Variation of Microsatellite Markers in a Collection of Lao`s Black Glutinous Rice (Oryza sativa L.)
N.R. Sackville Hamilton
The genetic diversity of 74 genotypes, including Black Glutinous
Rice (BGR) from Lao`s germplasm was assessed using 24 microsatellite markers.
A total of 75 alleles were detected at the 24 microsatellite markers.
The number of alleles per marker varied from 2 to 7 with an average of
3.1 alleles per locus. The Gene Diversity (GD) and Polymorphism Information
Content (PIC) ranged from 0.18 to 0.79 and 0.17-0.76, respectively and
the Allele Frequency (AF) ranged from 0.36 to 0.90. The markers were able
to classify rice genotypes into four groups; indica rices were
put in the three groups while the other group consisted of tropical japonica
rice. The first indica group (G1) included 24 genotypes of BGR
and five genotypes of white rice. Most of genotypes in this group have
thick culms, broad leaf blades, large and bold grain shapes and some of
them have purple coloration on all vegetative parts. In other varieties
with black pericarp, all other plant parts are green. Indica group
3 (G3) included 25 genotypes of BGR. The special characters of this subgroup
were small and slender culms, narrow short leaves, purple leaf margins,
purple leaf tips and purple stripes on leaf blades and sheathes. However,
the markers used could not differentiate between LG 8215 and LG 7937.
Indica group four (G4) consisted of eight genotypes of white rice
and the four check varieties. Group (G2) consisted of five white rices,
four BGRs and three check varieties.
Rice belongs to the genus Oryza in the grass family (Gramineae).
There are 22 species in the genus, of which only two are cultivated: O.
sativa, which was domesticated in the humid tropics of South and Southeast
to East Asia and O. glaberrima, which was domesticated in the Niger
basin in Africa (Khush, 1997). O. sativa was domesticated in south
Asia at least 10,000 years ago (Zhang and Jiarong, 1998) and O. glabberima,
was domesticated in West Africa between about 1500 and 800 BC (Murrey,
South and Southeast Asia is generally accepted as the center of origin
and domestication of glutinous rice and glutinous rice varieties are grown
in most countries in these areas that have long history of rice cultivation.
Laos has the highest per capita production and consumption of glutinous
rice in the world (Schiller et al., 2006). Lao PDR proved to have
one of the richest biodiversities in rice and it appears to be the world`s
biodiversity centre for glutinous (sticky) rice varieties (Anonymous,
2007). In Lao PDR, traditional varieties of black glutinous rice are named
by farmers for a particular distinctive character or region and a single
name may encompass a genetically diverse set of varieties. For example,
rice varieties with coloured pericarps other than red are usually called
Khao Dam or Khao Kam (Dark Rice or Black Rice) (Appa et al., 2002a).
The black rices of Laos are less in production and consumption, but they
are not least in their importance. They have long been cultivated and
are widely grown in small plots throughout the country for making special
recipes in special occasions such as bamboo rice, rice cakes and special
alcohol drinks. BGR varieties can command a premium price in both the
international and domestic markets. Their superior characteristics, such
as high eating quality and grain characteristics and good general adaptability
to biotic and abiotic factors, make them popular among Lao farmers.
The Lao Ministry of Agriculture and Forestry (MAF) and the International
Rice Research Institute (IRRI) had jointly collected 13, 992 samples of
cultivated rice germplasm in Laos during 1995 to 2003 (Appa et al.,
2002a, b) and 431 accessions (3.07% of the total collections) were classified
as having coloured pericarps based on the information provided by the
farmers at the time of collection of the germplasm and on the characterization
data from the Agriculture Research Center (ARC), (ARC, 2003, 2004; Appa
et al., 2003). This germplasm is currently conserved in the Lao`s
national genebank and in the International Rice Germplasm Collection (IRGC).
In an attempt to best utilize this indigenous rice germplasm, several
activities for short-term application have been initiated. These activities
include primary characterization, evaluation in replicated yield trails
(Inthapanya et al., 1995). For the longer term utilization of the
germplasm, it is necessary to quantify genetic diversity among Lao BGR
varieties and to classify the genetic differentiation among them at molecular
level. Most studies have been conducted so far with non-glutinous rice,
fragrant rice and white glutinous rice using several types of molecular
markers. The lowest genetic diversity was observed among the traditional
basmati varieties, whereas the evolved basmati varieties showed the highest
genetic diversity by Simple Sequence Repeats (SSR) and Inter Simple Sequence
Repeats (ISSR) assays (Nagaraju et al., 2002). Bao et al.
