Identification Of QTL And Cloning Candidate Genes For Grain Size In Wheat

Wheat is an important food source for humans.Identifying QTLs of yield-related traits is an important prerequisite for candidate genes cloning and theoretical and technical supporting for wheat molecular breeding.In this study,the large-grain size wheat line P271 and the small-grain size Chinese spring（CS）were used as parents to construct the F₂ population.The high-density genetic map was constructed by SLAF sequencing and used to QTL mapping of grain length,grain width and thousand grain weight.Functional annotation and analysis were performed on the candidate segments to screen out the candidate gene CYP78A5 that may regulate grain size.The sequence differences of CYP78A5 gene were studied through amplification of CYP78A5 gene and promoter sequence in different grain size wheats.Real-time fluorescent quantitative PCR was used to analyze the expression pattern of CYP78A5gene in root,stem,leaf,young panicles and grain of P271 and CS.The candidate genes were transformed into Arabidopsis thaliana for functional verification.The results are as follows:1.A high-density linkage map of P271/CS was constructed using SLAF technology.5,177 polymorphic markers were mapped on 21 chromosomes.The total length of coverage was 3,170.61c M and the average distance of markers was 0.87c M.Nine QTLs controlling grain length,grain weight and grain width were indentified by QTL mapping.A QTL controlling thousand grain weight was located on 1B;2 control-grain lengths and 1controlling thousand grain weight were mapped on 2A;Two QTLs detected on 2B controlled the grain length and thousand grain weight.A QTL controlling grain length was located on 4A,a QTL controlling grain length was located on 7A,and a QTL controlling grain width was located on7D.Functional annotation of QTL mapping intervals and prediction of candidate genes,a candidate gene CYP78A5 of controlling grain size was indentified on chromosomes 2A and2B.2.The TaCYP78A5 gene was cloned from chromosomes 2A,2B and 2D in the wheat lines P271,2017VP074,2017P296,CS and 2015P24 using of homologous cloning.Lengths of the coding regions of the TaCYP78A5 gene on chromosomes 2A,2B and 2D were 1637,1637 and 1622 bp.The TaCYP78A5 gene sequences on different chromosomes showed a high degree of similarity of 98.06%,indicating that the TaCYP78A5 gene was relatively conserved in different grain types of wheat.TaCYP78A5 consists of two exons and one intron,and contain the conserved structural domains（Hydrophobic regions,oxygen binding motifs,and heme binding motifs）.To construct the phylogenetic tree of the CYP78A family,we found that TaCYP78A5 gene was conserved during evolution.3.The promoters of the TaCYP78A5 gene in CS and P271 were predicted and cloned.The results showed that the promoter lengths of 2A and 2D chromosomes in CS and P271were identical,being 2322bp and 1876bp respectively.However,the length on chromosome2B was inconsistent.The promoter length of CS was 2469bp and the length of P271 promoter was 2566bp.Promoter element domains were analyszed and revealed that both the CS and P271 chromosomes contain important promoter domains TATA-box and CAAT-box.The 2B chromosome of P271 contains three domains more than CS,and the three domains werw ACE,Sp1,and TCCACCT-motif domains.We speculate that ACE and Sp1 may be related to the difference level expression in CS and P271.4.The expression of TaCYP78A5 gene was analyzed by q RT-PCR.The relative expression levels of TaCYP78A5 gene in P271 rooting,stem,leaf,young panicles before pollination and grain development before 15th day were higher than CS.P271 grain was significantly larger than CS,which may be caused by the difference expression level of TaCYP78A5 gene.Expression of TaCYP78A5 has a positive correlation with the formation of wheat grain size.5.The TaCYP78A5 gene was transformed into Arabidopsis thaliana through recombinant binary expression vector p CAMBIA-1302.The positive plants exhibited delayed growth,leaves became narrow,multiplication were stopped at the top of the inflorescence,fruit clips become thicker,seeds become larger,the seed settling rate was reduced.Overexpression of the TaCYP78A5 gene promoted the increase of seeds.