A Novel Gene Mapping Of TGMS Line HD9802S In Rice

The strategy of increasing grain production includes the exploitation of germplasm resources and the utilization of new genes and heterosis.Self-pollination of rice has brought problems to rice hybridization,and rice male sterility has solved this problem very well.The advantages of two-line hybrid rice breeding are:One-line dual-use,full use of genetic resources,sterile traits for easy transferring,sterility of the male sterile line simplify the breeding program.The thermo-sensitive genic male sterile line of this study was derived from the HD9802S of the Genetics Research Department of Hubei University,and the nuclear sterility gene was named HDt,ms in the previous study.In this study two methods were used.The first method was to construct the genetic map by using SSR markers,the material is HD9802S/(HD9802S/R287)BCi population,then screen out the SSR marker that is closely linked to the sterility gene and can be used for molecular marker-assisted selection.The second method is based on HD9802S/R287 F2 population combined with BSA resequencing method,screen and analysis the SNP or InDel that can cause functional mutations in the candidate interval.The specific findings include the following:1.The pollen fertility of the HD9802S/(HD9802S/R287)BCi population was identified.Two standards were used to divide sterile and fertile plants.It was found that the separation of sterile and fertile plants all conform to the theoretical ratio of 1:1,indicating that the sterility of thermo-sensitive genic male sterility line HD9802S is dominated by a pair of recessive nuclear gene.It was also found that there were always plants distribution in each pollen stainable interval.Therefore,the sterility of HD9802S was considered to have modification of minor gene.At the same time,it can be clearly seen that there are two peaks of fertility,indicating that major gene plays an important role.2.48 SSR markers recommended by the Ministry of Agriculture for identification of rice varieties were used to screen markers distributed on 12 chromosomes of rice and the polymorphism was stable between the sterile line HD9802S and the restorer line R287.15 markers were screened,covering chromosomes 1,4,5,6,7,10,11,and 12.In order to obtain the SSR markers covering the short arms of the long arms of 12 rice chromosomes,7 SSR makers were added from the GRAMENE database(http://www.gramene.org/)and the RAP database(http://rapdb.dna.affrc.go.jp/Index.html).From the HD9802S/(HD9802S/R287)BCi population,64 DNA samples(32 highly sterile samples;32 highly fertile samples)were selected as templates for PCR then calculate the number and ratio of the four types of bands(high-sterility parental band,high-sterility recombined band,highly-fertile parental band,and highly-fertile recombined band).According to statistics,the ratios of the two markers(RM5897 and RM1358)on chromosome 2 do not conform to the 1:1:1:1 free combination law for the four types of bands,and the difference between the two markers on chromosome 2 is extremely significant.And the P values correspond to the two makers are 6.683 × 10-9,1.118 × 10-8,which proves that HDtms is located on chromosome 2.Then HDtms was used as the target gene marker,and the corresponding band type of 64 samples were entry to the QTL IciMapping 3.0 software for chromosome grouping.The HDtms was also divided into chromosome 2.3.SSR markers with stable polymorphism between HD9802S and R287 were screened every 10 cM on chromosome 2,and finally RM279,RM12601,RM12631,RM12694,RM5897,RM12783,RM71,RM1358,RM1313,RM5707 were obtained.Using these 10 SSR markers and 428 samples of the HD9802S/(HD9802S/R287)BCi population constructing genetic map.The length of genetic map is 112.21 cM and the average map length is 11.21 cM,and HDtryms was between RM5897 and RM12783.4.After anchoring HDtms between RM12694 and RM12783,SSR markers were screened every 5 cM in this interval,and the polymorphism of RM12720 were stable between HD9802S and R287.The recombination rate between RM12720 and HDtms is the lowest and the screening rate was the highest(91.818%)for sterility traits in 428 individuals of BC,population,which could preliminary be applied to molecular marker-assisted selection.5.Construct HD9802S/R287 F-2 population,extract DNA from sterile samples in F2 population,and construct 50,1 00,and 150 sterile samples of gene pools in equal amounts each sample for BSA genome resequencing.The number of SNP and InDel in 50,100,and 150 samples mixed gene pools were 72634 and 1940,63965 and 1564,62948 and 1564 respectively.The number of SNP in the 100 samples mixed gene pool compared with that of 50 samples mixed gene pool was reduced by about 12%,and the number of InDel was reduced by about 20%;the number of SNP and InDel varied little relative to the 150 samples mixed gene pool.In the BSA re-sequencing,the result of 100 samples mixed gene pool is accurate enough,not only can reduce the background noise but also save the cost.The results of this study are still based on the sample amount of 150.By screening sites which SNP-index>0.8,we found that there is a peak at 6-7.5 Mb on rice chromosome 2.The number of SNP that can cause functional changes in this interval is 173,the number of InDel is 2,and there are 36 corresponding annotation genes including BGIOSGA006970 to BGIOSGA007791.Among them,10 genes have not been studied yet.Among the 26 genes studied,BGIOSGA007782 encode RNAse Z and it's just tms5 of Annong S-1.HDtms and tms5 are most probably the same gene.6.The gene corresponding to SNP located on the chromosome 4 may be the minor gene influencing the fertility of HD9802S.The temperature of the starting point of HD9802S is low,while the minor gene influences the transferred temperature of the fertility.SNP which SNP-index on chromosome 4 was significantly higher than the expected locus ranged from 15.78 to 18.93 Mb.The number of SNPs that can cause functional changes in this interval is 33,the number of InDels is 1,and contains 20 annotated genes from BGIOSGA015234 to BGIOSGA016370.Among them,the functions of 11 genes have not been studied at present.The role of whether or not some of them are related to the transferred temperature of the HD9802S will be explored in further studies.