Map-based Cloning Of A Novel QTL QBN-1 Influencing Branch Number In Soybean (Glycine Max)

Branch number?BN?is a dynamic agronomic attribute related to the plant architecture,adaptability,and yield of soybean?Glycine max?L.?Merr.?.To date,few studies have been conducted on BN to elucidate its genetic background.In the present study,we aimed to localize the genetic factors affecting BN,a segregation population derived from a cross of two varieties with extreme branching phenotype was developed and QTLs related to the BN were identified by constructing genetic linkage map.Then candidate genes were determined by analysis of bioinformatics,sequence variation and gene expression.The main results are as follow:1.A segregation population derived from the cross of KN24 high branching and KF19 with low branching was developed.We observed a significant difference in BN between the two parents.In 2015,the average number of branches was 6.0 for KN24 and 0.6 for KF19,respectively.While,in 2018,the average number of branches was 7.9 for KN24 and 1.1 for KF19,respectively.A slight reduction in branch was observed in 2019,with an average BN 6.2 for KN24 and 0.8 for KF19,respectively.The phenotypic stability of BN for KN24 and KF19 in different years indicated that they were suitable to be used as parents to develop genetic segregating populations and identify QTL for BN.The BN that was investigated over five populations showed a continuous variation and followed the normal distribution.2.A total of 543 SSR markers were screened for polymorphism,only 250 markers distributed across 20 chromosomes were polymorphic between KN24,KF19 and two DNA pools derived from the F₂ population.Of those,on Chr.06,a genomic region of qBN-1,appeared to govern BN phenotype.We found out two SSR markers of BARCSOYSSR?06?0717 and BARCSOYSSR?06?1441 that associated with qBN-1.3.Further screening to identify and narrow down BN QTL in soybean,a physical map comprising 10 SSR markers has been constructed and 599 F₂ population were used to map QTLs for BN.As a result,qBN-1 were co-located flanked by BARCSOYSSR?06?0993 and BARCSOYSSR?06?1070.The QTL,qBN-1,which explained 1.84%of phenotypic variation,could be a target region for identifying genes associated with BN.4.To fine map qBN-1,the RIL population of F_2:7:7 with 599 lines was developed and screened by ten SSR markers showed polymorphism between two parents.Then the qBN-1 was located within a115.67 kb genomic region on Chr.06 between marker BARCSOYSSR?06?1048 and marker BARCSOYSSR?06?1053.Additional four SSR markers were used to genotyping F_2:8.Results showed that qBN-1 located to the same region with the highest R ² value?22.69%?and the highest LOD score?32.07?compared to that in F₂ and F_2:7.5.Two SSR markers of BARCSOYSSR?06?0993 and BARCSOYSSR?06?1035 were also used for genotyping 1712 BC₃F₂ and 1305 BC₂F₂ individuals.Then 870 recombinants between these two markers were genotyped by ten SSR markers.Further screening was conducted for 370 recombinants in the region between markers BARCSOYSSR?06?0993 and BARCSOYSSR?06?1070 for fine mapping qBN-1.As a result,the qBN-1 was further confirmed between SSR markers BARCSOYSSR?06?1048and marker BARCSOYSSR?06?1053.6.Within the fine-mapping region of qBN-1,only two candidate genes.One gene Glyma.06G208800 encodes a calcium-binding cml15-related protein,and the other gene Glyma.06G208900 encodes a phospholipid-translocating ATPase.7.The expression pattern using qRT-PCR reveals that,compared to Glyma.06G208800 gene expression,Glyma.06G208900 had a much higher expression level and showed significantly different between high and low branching genotypes in either axillary meristem or shoot apical meristem,and the expression pattern was contrary between the two tissues at V4 and R1 stages.8.After comparing sequences of two genes between parents,Glyma.06G208900 had one variation SNP resulted in one amino acid change from GLY in KN24 to ASP in KF19 while,Glyma.06G208800 had no variation in the codon region.9.SNP marker was developed to identify 599 individuals from the F_2:7:7 generation of the KN24×KF19 population,the results showed that the average BN containing the T-type allele was 4.7;the average of BN lines containing the C-type allele 2.4;the average BN of heterozygous individuals is 4.2.Further analysis showed there was a significant difference between the BN containing C-type allele materials and T-type allele materials and heterozygous materials,while no significant differences between T-type allele materials and heterozygous materials.The results of this study provide the foundation of cloning and functional analysis of the qBN-1gene and improving BN through marker-assisted selection in soybean breeding.