Genetic Variation Of Aluminum Tolerance In The Chinese Cultivated Soybean Germplasm And Selection Of Candidate Genes For Aluminum Tolerance

A large area of soil in the world including China is acidic.Aluminum?Al?toxicity is a primary limitation to crop productivity on acidic soil.It can damage the root of the plants and reduce crop production.Soybean is an important legume grown on acidic soil and has moderate aluminum tolerance.There is obvious difference in aluminum tolerance among soybean varieties.The soybean tolerance to aluminum toxicity is controlled by quantitative trait loci.Therefore it is important to understand the genetic variation in aluminum tolerance of Chinese cultivated soybean germplasm,and to identify loci and candidate genes that are significantly associated with Al-tolerance.In this study,we screened the tolerance to aluminum toxicity in 387 Chinese cultivated soybean by a simple hydroponics method.We obtained high-density genome-wide SNP markers by using simplified genome re-sequencing technology?RAD-seq?to perform Genome-Wide Association study?GWAS?on soybean tolerance to aluminum toxicity,and selected candidate genes in significantly associated loci.The main results were as follows:1.A screening method for soybean resistance to aluminum toxicity was established.When we used the simple hydroponic solution of 0.5 mM CaCl2 containing 25 ?M AICl3 with pH 4.3 during the pre-treatment period,the phenotypic?relative root growth,RRG?differences was larger among the tested soybean varieties and the heritability was higher compared to other AICl3 concentrations.Then 13 soybean varieties were further tested for tolerance to aluminum toxicity using this hydroponic solution.We found that the relative aluminum content?depth of the color after hematoxylin staining?in soybean root tip was negatively correlated with RRG?P<0.0001?.The result showed that the less absorption of aluminum ions in root,the more Al-tolerant was the soybean variety?larger RRG?.2.We screened the Al-tolerance of 387 Chinese cultivated soybean germplasm that are originated from six major ecological regions using above method?two biological replicates with 6 plants in either of the control group or treatment group within each replication?.Analysis of variance showed that there is no significant difference between two biological replicates?P>0.05?,but extremely significant difference in soybean varieties?P<0.0001?.The range of relative root growth?RRG?of the soybean taproot is between 0.12 to 1.03,and it showed a normal distribution,with the mean RRG is 0.60 and the heritability is 90.01%.There are 26 soybean varieties showed tolerence to aliminum toxicity?RRG>0.80?and 26 soybean varieties showed sensitive to aliminum toxicity?RRG<0.40?.There is no significant correlation between RRG and eco-region?r2 =-0.03,P=0.55?or soil pH?r2=0.01,P=0.83?.There is no correlation between RRG and root growth-control?RG-c??r2=0.01,P>0.05?,while RRG and root growth-treatment?RG-t?is significantly correlated?r2=0.70,P<0.01?.3.We obtained 145558 SNPs by RAD-seq of 387 Chinese cultivated soybean germplasm,and 70203 SNPs whose minimum allele frequency?MAF?is greater than 0.05 were used for further analyses of population structure and Linkage disequilibrium?LD?.This population can be classified into four subgroups,and the LD decay distance?r2 decreased to half of the maximum value?is 621 kb.4.We carried out GWAS for the average RG-c?RG-t and RRG of 387 Chinese cultivated soybean with 70203 SNPs?MAF>0.05?.Using significant level of P?10-9 in general linear model?GLM?,no SNP is significantly associated with RG-c,51 SNPs were significantly associated with RG-t,and 97 SNPs were significantly associated with RRG.Using significant level of P? 10-4 in mixed linear model?MLM?,10 SNPs showed significant association with RG-c,4 SNPs were significantly associated with RG-t,and 19 SNPs were significantly associated with RRG.Considering the population is large?n = 387?and low false positive rate by using MLM,we carried out GWAS for RRG with 104914 SNPs?MAF>0.01?in order to identify the rare alleles.The results showed 31 SNPs were significantly associated with RRG.Six SNPs were significantly associated with RRG?P<0.05?in GLM multi-loci model,while four SNPs were significantly associated with RRG?P<0.05?in MLM multi-loci model.Three SNPs showed significant in both models,including Gmll₅374534,Gm03₃488621,and Gm17₃9638460.Among these,Gm17₃9638460 has a MAF of 0.37,and it was significantly associated with RG-t and RRG?P<0.01?.There are 16 candidate genes locate within 100 kb upstream or downstream of this SNP,including five genes related to stress tolerance,GIyma17g35580?Glymal7g35610?Glyma17g35620?Glymal7g35671 and Glyma17g35720.5.There are 72 candidate genes in the 100 kb upstream or downstream of above selected 31 SNPs that showed signficant association with RRG.One gene,Glyma18g09000,encodes a multi-drug resistant protein,which is likely to confer plant Al-tolerance based on previous study on its homologous gene.We developed a dCAPs marker for the SNP?Gml8₇746350?within Glymal8g09000,and genotyped 97 soybean varieties by this marker.However,the result showed that there was no significant correlation between the dCAPs marker?SNP Gml8₇746350?and Al-tolerance among the 97 soybean varieties.There are many possible reasons for this,such as small population size?n = 97?,non-functional SNP,and low contribution to the phenotypic variation.