Genetic Diversity Of TMS5 In Rice And Primary Study On The Creation Of Thermo-sensitive Genic Male Sterile Resources

Thermo-sensitive genic male sterile lines(TGMS)are widely used in two-line hybridization of rice.However,the origin and molecular evolution mechanism of the key gene TMS5,which regulates the thermo-sensitive male sterile traits,remains unknown.Studies showed that although cultivated rice was composed of different genetic groups,the key genes controlling important traits of rice were highly conserved in different genetic groups,and this result provides a new perspective for exploring the origin and evolution of cultivated rice.In this paper,the diversity of thermo-sensitive genic male sterile gene TMS5 was analyzed,and the phylogenetic tree was constructed to explore the origin and molecular evolution mechanism in cultivated rice.Meanwhile,TMS5 was edited by CRISPR/Cas9 technology,and we obtained tms5 knockout mutants.The mutants had typical thermo-sensitive genic male sterile characters,which provides a technical support for large-scale cultivation of new thermo-sensitive genic male sterile lines.The main results are as follows:1.We analyzed the diversity of TMS5 promoter,coding region and 3'end sequence of 72 accessions of cultivated rice and 25 accessions of common wild rice from different regions.The study of TMS5 diversity showed that this allele was highly conserved.Despite multiple nonsynonymous substitutions existed,the functional nucleotide polymorphism was not detected in all rice accessions.The genetic diversity analysis showed that common wild rice had more gene diversity than cultivated rice and the gene diversity of indica in group ?was obviously higher than that of japonica in group ?.2.We constructed the phylogenetic tree based on the diversity of TMS5 gene was.The results showed that the cultivated rice was divided into four groups,namely,indica,japonica,aromatic and aus rice.Wild rice was classified into the four groups,and the average number of nucleotide differences for each group of wild rice and cultivated rice showed that the genetic relationship between wild rice and the same group of cultivated rice was very close,indicating that the cultivated rice might independently originate from different wild rice ancestors,that is,multiple origins.Intriguingly,some of the aromatic rice was classified into group III,and the rest of aromatic rice were divided into other groups,suggesting that aromatic rice was also likely to evolve from rice varieties of different regions,which was different from the traditional view that aromatic rice originated from a single region of japonica rice.3.The results of neutrality test indicated that TMS5 gene in japonica underwent a higher degree of negative selection than indica and wild rice during the long-term domestication.Furthermore,from the values of Tajima's D in different region of TMS5,we only found the promoter and gene region in japonica were negative and significantly deviated from neutral model.4.We edited TMS5 gene by CRISPR/Cas9 system and obtained the tms5 knockout mutant.Through observation of the mutant pollen fertility under different temperature conditions,we found that tms5 knockout mutant exhibited partial or complete pollen abortion at the high temperature,and at the low temperature,these plants completely restored male fertility.These results showed that the mutant obtained by knockout of TMS5 gene was a thermo-sensitive sterile line in rice.