Assessment of Genetic Diversity in Wild Rice of Eastern India Using SSR Markers

Wild rice is an important reservoir of valuable and useful genes. O. rufipogon and O. nivara contain AA genome andare the progenitor of cultivated rice which makes them compatible with the cultivated rice for cross breeding to incorporate the genes for stress tolerance. SSR markers were used to assess the extent of diversity of 26 accessions of O. rufipogon and O. nivara collected from different districts of Orissa, West Bengal and Tripura. The Principal Co-ordinate Analysis (PCA) clearly indicates the clustering pattern and inter-relationships among different accessions. Mantel Z-testexhibitedacorrelation between cophenetic matrix and Jaccards’ similarity coefficient in 26 accessions and 4 CRRI released varieties using 54 STMS (SSR) markers which showed significant correlation (r = 0.8249) between them. O. nivara accessions and O. rufipogon accessions were grouped different clusters. O. nivara collected from Midnapore is placed in different cluster. It is concluded that the SSR markers used were found to be equally informative for the genetic diversity study between and among the accessions of two wild species such as O. rufipogon and O. nivara collected from different locations of Orissa, West Bengal & Tripura. Highlysignificant morphological variations were also observed among O. nivara and O. rufipogon accessions.


Introduction
Rice is the world's most important cereal crop and is a primary source of food for more than half of the world's population (Khush, 1977).More than 90% of the world's rice is grown and consumed in Asia.Oryza sativa L. is a diploid species having 24 (2n = 24) chromosomes.It belongs to genus Oryza of family Poaceae.The genus Oryza includes 24 species of different genome (AA, BB, CC, DD, EE, FF, GG, HH) of which 22 are wild species (2n = 24, 48) and two species namely Oryza sativa and Oryza glaberrima are cultivated in Asia and Africa respectively.The Asian cultivated rice (Oryza sativa) originates in South and South-East Asia (Chang, 1985) and is grown world wide.Where as the African cultivated rice, Oryza glaberrima, is domesticated in West Africa.All the wild species played a significant role in rice breeding by contributing genes valuable to resistance for diseases, insect and pests and tolerant to abiotic stresses (Chang et al., 1975;Stich et al., 1989;Khush, 1977).A number of useful traits such as Cytoplasmic male sterility, resistant to Grassy stunt virus, Bacterial blight, Blast and Brown Plant blast hopper, have been introgressed from wild species into cultivated rice (Brar & Khush, 1997;Yuan, 1993).The wild species exhibit tremendous diversity in morphological traits.Besides these, it constitutes an exceptionally valuable genepool for rice improvement (Lu, 1996;Bellan et al., 1988;Zhong et al., 2000).Recently many diseases and pests resistant genes, high yielding genes and abiotic tolerant genes have been found in wild Oryza species (Khush et al., 1990;Jena & Khush, 1990;Brar et al., 1996).
All the AA genome species are the most accessible genetic resources because transfer the alien gene to O. sativa can be achieved through sexual hybridization (Jena & Khush, 1990).The fuller exploitation of the wild rice species gene poolessentially relies on the better understanding of the genetic diversity and relationship of the rice production (Sharma, 1983) through assessment of genetic diversity and relationship of rice and its wild relatives.O. rufipogon and O. nivara are the closely related species of Asian cultivated rice and considered as the progenitor of cultivated rice (Okha, 1988;Khush, 1997).O. rufipogon is perennial, photoperiod sensitive, largely cross fertilized and distributed in South and South East Asian countries.It grows in swamps, and lakes year round.In contrast, O. nivara is an annual photoperiod insensitive, which is believed to be evolved from O. rufipogon because of habitat shift.It is distributed in Southeast Asia adapted to dry habitat (Vahugan & Morishima, 2003;Sang & Ge, 2007).The two species are cross compatible and exhibit narrow genetic differentiation (Oka, 1988;Zhu et al., 2007).O. rufipogon and O. nivara are treated as two ecotypes (Oka, 1988).Morphological variations are very high among the population and between the populations of O. nivara in Eastern India (Subudhi et al., 2004).
There is a remarkably rich diversity in cultivated rice and wild rice of Eastern India (Subudhi et al., 2002).However, a series of biotic and abiotic stresses continue to limit its productivity.Thus there is an urgent need to identify diverse sources of genes for tolerance to various stresses and broaden the rice gene pool.Wild rice with AA genome has been used as genetic resources to develop modern cultivars.
Measurement of morphological and biochemical characteristics has been commonly used method to arrive at an estimate of genetic diversity in parental stock material (Second, 1982).However, these characters can be influenced by environmental factors.Molecular markers may avoid many of these complications by looking directly at the genetic material itself.So in recent times Molecular markers represent a powerful and potentially rapid method for characterizing diversity.

