Mapping And Functional Analysis Of Rice Fertility Related Genes In Thermo-sensitive Genic Male Sterile Lines

Thermo-sensitive genic male sterile rice plays an irreplaceable position in two-line rice breeding.Related research on male sterility in rice is a hot spot for rice breeding workers.Fertility traits of thermo-sensitive genic male sterile rice vary with external temperature.When the external temperature was higher than the critical temperature,the TGMS showed sterile traits,and when the external temperature was lower than the critical temperature,it showed fertile traits.HD9802 S has the advantages of low critical sterility-inducing temperature and stable fertility(the average daily temperature is below 23 C for more than 5days before transfer).The critical sterility-inducing temperature of HD9802-7S and HD9802-9S lines crossed with HD9802 S as female parent and Guyou 12 as male parent was higher than that of HD9802 S.We predicted that there were minor genes differentiated from HD9802 S in these two lines to regulate their fertility.In our previous studies,SSR molecular markers were used to locate the sterile gene of HD9802 S.Os02g0221300 gene and Os02g0221400 gene on chromosome 2 were noted.BSA re-sequencing revealed that the gene was located near the main gene tms5,which regulates HD9802 S fertility on chromosome 2.Os02g0221300 gene interference vector was designed to transform 93-11.All transformed seedlings showed fertile traits,and 93-11 filial generation with sterile traits were isolated from T1 generation.It is predicted that this gene is associated with fertility regulation in rice.We have done some research on it.The concrete results are as follows:1、Three populations,HD9802-9S/R446F2,HD9802-9S/R144F2 and Ghuangzhan63-4S/R66F2,were constructed,with 1000 plants in each population.100 sterile plants and100 highly fertile plants were randomly selected from each population to construct mixedpool.Six extreme pools and five parent pools of three populations were constructed by extracting rice genomic DNA,and 11 pools were re-sequenced by BSA.2、The target intervals of BSA re-sequencing results for HD9802-9S/R446F2 and HD9802-9S/R144F2 populations were consistent.BSA re-sequencing located the sterile gene of HD9802-9S on chromosome 2 and 7,while the sterile gene of 63-4S was widely located on chromosome 2.At 95% confidence intervals.The SNP mutations witch was related to HD9802-9S sterility traits were located in the 4590000-10180000 interval of chromosome 2and 205400-21240000 interval of chromosome 7.These SNPs were distributed in 84 candidate genes on chromosome 2 and 4 candidate genes on chromosome 7.The SNP mutations witch was related to Ghuangzhan 63-4S/R66F2 sterility traits were located in the1840000 ～ 19480000 interval of chromosome 2.These SNPs were distributed in 392 candidate genes on chromosome 2.At 99% confidence intervals,The SNP mutations witch was related to HD9802-9S sterility traits were located in the 5130000 ～ 9950000 interval of chromosome 2 and 20580000 ～ 20730000 interval of chromosome 7.These SNPs were distributed in 64 candidate genes on chromosome 2 and no candidate genes on chromosome7.The SNP mutations witch was related to Ghuangzhan 63-4S/R66F2 sterility traits were located in the 1970000 ～ 19270000 interval of chromosome 2.These SNPs were distributed in 375 candidate genes on chromosome 2.3、Two target sites were designed on exon 1 of Os02g0221300 gene,and a bi-target vector of Os02g0221300 CRISPR-CAS9 was constructed.The vector was used to transform Nipponbare(Japonica)and 93-11(Indica).Twenty-nine Nipponbare positive plants and nine93-11 positive plants were obtained by PCR.Microscopic identification and statistics of pollen fertility of all plants.When the gene Os02g0221300 was knocked out,the pollen fertility of Nipponbare and 93-11 positive plants was affected.The pollen fertility probability of 29 positive plants of Nipponbare was 20%-55%.Semi-sterile or sterile traits were found in all the plants when the Nipponbare were harvested.The pollen fertility probability of two93-11 plants was 60% and 1 was 95%.Two of 93-11 plants showed sterile traits and one showed skipping seed when the 93-11 were harvested.4、 Os02g0221400 is an RNA gene.It has one transcript at the 5’ site and 3’ site of the gene,and can transcribe a long non-coding RNA.Interference fragment(433 bp)wasdesigned on the 5’transcript of Os02g0221400 gene to construct Os02g0221400 RNAi vector.and transform it into indica rice 93-11,Nipponbare and sterile line HD9802 S,respectively.Thirteen Nipponbare positive plants and ten 93-11 positive plants and seventeen HD9802 S positive plants were obtained by PCR.Microscopic identification and statistics of pollen fertility of all plants.When the expression level of Os02g0221400 gene was interfered,Nipponbare and 93-11 pollen fertility rate decreasing.Microscopic examination of 13 pollens produced by Nippon Sunshine showed that the quantity of pollens was small and mostly sterile,and the seed setting rate decreased from 86.5% of wild type to 20%～80%.Pollen microscopy and statistics of four 93-11 plants showed that the quantity of fertile pollens accounted for 45%-75% of the total quantity of pollens.When all 93-11 plants were harvested,the semi-sterile(slightly skipping seeds)trait was found in the plant No.7,and the Os02g0221400 gene expression level of the plant was 0.42229 times of that of the wild type93-11.The plants numbered 8,9,and 10 showed sterile traits,and the expression levels of Os02g0221400 gene were 0.44290 times,0.17184 times,and 0.13504 times,respectively,of the 93-11 wild type.All HD9802 S were transplanted to Hainan for growth.Because the daily average temperature of HD9802 S is higher than the critical sterility-inducing temperature of HD9802 S in Hainan during its growth and development period.HD9802 S seeds can not be harvested.