A Study On The Saponin Composition Of Subgenus Soja In China

Soybean[Glycine max（L.）Merr.]contains not only large amount of nutritional components but also a number of health functional components,of which soybean saponin is one.Soybean saponins,which have been found more than 100 different components,show lots of health functionalities for human health affected by their aglycone structures and sugar compositions and are therefore of great value in soybean nutritional breeding,which can improve soybean taste and nutrition for human health.However,the group A saponins are thought to be the cause of bitter and astringent tastes in processed foods of soybean,and the elimination of group A saponins is an important breeding objective.Wild soybean（Glycine soja Sieb.&Zucc.）is the progenitor of cultivated soybeans and contains many useful genes and characters,and is therefore an important genetic resource for soybean breeding.Soybean saponin components in subgenus Soja have been studied in Japan and South Korea,and even,useful mutants have also been utilized in soybean breeding.However,the situation of soybean saponin composition in Chinese subgenus Soja is yet unclear.Therefore,the objective of this research was to（1）identify the saponin composition and genetic variation in Chinese subgenus Soja;（2）investigate their genetic diversity and geographical distribution;（3）determine the saponin concentration and（4）to understand the level of genetic variation of saponins and mine useful gene resources for the future soybean breeding programs that aim to improve soybean nutritional levels and quality.The main results were as follows:1.A total of 3805 wild soybean accessions,collected from almost all the areas where wild soybean grows in China,were analysed using thin-layer chromatography（TLC）.Twenty-four saponin components were distinguished including four new components and these components were divided into six saponin groups（A,DDMP,B,E,?andβ）,composing 32 phenotypes.The four new components included saponin K-αg（groupα）in 18 accessions,HAb-αg（groupβ）in an Ab type wild accession,A-αg（group A）,a biosynthetic precursor compound of the group A saponin and its derivative KA-αg（groupβ）in the sg-7 mutant that lacked group A saponin.Eight A0 variant accessions without the second sugar（xylose or glucose）at the C-22 position and two high-content group E saponin Bd and four high-content group A saponin Ae accessions without Aa andαg were also detected.The identified sg-7 and A0 accessions were useful gene resources for elimination of bitter and astringent tastes in soybean breeding.2.The molecular structures of four new saponins（A-αg,KA-αg,K-αg and HAb-αg）were analyzed.In this study,a new key locus Sg-7 was found for group A saponin biosynthesis,which is a critical step necessary for arabinose binding to the C-22 hydroxyl group in the biosynthetic pathway of group A saponins.This locus was discovered through a recessive mutant sg-7 in a wild soybean accession（No.5306）that lacked group A saponins,it caused the failed arabinose bonding of arabinosyl-transferase to the C-22 hydroxyl group,consequently leading to the accumulation of the component A-?g and the concomitanceofitsderivativecomponentKA-?gintheseeds.TheA-?g[3-O-（Glc-Gal-GlcUA）-soyasapogenol A（SS-A）]is a steady precursor compound in biosynthesis of group A saponins in the seeds of soybean plants.KA-?g as a by-product in the sg-7 mutant has one acetyl at the C-29 position of the aglycone in contrast to the A-?g.K-αg（C-29-acetyl H-αg）was identified in 22 wild soybean accessions,which is a group?component having an additional acetyl group（-COCH₃）in the C-29 position of soyasapogenol H.HAb-αg（C-29-hydroxyl Ab）was found in an Ab type accession;it has an-OH group at the C-29position,i.e.a C-29-hydroxyl SS-A.3.Seed saponin composition of 2959 Chinese cultivated soybean landrace accessions was examined by TLC.Thirteen components were distinguished in five groups A,DDMP,B,?andβ.The group?（H-αg,I-αg,and K-αg）and groupβ（KA-αg）were first found in cultivated soybeans.The frequencies of the Sg-1^a and Sg-1^b codominant alleles were 39.3%and 60.7%,respectively,and the frequency of Sg-4 allele was very rare（0.07%）.The recessive alleles,sg-3 and sg-7,were not found in this investigation of Chinese cultivated soybeans.The distribution of saponin composition phenotypes in botanically qualitative and quantitative characters indicated that the Sg-1 locus was closely related to soybean evolution and ecotypes;small seed,prostrate stem,black seed-coat,indefinite growth,broad-leaved shape,low-oil content,and spring-sowing types accumulated relatively higher frequencies of the Sg-1^a allele.The frequency of the Sg-1^a allele in soybeans were regionally higher（>60%）along the Yellow River valley.As a result of integrating archaeological documents,the frequency distribution of the Sg-1^a allele through China and the appearance of group?soybean