QTL Associated With Heavy Metal Accumulation In Tetraploid Wheat Grains And Related Gene Functional Analysis

Wheat is one of the most important cereal crops for human beings,and China is the largest wheat producer and consumer in the world.However,due to the excessive utilization of farmland,the application of fertilizers and agricultural chemicals,and the discharge of waste water and waste residues,the pollution of non-essential heavy metals and the lack of essential metal elements in the soils are becoming more and more seriously.These factors also lead to the production of wheat grains with excessive non-essential metal heavy metal content and deficiency of essential metal elements.Therefore,reducing the content of non-essential heavy metals(e.g.,Cd,Pb,Cr)and increasing the content of essential metals(e.g.,Mn,Fe,Zn)in wheat grains has become the focus and topical point for genetic improvement of common wheat(Triticum aestivum L.,2n=6x=42,AABBDD).Tetraploid wheat is a potential source of minor genes for genetic improvement of common wheat and is enriched with excellent traits.Jianyangailanmai(JAM,Triticum turgidum L.ssp.turgidum,2n=4x=28,AABB),a regional tetraploid wheat resource material in Sichuan,China,is genetically different from other tetraploid wheat.It possesses characteristics such as semi-dwarf,low Cd and high Zn accumulation in grains,short glumes,multiple flowers and multiple fruits,high spike number,and strong resistance to wheat stripe rust.Dwarf Polish wheat(DPW,Triticum polonicum L.,2n=4x=28,AABB)originated from Xinjiang,China,and it possesses traits such as semi-dwarf,long kernel,high thousand grain weight,and low Cd and high Zn/Fe accumulation in grains.Therefore,JAM and DPW are excellent resources for genetic modification of common wheat grains with low Cd and high Zn accumulation.However,their respective genetic characteristics are unknown.In the present study,recombinant inbred lines(RIL＿DJ)of JAM and DPW was genotyped using specific-locus amplified fragment sequencing(SLAF-Seq),and generated a high-density genetic map;then we analyzed the concentration of heavy metals such as Cd,Zn and Mn in the grains and related quantitative trait locus(QTL);and we also performed basic functional analysis of the genes encoding metal transporters in the important QTL regions.The main results are as follows:(1)Under four environments,Zn and Mn concentrations in grains of JAM were significantly lower than that of DPW.Cd concentration in grains of JAM was significantly higher than that of DPW.Mn,Zn and Cd concentrations in grains of RIL＿DJ showed a continuous normal or skew-normal distribution under all environments,and were significantly positively correlated among environments,with generalized heritabilities reaching 0.76,0.71 and 0.70,respectively.Therefore,Zn,Mn and Cd concentrations in grains of JAM and DPW were also controlled by genetic factors,and they are quantitative traits.Meanwhile,Zn,Mn or Cd concentrations in grains of RIL＿DJ population were negatively correlated with plant heights under all environments,and plant heights significantly affected the accumulation of seed Zn,Mn and Cd,respectively.(2)JAM,DPW and 171 RIL＿DJ individuals were genotyped by SLAF-seq,and a total of 8091 high-quality single nucleotide polymorphism(SNP)markers(aa×bb type)were screened in these 171 individuals.Then,1022 skeletal markers were selected for high-density genetic map construction.The total length of high-density genetic map was2201.36 c M,of which 1238.48 c M and 962.88 c M were for A and B genomes,respectively.The length of individual chromosomes ranged from 36.46 c M(7B)to 242.82 c M(5A).A and B genomes possessed 507 and 515 skeletal markers,respectively,with a maximum of120 skeletal markers on chromosome 7A and a minimum of 23 skeletal markers on chromosome 7B.The average marker density on individual chromosomes ranged from1.16 c M(4B)to 3.63 c M(6A),with an average marker density of 2.15 c M on a single chromosome.Numerous markers were mutually translocated between 1A and 7A.High degree of covariance between genetic and physical locations of markers with covariance coefficients ranged from 0.39-0.93.