Genetic Analysis Of Phosphorus Efficiency-Related Traits In Seedlings And Functional Study Of GmNLA1 In Soybean

Soybean[Glycine max(L.)Merr.]is one of the main crops and a major source of vegetable protein and oil.Two-thirds of China’s arable land is deficient in phosphorus(P),and low P is one of the main abiotic stresses that limit soybean yield.The total P content of soil in agricultural production is high,but the P that plants can absorb and utilize is very low.Therefore,understanding the mechanism of soybean P high-efficiency and cultivating P high-efficiency soybean are important method to increase the yield of soybean under low P condition.The traits associated with low P tolerance in soybean are complex quantitative traits and are susceptible to environmental factors.Traditional breeding progress is slow,but molecular marker-assisted breeding(MAS)can speed up breeding process.Soybean originated in China,and China has abundant soybean germplasm resources.In recent years,with the application of genome-wide association analysis(GWAS)in plants,the genetic mechanism of complex traits can be better resolved.In this study,to identify the genetic loci associated with P efficiency,GWAS was performed for five shoots mineral elements concentrations and four shoots P efficiency-related traits using a natural population containing 211 cultivated soybeans.And linkage analysis was performed for the same four shoots P efficiency-related traits using NJ(SP)BN population with 123 lines.Finally,functional studies were performed on identified candidate genes.The main results are as follows:Firstly,six mineral elements concentrations in shoots including shoot P concentration(SP),shoot Zn concentration(SZn),shoot Fe concentration(SFe),shoot Mn concentration(SMn),shoot Mg concentration(SMg)and shoot Ca concentration(SCa)of 211 cultivated soybeans were measured,and six mineral elements concentrations were used for GWAS using high-density NJAU 355K SoySNP array and four models(GLM、MLM、CMLM and MLMM).The results showed that six mineral elements concentrations in shoots had higher phenotypic variation,which showed a normal or near-normal distribution.Correlation analysis showed that there were positive correlations among the six elements under normal P condition.However,there was a significant negative correlation but a small correlation coefficient between SP and SMn/SMg/SCa under low P condition,and the other elements were positively correlated.GWAS results showed that the CMLM and MLM models had better results in four models.Except SP,19 SNPs and 29 SNPs were significantly associated with other five mineral elements concentrations in shoots.A total of 19 SNPs were identified using CMLM model,and 29 SNPs were significantly identified by MLM model.There were 10 SNPs associated by two models,which were mainly located on chromosome 2,3,4,5,6,8,14 and 19.Two SNPs were significantly associated with SMg and SCa,with AX-93722354 on chromosome 5 and AX-93889213 on chromosome 19.Among them,two SNPs fell within the QTL associated with soybean seed elements concentrations reported by previous studies,indicating that accumulation mechanism of shoot mineral element concentrations may be similar to the accumulation of elements in seed.Secondly,GWAS was performed using the average of four P efficiency-related traits including shoot dry weight(SDW),shoot P concentration(SP),P acquisition efficiency(PAE)and P use efficiency(PUE)of 211 cultivated soybeans and high-density NJAU 355K SoySNP array.The results showed that the four P efficiency-related traits had extensive phenotypic variation under two P conditions and showed a normal or near-normal distribution.Correlation analysis showed that there was a significant positive correlation between SDW and PAE/PUE under two P conditions,and SP and PAE were also significantly positively correlated,but the other traits showed a significant negative correlation.A total of 180 SNPs were significantly associated with four traits.SNPs significantly associated with SDW,SP,PAE,and PUE were 113,40,0,and 27,respectively,mainly located on chromosomes 6,10,and 11.The 113 SNPs significantly associated with SDW were all located on chromosome 11 and formed a SNP cluster.SNPs significantly associated with SP and PUE were mainly located on chromosome 10,and 10 SNPs were identified simultaneously in at least one trial of two traits.Among them,four SNPs were significantly associated on chromosome 10 associated with SP,PUE and their mean values,and formed a SNP clusters,namely AX-93932874,AX-93932863,AX-93636685 and AX-93636692.These four SNPs had a close physical location on chromosome in range of 36.64 kb.Among them,AX-93636685 fell in the intron region of Glyma.10g018200,AX-93932874 fell in the 5’UTR region of Glyma.10g018800,and the other two SNPs did not fall within the gene.Interestingly,these four SNPs fall within the QTL interval reported by previous researchers.Thirdly,linkage analysis was performed for the average values of the same four shoot P efficiency-related traits including shoot dry weight(SDW),shoot P concentration(SP),P acquisition efficiency(PAE)and P use efficiency(PUE)using recombinant inbred lines NJ(SP)BN.The results showed that four P efficiency-related traits in RILs had extensive phenotypic variation under both P conditions,showing a normal or near-normal distribution.Correlation analysis showed that there was a significant positive correlation between SDW/SP and PAE,and SP/PAE and PUE were significantly negatively correlated,under two P conditions.In NJ(SP)BN population,11 QTLs loci were identified mainly on chromosomes 1,2,5,7,11,13,15 and 19.There were five QTLs(qP1-1,qP2-1,qP15-1,qP15-2 and qP19-1)that fell within the previously reported QTL interval.Fourthly,functional verification of the identified candidate gene GmNLA1(Glyma.10g018800)was performed.Conserved domain analysis indicated that GmNLA1 contains SPX and RING domains and GmNLA1 belongs to SPX-RING families.Also GmNLA1 encodes E3 ubiquitin ligase.After 15 days of low-P treatment of Kefeng No.1(P high efficiency)and Nannong 1138-2(P low efficiency),the relative expression of GmNLAl in both accessions decreased significantly.Also,the relative expression of GmNLA1 in Kefeng No.1 was significantly lower than that of Nannong 1138-2 under two P conditions.However,the relative expression of Glyma.10g018200 was also significantly reduced under two P conditions,but there was no significant difference between two accessions.The relative expression of GmNLA1 in different tissues of soybean was found to be the highest in roots,followed by flowers and pods,which were almost undetectable in seeds,leaves and stems.Phylogenetic tree and gene structure analysis showed that GmNLA1 contains 6 exons and 5 introns in both monocotyledonous and dicotyledonous plants,dicotyledon is clustered into one branch,and monocotyledon is clustered into another branch.GmNLA1 is more closely related to legumes.Subcellular localization of GmNLA1 in tobacco was found to be mainly localized in cell membrane.To further understand the function of GmNLA1,an overexpression vector and an interference vector were constructed to transform soybean hairy roots.Positive transgenic hairy roots were screened by green fluorescent protein(GFP)to determine the relative gene expression and P concentration.It was found that the concentration of P in overexpression hairy roots was significantly decreased,while the concentration of P in interference hairy roots was significantly increased.These results indicated that GmNLA1 participates in soybean P homeostasis by regulating P accumulation in soybean transgenic hairy roots.