QTL Mapping And Candidate Genes Analysis Of Traits Related Low Phosphorus Stress And Aluminum Stress In Soybean

Phosphorus(P)deficiency and Aluminium(Al)stress are two adverse abiotic factors limiting the growth and productivity of crops in acidic soils.The P nutrient also limits crop productivity worldwide,principally because 80% of P is bound to soil particles and unavailable to plant.Moreover,the inhibition of root growth in plants influences the water and nutrient uptake,which aggravates the damage of low P stress.Soybean(Glycine max L.)is one of the most important sources of protein,oil and micronutrients for human and livestock consumption,and it is sensitive to low P stress and Al toxicity.With the untiring efforts,several soybean varieties adapted to acid soil have been developed and commercially released in South China.Fine-mapping and cloning of quantitative trait loci(QTLs)provides an effective tool in analysing the genetic mechanisms underlying low P stress and Al tolerance and in breeding low P-tolerant and/or Al-tolerant soybean varieties.The Huachun2 is a soybean variety bred in South China,which has strong adaptability in acidic soils.In the present study,the mapping population consisting of 196 RILs was developed from a‘Huachun2’ and ‘Wayao’ cross was used to mapping low P-tolerant and/or Al-tolerant QTL/genes.The main results obtained are as follows:To elucidate the genetic mechanisms for low-P tolerance in soybean,quantitative trait loci(QTL)related to root morphology responses to low-P were identified via hydroponic experiments.A total of 107 QTL associated with these traits were obtained in the RIL population of Huachun 2×Wayao.Fourteen hot spots were further considered to be the m major loci for low phosphorus tolerance.Among them,Six major loci(qP1,qP2,qP8-2,qP10-2,qP11 and qP13)were co-localized with previously identified root-related or P efficiency related QTLs,and eight loci(qP5,qP6,qP8-1,qP8-3,qP10-1,qP14,qP16-1,and qP16-2)were novel.The qP10-2 on chromosome 10 displayed highest LOD score of 7.43 and explained highest phenotypic variance of 13.98%.The q RT-PCR assay was performed to obtain the candidate genes within fourteen major QTL.Three phosphatase encoding genes(Glyma08g20800,Glyma08g20810 and Glyma08g20820)in qP8-2 were up-regulated significantly induced by low-P,indicating they may candidate genes.Similarly,GmHAD1 belonged to HAD superfamily encoding a phosphatase gene and was identified in qP10-2and significant up-regulated in Huachun 2,which indicated the GmHAD1 may be a key candidate gene.The promoter and full-length DNA sequences of the phosphatase encoding gene GmHAD1 in the qP10-2 were cloned and analyzed.Combined with the analysis of functional cis-elements and domains,multiple stress-related cis-elements was found in the promoter region.Analysis of induced expressed protein in vitro showed that expressed protein has the ability to hydrolyze the ρ-nitrophenol phosphate(ρ-NPP)with higher hydrolysis activity under acidic conditions.Transgenic Arabidopsis and soybean hairy roots overexpressing GmHAD1 were created to further explore the function of the GmHAD1.Under the low phosphorus medium and soil conditions,the transgenic Arabidopsis grew better and displayed higher content of total phosphorus compared to wild-type plants.Under the condition of a single phosphorus source(ρ-NPP),the phosphorus content in transgenic hairy roots was 38.2-50.0% higher than that of wild types and the utilization rate of phosphorus increased by 8.4-16.5%.Overexpression of GmHAD1 in both Arabidopsis and soybean hairy roots can significantly improve their growth abilities under low phosphorus conditions,indicating that GmHAD1 was the major candidate gene at the qP10-2.A total of five Al tolerance related QTL(qRRE06,qAl06,qRRE15,q Al-HC2 and q RRE18)were obtained by performing QTL mapping with the RIL population of Huachun2×Wayao.Among them,the q Al06 was associated with root-tip Al content,which could explain 5.89% of phenotypic variance.The expression of GmGSTU9 in root tips was upregulated by Al treatments in Huachun2 and Wayao over a period of 24 h.However,the expression level of GmGSTU9 in Al-tolerant parent Huachun2 was higher than that in Alsensitive parent Wayao during six to ten hours.To evaluate whether GmGSTU9 in q Al06 were correlated with Al tolerance,the hairy roots induced by the K599 strain carrying RNAi,overexpression and no vector were used for Al treatment.The overexpression and silencing experimental results suggest that GmGSTU9 positively regulates Al toxicity in the hairy roots by regulating GST activity.Besides,three promising target genes(GmPrx143,144 and 145)encoding peroxidase identified in q Al-HC2.Further analysis found that three peroxidase genes with higher expression in root,GmPrx143,GmPrx144 and GmPrx145,were induced by Al stress in two parents.Overexpression of GmPrx145 in hairy roots resulted in high antioxidant activity that improved the tolerance to aluminum,while inhibition of GmPrx145 reduce the tolerance of hairy roots to aluminum,indicating that GmPrx145 was the major candidate gene conferring Al tolerance in q Al-HC2.Based on the mapping of low-P and Al-resistant QTL markers,nine copies of potential inbred lines that resistance to both low-phosphorus and aluminum were obtained from RIL population(RIL43,RIL97,RIL98,RIL123,RIL131,RIL160,RIL166,RIL181 and RIL182).The results of both low-P and Al stress showed that the relative total root length and relative root surface area of RIL43,97,98,123,131 and 166 were slightly larger than those of Huachun 2,and the total root length was similar to that of Huachun 2.In addition,the growth index of RIL160,181 and 182 were worse than Huachun 2,but significantly better than Wayao.In summary,the major QTL for low-P and Al resistance traits were obtained by perfoeming QTL mapping using population of Huachun 2×Wayao.Candidate genes within the physical QTL confidence intervals were screened using the method of prioritization,followed by the use of the variation and expression data to further confirm the valuable genes.Three candidate genes,including GmHAD1 within the qP10-2,GmGSTU9 within the q Al06 GmPrx145 within the q Al-HC2 QTL were selected to further verify the function by using transgenic hairy roots and/or transgenic Arabidopsis.The SNP markers in the QTL and candidate genes will be potentially utilized to overcome limitations in the selection of soybean genotypes in acidic-soil-limited environments through MAS.Moreover,RIL43,97,98,123,131 and 166 obtained in this study may be potential germplasm resources on the acid soil.