(2006) reported that high levels of AFLP (78.3%) and ISSR (92.2%) polymorphism
were found in 56 waxy (glutinous) rice accessions. In general, gene diversities
in rice populations (landraces, old varieties, newly developed varieties,
wild species and parental lines of hybrids) as measured by different types
of molecular markers ranged from 32.0 to 70.0 depending on methods used
and rice populations under study (Giarrocco et al., 2007; Pessoa
et al., 2007; Xu et al., 2004; Yu et al., 2003;
Coburn et al., 2002; Ni et al., 2002) except for very low
genetic diversity (PIC = 0.001) of landrace accessions in high altitude
regions of Nepal (Bajracharya et al., 2006).
Most studies identified indica-japonica pattern of genetic diversity
of Asian rice. In a study on a study of a wide range of rice genotypes
using nuclear SSRs and two chloroplast loci, Garris et al. (2005)
found five distinct groups corresponding to indica, aus,
aromatic, temperate japonica and tropical japonica
rices. They also found that indica was closely related with aus,
whereas tropical japonica, temperate japonica and aromatic
rices were closely related. In a study in Indonesia conducted on traditional
and improved varieties, Thomson et al. (2007) found that indica
rice (68%) predominated over tropical japonica rice (32%) in total
number of accessions studied but gene diversities were rather similar
(0.53 and 0.56 for indica and tropical japonica, respectively).
Using combined AFLP and SSR markers, Bao et al. (2006) could clearly
differentiate indica and japonica groups of waxy rice accessions.
Unfortunately, this very important information for black glutinous rice
is not available in the literature. This is because most rice breeding
programs have been focused on non-glutinous rice and breeding of glutinous
rice especially for black glutinous rice is far behind. The objective
of this study was to use SSR markers to quantify genetic diversity among
Lao BGR varieties and classify the genetic differentiation among them.
The information will be useful for future breeding program, conservation
and effective management of black rice genetic resources.
MATERIALS AND METHODS
Plant materials: The research was undertaken in 2006 at the
IRGC at the International Rice Research Institute (IRRI), Philippines.
Rice varieties used for this study consisted of 56 varieties of Oryza
sativa provided by the Lao`s national genebank (Table
1) and 18 varieties of standard white rice supplied by IRGC. Fifty
three varieties out of the total of 74 varieties were tested and classified
into black rice. The remaining consisted of tropical japonica,
indica rice and white glutinous and non-glutinous rice.
DNA extraction: Total genomic DNA was extracted from young leaves
using a modification of the method described by Fulton et al. (1995).
Approximately 5 mg of young leaf tissue was frozen in liquid nitrogen,
followed by grinding using a plastic bit attached to an electric drill
before addition of extraction buffer, which was different from
that of Fulton et al. (1995) who used mortar and pestle. Other
steps in extraction followed Fulton et al. (1995). After extraction,
pellets were dissolved in 150 μL TE (10 mM Tris, 1 mM EDTA, pH 8).
DNAs were quantified by gel densitometry on agarose gels using Lambda
DNA as a standard followed by normalization to concentration of 5 ng μL-1
prior to use in PCR.
PCR amplification: Polymerase chain reactions using microsatellite
primers were carried out in a final volume of 10 μL. The PCR reaction
mixture contained 1 μL 10xTBE buffer (containing 100 mM Tris-HCl
pH 8.5, 50 mM KCl), 1 μL 15 mM MgCl2, 1 μL of 5 mM
dNTP mixture, 1 μL each of 10 mM forward and reverse primers, 1 unit
(0.5 μL) of Taq DNA polymerase, 10 ng of DNA template and ddH2O
to reach the final reaction volume of 10 μL. Reactions were overlaid
with mineral oil.
||Some agronomic characters of 53 accessions of lowland black glutinous
rice and three varieties of Lao white rice (6732 = Kai Noi Leuang,
1655 = Hom Nang Nouan and TDK5)
To optimize the PCR amplification conditions, experiment were carried
out with varying quantity of MgCl2 (15 mM) and Taq DNA polymerase,
two different quantity of MgCl2 and Tag polymerise were used
(1-1.5 and 0.5-1 μL) respectively.
