Characterization Of Genomic Variations And Artificial Evolution In Soja Subgenus

The genus Glycine is composed of two subgenera,Soja and Glycine.Soja subgenus includes cultivated soybean Glycine max?L.?Merr.and its annual wild relative Glycine soja Sieb.et Zucc..It is generally acknowledged that G.max was domesticated from G.soja in China.There are a larger number of Soja subgenus germplasm with a high level of genetic diversity generated over a long time of natural and artificial selection.These diverse soybean with phenotypic variations adapt to various environments,climate and cultivation conditions,which reflecting and affecting abundant genetic variation in the genome.Due to its high contents of protein and oil in seeds for human and animal as well as for industry,soybean is one of the most important crops in the world.The agronomic traits of soybean seed are the most striking quantitative traits to be subjected with the artificial selection and improvement,and being under the controlling of multiple loci,so uncover the genetic basis of seed traits is important to soybean genetic research and breeding.In this study,'a large number of nationally representative samples of Soja subgenus in China were selected to establish the experimental population,and constructed a high-density haplotype map of soybean genome by using restriction-site associated DNA-sequencing strategy,in order to investigate the genetic variation of populations.Based on these,the accurately improved genome-wide association study method was applied to dissect the genetic basis of seed traits with different evolutional traces,and traits associated QTL-Haplotype matrixes were established by allele compostion dissecting of QTLs.And finally,the comprehensively genetic bases of traits with evoultional traces under different intensity and duration time were deeply explored from QTL-Haplotype matrix level.The main results were as follows:1.In addition to objective existence of the "genetic bottlenecks",new haplotypes were emerged during the domestication stages of Soja subgenusA database with total number of 145,558 SNPs were obtained from restriction-site associated DNA-sequencing of the experimental population of Soja subgenus with 203 annual wild soybean?WA?accessions,375 cultivated soybean landrace?LR?accessions and 446 released cultivar?RC?accessions from six eco-types of China.Nucleotide diversity parameters ? was assessed based on the SNP database identified a lower level of genetic diversity in cultivated soybeans compared to wild soybeans?the ? value was 1.168 × 10-3 for WA,0.787 × 10-3 for LR and 0.712 × 10-3 for RC?.At the same time,in consideration of most loci are multiple allelic in natural population,so we composed biallelic SNPs into discrete haplotype blocks based on the definitions of minimal linkage disequilibrium range between two SNPs?D'?0.7?.Consequently,the database with 36,952 SNPLDB?SNP linkage disequilibrium block?was constructed.The haplotype flow at the two artificial evolutionary processes showed the haplotype number shrinkage from WA?97,532 haplotypes with Simpson index His 0.590?to LR?84,496 haplotypes with Simpson index H is 0.398?and then to RC?79,221 haplotypes with Simpson index H is 0.362?.These trends all reflected the traditional "genetic bottleneck" in process of domestication.Based on the haplotype analysis,the manifestly expansion of newly emerged haplotypes were discovered along with the bottlenecks.That means there are 74,294 commonly haplotypes for three populations;7,936 commonly haplotypes for two populations of WA and LR;12,855 haplotypes were only detected in WA?lost after the bottleneck?;in LR population there are 2,266 newly emerged haplotypes and 2,186 of them were transmitted to the RC;and there are 294 haplotypes newly emerged for the RC.That means it spent 5000 years to get 2,266 new haplotypes,but 100 years to get 294 new haplotypes for the 36,952 genome wide SNPLDBs,2.A larger number of domestication associated regions were accompanied with the differentiation of three populations in Soja subgenus;and the South-Central&Southwest China wild soybean ecotype?IV G.soja?was deduced to be the common ancestor for cultivated soybeansThe clustering based on principal component analysis and genetic distance of three soybean populations were both separated 1024 accessions into three distinct clusters.The linkage disequilibrium patterns were also different?the average distance over which LD decays to half of its maximum value was-54.60 kb for WA,114.50 kb for LR and 153.87 kb for RC?.They were both indicated the three populations have individual genetic bases and there were two evolutionary stages for three populations.Furthermore,the FST of domestication stage?0.335?is much higher than improvement stage?0.053?.A total of 1053 regions?100 kb windows?,contained