Identification, Characterization Of Photoperiod-and Thermo-Sensitive Genic Male Sterility Genes In Rice

Rice is one of the most important crops in the world, and hybrid rice has made a tremendous contribution in increasing rice productivity. As a kind of self-pollination crop, the success of hybrid rice is benefited from utilization of male sterility, including cytoplasmic male sterility (CMS) and genic male sterility (GMS). The discovery of a spontaneous photoperiod-sensitive genic male sterility (PGMS) mutant, Nongken58S, established a new paradigm in hybrid rice breeding and production: two-line hybrid rice system. Since then more than a hundred of environment-conditioned GMS (EGMS) rice lines have been developed, mainly from three independent EGMS progenitors (Nongken58S. Annong S-1and Zhu1S). Two-line hybrid rice system has played a major role in rice production. However. the identity and relationship of sterility genes in those EGMS lines is largely unknown. w-hich is hindering two-line hybrid rice breeding and production to a certain extent.In the present study, the TGMS gene of a widely used line. i.e. Zhu1S was preliminarily mapped to a region overlapping that delimited for another PTGMS line Guangzhan63S. The candidate gene for TGMS was identified and that had been or are been using a large number of EGMS and WT rice. Functional molecular markers were developed to indetify male sterility gene of EGMS lines used in the two-line hybrid rice which provide a comprehensive understanding for the history and status quo of sterility gene utilization in hybrid rice. Furthermore, the molecular characterisitic and function of the TGMS candidate gene was also studied by sub-celluar location and RNAi technology. The main results are summarized as following:1. The TGMS gene of Zhu1S was mapped on chromosome2between microsatellite markers RM12521and RM12721. This region coincided with the delimited segment for ptgms2-1of Guangzhan63S, and allelism test showed Zhu1S and Guangzhan63S are allelic. So it is speculated that Zhu1S and Guangzhan63S have same mutation in RNaseZ (ribonuclease Z). 2. Sequencing the RNZ for14commercial EGMS and21non-EGMS lines revealed that the RNZ allele of EGMS lines contain a premature stop codon TAG (nucleotides70-72from translation starting site), hence is referred to as RNZ", while wild type varieties have nucleotide of TCG (RNZC) or GCG(RNZgc). Beside, the sequence of RNZtc and RNZ"’were identical except the mutation site, while RNZgc type had other SNPs in the intron and could be classified into six haplotypes.3. Two dCAPS markers were developed for distinguishing RNZ alleles and were used to detect RNZ alleles among32EGMS and310non-EGMS lines. The results demonstrated that the RNZm allele existed exclusively in EGMS lines only, which means it was not detected among331non-EGMS lines at all. Other two alleles. RNZgc and RNZtc. are variable among non-EGMS lines. RNZgc type is more commonly detected than RNZtc.4. By use of functional molecular markers developed for EGMS genes, it is revealed that all of the EGMS lines derived from the TGMS line Annong S-land Zhu1S carry TGMS gene (RNZm):while EGMS lines derived from PGMS line Nongken58S either carry PGMS gene (lncRm) or have TGMS gene (RNZm). two lines carry both genes. while the remaining four lines contain none of them.5. A pedigree map with P/TGMS genes was drawn based on lncRm and RNZm identification data for92EGMS lines, including derivatives from Nongken58S, Annong S-1. Zhu1s and others. The study demonstrates the presence of discrepancy between pedigree of EGMS lines and the P/TGMS gene map; the spontaneous emergence of RNZm can be the reason for the transition from PGMS to TGMS in EGMS lines derived from Nongken58S the past two decades. Meanwhile, the status quo and transition of EGMS genes in two-line hybrid rice production in China (1993-2012) was also analyzed. The map clearly showed that the transition from PGMS to TGMS gene in two-line hybrid rice production after examination of statistics data of the two-line hybrid rice planting area during1993-2012, and TGMS lines basede hybrids currently occupy>95%planting area in the two-line hybrid rice production. TGMS lines currently dominate the two-line hybrid rice production in China. 6. High-throughput HRM markers were developed according to PGMS and TGMS mutation information for efficient genotyping all potential genotypes for the loci of incR and RNZ, and explored the capacity of four approaches in high resolution melting (HRM) curve analysis, i.e. 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 results showed that CADMA approach was able to discriminate all possible genotypes of both genes. Established a CADMA-based HRM analysis method for high throughput P/TGMS genotyping, which can be applied for not only marker assisted selection of PGMS and TGMS rice but also for seed purity test in the two-line hybrid rice system.7. Sub-cellular localization vector was constructed for RNZ gene, and transiently expressed in rice protoplasts. The result demonstrated that RNZ protein was localized in chloroplast.8. RNA silencing expression vector was construsted for RNZ gene and transgenic rice plants were produced by Agrobacterium-mediated transformation. Leaf GUS assay and PCR analysis confirmed two lines were transgenic positive lines. qRT-PCR analysis showed that significant reductions (35%-45%) of target gene expression were proven in the trangentic positive lines.