Competitive amplification of differentially melting amplicons facilitates efficient genotyping of photoperiod- and temperature-sensitive genic male sterility in rice

Genic male sterility conditioned by either photoperiod (PGMS) or temperature (TGMS) is the key trait that has enabled the establishment of the two-line hybrid rice (Oryza sativa L.) system. The PGMS trait is known to result from a C→G mutation in a long noncoding RNA gene (lncR) and the TGMS from a premature stop codon mutation (TC/GC→TA) in an RNase Z gene (RNZ). To develop an efficient genotyping method capable of distinguishing all potential genotypes for the loci of lncR and RNZ, the present study explored the capacity of four approaches in high-resolution melting (HRM) curve analysis, viz. amplicon scanning, amplicon scanning with pre-PCR addition of mutant DNA template or inclusion of an unlabeled oligonucleotide probe, and melting analysis by competitive amplification of differentially melting amplicons (CADMA). The former three approaches were demonstrated to be able to differentiate only a few genotypes; in contrast, CADMA was shown to be able to discriminate all possible genotypes of both genes. For the lncR locus, HRM curves of three possible genotypes (PGMS, wild-type line and their hybrid) had peak fluorescence differences (ΔFs) of ~0.075 to ~0.20, significantly greater than the threshold (>0.05) for differentiation. For the RNZ locus, the five genotypes that would be of interest to breeders and seed technologists were separated according their HRM curves with ΔFs > ~0.1 between any two genotypes. The usefulness of the CADMA-based approach was further validated by analyzing a number of uncharacterized genotypes including P/TGMS lines, F1 hybrid seeds and F2 plants derived from crosses of P/TGMS lines with wild-type lines. Taken together, the present study proved the usefulness of CADMA in plant genotyping and established a CADMA-based HRM analysis method for high-throughput P/TGMS genotyping, which can be applied not only to marker-assisted selection of PGMS and TGMS rice but also to seed purity testing in the two-line hybrid rice system.