Abstract: 【Objective】The objective of this study is to identify and reveal the transition of the male sterile gene(s) in photoperiod- and temperature-sensitive genic male sterile (P/TGMS) lines utilized in the two-line hybrid rice system in China.【Method】A total of 90 environment-conditioned genic male sterile (EGMS) lines including descendents of Nonken 58S, Annong S-1 and Zhu 1S, were used in the present study. Genomic DNAs were extracted from rice leaves by modified CTAB. One functional CAPS marker based on the C to G mutation in the long non-coding RNA (lncR) gene was designed for PGMS genotyping; Namely, a pair of primers NK-F (5′-ATCCCACAAATCCTTTAGCA-3′) and NK-R (5′-CCGTTATAGATAGACCCGAGA-3′) were used to amplify segments harboring the mutation site, followed by digestion overnight at 37℃ with restriction endonuclease RsaⅠ and separation on 1% agarose gel electrophoresis. Homozygous PGMS allele (lncR^m) (329 bp) can be readily distinguished from homozygous wild type (lncR^wt) (414 bp) and heterozygous type (lncR^m/lncR^wt) (414 and 329 bp) based on the sizes of digestion products. For TGMS genotyping, functional dCAPS markers were deployed with the following steps: Two pairs of primers RNZ1F (5′-ACCGCGCCGCCACCGGGTCGGCCGGAG-3′)/RNZR (5′-TGAAGAGGAACTCCTGCGAGACGG-3′), RNZ2F (5′-ACCGC GCCGCCACCGGGTCGGCCCAAG-3′)/RNZR were used to amplify segments harboring the mutation site (SNP-^+70TA/TC/GC); Amplified products were digested overnight at 37℃ with restriction endonucleases HinfⅠand StyⅠ, respectively, and separated on 8% polyacrylamide gels. Homozygous lines with the TGMS allele (RNZ^m) cannot be digested by these two restriction enzymes; on the other hand, homozygous wild type lines with the alleles of RNZ^tc or RNZ^gc, can be digested completely by HinfⅠ and StyⅠ, respectively; heterozygous genotypes, RNZ^m/RNZ^tc and RNZ^m/RNZ^gc, can be digested incompletely by HinfⅠand StyⅠ, respectively. By using these functional molecular markers, the PGMS (lncR^m) and TGMS (RNZ^m) genes were identified in commercial EGMS lines that had been utilized in the two-line hybrid rice system. Meanwhile, the transition of P/TGMS genes utilized in two-line hybrid rice production in China (1993-2012) was analyzed according to the information of pedigree and growing area. 【Result】Out of the 47 EGMS lines derived from the PGMS line Nongken 58S, 12 lines carry the PGMS gene lncR^m, 29 lines have the TGMS gene RNZ^m, two lines carry both genes, while the remaining four lines contain none of them. All 18 lines derived from Annong S-1 and Zhu 1S carry RNZ^m gene. All P/TGMS descendants from crosses between a Nongken 58S derivative (Pei’ai 64S) and Annong S-1 carry RNZ^m gene. In the two lines derived from Pei’ai 64S and Zhu 1S, one carries both genes, the other has RNZ^m gene. In addition, in 16 EGMS lines with EGMS progenitors independent from Nongken 58S, Annong S-1 and Zhu 1S, six of them have lncR^m, nine of them have RNZ^m gene, while one of them contains neither lncR^m nor RNZ^m gene. A pedigree map with P/TGMS genes was drawn for 92 EGMS lines, including derivatives from Nongken 58S, Annong S-1, Zhu 1S and others. Furthermore, the transition from lncR^m-based to RNZ^m-based two-line hybrid rice production was shown after examination of statistics data of the two-line hybrid rice planting area during 1993-2012, with the RNZ^m-based hybrids occupying ＞95% planting area in the two-line hybrid rice production in 2012. 【Conclusion】The study systematically revealed the P/TGMS genes in commercial EGMS rice and the presence of discrepancy between pedigree of EGMS lines and their P/TGMS gene. The spontaneous emergence of RNZ^m can be the reason for the transition from PGMS to TGMS in some EGMS lines derived from Nongken 58S. EGMS lines with RNZ^m currently dominate the two-line hybrid rice production in China.

Key words: hybrid rice, PGMS, TGMS, cultivar transition, molecular marker