The 35 SSR markers were screened in this study. Eleven SSR markers were
not polymorphic and discarded. Twenty four polymorphic markers were used
to amplify DNA products (Table 2). These markers were
reported previously (Temnykh et al., 2000). Amplifications were
carried out using MJR DNA Engine Dyad® thermal cyclers.
The PCR programs consisted of the following steps: initial denaturation
for 5 min at 94°C, followed by 40 cycles of 1 min denaturation at
94°C, 1 min annealing at 55°C, 2 min extension at 72°C, followed
by a final extension of 5 min at 72°C and holding at 15°C until
recovery. For some specific microsatellites, a different annealing temperature
of 53, 57, 61 or 63°C was used as indicated in Table
||Total number of alleles, allele length in bp, gene frequency and
gene diversity of 24 SSR markers assayed in 74 rice genotypes
|Remark: AF = Allele Frequency, AL = Allele Length in
bp, SS = Sample Size, AT = Annealing Temperature and PIC = Polymorphic
Amplification products were separated by non-denaturing PAGE on mini
vertical units using either 6.5 or 8% polyacrylamide gels with 1x TBE
buffer at 100 volts for 1.5 h. After electrophoresis, gels were stained
with ethidium bromide and visualized by UV illumination. Gel images were
photographed using Bio-Rad GelDoc system. The molecular weights of PCR
products were estimated relative to a 1 kb ladder that served as the size
Data analysis: Summary statistics (PIC, GD and AF) on the marker
data were calculated using PowerMarker 3.25 (Liu and Muse, 2005).
The allelic state encoded SSR data were used to calculate a matrix of
genetic distances between all pairs of varieties using simple matching
coefficient under DARwin® 4.0 Release (2007) (Perrier et
al., 2003). Cluster analysis was accomplished on this matrix by the
unweighted neighbor joining option.
Twenty-four SSR markers were used for amplifying DNA segments from
genomic DNA of 74 genotypes. A total of 75 alleles were scored from the
24 SSR markers. The number of alleles per primer ranged from 2 to 7. On
average, 3.1 alleles per locus were observed. The Gene Diversity (GD)
ranged from 0.18-0.79. The Polymorphism Information Content (PIC) ranged
from 0.17 to 0.76 and the Allele Frequencies (AF) ranged from 0.36 to
0.90 (Table 2). RM259 SSR was the highest polymorphic
marker with 7 alleles. The size of the amplified segment ranged from 100-150
bp for RM161 to 100-550 bp for RM259. Number of genotypes from 24 SSR
markers ranged from 2-13. The PCR products amplified by RM259, RM307 and
RM162 are shown in Fig. 1. The number of alleles detected
by RM171, RM271 and RM259 was slightly higher than the average with 4-7
The microsatellite markers were able to distinguish between different
rice genotypes. The high degree of polymorphism of microsatellite markers
allows rapid and efficient identification of rice genotypes. The microsatellite
markers classified the rice genotypes into four groups; indica
accessions were put in the three groups while the other group consisted
of tropical japonica rice. Genotypes in indica group one
(G1) included 24 genotypes of BGR and five genotypes of white rice. Most
of genotypes in this group have thick culms, broad leaf blades, large
and bold grain shapes and some of them have purple coloration on all plant
parts. In other varieties with black pericarp, other plant parts including
glumes and leaves are green color. Indica group 3 (G3) included
25 genotypes of BGR. The special characters of this subgroup were small
and slender culms, narrow short leaves, purple leaf margins, purple leaf
tips and purple stripes on leaf blades and sheaths. However two genotypes
were not distinguished from each other (LG 8215 and LG 7937), these might
be of the same genotype. Indica group four (G4) consisted of eight
genotypes of white rice, which were green color with tall plant parts
and included the four check varieties (Hom Nang Nouan, TDK5, IR64 and
Shan-Huang Zhan-2). Tropical japonica group (G2) consisted of 5
genotypes of Lao white rice varieties and 4 genotypes of BGR and three
check varieties (Khao Kai Noi Leuang, Azucena and Li-Jiang-Xin-Hei-Gu).
