Identification Of Locus Associated With Crinkled Leaf Phenotype In A Soybean(Glycine Max L.)Mutant By BSA-Seq Technology

Leaf is an important organ of plant growth and development.The crinkled leaf soybean has not been well studied.Therefore,detailed information is required in order to understand the genetic basis of crinkled leaf phenotype.In the present study,the bulk segregant analysis(BSA)method was employed to identify genomic regions associated with crinkle leaf phenotype in a soybean mutant 16M0169-72 and to find the most promising candidate gene.The main results were as following:Leaf form of the F₂ population consisting of 679 individuals derived from a cross between a normal leafy type soybean cultivar,Zhongpin661 and a crinkled leaf mutant 16M0169-72 was investigated.A segregation analysis revealed 3:1 ratio of mutant to wild type indicating that the crinkled leaf phenotype was controlled by a single dominant nuclear mutation.Other agronomic traits associated with the crinkled leaf mutant included reduced plant height,breached leaves and short petiole.The genomic DNA samples of individuals plants with extreme phenotype were pooled to form two bulks consisting of normal leaves and crinkled leaves for Illumina sequencing.A total of 142.41Gbp of Clean Bases and 80%of Q30 were obtained and the average alignment efficiency to the reference genome was98.93%with average coverage depth of 63.00X and 99.53%genome coverage.The total of 11,493 SNPs and 10,437 small In Dels were detected between the pools.Based on the association analysis,the ED correlation algorithm indicated a total of 12 candidate genomic regions with a total length 20.32 Mb found related to the trait.Within the mapping region,the gene Glyma.19G207100 was identified as the most promising candidate related to the crinkle leaf mutant(16M0169-72).The gene Glyma.19G207100 is a homolog of A.thaliana gene(AT2G47890)and encodes B-box type zinc finger protein with CCT domain.A non-synonymous coding mutation resulted to a single nucleotide substitution mutation of G to A(Gcg/Acg),causing crinkle leaf phenotype.The sub-cellular localization of the candidate gene(Glyma.19G207100)was detected only in the nucleus.The haplotypes of Glyma.19G207100 across a total of 2000 soybean accessions were analyzed;with two accessions(QLJ31 and QLJ 32)found carrying the same mutation as the mutant(16M0169-71)at locus Chr19:46245277.Phenotypic assaying revealed that the two accessions had the same phenotypes of crinkled leaf as 16M0169-72,which provides evidence of the Glyma.19G207100 as the candidate gene underlying the crinkled leaf phenotype.These findings hold enormous potential for laying the foundation for cloning the gene and broadening our understanding on the developmental regulation of soybean leaf as well as contributing towards a more efficient soybean breeding system for yield improvement.