Candidate Gene Analysis And Fine Mapping Of The Major QTL QGK1/qTGW1.2 For Tartary Buckwheat Thin Shell And 1000-grain Weight

Tartary buckwheat（Fagopyrum tataricum）is one of the important corn crops in our country,which is divided into thin shell types and thick shell types according to the different shell types,the different shell types lead to different husking efficiency and rice yield.Grain weight is an important factor affecting yield and quality of Tartary buckwheat.Therefore,locating the thin-shell genes and grain weight QTLs of Tartary buckwheat,mining their candidate genes and closely linked molecular markers are helpful to elucidate the thin-shell genetic mechanism of Tartary buckwheat and breeding high-yield and high-quality Tartary buckwheat varieties.In this study,the thin-shell gene and the thousand-grain weight dominant QTL qGK1/q TGW1.2 were used as targets to excavate SSR markers closely linked to thin-shell,analyze candidate genes and fine-locate them.The specific research results are as follows:（1）A total of 286 SSRs were detected in the genomic region of qGK1/q TGW1.2（Ft1:5,690,284 bp-7,065,602 bp）,and 186 pairs of SSR primers were synthesized successfully.Linkage analysis of 5 pairs of polymorphic SSR markers and morphological markers（a7GK）showed that SSR markers Ftg GKssr-149 and Ftg GKssr-175 were most closely associated with shell types.Correlation analysis of SSR polymorphic markers with shell rate,1000-grain weight and grain shape traits in32 Tartary buckwheat varieties（lines）showed that markers Ftg GKssr-147,Ftg GKssr-149 and Ftg GKssr-175 were significantly correlated with shell rate,1000-grain weight and grain length.（2）There were 199 annotated genes in the region of Ft1:5,690,284 bp-7,065,602bp.Among them,15 genes were homologous to 10 reported genes controlling grain weight or grain type（AGB1,BRI1,DASH,GSK2,IKU2,SMG2/Os MKKK10,ANT,LP1,MADS1,and CYP78A9）.Totally,14 genes were identified with non-synonymous SNP variations between the two parents of RILs population,"Xiaomiqiao"and"Jinqiaomai2".Of the 14 genes,four genes,Ft Pin G0001418200.01,Ft Pin G0001418500.01,Ft Pin G0001428300.01 and Ft Pin G0001423200.01,had significant or extremely significantly differential expression levels between the two parents,indicating that these genes may play important roles in Tatary buckwheat grain weight/size control.of the four genes,Ft Pin G0001423200.01 was the homologue of grain weight/size gene BRI1 reported in Arabidopsis thaliana and rice.Hereafter it was named FtBRI1.（3）The FtBRI1 gene was cloned from the developing seeds of"Xiaomiqiao"and"Jinqiaomai 2".Sequence analysis showed that the full-length length of FTBRI1 in"Xiaomiqiao"and"Jinqiaomai 2"span 2,576 bp and 2,573 bp,and the CDS length was1,920 bp and 1,917 bp,both containing only one exon and encoding 639 and 638 amino acids,respectively.Compared with"Jinqiaomai 2",3 bases,GAA,were inserted in"Xiaomiqiao"at 1,903 bp,resulting in an extra glutamic acid after the 635th amino acid.Bioinformatics analysis showed that FtBRI1 protein was not in the conserved domain at the differentially located sites between parents,so it was speculated that the difference in gene expression might be the main reason for the difference in parental grain weight.Multiple alignment of amino acid sequences and evolutionary relationship analysis showed that FtBRI1 was closely related to Arabidopsis thaliana,but far related to wheat and maize.The overexpression vector p BI121-FtBRI1 was successfully constructed,which established the experimental basis for further study of its function.（4）Eight SNP markers are encrypted in the genetic region of Ft1:5,690,284 bp～7,065,602 bp.Five residual heterozygous lines（RHLs）were screened based on the shell phenotype and SNP genotype.Five RHL-F₂ populations were genotyped by SNP markers.Combined with the shell phenotype and SNP genotypes,the thin shell gene was located between 6,754,575 bp and 6,938,819 bp,and the interval was narrowed from 1.3 Mb to 184 Kb.The 1000-grain weight and grain shape traits showed continuous distribution in the five RHL-F₂populations.The correlation analysis between grain genotype and phenotype in the five RHL-F₂ populations showed that the fine mapping interval of thin-shell genes also has significant influence on 1000-grain weight,grain length and grain width,and the three grain traits was mainly controlled by dominant effect.There were 20 annotated genes in this region,among which Ft Pin G0002494500.01 had 2 SNPs between parents.Seven genes,Ft Pin G0002495300.01（MADS1）,Ft Pin G0002493300.01（LP1）,Ft Pin G0002493000.01（IKU2）,Ft Pin G0002492500.01（GSK2）,Ft Pin G0002492200.01（BRI1）,Ft Pin G0002491900.01（SMG2/Os MKKK10）and Ft Pin G0002491700.01（IKU2）,were homologous to six genes related to grain weight/grain type.These eight genes are the key genes in controlling the thin shell and grain development of Tartary buckwheat.