Germplasm Screening,transcriptome Analysis And CRISPR/Cas9-mediated Mutagenesis Of J Gene For Long Juvenile Trait In Soybean

Soybean is one of the major oil crops and protein resources in the world with increasing demand.The long juvenile(LJ)trait influences the yield of soybean production in tropical regions.The trait delays flowering under short day(SD)condition and ensure sufficient vegetative growth before flowering leading to increase in grain yield.The J/Gm ELF3 gene has been recently identified to control the LJ trait under SD.In the study we screened 159 tropical soybean genotypes from different tropical regions to identify genotypes with the LJ trait and the loss of function alleles responsible for the trait.The 159 genotypes were evaluated on the field for two years in a tropical environment,Sanya-Hainan Island.The causative gene gene for the LJ trait was cloned in all the 159 genotypes.A total 53 genotypes were identified to delayed flowering based on the phenotypic data.Haplotype analysis identified 7 haplotypes of which 3 resulted in the loss of function alleles of the gene.Six genotypes were identified with the loss of function alleles for the J gene.We also profiled the transcriptome of a LJ soybean variety Huaxia-3(HX3)with the non-functional J gene under both long day(LD)and SD condition.This was to help us elucidate the biological mechanism and the flowering related genes involved in the LJ trait.A total of 31 and 2311 genes were differentially expressed in the LJ variety relative to the transgenic variety overexpressing the J gene under SD and LD condition respectively.The circadian rhythm pathway was the most significantly enriched pathway in the HX3 under SD condition.The Gm ELF3a(Glyma.04G050200)and FLOWERING LOCUS T(FT)genes such as Gm FT2a(Glyma.16G150700)and Gm FT5a(Glyma.16G044100)were down-regulated,whiles Gm FT4(Glyma.08G363100)was up-regulated in the HX3 under SD.Under LD FT homologs Gm FT4(Glyma.08G363100)was down-regulated and Gm FT1a(Glyma.18G299000)was up-regulated.Our study suggests that these FT homologs may be involved in delayed flowering of LJ soybean under SD through the circadian clock pathway.Finally,we made use of CRISPR/Cas9 genome editing technique to mutate the J gene in a soybean variety Jack with a functional J gene to delay flowering under SD.The mutation efficiency was 18% but not all the homozygous mutations were transferable from T0 to T1 generation.Evaluation of the mutant’s lines under SD and LD condition resulted in delayed flowering compared to the wild type(WT).However,the delayed flowering was more obvious under LD than SD.This may be attributed to genetic background of the Jack variety used in the experiment.The Jack variety has e1 as background,previous studies indicated J gene may depend on E1 gene for its full activity under SD.Taken together this study contributes to the knowledge gap in soybean flowering under SD and serve as basis for future studies.The identified LJ genotypes and the mutants will be useful in tropical soybean improvement programs.