Genetic Mapping,Function Identification And Molecular Mechanism Of Seed Coat Color Gene In Soybean

Soybean seed coat color often plays an important role in breeding selection and utilization as an important morphological marker due to its ease of observation and discrimination.Influencing changes in soybean seed coat color are generally flavonoids such as anthocyanins,which not only have important nutritional and medicinal values in terms of antioxidant properties in humans,but also play an important role in plant resistance to stress.Therefore,research on soybean seed coat color-related genes is beneficial for breeding soybean germplasm material with excellent appearance quality and intrinsic nutrient enrichment.In this study,we used brown seed coat（bsc）mutant material and Williams82sequenced varieties to genetically localize the genes controlling soybean seed coat color and to investigate the function and molecular mechanism of the target genes controlling soybean seed coat color.The main findings and conclusions are as follows:（1）Using the Williams82 sequenced variety and the bsc mutant material to cross the population,the F₁ seed coat of the hybrid showed the same yellow seed coat as Williams82,indicating that the brown skin mutation of the bsc material is a recessive mutation.the F₁ self-cross produced F₂ generation seed coat color phenotypes that segregated and produced a total of four types of seed coat color-related phenotypes,namely yellow skin black hilum,yellow skin brown hilum,black skin black hilum and brown skin brown hilum,The genetic segregation ratio was 9:3:3:1,indicating that the brown seed coat of bsc material was caused by two mutually independent genes,which were named a gene controlling seed coat pigment color and b gene controlling seed coat pigment bearing region.（2）Using the F₂ population to construct a hybrid pool,the a gene controlling seed coat pigment color was localized by BSA resequencing to a physical interval of approximately2.34 Mb on chromosome 6 and the b gene controlling the pigmentation region was localized to a physical interval of approximately 2.89 Mb on chromosome 8,respectively.Subsequently,molecular markers were designed within the candidate intervals and finely positioned to locate the a gene in a physical interval of approximately 200 Kb on chromosome 6 and the b gene in a physical interval of approximately 236 Kb on chromosome 8,respectively.（3）Quantitative expression analysis of candidate genes within the a locus in different tissues of the parental material revealed that only the a-6 gene was specifically expressed in different tissues,indicating that the a-6 gene may be the target gene responsible for the brown seed coat of the bsc material.Based on the sequencing results of the a-6 gene in Williams82and bsc material,it was found that the a-6 gene in bsc material had a single base deletion in the coding region,resulting in early termination of protein translation.Genome-wide BLAST analysis of the a-6 gene revealed that the gene belongs to the monooxygenase gene of the cytochrome P450 superfamily,and thus it was named GmCYP75.The seed coat color of the Arabidopsis tt7（CYP75 loss-of-function）mutant was significantly lighter compared with the wild-type COL,further suggesting that GmCYP75 may be the target gene controlling the brown seed coat of soybean bsc material.Bioinformatic analysis revealed the presence of growth hormone response elements（Aux RE）in the upstream promoter region of both GmCYP75 and the Arabidopsis homolog TT7,suggesting that the CYP75 gene may play a role in plant flavonoid biosynthesis and plant seed coat coloration by responding to growth hormone response elements.（4）The GmCYP75 gene was edited in Williams82 using CRISPR-Cas9 technology,resulting in two mutant types of edited materials,gmcyp75-3 and gmcyp75-7,respectively.and the hilum of both edited materials showed brown color compared with Williams82.Meanwhile,the overexpression of GmCYP75 gene in the bsc material was restored and three overexpression lines,OE-1,OE-3,and OE-7,were obtained,which had different degrees of black ingrowth on the seed coat（black seed hilum,black seed coat,and black and brown co-existing fancy seed coat）,respectively.Therefore,it is sufficiently certain that the GmCYP75gene is the target gene controlling the brown seed coat of bsc material.Under normal conditions,this gene promotes the change of soybean seed coat to black,whereas when this gene loses its function,it causes the change of soybean seed coat color to brown.（5）In order to investigate the molecular mechanism of the GmCYP75 gene causing the color change of soybean seed coat,we firstly performed subcellular localization of GmCYP75protein and found that GmCYP75 protein was expressed in the endoplasmic reticulum of tobacco cells.Subsequently,a yeast two-hybrid technique（Y2H）was used to screen the interaction protein of GmCYP75 as GmCSN.Protein structural domain analysis revealed that the GmCSN protein contains an MPN structural domain,and the interaction between GmCYP75 and GmCSN protein is realized through the MPN structural domain.（6）Based on previous studies,it was found that CSN can interact with ascorbate synthase VTC1 and degrade its ubiquitination,leading to a decrease in ascorbic acid（AsA）content in plant tissues.In this experiment,we used luciferase complementation assay（LCA）to demonstrate that GmCYP75 competitively bound GmCSN protein with GmVTC1,which reduced the degradation of GmVTC1 protein by GmCSN and eventually increased the AsA content in soybean tissues.This suggests that GmCYP75 protein regulates the synthesis of flavonoid compounds such as anthocyanins by promoting the accumulation of AsA in soybean seed coat during seed development,and ultimately controls changes in soybean seed coat color.In this study,a soybean brown skin mutant material was used as an entry point.The genetic analysis and fine localization of GmCYP75 gene,which controls the change of soybean seed coat color from brown to black.The Y2H and LCA experiments demonstrated that GmCYP75 interacted with GmCSN protein,and this interaction effect reduced the degradation of ascorbic acid synthase GmVTC1 by GmCSN and promoted the accumulation of AsA in soybean seed coat.On the one hand,the results of this study reveal a new mechanism for the GmCYP75 gene to regulate changes in soybean seed coat color,and also complement the pathway for AsA synthesis in plants,providing a theoretical basis and breeding material for breeding soybean varieties with excellent appearance quality and intrinsic nutrient enrichment.