Genome-Wide Association Analysis Of Highland Barley Grain Traits And Related Gene Differential Expression

Qingke,Hordeum vulgare L.var.nudum Hook.f.,also called hulless barley or naked barley,which is an important grain and forage crop in the Qinghai-Tibet Plateau.Development and application of eilite Qingke cultivars provides important support for Tibet food security and animal husbandry development.Spikes,grains per spike and thousand kernel wight are three factors controlling Qingke yield.Genetic dissection of seed traits is important for Qingke improvement.Seed size was controlled by seed length,width,length / width,thickness and quality,which was widely studies in plants.Many progresses have been made in rice,Arabidopsis and wheat,while few studies were made in Qingke.In this study,a set of core germplasm including 310 landraces from all ecological regions of Tibet were used for seed morphology and quality investigation in order to reveal the genetic and variation characteristics of Qingke seed traits,identify key SNPs significantly associated with its seed traits via genome-wide association analysis(GWAS)and find differentially expressed genes via RNA-seq,this study also aims to identify Qingke germplasms with good seed traits for Qingke improvement.The major results was as following:(1)Based on two year two location evaluation,7 seed traits of the core germplasm of Qingke were investigated and the results indicated all traits varied significantly among different landraces,among them four traits including seed length,thickness,TGW and starch content showed higher heritability,while protein and fiber content showed lower heritability.There was significant correlations among the 7 traits.Additionally,seed hardness,waxy and grain color were also identified,among them hard Qingke accounted for 94.23% and soft ones only 0.64%.5.16% Qingke were waxy while 92.58% were non-waxy,seed color included black,purple,blue,red and yellow,blue Qingke were the most(42.26%),followed by purple(22.90%),yellow(20.97%),red(5.48%)and black(4.19%).(2)The core germplasm included six subgroups of Qingke landraces(WT1,WT2,WT3,ET1,ET2 and IL).Based on 46719 high quality SNP after t GBS,genome wide association study were conducted on the 10 seed traits.A total of 338 significant SNP loci(P ≤ 0.001)were detected,of them 66 SNPs associated with grain size,protein content,starch and cellulose content in more than 2 replicates.12 SNPs each associated with two traits,including grain length and grain width,starch and protein content,starch and fiber content.5 SNPs were detected in twoyear environment,43 SNPs associated with waxy traits and 5 associated with hardness.A total of 307 proteins genes were identified in the peak regions covering all chromosomes except 4H.Among them,HORV7Hr1G112710 encoded Wax2＿C-domain protein,controlling waxy traits,while five other genes including HORVU1Hr1G011800(AP2/ERF),HORVU2Hr1G096350,HORVU7Hr1G101270,HORVU7Hr1G101040(b HLH),HORVU2Hr1G096550(NAM)were consistent with the previous reports,which were possible candidate genes controlling TGW,grain width and starch content.(3)Based on TKW difference,small grain inland barley DM106 and large grain Qingke landrace ZDM07486 and their F1 hybrids were investigated and used for RNA-seq at milk-ripe stage.The heterosis index of TKW was 17.38%.A total of 5394 differentially expressed genes(DEG)were identified between two parents.GO analysis showed that these genes involved in all of cellular components,molecular function,biological process function.KEGG analysis revealed that pathways of DEGs mainly concentrated in amino acid metabolism and plant hormone regulation.Allele-specific expression analysis(ASE)found that F1 allele expression was biased towards female parent DM106 with 1644 genes in total,while874 genes biased towards male parent ZDM07486.Genes biased towards large grain ZDM07486 were annotated where encoded proteins regulating grain size,such as b HLH,AAI,AP2 /ERF,WRKY,NAC.Among them b HLH,AP2 /ERF and NAC was consistent with the GWAS results of gene annotation,which were possible candidate genes controlling Qingke grain size and require further validations.(4)five landraces with higher TGW,ZDM05698 and ZDM07262 were over 48.5g,Six with higher seed width,Sixteen waxy Qingke,five higher fiber were identified,ZDM05154,ZDM05169 and ZDM04214 were over 7.85%,ten higher protein,ZDM07572 and ZDM07621 were over18%,which provide important germplasms for Qingke improvement.