Fine Mapping Of QBp.caas-3BL For Black Point In Wheat

Wheat is an important staple crop that provides about 20% of calories for humankind.Black point(also known as kernel smudge)is a serious disease characterized by dark discoloration at the embryo end of wheat grains and occurs in most wheat-growing regions of the world.It causes inferior grain appearance,processing and nutrient quality as well as lower seed vigor.Mining and characterization of genetic loci for black point resistance is helpful for breeding cultivars with resistance to the disease.We previously identified a major QTL QBp.caas-3BL for black point,explaining 6.0-12.2% of phenotypic variation in a recombinant inbred line(RIL)population of Linmai 2/Zhong 892 across five environments.Here we achieved fine mapping and candidate gene prediction of QBp.caas-3BL through creation of secondary mapping population,development of molecular marker and differential expression assays.These findings laid a solid foundation for map-cloning and molecular mechanism dissection underlying QBp.caas-3BL.The major progress in QBp.caas-3BL was made as follows.(1)Confirmation of QBp.caas-3BLTo confirm the presence of QBp.caas-3BL,we re-investigated black point response of the RIL population in two additional environments(2019 Anyang and 2019 Suixi).Based on the phenotypes in these two environments,the QTL QBp.caas-3BL was confirmed with a consistent genetic location across environments,explaining 6.6-12.4% of the phenotypic variance.These results also confirmed that QBp.caas-3BL was a stable QTL for black point.(2)Fine mapping of QBp.caas-3BLTo narrow down the target interval of QBp.caas-3BL,we created secondary mapping populations using RILs with residual heterozygosity in the QBp.caas-3BL target region.Four sets of QBp.caas-3BL near isogenic lines(NILs)were selected and used to create secondary populations for fine mapping.Six chip-based SNPs that were identified previously as flanking markers of QBp.caas-3BL were converted into genotyping-by-sequencing-based(GBSB)markers.We also identified polymorphic sites of six genes within the target region based on the IWGSC Ref Seq v1.1 and developed gene-specific GBSB markers.These markers defined QBp.caas-3BL in a physical interval between 638.9-640.6 Mb in the chromosome 3B.The physical interval spanned approximately 1.7 Mb,including five high-confidence annotated genes,Traes CS3B02G404300,Traes CS3B02G404400,Traes CS3B02G404500,Traes CS3B02G404600 and Traes CS3B01G404700 according to Ref Seq v1.1.(3)Prediction of candidate genesTo determine the candidate genes in QBp.caas-3BL,we compared the open reading frames(ORFs)of the five genes in parents Linmai 2 and Zhong 892 by sequencing.The ORFs of Traes CS3B02G404600 and Traes CS3B02G404700 contained polymorphic sites between the parents.The SNP in Traes CS3B02G404600 was a synonymous mutation,whereas that of Traes CS3B02G404700 was a nonsynonymous mutation causing a serine-to-alanine substitution.We also compared the transcriptional levels of the five genes in developing grains using q PCR assays and observed that Traes CS3B02G404300,Traes CS3B02G404600 and Traes CS3B02G404700 were differentially expressed between the parents.Collectively,each of Traes CS3B02G404300,Traes CS3B02G404600 and Traes CS3B02G404700 might be the causal gene of QBp.caas-3BL based on sequence polymorphism and differential expression.(4)Development and validation of a KASP marker for QBp.caas-3BLTraes CS3B02G404700 was identified as a candidate gene for QBp.caas-3BL,with a SNP in its ORF between the parents.To develop a breeding-applicable marker for QBp.caas-3BL,we converted the SNP into a cost-effective,high-throughput KASP marker and validated it in a panel of 166 wheat cultivars from the Huang-Huai Wheat Region,the largest wheat area in China.Association analysis indicated that the sequence variation in Traes CS3B02G404700 was significantly associated with black point response,suggesting that the KASP marker was an effective tool of marker-assisted selection for QBp.caas-3BL in wheat breeding.