Identification of informative SSR markers capable of distinguishing hybrid rice parental lines and their utilization in seed purity assessment

With the objective of identifying SSR markers that can distinguish parental lines of rice hybrids, we characterized 10 each of cytoplasmic male sterile (CMS) and restorer (R) lines along with 10 popular Indian rice varieties using a set of 48 hyperpolymorphic SSRs distributed uniformly across the rice genome. All the SSR markers were polymorphic, amplifying a total of 163 alleles, with an average of 3.36 +/- 1.3 allelic variants per locus. Twenty-seven SSR markers showed amplification of an allele, which was very specific and unique to a particular parental line and not amplified in any other rice genotype tested. Through multiplex PCR, SSR marker combinations that were unique to a particular parental line or hybrid were also identified. With a set of 10 SSR markers, all the public bred Indian rice hybrids along with their parental lines could be clearly distinguished. To utilize these SSR markers effectively for detection of impurities in parental lines, a two dimensional bulked DNA sampling strategy involving a 20 x 20 grow-out matrix has been designed and used for detection of contaminants in a seed-lot of the popular CMS line IR58025A. We have also designed a multiplex PCR strategy involving single tube analysis using 2-3 markers for hybrid seed purity assessments and demonstrate its superiority over single marker analysis in accurate detection of impurities in hybrids. Implications of parental and hybrid specific SSR markers and strategies to utilize the informative SSR markers for detection of contaminants in a cost effective manner are discussed.