Genetic Dissection And Functional Analysis Of The Candidate Gene GmABCC3 For Aluminum Tolerance In Soybean

Soybean[Glycine max（L.）Merr.]is widely grown in the world as an important economic and oil crop.It is widely utilized in food,feed and industrial production because it provides rich vegetable protein and essential fatty acids as edible oil for human body.In recent decades,the predatory exploitation and destruction of natural resources and the environment,have led to a rapid deterioration of the global climate and environment,with rising temperatures,melting icebergs,and increasingly frequent acid rain.There are about 3.95 billion hm² of acidic soils globally,accounting for 50%～70%of the world’s potentially arable lands.About 35%of the soybean growing areas in the world are also in acidic soil.In acidic soil,aluminum（Al）toxicity is one of the most serious abiotic stresses that harm crop growth and development,and limit crop yield.At present,identification of quantitative trait loci（QTL）/genes for Al tolerance and exploring the molecular mechanism of Al tolerance in soybean have little progress.Therefore,screening Al-tolerant soybean germplasm resources,mining Al tolerance QTL and superior genes by QTL linkage mapping and GWAS,and exploring the molecular mechanisms of Al tolerance QTL and genes in soybean are of significance for providing material and theoretical basis for Al-tolerance breeding in soybean and other crops,improving acidic soil utilization and ensuring food security.In this study,the genetic dissection of soybean Al-tolerant traits was performed by linkage mapping and GWAS,and candidate genes were analyzed in the major QTL.At first,159 recombinant inbred lines（RILs）from Magellan×PI 567731 were used to map Al-tolerant QTL in soybean seedlings by three root indexes of Al tolerance,and near isogenic lines（NIL）were constructed for two major QTL for Al tolerance confirmation and Al tolerance mechanism exploration.We also conducted a GWAS for Al tolerance in soybean seedlings by 387 Chinese soybean accessions.The candidate genes were screened in the most significant loci region associated with Al tolerance.The expression pattern analysis,sequence variation analysis,haplotype analysis,and molecular marker development of the candidate gene GmABCC3（ABC TRANSPORTER C FAMILY MEMBER 3-RELATED）were performed.At the same time,the Al tolerance capability of different GmABCC3haplotypes and Al tolerance mechanism of GmABCC3 were also investigated.The main results obtained from this study are as follows:1.Two major QTL were mapped for Al tolerance in recombinant inbred lines（RIL） populationFirstly,the Al tolerance capability of four pairs of parental materials were estimated rapidly.The largest difference in Al tolerance was found between Magellan and PI 567731（P<0.01）.Then,a comparison was porfermed between Magellan and PI 567731 in relative primary root length（PRL＿Ratio）,relative total root length（TRL＿Ratio）,and relative root tip number（RTN＿Ratio）after 3,7,and 10 days’treatment under aluminum toxicity versus（vs.）control conditions.It was found that Magellan showed no necrosis of primary root tips,while PI 567731 showed severe necrosis of primary root tips after 7 days of Al treatment.There were also significant differences（P<0.01）between Magellan and PI 567731 in three Al-tolerant indexes of PRL＿Ratio,TRL＿Ratio,and RTN＿Ratio after 7 days of Al treatment.159 RILs from Magellan×PI 567731 were evaluated for Al tolerance using the above three indexes.The results showed that all three indexes of PRL＿Ratio,TRL＿Ratio,and RTN＿Ratio exhibited highly significant differences（P（_G）≤4.90E-3）and with broad sense heritability（H²）of 60%～73%among the 159 RILs.Correlation analysis detected a significant positive correlation between TRL＿Ratio and PRL＿Ratio,also TRL＿Ratio and RTN＿Ratio（r=0.42～0.55,P<1.00E-3）.QTL mapping was performed in 159 RILs for Al tolerance.Three QTL for Al tolerance,located on chromosomes（Chr.）3,13,and 20,respectively,were mapped and superior alleles of all three QTL are from the parental Magellan.The major effect loci of qAl＿Gm13 and qAl＿Gm20 were also detected by more than two traits,and qAl＿Gm13 and qAl＿Gm20 explained a maximum of 13.1%and 27.4%of the phenotypic