Development and validation of candidate gene-specific markers for the major fertility restorer genes, Rf4 and Rf3 in rice

Two major nuclear genes, Rf3 and Rf4 , are known to be associated with fertility restoration of wild-abortive cytoplasmic male sterility (WA-CMS) in rice. In the present study, through a comparative sequence analysis of the reported putative candidate genes, viz. PPR9 - 782 - (M,I) and PPR762 (for Rf4 ) and SF21 (for Rf3 ), among restorer and maintainer lines of rice, we identified significant polymorphism between the two lines and developed a set of PCR-based codominant markers, which could distinguish maintainers from restorers. Among the five markers developed targeting the polymorphisms in PPR9 - 782 -( M,I ), the marker RMS-PPR9-1 was observed to show clear polymorphism between the restorer ( n = 120) and maintainer lines ( n = 44) analyzed. Another codominant marker, named RMS-PPR762 targeting PPR762 , displayed a lower efficiency in identification of restorers and maintainers, indicating that PPR9 - 782 -( M,I ) is indeed the candidate gene for Rf4 . With respect to Rf3 , a codominant marker, named RMS-SF21-5 developed targeting SF21 , displayed significantly lower efficiency in identification of restorers and non-restorers as compared to the Rf4 -specific markers. Validation of these markers in a F 2 mapping population segregating for fertility restoration indicated that Rf4 has a major influence on fertility restoration and Rf3 is a minor gene. Further, the functional marker RMS-PPR9-1 was observed to be very useful in identification of impurities in a seed lot of the popular hybrid, DRRH3. Interestingly, when RMS-PPR9-1 and RMS-SF21-5 were considered in conjunction with analysis, near-complete, marker–trait co-segregation was observed, indicating that deployment of the candidate gene-specific markers both Rf4 and Rf3 , together, can be helpful in accurate identification of fertility restorer lines and can facilitate targeted transfer of the two restorer genes into elite varieties through marker-assisted breeding.