Identification Of QTLs Associated With Cd Accumulation And Tolerance,and Functional Characterization Of A Candidate Gene HvPAA1 In Barley

Cadmium(Cd)contamination in soil has posed a serious problem for safe food production and become a potential agricultural and environmental hazard worldwide.For fields contaminated with mild to moderate Cd,it is one of the vital ways to breed new cultivars with low Cd accumulation through exploring genes associated with Cd tolerance and accumulation,in which way we can effectively use natural resources and ensure food safety.Identification of genes underlying Cd tolerance/accumulation quantitative trait loci(QTLs)will facilitate understanding of molecular mechanisms of Cd tolerance/accumulatior,and will promote genetic improvement of barley through marker-assisted selection.In this study,we developed a doubled haploid(DH)barley population that was derived from a cross of Cd-tolerant and Cd-sensitive parents to identify genes associated with Cd tolerance and accumulation,and a candidate gene HvPAA1 was then characterized in terms of function.The main results are as follows:1.A population of 108 DH lines.,derived from a cross between the Suyinmai 2(Cd-sensitive)and Weisuobuzhi(Cd-tolerant),and the two parents were used for QTL analysis.Within the genetic map,a total of 1572 markers were successfully assigjned to the seven barley chromosomes.The total length of the map was 1134.5 cM(centimorgan),and the average distance between two adjacent markers was 0.72 cM.2.The pot experiment of 15 mg kg-1 Cd artificial soil-Cd-contamination was conducted to study QTLs related to Cd tolerance and accumulation in barley grains.Five QTLs were detected and qKCd5H associated with kernel Cd concentration was located on chromosome 5H.This QTL can explain 10.5%of phenotypic variation.The interval of qKCcd5H region included approximately 31 genes that were annotated in the Barley Map database.Among them,HORVU5Hr1G118750 gene with annotation of"Zinc finger protein 668"was selected as candidate gene.3.The hydroponic experiments were carried out for study to identify QTLs related to Cd tolerance and accumulation during barley seedling stage.A total of 24 QTLs were detected from this study.Among them,a single QTL,designated asqShCd7H,for Cd concentration in shoots was identified on chromosome 7H and can explained 17%of phenotypic variation qShCdlH was mapped to a 2.6 cM region and approximately 60 genes that were annotated in the Barley Map and IBCS genome database.As a result,AK355848.1 with the annotation"P-type ATPase,cation/copper-transporter,was selected as the candidate gene for further study.4.The full length CDS of AK355848.1 was cloned from the three barley genotypes(c.f.Suyinmai 2,Weisuobuzhi and Zhenong8).Polygenetic analysis showed that the cloned sequence of AK355848.1 was mostly similar to that firom OsPAAl(Oryza sativa L.).As a result,we designated this gene as barley HvPAAl.The DH parents(c.f.Suyinmai 2,Weisuobuzhi)and Zhenong 8 shared the same sequences of amino acid and CDS.The alignment result showed that Weisuobuzhi and Suyinmai 2 contain 4 single nucleotide polymorphism sites(SNPs),all of the 4 SNPs are located in intron.HvPAA1 was mainly expressed in shoots,and 5?M Cd stress significantly induced the relative expression of HvPAA1 transcript in shoots.Based on GFP signal.HvPAA1 protein was localized to plasma membrane.5.To clarify the function of HvPAA1,the BSMV-VIGS-based(Barley stripe mosaic virus induced gene silencing)gene silencing technology-was used to determine if the silencing of this gene at the mRNA level affected Cd concentration in barley.The presence of 5 ?M Cd resulted in decreases in shoot dry weight,especially at 10 dpi(days post infection),in BSMV:HvPAA1-inoculated plants compared with the BSMV:y-inoculated plants.As to Cd concentration in mock-and BSMV:HvPAAl-inoculated seedlings exposed to 5 ?M Cd,shoot Cd concentration showed no significant differences between mock-and BSMV:HvPAA1-inoculated seedlings at 1 dpi,while root Cd concentration in BSMV:HvPAA1-inoculated seedlings was notably higher than in mock-inoculated seedlings.At 5 dpi,silencing of HvPAA1 significantly increased both shoot and root Cd concentrations by 48.3%and 62.2%,respectively,compared with the mock-inoculated seedlings.Meanwhile.BSMV:HvPAA1-inoculated seedlings accumulated more Cd than mock-inoculated seedlings.In summary,the silencing of HvPAA1 resulted in growth inhibition,greater Cd accumulation,and a significant decrease in Cd tolerance under Cd stress.6.Based on the new genetic map,the data obtained from our previous experiments were used for QTL re-analysis.A total of 31 QTLs were detected.In hydroponic experiments under 50 ?M Cd,only six QTLs were found.In pot experiment under 25 mg kg-1Cd stress,totally 25 QTLs were identified.The QTL named qSLCd2H on chromosome 2H was associated with leaf Cd concentration in seedling stage,which was overlapped with qELCd2H,a QTL account for leaf Cd concentration in elongation stage.In the overlapped QTL interval in chromosome 2H,32 candidate genes were annotated in the QTL interval in approximately 3.92 cM.