variation,respectively.2.Confirmation of two major QTL for Al tolerance in near isogenic lines（NILs）and investigating its Al tolerance mechanismNILs of qAl＿Gm13 and qAl＿Gm20 were constructed（the genotype of qAl＿Gm13 and qAl＿Gm20 were set as as Aa and Bb,respectively.）,and Al tolerance was verificated in NILs by three indexes of PRL＿Ratio,TRL＿Ratio,and RRG.The value of above three Al-tolerant indexes in NILs seedlings were ranked as follows,seedlings with genotype of AABB>seedlings with genotype of aa BB>seedlings with genotype of AAbb>seedlings with genotype of aabb.Moreover,a significant epistasis between qAl＿Gm13 and qAl＿Gm20（P≤0.01）was detected.In comparison of primary root tip necrosis rates,the seedlings with genotype of AABB had the primary root tip necrosis rate of 8.00%;genotype of aa BB had the primary root tip necrosis rate of 12.50%;genotype of AAbb had the primary root tip necrosis rate of 70.59%;and the seedlings with genotype of aabb had the highest primary root tip necrosis rate of 96.15%（25/26）.In order to investigate the possible mechanisms of the qAl＿Gm13和qAl＿Gm20.The Al concentration in roots and shoots（stem and leaf）was measured in NILs of qAl＿Gm13 and qAl＿Gm20 and the parent of Magellan and PI 567731.The results showed that there was no significant difference in Al concentration among the the seedlings with genotypes of AABB,aa BB,AAbb,and aabb in roots and shoots（stem and leaf）before and after 3 and 7 days of Al treatment.There was also no significant difference in Al concentration detected between Magellan and PI567731 in roots and shoots（stem and leaf）.These results suggest that qAl＿Gm13 and qAl＿Gm20 possibly carry an internal detoxification mechanism of Al tolerance.3.Dissecting natural variation of Al tolerance and mining genes for Al tolerance by387 Chinese cultivated soybean accessionsA total of 387 Chinese soybean accessions,including 204 Glycine max landraces and183 Glycine max released cultivars,were tested for Al tolerance by using RRG（24 h）index.The results showed that the 387 soybean accessions showed a significant range of variation in Al tolerance（P<1.00E-04）,and RRG ranged from 0.19 to 1.03,with mean RRG of 0.60and the broad sense heritability of 94%.The mean RRG of 204 Glycine max landraces was0.56,while the RRG of 183 Glycine max released cultivars reached 0.64,and there was a significant difference in RRG between these two subpopulations（P=4.22E-11）.The mean genomic linkage disequilibrium（LD）decay distance of the 387 soybean accessions was203 kb,and the analysis of the subpopulations showed that the LD decay distance was 196kb for 204 Glycine max landraces and 218 kb for 183 Glycine max released cultivars.Principal component analysis（PCA）,phylogenetic tree,and population structure analysis all revealed that the 387 soybean accessions were mainly classified into two subpopulations.A GWAS for Al tolerance（RRG）was perfomed in 387 soybean accessions.The result showed that the loci significantly associated with RRG(-log₁₀（P-value）>7)were on chromosome 18,with a total of six SNPs,all of which were 116.5 kb apart overall.The phenotypic variance explained by individual SNPs was 7.62%～8.53%.The SNP,most significantly associated with RRG（lowest P-value）was Gm18＿7853444.Based on the genomic LD decay distance of 203 kb for the genome of 387 soybean accessions,candidate genes were screened within 203 kb upstream and downstream of the physical position of Gm18＿7853444.Since most known Al-tolerant genes are induced by Al treatment.In q RT-PCR result,Only GmABCC3（Glyma.18G080900）was found to be up-regulated by Al treatment at 6,12,and 24 h in Al-tolerant accession（644）among the all 26 genes.At the same time,GmABCC3 was not induced by Al treatment in the Al-sensitive accession（84）.Therefore,the role of GmABCC3 in soybean Al tolerance was further analyzed.4.Natural variation and functional analysis and mechanism investigation of GmABCC3 for Al toleranceThe coding sequence（CDS）and promoter of GmABCC3 were sequenced and analyzed in 24 Al-tolerant and 22 Al-sensitive soybean accessions.Hap1 to Hap9,total of nine haplotypes were genotyped by nine Tag-SNPs/In Dels in GmABCC3 that associated with RRG in all 46 soybean accessions.Among them,most accessions carried Hap1（Pro1+CDS1）and