An understanding of genetic diversity among parental lines is useful in hybrid rice breeding through informed selection of the parental lines to maximize heterosis. Diverse data sets including morphology (Bar-Hen et al. 1995), isozymes (Hamrick and Godt 1997) and storage protein profiles (Smith et al. 1987) have been used to assess genetic diversity among parental lines. Recently, the utility of DNA markers has been suggested for precise and reliable characterization and discrimination of genotypes (Karkousis et al. 2003). Among different classes of molecular markers, SSRs are the most suitable for such applications because of the ease in handling, reproducibility, multiallelic nature, codominant inheritance, relative abundance and genome-wide coverage (Powell et al. 1996). Recently, due to the availability of enormous data for expressed sequence tags (ESTs) in public domain, emphasis has shifted from genomic SSRs to EST–SSRs, which belong to transcribed region of the genome and may have a role in gene expression or function. The objective of present study was to assess the genetic diversity among 41 hybrid rice parental lines (9 CMS and 32 restorer lines) along with three indica and three japonica cultivars using 25 EST-derived and 25 non-EST SSR markers distributed uniformly across the rice genome. Most of the EST-derived SSR Markers (RMES markers) were designed based on information available at http://wheat.pw.usda.gov/ITMI/EST-SSR/LaRota. In addition, a few ESTSSRs (RM101 to RM199) were also considered for analysis (McCouch et al., 2002). The non-EST SSR (RM markers) markers were selected based on their high Polymorphism Information Content (PIC) value available at http://gramene.org. It was ensured that at least two markers were designed/selected from each chromosome for each category. PCR was carried out using the total genomic DNA as per Chen et al. 1998. The amplified products were resolved in 5% denaturing poly-acrylamide gels and visualized after silver staining. The marker alleles were converted in to binary scores and analyzed using TREECONW software (Peer and Wachter, 1994). The PIC values were calculated using the online software ‘Polymorphism Information Content Calculator’ available at http://www.agri.huji.ac.il/~weller/Hayim/parent/PIC.htm.

A total of 179 alleles were amplified using 50 primer pairs among 47 genotypes analyzed. The EST-derived SSR marker RMES9-2 amplified maximum number of seven alleles while non EST-SSR markers, RM7279, RM6697 and RM6925 amplified maximum number of six alleles. The EST-derived SSR markers showed maximum PIC value of 0.73 (RMES 9-2) and minimum value of 0.10 (RM161). Among non EST-SSR markers, RM6697 showed maximum PIC value (0.78) while RM3459 showed minimum PIC value (0.04). The average PIC value for EST-derived SSR markers was 0.45 while for non EST-SSR markers it was 0.36. Out of 50 markers analyzed, 5 best polymorphic markers each for EST-derived and Non EST-SSRs that were polymorphic between parental lines of nine released and commercially grown hybrids in India have been identified (Table 1).

A dendrogram constructed based on dissimilarity matrices using data derived from all the 50 SSRs grouped 47 genotypes in to two major clusters with 70% dissimilarity among them (Fig. 1). The first major cluster consisted of japonica varieties Azucena, Nipponbare and Taipei 309. The second major cluster was divided in to two sub groups. Group I consisted of the two-indica varieties Pokkali and W1263 with a genetic dissimilarity of 45%. Group II consists of 7 sub-groups. The first sub-group consisted of 2 restorer lines (111-3 and 611-1). The second sub-group consisted of 13 restorer lines (Fig. 1). Third sub-group consists of 3 restorer lines (612-1, C20R and RWC15) while fourth sub-group consisted of 5 restorer lines and one indica variety (Jalamagna). Fifth sub-group possessed 5 CMS lines while sixth sub-group had 5 restorer lines. The last sub-group possessed 4 restorer lines and 4 CMS lines. The dendrogram clearly indicated that most of the CMS lines and restorer lines formed distinct clusters with the exception that the restorer lines NDR3026, BCW56, BR-827-35 and Salivahana were grouped with the CMS lines. Grouping of CMS and restorer lines in to distinct clusters was also reported by Xu et al. 2002.

We also made an attempt to utilize the EST and non-EST based SSR markers to predict heterosis of eight released public bred Indian rice hybrids. SSR markers (6 EST and 6 non-EST SSRs) with polymorphic information content (PIC) value ≥ 4 were used for this purpose. The coefficient of polymorphism was calculated for these markers, which was used to correlate with heterosis (standard heterosis) for grain yield of the hybrids. The results are given in Table 2. EST-SSRs showed a higher positive correlation with heterosis as compared to non-EST SSRs. This may be due to the fact that the EST-SSRs amplify portions of expressed sequences in the rice genome which may be functionally associated with component traits of yield as compared to the non-EST SSRs which may be randomly distributed across the genome. Studies carried out in Grapes (Scott et al. 2000), Sugarcane (da Silva 2001) and Wheat (Eujay et al. 2002), indicate that EST-SSRs are highly useful due to their high polymorphism, cross transferability across species and most importantly due to their association with sequences coding for function.

In conclusion, a set of 41 hybrid rice parental lines were grouped in to distinct clusters using EST and Non EST-SSR markers. EST-SSR makers were highly polymorphic compared to non EST-SSRs as revealed by high average PIC value and also were a better predictor of yield heterosis. EST-SSR markers are thus more informative than non EST-SSR markers for genetic diversity studies. The ESTderived SSRs polymorphic between the parental lines of rice hybrids identified in the present study can also be used for testing seed genetic purity of these hybrids.

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