Natural Variation Analysis And Candidate Gene Ldentification Of Tocopherol Compositions In Soybean

Soybean[Glycine max（L.）Merrill]is one of the most widely traded legume crops in the world,and it contains a wide range of bioactive components in addition to protein and oil.Tocopherols are one of the most important bioactive compounds,having strong antioxidant properties.It is the most common form of vitamin E and a crucial element of soybean seed oil.Soybean seeds are primary sources of natural tocopherols used by the food and pharmaceutical industries owing to their beneficial impacts on human health.Tocopherols are naturally occurring powerful lipophilic antioxidants produced solely by photosynthetic organisms such as higher plants,algae,and some cyanobacteria.Firstly,seed tocopherol compositions in a soybean mini-core collection of 1551 accessions grown in three locations for two years in China were determined in this study by utilizing the HPLC method.Secondly,the content and composition of tocopherols in soybean germplasm from different countries（including China,the United States,Japan,and Russia）and maturity groups（MGs）were investigated.Finally,the BSA-seq technique based on two bulk pools was performed to determine the genomic loci and candidate genes associated with tocopherol composition in diverse soybean accessions.Therefore,the present study was conducted to determine the content of tocopherols in Chinese soybean accessions that originated from different ecoregions of China,to evaluate the impact of MGs on seed tocopherol composition of soybeans from different origins and to identify the genomic loci and candidate genes controlling the tocopherol content in soybean,in order to provide resources and theoretical support for soybean tocopherol breeding.The main results are as follows:1.The genetic variation and geographical distribution characteristics of tocopherols in Chinese soybean germplasm were examinedAnalysis of variance revealed significant differences（p<0.001）in tocopherol content among the accessions,ecoregions of origin,and accession types.Total tocopherol content in soybean accessions ranged from 113.97μg g^-1 to 361.07μg g^-1,which represents a 3.17-fold difference.The highest average contents ofα-andγ-tocopherols(15.43μg g^-1 and 151.44μg g^-1,respectively)were observed in the Northern Region（NR）accessions of China.Moreover,α-,γ-,and total tocopherol contents were higher in cultivars than in landraces.The correlation analysis showed that total tocopherol has significant and positive correlation with oil content（r=0.36***）,whereas negative correlation with crude protein（r=-0.30***）,total isoflavone（r=-0.15***）,and linolenic acid content（r=-0.11***）.α-andγ-tocopherols had significant positive correlations with latitude and longitude,whileδ-tocopherol showed a contrary trend,indicating that the geographical origin of accessions had a pronounced effect on seed tocopherol composition.Taken together,we identified novel soybean accessions that can be utilized as raw materials for food industries and in breeding programs to improve the content of tocopherols in soybean seeds.2.The effects of different MGs on the content and composition of tocopherols in soybean germplasm were investigatedSignificant differences（p<0.001）were observed among the accessions and origins for individual and total tocopherol contents.The total tocopherol content ranged from 118.92μg g^-1 to 344.02μg g^-1.Accessions from the USA had the highest average concentration ofγ-and total tocopherols(152.92 and238.21μg g^-1,respectively),whereas a higher level ofα-tocopherol(12.82μg g^-1)was observed in the Russian accessions.The maturity group of the accession significantly（p<0.001）influenced all tocopherol components,and higher levels ofα-,γ-,and total tocopherols were observed in early maturing accessions,while late-maturing accessions exhibited higher levels ofδ-tocopherol.The inclination of tocopherol concentrations with various MGs provided further evidence of the significance of MG in soybean breeding for seed tocopherol components.3.Using the BSA-seq technique,candidate genes regulating tocopherol content in soybean were identifiedA total of 1551 soybean accessions grown in three locations over two years were profiled for seed tocopherol content and used to create two extreme pools for conducting a BSA-seq study.A total of 144variant sites and 109 predicted genes related to tocopherol content in soybean were identified.These genes were distributed across the entire 20 chromosomes of soybean.The 109 predicted genes consisting of 144 variant sites,were subjected to gene ontology（GO）analysis,in which a total of 83 genes were annotated by the GO functions.Further,13 enriched terms（p<0.05）were detected with four of them found highly enriched:（1）response to lipid,（2）response to abscisic acid,（3）transition metal ion transmembrane transporter activity,and（4）double stranded DNA binding.Based on ANNOVAR analysis,genes containing mutations that occurred in the exonic region including ten non-synonymous,one stop gain,and two frameshift variants were preferentially selected as candidate genes related to tocopherol content.A total of six additional genes（Glyma.05G243400,Glyma.05G243300,Glyma.05G244100,Glyma.05G244200,Glyma.05G243800,and Glyma.05G244000）were detected on chromosome 5 at 41.8 Mb-41.9 Mb genomic hotspots,which were predicted to be related to tocopherol content in soybean.Among the genes detected on chromosome 5,only Glyma.05G243400,an ortholog of Arabidopsis thaliana gene AT1G18070 was annotated to carry a mutation（T>G）with a genomic variant of Chr05＿41807338 that induced a non-synonymous mutation encoding“translation elongation factor EF1A/initiation factor IF2gamma family protein”.Analysis of haplotype variation also confirmed that Glyma.05G243400 exhibited highly significant variations in terms of tocopherol content across the experimental locations.Although,many genes were predicted to be related to tocopherol content in soybean seed,Glyma.05G243400 was considered the most promising candidate genes for further analysis.The present findings hold prospects for molecular marker identification and marker-assisted selection of soybean genetic resources endowed with improved tocopherol content.