Natural Variation Analysis And Candidate Gene Identification Of Carotenoid And Chlorophyll Compositions In Soybean Seeds

Soybean[Glycine max（L.）Merrill]seeds are rich sources of secondary metabolites including isoflavone,carotenoids and among others.Carotenoids are significantly associated with human health and nutrition owing to their provitamin A,antioxidant and anti-inflammatory activities.Carotenoids and chlorophylls both involve in different functions,such as light harvesting,energy transfer,photochemical redox reaction,and photoprotection.The distribution and variation of soybean seed metabolites are influenced by plant genetic characteristics and environmental factors.However,there is scarce information pertaining to carotenoid content of diverse soybean accessions,implying that carotenoids in the soybean seed have not been given more attention in breeding research programs,despite of its health nutrition.A high genetic diversity provides an opportunity for plant breeders to develop cultivars with traits of interest.In the present study,therefore,a collection of 1553 soybean accessions,which comprised landraces and cultivars with various seed coat color,maturity group,and diverse origins including China,Russia,Japan and the USA,were deployed.In addition,BSA-seq was conducted to identify candidate gene underlying accumulation of carotenoids in soybean seeds.The main findings in each experiment were abstractly highlighted as follows:1.Simultaneous Determination of Carotenoids and Chlorophylls by HPLC-UV MethodA simple,reliable,rapid and selective reversed phase high performance liquid chromatographic method was developed and validated for the simultaneous analysis of lutein,zeaxanthin,α-carotene,β-carotene,β-cryptoxanthin,chlorophyll-a and-b in soybean seeds using ethanol-acetone（1:1）extractant solvents and effectively separated using the mobile phase of methyl tert-butyl ether,methanol-10m M ammonium acetate and water delivered at 0.9 m L min^-1 flow rate through C30YMC carotenoid column coupled with UV-Vis detector at 450 nm.This method was also successfully implemented in other important legume seeds（such as Lablab purpures,Vigna radiate,Vigna unguiculata,Phaseolus multiflorus,Pisum sativum,Vigna angularis,and Vicia faba）.2.Natural Variation and Geographical analysis of Carotenoids and chlorophylls in Chinese Soybean GermplasmsBased on this method,we analyzed the carotenoid and chlorophyll contents and geographical distributions of 1167 Chinese soybean accessions collected from North,South and Huang-Huai-Hai Valley ecoregions.The results showed that genotype,planting year,accession type and ecoregion of origin significantly influenced the accumulations of carotenoids and chlorophylls.The landraces contained 1.20 times higher total carotenoid contents than that of cultivars.The concentrations of carotenoids and chlorophylls varied based on the geographical origin of soybeans because of their various environmental and climatic conditions.Soybean carotenoid contents were geographically distributed across the main ecoregions with a decreasing trend towards south.The components of carotenoid and chlorophyll showed significant correlations with other quality traits（including isoflavone,tocopherol,oil,proteins,and fatty acids）,helping to set breeding strategies for enhancing soybean carotenoids without affecting other components.Collectively,we demonstrated that soybean seeds are good sources of carotenoids(mean total carotenoid ranged from 8.15-14.72μg g^-1),and the identified novel germplasms with the highest total carotenoid content across the ecoregions of China should be preferably selected in breeding specialty soybeans for soy food,animal feed and other industrial products to satisfy consumers’demand.3.Origin,Maturity Group and Seed Coat Color Influenced Carotenoid and Chlorophyll AccumulationAnother experiment on the effects of germplasm origin,genotype,seed coat color and maturity group（MG）on the concentration variation of carotenoids and chlorophylls in 408 soybean accessions showed that these factors influenced carotenoid and chlorophyll accumulations in soybean seeds.Russian soybeans yielded 1.3-fold higher total carotenoids compared with Chinese and Japanese soybeans.Soybeans with black seed coat color contained abundant concentrations of carotenoids,with mainly lutein(19.98μg g^-1),β-carotene(0.64μg g^-1)and total carotenoids(21.04μg g^-1),implying that black soybeans are rich sources of lipophilic pigments.Moreover,contents of carotenoids and chlorophylls generally decreased towards late MG soybeans.Additionally,the unique accessions with prominent content of carotenoids identified in this study can be used to strengthen the soybean breeding programs for meeting various human nutrition patterns around the globe.4.Candidate Genes Underlying Carotenoid Accumulation in Soybean Seeds based on BSA-seqThe genomic DNA samples of individual plants with extreme phenotype were pooled to form two bulks with high and low carotenoid contents for Illumina sequencing.A total of 125.09 Gbp of clean bases and 89.82%of Q30 were obtained and the average alignment efficiency was 99.45%with average coverage depth of 62.20X and 99.75%genome coverage.Based on the G’method analysis,a total of 16candidate genomic loci with a total length 20.41 Mb were found to be related to the trait.Of these loci,the most significant region displaying the highest elevated G’-value was found in Chromosome 06 at a genomic region interval of 18.53-22.67 Mb,and preferably taken to identify the key candidate gene.In this interval,144 predicted genes were found and in which,94 genes had gene ontology（GO）annotation functions as well as 29 significantly enriched（p<0.01）GO terms.Based on ANNOVAR analysis,12 genes with non-synonymous and one with stop gain mutations were selected as potential candidate genes.Following its gene annotation function and significant haplotype variation in various environments,the gene Glyma.06G211900,encodes tetratricopeptide repeat（TPR）-like superfamily protein,was identified as the most promising candidate gene regulating soybean seed carotenoid accumulation.It carried a non-synonymous mutation（C>T）in the first exon at the genomic variant of Chr06＿21252325.Therefore,we found a successful application of NGS-based BSA approach for identifying key genomic regions governing seed carotenoids.