 5 rice varieties
including indica control and 4 Myanmar rice varieties with maximum genetic similarity of 0.520.
The group-II was formed cluster by 48 Myanmar rice varieties together with temperate japonica
control and tropical japonica control. All the rice varieties in group-II possess japonica type DNA
in both nuclear and chloroplast genomes. Therefore, it is assumed that the rice varieties from thegroup-I are genetically closer to indica and the group-II closer japonica.
Table 2 Number of alleles and gene diversity of rice landraces from Myanmar andstandard strains observed for 10 SSR loci
Locus
RM1
RM7
RM19
RM20A
RM20B
RM120
RM164
RM167
RM241
OSR21
Average

Alleles/locus
MyanmarStandard
9
4
18
9
10
11
15
10
18
8
11
5
17
12
17
10
15
10
20
14
15
9.3

Gene diversity
MyanmarStandard
0.876
0.718
0.939
0.839
0.930
0.919
0.877
0.911
0.918
0.888
0.885
0.851
0.919
0.904
0.908
0.888
0.879
0.888
0.922
0.900
0.905
0.871

According to Htay Htay Aung (2007), SSR analysis revealed high variation among the
Myanmar rice varieties. There are 202 alleles in 132 accessions using 34 SSR loci, the Myanmar
rice varieties showed 197 alleles. The average alleles per locus for all accessions and Myanmar rice
varieties are 5.94, and 5.79, respectively. The average gene diversity for all accessions is 0.71
while for the Myanmar rice varieties, it is 0.70. This indicates that high gene diversity exists among
Myanmar rice varieties. The dendrogram based on cluster analysis of SSR markers generated two
major clusters. Cluster I consisted of mostly japonica, javanica and high quality aromatic rice types.
Cluster II corresponded to the indica group. Glutinous rice varieties are mostly scattered in cluster
II with a few included in cluster I. It has been indicated that most glutinous cultivars arose
independently from indica types. Myanmar rice varieties represented a genetically diverse andheterogeneous group with multiple alleles at many of the SSR loci. Genetic polymorphism of ten
microsatellites loci were examined in 100accessions of rice including 77 landraces from Myanmar,
3 cultivated varieties from Japan, 4 from China, 1 from Vietnam, 4 from Philippines, 5 from
Taiwan area, 6 from India and 2 from Indonesia (San San Yi et al., (2005). Those other varieties
were representative strains for each country (Oka, 1958). In this study, RM 1 produced the smallestⒸ ISERD

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number of alleles per locus and OSR 21 generated the largest number in both samples. Range ofalleles per locus is 9-20 and totally 150 alleles in Myanmar strains and 4-14 and totally 93 alleles
for standard strains. Therefore, in Myanmar samples, total and average numbers of alleles arehigher than those in standard strains. Gene diversity of Myanmar strain is also greater than standard
strains from different Asia countries (Table 2). Oka (1988) also stated that the diversity of Oryza
sativa is the highest in Myanmar compared with other Southeast Asia countries because thesestandard strains are core collection of landraces of Asian rice that seeds were donated from
National Institute of Genetics (Japan). In cluster analysis, it produced 5 main clusters. 4 maingroups are indica and the other one is japonica rice group.
Table 3 Informative primers
SSR locus
RM1
RM263
RM7
RM241
RM164
OSR21
RM11
RM72
RM3164
RM271
RM167
RM247

Chromosome
1
2
3
4
5
6
7
8
9
10
11
12

No. of alleles
12
17
18
15
17
20
9
11
8
8
17
9

Gene diversity
0.90
0.62
0.94
0.88
0.92
0.92
0.80
0.81
0.80
0.84
0.91
0.77

CONCLUSION
Overall we can conclude that microsatellites are useful markers and they detect a high level ofallelic variation and give an understanding of the genetic relationships and diversity among the
Myanmar rice varieties. SSRs can also be used not only in rice germplasm evaluation but also
effectively in indica-japonica differentiation. According to the survey articles, the genetic diversityof over 600 Myanmar landraces have been evaluated by using 148 SSR loci, which are distributed
all over the 12 chromosomes. Despite this, the publications involving genetic diversity
measurement using microsatellites are still rare in Myanmar rice varieties because around 7,000
accessions are kept in the Seed Bank at the Department of Agricultural Research, Myanmar. Basedon the level of polymorphism detected by individual primers, the most informative primers wereidentified with different polymorphic bands (Table 3). Therefore, future research studies of genetic
variation should be performed on Myanmar rice landraces through the use of those highpolymorphic SSR loci.
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