germplasm with a relatively higher frequency observed in the northwest,a single area of origin for soybeans is proposed as the middle and lower reaches of the Wei River valley,i.e.the middle-upper reaches of the Yellow River valley in China.The existence of group?saponins in cultivated soybeans suggests that this group of saponin is safe for humans because these saponins have been ingested for several thousand years in China.4.The geographical distribution and genetic differentiation of saponin phenotypes and genotypes were evaluated in Chinese wild soybeans.Thirty-two saponin composition phenotypes were identified in 3805 wild soybean accessions and their genotypes were assigned based on ten known or putative loci.Four composition genes had high frequencies:Sg-1^a allele accounted for 78.8%and its codominant allelic Sg-1^b was 21.0%at Sg-1 locus;Sg-4 and Sg-6e reached 30.7%and 13.7%in respective loci.Northeast and South China showed high genetic diversity,however,the Northeast had more novel variant types(AuAe,A0,A0Bc,Bd,α_H,α_Iα_J,α_K and Abβ_HAb).Rare Ae-accumulation（AuAe）type is caused by a sg-3 allele.Usually Ae component is a minor one in some plants.However,rare types with a high Ae concentration were identified in four accessions northeastern wild soybeans.Gene differentiation(F_st)analysis suggested that Sg-4 and four group?saponin loci Sg-6e,Sg-6h,Sg-6i and Sg-6j were important factors influencing on the geographical genetic structures and differentiation in Chinese wild soybeans.The southern regional populations of Chinese wild soybeans contained high frequencies of Sg-4 gene.Chinese wild soybeans were distinctly clustered into two primary geographical groups of northern and southern ones based on saponin gene frequencies;genetic differentiation was characterized mainly by lengthwise latitudinally genetic differences across the geographical areas.Chinese wild soybean differs from Japanese and South Korean ones in genetic structure on the basis of saponin chemical composition and two latter ones are likely to be spread from southern China during certain glacial stages of several times.5.Nucleotide sequence analysis was performed to reveal the molecular structural features of allelic polymorphism at the Sg-1 locus and the mutant mechanisms of the abiogenetic absence of group A（i.e.A0-type）,Eight A0-type mutant plants,72 Aa and 74 Ab type accessions and one sg-7 mutant(Aβ_KAA phenotype)were used.The results showed that Sg-1 alleles had a lower conservatism in the coding region;eight allelic sequences were detected from Aa type and ten from Ab type Chinese wild soybeans.Sg-1^a and Sg-1^b alleles were characterized by eight synonymous codons in addition to nine characteristic amino acid substitutions.Two evolutionarily transitional allelic sequences(Sg-1^a7 and Sg-1^b2)were detected from Sg-1^a toward Sg-1^b.The A0 phenotype was revealed to be caused by at least three kinds of mechanisms involving four allelic variants(sg-1^0-b2,sg-1^0-b3,Sg-1^b-0-0 and Sg-1^b-01).The sg-1^0-b2 had two nucleotide deletions at positions+72 and+73.The sg-1^0-b3 contained a stop codon（TGA）at the 254th residue.The Sg-1^b-0 and Sg-1^b-01 were two newly-found novel A0 type mutants,which carried two normal codominant alleles for Sg-1^b type,nevertheless did not encode Ab saponin due to unknown mutations beyond the normal coding regions.The conjectures on the reasons for the absence of Ab-series saponins in the two A0 accessions(Sg-1^b-0 and Sg-1^b-01)need to be resolved in future studies.The Phenotypic mutants sg-7 and A0 were important gene resources for improving bitter and astringent tastes of soybeans6.Relative saponin concentration was analyzed for total 106 accessions of Chinese wild soybeans with 27 different saponin phenotypes in hypocotyls and cotyledons.Results showed that typical wild soybeans had a higher total saponin content than semi-wild soybeans,and that group?-absent type wild soybeans had higher content of group A and relatively lower contents of groups B and E components in the hypocotyls and cotyledons,group?-present type did contrariwise.These results suggested that the groupαsaponins were synthesized from group A-series rather than group B saponin.Based on these results together with the composition identification in the saponin biosynthetic pathways,five putative loci Sg-6e,Sg-6h,Sg-6i,Sg-6j and Sg-6k were hypothesized to involve in biosynthesis of groupαsaponins.Concentration determination confirmed that AaBcE?_J and AbE?_IJ were indeed two Bd-accumulating mutants.The mechanisms of the high Bd accumulation and the mechanism of the differential expressions of group A between cotyledons and hypocotyls were proposed on the basis of the output of various components in the seeds.Eleven useful wild soybean accessions,with phenotypes of A0,A0Bc,AaBcEα_J,AbEα_IJ and Aβ_KA,containing low group A and high group B+DDMP components,were identified.