Hence, the four BGR genotypes (LG6730, LG7260, LG8140 and LG13259) in
this group should be tropical japonica types (Fig.
||SSR banding patterns of 53 black glutinous rice and 21 white rice
generated from RM162 (A and B) and RM259 (C and D)
||Dendrogram of 74 rice genotypes, constructed using UPGMA based on
simple matching coefficient using allelic states
Glutinous rice is a staple food for Lao people and black glutinous
rice is also important as food for special occasions and special recipes.
Black glutinous rice also serves as a valuable genetic resource for genes
with economic importance such as resistance to abiotic and biotic stress,
table quality and starch quality for a variety of value-added products.
As a part of the center of origin and domestication of glutinous rice,
high genetic diversity of this type of rice would be expected in the samples
of total accessions collected in different regions of the country. The
main objective of this research was to understand genetic variability
of the indigenous black rice germplasm. The other objectives were to select
parental genotypes for potential use in future breeding program.
Out of 35, 24 SSR primers revealed rather moderate genetic diversity
of black glutinous rice as indicated by relatively low alleles per locus
(3.1), GD (0.50) and PIC (0.44), when compared with the results of those
available in the literature. By using 26 SSR markers, Giarrocco et
al. (2007) found high alleles per locus (8.4) low pair-wise genetic
similarity (0.32) and high PIC (0.69) in 69 rice cultivars with historical
importance in Argentina. By using 47 RAPD markers, Singh et al.
(2003) obtained a total number of 275 alleles (75 allels in present study)
in 21 rainfed lowland rice genotypes consisting of historic and modern
cultivars. In general, genetic diversities reported in previous studies
(Xu et al., 2004; Bao et al., 2006; Pessoa et al.,
2007; Thomson et al., 2007) were higher than this study but the
results cannot be compared directly because neither of the previous research
was conducted with black glutinous rice. However, Bajracharya et al.
(2006) found low diversity in landrace population in Nepal with diversity
index of 0.23 and these genotypes might derive from single origin. The
lower genetic diversity might be caused by the following reasons. First,
the samples represented only lowland black glutinous rice excluding upland
black glutinous rice. Second, the samples are not well represented the
total genetic variability because of low accessibility to localized areas
and, if this is the case, resampling in remote sites is recommended to
collect additional samples. Third, there are differences in rice populations
and methods used in many studies.
Out of 35, 11 SSR primers failed to differentiate black glutinous rice
samples indicated that genetic variability was not high and a large number
of randomly distributed SSR primers are required to identify the genotypes.
Ni et al. (2002) suggested that 30 SSR primers were needed to stabilize
the dendrogram of 38 rice accessions. The other possible reasons for low
genetic diversity in black glutinous rice are that preferential selection
of farmers and genetic erosion by introduction of modern varieties might
also be the causes. This highlights the importance of genetic conservation
of black glutinous rice germplasm for future use. From the preliminary
study on agronomic characters (Table 1), the accessions
of BGR had high variations in many characters such as yield, days to flowering,
panicle length and culm length. Improvement of economically important
characters of this germplasm can be achieved but might be in limited extent
because of low genetic diversity. Introduction of genetic materials from
diverse sources is required.
Typical indica-japonica grouping pattern was clearly identified
by the SSR dendrogram. Three groups formed an indica main group,
whereas only one group represented japonica type. Higher diversity
was found in idica rice than in tropical japonica rice.
The results corresponded to the widely accepted grouping pattern of Asian
rice (Giarrocco et al., 2007; Yu et al., 2003; Ni et
al., 2002; Glaszmann, 1987) in which six groups constitute O. sativa
species (Glaszmann, 1987). The results were in agreement with the study
of Prathepha and Baimai (2004) who found that almost all of glutinous
upland rice genotypes grown by various ethnic groups in northern Thailand
were characterized as japonica type, whereas most of rainfed glutinous
lowland varieties from other regions of Thailand were indica. A
study with Thai and Lao black glutinous rice found that based on two growing
conditions, rainfed lowland and rainfed upland, chloroplast DNA type was
distinct from each other (Prathepha, 2007).
To better utilize black glutinous rice in the country and achieve breeding
goal, genetic diversity of upland glutinous rice should be studied and
compared with this study. Black glutinous rice in other countries should
be studied and introduced to broaden germplasm base of black glutinous
We thank Mr. Phumi INTHAPANYA Director of Agricultural Research
Center of Lao PDR, for seed samples of Lao accessions. Financial support
from IRRI`s Training Center is gratefully acknowledged.
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