Materials and Methods
Seeds from 12 accessions of Oryza rufipogon, 14 accessions of Oryza nivara and 4 CRRI released varieties were collected from NRRI Rice Gene Bank.The seeds were dried and germinated in petriplates by keeping seeds in a moist germination paper.Then the petriplates were kept in dark.Four days later the germinated seeds were transferred to individual pots.After three weeks, 3 grams of young leaves were collected from each accessions for genomic DNA isolation.The plants were maintained in pots.Some morphological observations were also recorded.The detailed passport information was given in Table 1.morphological data like Plant height, EBT/mt2, Leaf length, Leafwidth, Paniclelength, Panicleweight, Grain weight, Days to fifty percent flowering etc were recorded in selected five plants.
Young leaves were used for genomic DNA isolation.Total genomic DNA was extracted from young leaves by modified CTAB method (J.J. Doyle & J. L. Doyle, 1987).The quality of total DNA isolated was checked by adding 2 µl of 6X orange loading dye (Fermentas, USA) to 4 µl of isolated DNA.Six micro liter of this was loaded in a well of 0.8% (w/v) agarose gel (SRL) containing 0. 05 µg/ml of ethidium bromide PCR reaction, the optimum concentration (25 ng/µL) of the genomic DNA was used for the SSR markers taken for study.To bring the concentration of the sample genomic DNA into 25 ng/µl the sample DNA were diluted accordingly by adding TE buffer.
Microsatellite analysis was carried out using 60 (5 of each chromosome) mapped SSR markers distributed on all the 12 chromosomes (Temnykh et al., 2000) to assess the genetic relationship/variability of rice varieties (Table 3).The PCR reaction mixture contained 25 ng template DNA, 10 pmole of each of the primers, 0. 25 mM dNTPs (Fermentas), 1X PCR buffer (Biotool B&M Labs, Spain) (75 mM TrisHCl (pH 9.0), 50 mMKCl, 20 mM (NH4) 2 SO 4 and 2 mM MgCl 2 ) and 1 unit of Taq DNA polymerase (Biotool B&M Labs, Spain) in a volume of 10 l.The reaction mixture was initially denatured for 5 min at 94 o C, and, then, subjected to 35 cycles of 30 sec denaturation at 94 o C, 30 sec annealing at 55 o C and 1 min.extension at 72 o C; and a final extension for 10 min at 72 o C. Two microlitres of 6X loading dye was added to ten microliters of amplified products of each sample and was loaded on an ethidium bromide stained 2.5% agarose gel in 1X TBE buffer to separate the amplified fragments.The electrophoresis was done for about 3 hours at 100 volts.sp

Morphological Study
The data on eight morphological characters like plant height, ear bearing tillers, leaf length, leaf width, panicle length, panicle weight, 100 grain weight and days to fifty percent flowering etc. were recorded in 5 well grown plants for each genotype to assess the phenotypic variation of 26 wild rice accessions.The analysis of variance was given in Table 2.The plant height varied from 54.3 cm.(ac100228) to 177 cm (ac100165).Similarly, Ear bearing tillers ranged from 5.0 (ac100331) to 13.5 (ac100343 Note.PH = plant height; EBT/PL = ear bearing tiller; LL = leaf length; LW = leaf width; PL = panicle length; PW = panicle weight; GW = grain weight; DFF = days to fifty percent flowering.

Statistical Analysis
The data were scored as 1 for the presence and 0 for the absence of the band for each primer-variety combination for SSR analysis.Resolving power of the primer/primer combination was calculated as per Prevost and Wilkinson (1999) is: Where, P is the proportion of the 30 varieties containing the band.
The Primer Index was calculated from the polymorphic Index.The Polymorphic Index (PIC) was calculated as PIC = Σ P i 2 , where, P i is the band frequency of the ith allele.Here, the PIC was considered to be 1 -p 2 -q 2 , where, p is the band frequency and q is no band frequency (Ghislain et al., 1999).PIC value was then used to calculate the SSR Primer Index (PI).PI is the sum of the PIC of all the markers amplified by the same marker.The term polymorphism information content (PIC) refers to the value of a marker for detecting polymorphism within a population, depending on the number of detectable alleles and the distribution of their frequency.In the present study, PIC value of a marker was calculated according to a simplified version of Anderson et al. (1993): Where, P ij is the frequency of the jth allele for the ith marker, and summed over n alleles.
Jaccard's coefficient of similarity (Jaccard, 1908) was measured and a dendrogram based on similarity coefficients generated by unweighted pair group method using arithmeticaverages (UPGMA) (Sneath & Sokal, 1973), and the SHAN (Sequential AgglometricHierarchial and nested) clustering was obtained.The entire analysis was performed using the statistical package NTSYS-pc 2.02e (Rohlf, 2000).dy

Molecular Study
genotypes for the DNA sam TMS (SSR) ma ds were amplif rker wise resu er index and PI all traits, indi mples of 26 acc arkers showed fied ( Fifty four STMS markers produced a total of 165 bands, out of which 135 bands (81.81%) were polymorphic in nature.The maximum number of total bands (6) was amplified with RM341, RM17201, RM27201 and RM24717 while RM13357, RM14981, RM14860 and RM18691 produced the lowest number (1) of total bands.The amplicons were observed in the range of 80 to 800 bp.The highest and lowest amplicon size was found in RM27635 and RM26579 respectively.The highest number of polymorphic bands (5) was produced with RM 341 and RM24717 markers, whereas the lowest number (0) was amplified in RM13357, RM14981 and RM18691 markers.The Resolving Power (RP) of the markers varied from 0.333 to 5.400, while the marker index varied from 0.153 to 0.500.Best Resolving Power was observed in the marker RM18065.The maximum Marker Index (MI) 0.500 was observed with the markers RM5443, RM11111, RM25817, RM1127, RM18360, RM26579and RM22824 and the minimum (0.153) was in RM19844 and RM14320.The maximum PIC value (0.999) was observed with the marker RM28464 followed by 0.997-900 in RM17201, RM 20368, RM24717, RM20615, RM27507 and RM 15981 while RM19844 showed the minimum value (0.159) jas.ccsenet.

Table 2 .
Source of variation of 8 morphological traits in 26 wild rice accessions

Table 3 .
Sequence and Chromosome position of SSR (STMS) markers screened