Allelic Polymorphism Analysis And Functional Identification Of Drought Tolerance Related Genes In Tibetan Wild Barley

Drought is a main meteorological disaster that limits crop production worldwide.Therefore,an improvement in drought tolerance in crops is prerequisite for fully utilizing natural resource and achieving economic gains.The best and most effective approach in this regard is to develop drought tolerant crop varieties.It is therefore important to identify the genetic resources with high tolerance and to understand the mechanisms of drought tolerance in plants.Tibetan annual wild barley(Hordeum vulgare L.ssp.spontaneum and H,vulgare ssp.agriocrithum)is the precious germplasm resource,and it is rich in genetic diversity and elite alleles for crop improvement.In this study,we used the natural population of Tibetan annual wild barley to explore the genetic diversity of these germplasms and develop new EST-SSR markers.Then we performed genome-wide association analysis(GWAS)with DArT markers and agronomic and physiological traits of wild barley under drought stress.According to the results of GWAS,we selected two drought tolerance related genes HvHAK1 and HvAKT1 to study their function in drought tolerance.The main results are as follows:1.By searching online for EST sequence and SSR loci,performing PCR and capillary electrophoresis analysis,we developed 49 novel EST-SSRs and confirmed 20 genomic SSRs for 80 Tibetan annual wild barley and 16 cultivated barley accessions.A total of 213 alleles were generated from 69 loci with an average of 3.14 alleles per locus.The trimeric repeats were the most abundant motifs(40.82%)among the EST-SSRs,while the majority of the genomic SSRs were di-nuleotide repeats.Overall,the polymorphism of genomic SSRs was higher than that of EST-SSRs.Furthermore,the number of alleles and the PIC of wild barley were both higher than that of cultivated barley,indicating more polymorphism existed in the Tibetan wild barley than in cultivated barley.The 96 accessions were divided into eight subpopulations based on 69 SSR markers,and the cultivated genotypes can be clearly separated from wild barleys.A total of 47 SSR-containing EST unigenes showed significant similarities to the known genes.2.Greenhouse hydroponic(drought stress,keeping roots in air 6 h/d for 7 d)and pot experiments(drought stress,soil moisture at 4%after stopping watering for 30 d)were undertaken,respectively,using 166 Tibetan annual wild barley accessions,to study the genotypic difference in response to drought stress in the agronomic traits,and perform GWAS.The results showed that there was significant genotypic variance among wild barley in root length,ear length,awn length,internode length and grain weight per spike under drought stress.The largest variance existed in grain weight per spike(37.4%),and the smallest variance in shoot height and awn length(8.4%).Population structure analysis revealed 3 subpopulations.The average LD decay distance was 5.16 cM,with the minimum on 6H(0.03 cM)and the maximum on 4H(23.48 cM).A total of 33 DArT markers were identified to be associated with different morphological traits.The individual contribution of each of the 11 DArT markers associated with root dry weight to the phenotypic variation was ranging 9.8%-15.5%.The marker bpb-3653 was associated with shoot dry weight,contributing to 9.4%of phenotypic variation.There were 4 and 2 markers associated with shoot height and root length,and they contributed to 9.1%-10.5%and 8.9%?12.3%of phenotypic variation,respectively.Among the 14 markers associated with awn length,bpb-0068 can contribute to 19.8%of phenotypic variation.Furthermore,bpb-4184 was significantly associated with both awn length and internode length,contributing to 12.4%and 9.7%of phenotypic variation,3.Drought-stress-hydroponic/pot-experiments were performed,and the physiochemical characteristic of the 166 Tibetan wild barley genotypes were compared,and GWAS was performed.The results showed that there was large genotypic variation among the wild barley genotypes for the activities of POD and CAT in leaves,MDA accumulation in leaves,H+K+-ATPase activity in leaves and roots,potassium content in leaves and roots,and soluble protein content in leaves.The largest genotypic variance existed in root H+K+-ATPase activity,and that in leaf CAT activity and MDA content were smaller,with the increase/decrease percentage ranging-93%?+256%,-66%?+599%and-80%?+351%over the control,respectively,and the coefficient variance being 75.9%,74.7%and 71.6%.DArT markers of 22,2,and 14,2,4,10,3,1 were identified to be associated with root H+K+-ATPase activity,K+ concentration,and leaf CAT,POD,H+K+-ATPase activities,K+,soluble protein,MDA contents.Among these markers,both bpb-7069 and bpb-7063,which were associated with CAT activity,can contribute to 25.7%of phenotypic variation,respectively.It is interesting to find that some traits shared the same loci and some different loci located at the same positions of barley genome.Markers bpb-5755(44.79 cM,21H)and bpb-4877(4738 cM,2H)were located closely to potassium channel HvAKT1(45.5 cM,2H)and potassium transporter HvHAKl(58cM,2H).4.We measured the net ion flux(K+,H+,Ca2+ and Cl-)of Arabidopsis transiently expressed HvAKT1 and HvHAK1 using MIFE technology.Under drought stress(5%PEG),the K+ influx in the Arabidopsis expressed HvAKT1(AKT1)increased significantly,compared with Col-0 and GV3101,while that in Arabidopsis expressed HvHAK1(HAX1)decreased significantly.ForH+,the H+ efflux of AKTI under drought stress had no significant variance,compared with Col-0 and GV3101,while the H+ efflux of HAK1 increased significantly.Compared with GV3101,the influx of Ca2+ and efflux of Cl-in AKT1 decreased significantly,but there was an opposite trend in HAK1.The subcellular localization assay showed that both HvAKT1 and HvHAK1 were located on the plasma membrane.Our findings provide an insight into gene discovery and the molecular mechanisms related to drought tolerance in Tibetan wild barley.