Hap2（Pro2+CDS2）,and the Al-tolerant index of RRG was significantly higher in soybean accessions carrying Pro2,CDS2,and Hap2 than in soybean accessions carrying Pro1,CDS1,and Hap1（P value was 3.51E-08,2.43E-04,and 6.08E-07,respectively）,respectively.Pro＿-1213（Tag-In Del in promoter）,the most significantly associated with Al tolerance in GmABCC3,was a 23-bp In Del.The molecular marker of M＿-1213 was developed based on Pro＿-1213,and the genotyping results were consistent with the sequencing results in 36 soybean Al-tolerant/sensitive accessions.Therefore,M＿-1213 could be used for molecular marker-assisted selection（MAS）of Al tolerance in soybean in furture.In transgenic soybean hairy roots and transgenic Arabidopsis,overexpression of both GmABCC3^CDS1and GmABCC3^CDS2significantly increased the Al tolerance in the root indexes of relative root length,RRG,and relative total root length when compare with empty vector transformed soybean hairy roots and wild-type（Columbia-0,Col-0）,respectively（P<0.01）.The results also showed significant reduction in relative root Al concentration after Al treatment（P<0.05）by overexpression of both GmABCC3^CDS1and GmABCC3^CDS2in soybean hairy root tips.In transgenic Arabidopsis,overexpressing of GmABCC3^CDS2also significantly reduced the root Al concentration when compare with Col-0（P<0.05）after Al treatment,whereas overexpressing of GmABCC3^CDS1 relatively reduced the root Al concentration when compare with Col-0,but not reach the significant level.Constructs of GmABCC3^Pro1/2:GUS and GmABCC3^Pro1/2:GmABCC3^CDS2 were used to analyze the difference between GmABCC3^Pro1 and GmABCC3^Pro2 by transgenic soybean hairy roots.The result showed the activities of GmABCC3^Pro2 were higher than GmABCC3^Pro1 under both control and Al treatment conditions（P<0.01）,and the Al-induced expression of GmABCC3 was significantly higher in transgenic soybean hairy roots with GmABCC3^Pro2:GmABCC3^CDS2 than with GmABCC3^Pro1:GmABCC3^CDS2（P<0.01）.At the same time,GmABCC3^Pro2:GmABCC3^CDS2 transformed soybean hairy roots also showed higher Al tolerance in root index than GmABCC3^Pro1:GmABCC3^CDS2 transformed soybean hairy roots（P<0.01）,and GmABCC3^Pro2:GmABCC3^CDS2 transformed soybean hairy root tips had lower Al concentration（P<0.01）.There were also some unique cis-elements involved in the phytohormone and Al stress responsiveness detected in promoter of GmABCC3 in Al-tolerant accession of 644(GmABCC3^Pro2)instead of Al-sensitive accession of 84(GmABCC3^Pro1).These results indicate that GmABCC3^Pro2was superior allele promoter for Al inducible.The GmABCC3 contains two nucleotide binding domains（NBDs）and two trans membrane domains（TMDs）,belongs to the full-size ABC transporter.Subcellular localization result showed that GmABCC3 is mainly expressed on the plasma membrane,which is also similar to the reported Al tolerance related half-size ABC transporter of At ALS3.At ALS3 may belong to the exclusion mechanism of Al tolerance by transporting Al³⁺out of the cell.In present study,GmABCC3^Pro2:GUS was found to be expressed mainly in root tips and phloem of vasculature,which was also similar to the expression pattern of At ALS3.Moreover,GmABCC3 has the function of reducing the Al concentration in the roots.Therefore,GmABCC3 may transport Al³⁺out of soybean root cells through exclusion mechanism,and reduce the Al concentration in soybean roots.As a result,GmABCC3alleviate the toxicity of Al³⁺to root cells and exhibition of Al³⁺to root growth,and improve the Al tolerance in soybean.In summary,genetic dissection of soybean Al tolerance by QTL linkage mapping and GWAS,four Al tolerance loci were identified and three of them were reported for the first time.Two major QTL from linkage mapping were verified by NILs for Al tolerance.The candidate gene GmABCC3 was screened in the LD interval of the new Al tolerance locus by GWAS and its Al tolerance function was verified,and the relationship between the superior allele of GmABCC3 and Al tolerance was also analyzed.This study can provide elite germplasm and gene resources for Al tolerance breeding in soybean.The molecular markers tightly linked to Al tolerance loci and genes can be apply to marker-assisted selection and breeding in soybean.