(3)A total of 15,9 and 11 QTLs controlling Mn,Cd and Zn accumulation in grains were detected by QTL analysis,respectively.Among these QTLs,q GMn.sicau-7A-1,q GZn.sicau-4B-1 and q GCd.sicau-4B-1 were detected under all environments,which controlled Mn,Zn and Cd accumulation in grains,explaining 4.32-14.78%,3.56-17.33%and 4.79-25.93% of phenotype contribution,respectively.Four QTLs for plant heights were detected under four enviroments,of which,q GZn.sicau-4B-1 and q GCd.sicau-4B-1were major QTLs.The physical intervals of q GZn.sicau-4B-1,q GCd.sicau-4B-1overlapped with q PH.sicau-4B-1 on 4BS,and the physical intervals of q GMn.sicau-7A-1overlapped with q PH.sicau-7A-1 on 7AS,and their candidate genes are Rht-B1 b and Rht22,respectively.Therefore,Rht-B1 b significantly affects Cd and Zn accumulation in grains,while Rht22 significantly affects Mn accumulation in grains.(4)RNA-seq analysis was performed in the near-isogenic lines of tall Polish wheat(TPW,Triticum polonicum L.,2n=4x=28,AABB)and JAM,and the differential genes were mainly involved in hormone synthesis and signal transduction,cell wall metabolism,and cell proliferation pathways at the heading stage.The number of cells in stems of semi-dwarf wheat was significantly reduced compared with that of tall wheat.The polysaccharide content in stems of semi-dwarf wheat was decresed,and the cell wall binding content of heavy metals such as Mn was reduced,leading to an increase in free heavy metals content that could be transported into graisn by both active and passive transport ways.(5)Natural resistance-associated macrophage protein 2(NRAMP2)in the candidate interval of grain Mn accumulation q GMn.sicau-4A-1 was analyzed.Tp NRAMP2-4A has produced two types in DPW,namely Tp NRAMP2.1-4A and Tt NRAMP2.2-4A,repectively.The amino acid sequence of Tp NRAMP2.1-4A was consistent with that of Tp NRAMP2.1-4A,while Tt NRAMP2.2-4A had a deletion at the N-terminal end of the amino acid coding sequence.The expression level of Tt NRAMP2-4A in grains was significantly higher than that of Tp NRAMP2-4A at grain filling stage.Protoplast subcellular localization indicated that Tt NRAMP2-4A was expressed in the endoplasmic reticulum and plasma membrane.Expression of Tt NRAMP2-4A in yeast significantly reduced the accumulation of Mn and Cd.Overexpression of Tt NRAMP2-4A in Arabidopsis significantly reduced Mn and Cd concentrations in roots,shoots,and whole plant.Transcriptome analysis revealed that Tt NRAMP2-4A-expressing negatively regulated a Mn uptake gene iron-regulated transporter1(IRT1).These results suggest that Tt NRAMP2-4A is involved in the efflux of Mn and Cd,and the differential expression level of NRAMP2-4A in grains is one of the reasons for differential Mn accumulation in grains between DPW and JAM.(6)Bioinformatic approach has been used to obtain eight NRAMP members basd on the reference genome of Triticum dicoccoides.Four NRAMP members,Tp NRAMP1-7A,Tt NRAMP4-6B,TtNRAMP6-3B and Tt NRAMP7-5A,were successfully cloned in tetraploid wheat.Expression pattern analysis showed Tt NRAMP1-7A and Tt NRAMP7-5A were mainly expressed in leaves,and TtNRAMP6-3B was mainly expressed in roots at all stages.Expression of TtNRAMP6-3B increased the sensitivity of yeast to Cd.Overexpression of TtNRAMP6-3B significantly increased Cd concentration in roots,stems,leaves and whole plant in Arabidopsis.Thus,TtNRAMP6-3B encodes a Cd transporter.