QTL Mapping And Candidate Gene Cloning Of Grain Protein Concentration In Barley(Hordeum Vulgare L.)

Grain protein concentration(GPC)is a major determinant of quality in barley(Hordeum vulgare L.).The identification of quantitative trait loci/gene controlling grain protein content will facilitate the genetic improvement for grain protein concentration in barley.The aim of the present study was to identify quantitative trait loci(QTL)for GPC that could be detected under multiple environments using a population of 190 recombinant inbred lines(RILs)deriving from a cross between ZGMLEL and Schooner.Meanwhile,three genomic regions harboring stable QTL were further validated using the near genetic lines(NILs).A stable QTL,QGpc.ZiSc-6H.3,was fine mapped using the recombinants deriving from the NILs.The main findings are as follows:1.A high-density genetic linkage map containing 21 SSR markers and 1473 SNP markers was constructed.The genetic linkage map spanned 2353.48 cM in length with an average locus interval of 2.33 cM.2.Composite interval mapping(CIM)was performed to detect QTL for GPC under six environments.Meanwhile,the best liner unbiased prediction of GPC across all the environments was also analyzed by CIM mapping.A total of 17 QTLs were detected for GPC,which are randomly distributed among chromosomes 2H(3 QTLs),4H(3 QTLs),5H(3 QTLs),6H(4 QTLs),7H(4 QTLs).Among which,six were environmentally stable QTLs,with the GPC contribution of single QTL vary from 4.20%to 17.90%.Furthermore,three stable QTL segments(Region 2H,Region 6H and Region 7H)were validated using the NILs.4.QGpc.ZiSc-6H.3 We further fine mapped using the recombinant individuals.Finally,the locus was delimited in a 31.7 Kb region.The gene annotation revealed that only one open reading frame was detected in this genomic region,and we named it GPC-6H,which encodes a NAC transcription factor.To confirm the effect of GPC-6H,Z-GPC-6H was introgressed into Schooner using marker-assisted background selection.Compared with the NIL harboring S-GPC-6H segment,the NIL with Z-GPC-6H showed higher GPC,but lower thousand grain weight,indicating that GPC-6H affects grain quality and yield.Genomic sequence analysis of Schooner and ZGMLEL revealed that a single-nucleotide transition(C-A)at the first exon in GPC-6H led to the amino acid transition(P-Q),which belongs to the conserved amino acid in the NAC domain.Also,we examined the expression of GPC-6H in grain pericarp,endosperm and flag leaf using qRT-PCR,and GPC-6H was expressed mainly in pericarp,especially at the stage of 6 days after pollination.Comparised with Schooner,ZGMLEL showed a significantly higher expression in GPC-6H.We further confirmed the tissue-specific expression of GPC-6H using RNA in suit hybridization analysis.GPC-6H was predominantly in outer integument cells.Reciprocal crossing experiments indicated that the effect of GPC-6H on GPC and seed size is strictly sporophytic maternal.