Studies On The Methods For Mapping Major Genes Using BSA-seq

The approach of bulked segregation analysis combined with high throughput sequencing technology(known as BSA-seq)can quickly map genes associated with specific traits.BSA-seq has been widely used in single gene mapping,but still lack systemic integration and research.In this thesis,based on the previouexperimental s studies,a set of BSA-seq methods for single gene mapping under two different crossing designs is proposed,hoping to provide reference for practical applications.One design is outcrossing,namely,to create a segregating population by crossing the single gene mutant with an unrelated wild-type variety,from which a very high density of markers can be obtained.Therefore,the target gene can be precisely mapped using these markers.In addition,the candidate gene can be further identified by searching for the mutant site within the target region and referring to the gene annotations.The other design is inbreeding,namely,to create a segregating population by crossing the single gene mutant with its original wild-type variety or selfing the heterozygote of the mutant locus.The inbreeding population has a uniform genetic background with only a small number of markers.Therefore,it is difficult to precise map the target gene with the help of markers.However,it is possible to identify the mutant site by scanning the whole genome.If the mutant site is not found,it can be tried to identify structural variation as the possible cause of the mutation by examining breakpoint.The above method has been used to analyze two practical examples in rice.The main results are as follows:Example 1:outcrossing design.The mutant srtl from indica variety MH86 was crossed with another indica variety 9311 to create an F2 population,and BSA-seq was performed to map the SRTl gene.A total of 656,901 markers were identified,with which SRTl was mapped on the terminal region of the long arm of chromosome 4,and the candidate gene was identified as LOC_Os04g56780.The mutant allele srtl was the result of a 21-bp deletion in the first exon of this gene.Example 2:inbreeding design.Seven different mutants(moc、mp2、mp3、psd5、psd6、sp、sg)generated from MH86 by radiation were jointly analyzed using this design.Six genes were successfully mapped and their candidates were identified.Among them,four genes have been reported before,and their mutations were missense mutation(mp3,psd6),frame shift mutation(sg)and splicing site mutation(sp),respectively;two genes were new(moc,psd5),both resulting from structural variations.These two examples suggested that the BSA-seq method proposed in this thesis is a rapid and accurate gene mapping method,which can detect not only common mutations,but also structural variations,which have not been reported before.Therefore,this method could be widely applied to major genes mapping.