Euphytica (2008) 163:215–224 DOI 10.1007/s10681-007-9630-0 123 IdentiWcation of informative SSR markers capable of distinguishing hybrid rice parental lines and their utilization in seed purity assessment R. M. Sundaram · B. Naveenkumar · S. K. Biradar · S. M. Balachandran · B. Mishra · M. IlyasAhmed · B. C. Viraktamath · M. S. Ramesha · N. P. Sarma Received: 8 July 2006 / Accepted: 30 November 2007 / Published online: 15 December 2007  Springer Science+Business Media B.V. 2007 Abstract 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 pop- ular Indian rice varieties using a set of 48 hyperpoly- morphic 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 ampliWcation of an allele, which was very speciWc and unique to a particular parental line and not ampliWed in any other rice genotype tested. Through multiplex PCR, SSR marker combinations that were unique to a particular parental line or hybrid were also identiWed. 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 eVectively for detection of impurities in parental lines, a two dimensional bulked DNA sam- pling strategy involving a 20 £ 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 speciWc SSR markers and strategies to utilize the informative SSR markers for detection of contaminants in a cost eVective manner are discussed. Keywords Hybrid rice · SSRs · Grow out test · Genetic purity assessment Introduction Tropical rice growing countries including India need to step up their rice production because of increasing pop- ulation and decreasing land and water resources. Hybrid rice cultivation oVers an opportunity to increase rice yields and thereby ensure a steady supply of rice (Virmani and Kumar 2004). Since the develop- ment of the Wrst CMS line in China (Yuan 1977), many hybrids have been released in China, India, Vietnam, Philippines, Indonesia, and Bangladesh. These hybrids have recorded a yield advantage of 15–20% over semi dwarf high-yielding varieties (HVYs) in farmers’ Welds (Rangaswamy and Jayamani 1996; Mishra et al. 2003). The success of hybrid rice technology beside other fac- tors, depends on the production and timely supply of genetically homogenous seeds to farmers. This ensures that the gains of heterosis can be harnessed through enhanced yields by growing a genetically pure hybrid R. M. Sundaram · B. Naveenkumar · S. K. Biradar · S. M. Balachandran · B. Mishra · M. IlyasAhmed · B. C. Viraktamath · M. S. Ramesha · N. P. Sarma (&) Crop Improvement Section, Directorate of Rice Research, Rajendranagar, Hyderabad 500030, India e-mail: npsarma@lycos.com