loci with high average fixation indices?FST?0.60?for the domestication stage;while for improvement stage,just a total of 32 regions have relatively high average fixation indices?FST?0.25?.These regions may contain loci associated with domestication from wild soybean for five thousand years,and with selection imposed during 100 years of released cultivars breeding.The results of Analysis of Molecular Variance?AMOVA?among and within ecotypes of three populations indicated that significant portions of the variation existed in both inter-species?30%?and intra-species?70%?among geographic ecotypes.It implies that the geographic differentiation of three soybean types is related to their genetic differentiation.On the other hand,both the population pair-wise FST and genetic distances among ecotypes of three populations interpreting the evidence of the high degree differentiation between the ecotypes,and the lowest levels of differentiation were observed in pairwise comparisons of the South-Central&Southwest China G.soja ecotype with all the four cultivated soybean landrace ecotypes,which indicated that the South-Central&Southwest China G.soja ecotype had the closest genetic relationship with all the cultivated ecotypes,and can be confirmed as the common ancestor of the cultivated soybean.3.The genetic compositions indicated the recombination and mutation are the power for formation and evolution of different types of artificial evolutionary traits in Soja subgenus,and the recombination has great potential for traits improvement,meanwhile the mutation emergence have ecological specificityWith the field experiment and analysis of 1,024 accessions of Soja subgenus,the results reveal that during the two evolutionary stages of from WA to LR and from LR to RC,three traits showing progressively increasing trend and can be defined as "Positive evolution traits",i.e.100-seed weight?from 2.41g to 12.28g,and then to 16.95g on average?,seed oil content?from 12.12%to 16.98%,and then to 19.43%on average?,and seed oleic acid content?from 14.98%to 21.24%,and then to 26.42%on average?.This coincided with two traits showing progressively declining trend during two evolutionary stages,and can be defined as "Negative evolution traits",they are seed linolenic acid content?with an average from 12.98%to 8.92%,and then to 7.18%?and seed linoleic acid content?with an average from 56.19%to 55.14%,and then to 51.61%?.Meanwhile,other two traits of seed palmitic acid content has an average value of 12.61%in WA,11.16%in LR and 11.23%in RC,and seed stearic acid content has an average value of 3.33%in WA,3.48%in LR and 3.50%in RC,they are both "Neutral evolution traits" and present as fluctuating trend during two evolutionary stages.In this study,these three types of traits were dissected with restrictive genome wide association mapping analytical method.Finally,many traits associated genes were discovered,such as genes of drought resistance,cold resistance,salt tolerance,and other regulatory factors,these genes establish a well foundation for our future works of cloning and functional identification.The deeply exploration of the haplotype construction of traits associated SNPLDB?or alleles of traits associated QTL?showed that:Firstly,both the positive and negative alleles are existing in superior or inferior materials for all traits,but with significant difference for three types of traits.Secondly,both the positive and negative evolution traits have alleles in opposite direction,mainly the allele in wild soybean population and haven't been taken advantage in cultivated soybean populations,which can be used for genetic improvement in future by hybrid with wild soybean for cultivated soybean.Thirdly,a larger number of newly emerged mutations with both the positive and negative alleles are detected.This means that recombination and mutation are two key evolutionary dynamics for soybean domestication.At last,two positive effect haplotypes of 100-seed weight associated QTLs?Gm09_SNPLDB₁527-AGACGCATGGC and Gm04_SNPLDB₉11-C?,two positive effect haplotypes of seed oleic acid associated QTLs?Gm20_SNPLDB 1614-A and Gm15_SNPLDB1579-T?and one negative effect haplotype of seed linoleic acid associated QTLs?Gm02_SNPLDB₈33-C?are first emerged within the northeast and Huang-Huai-Hai Valleys ecotypes,where the soybean breeding activities are frequently?under the larger artificial selection strength?.Here in this study,the alleles with frequency ?0.01 were used for analysis,the rare alleles?frequency ?0.01?need further investigation.These studies provide a fundamental resource for soybean genetics research and breeding,and offered a new approach for dissecting directional evolution of seed traits,the allele exploration of traits associated QTLs will take great advantage to future genetic improvement in soybean.