| Germplasm diversity investigation and functional loci identification are not only helpful to deeply understand the genetic basis of wheat agronomic traits,but also crucially beneficial to wheat breeding.In this study,exome sequencing of 287 wheat accessions representing a collection of the past 100 years in China was used to identify whole-genome exonic variations.Based on these variations,the population structure,genetic diversity and genome-wide selection sweeps of Chinese mini-core collections were dissected in detail.Genome wide association study(GWAS)was then conducted for 11 important agronomic traits to systematically identify genome-wide functional loci.Combined with the breeding process and breeding history from landraces to cultivars,the synergistic pyramiding effect of functional loci was explored and studied,and a batch of elite synergistic loci that impacted-multiple traits were obtained that may be helpful for breeding.The main results are as follows:1.Population diversity:A total of 983,262 SNPs and 76,952 In Dels were identified by exon capture which provided extensive variation in wheat exonic regions.Many SNPs and In Dels caused frameshift mutations,non-synonymous mutations and stop codon gain or loss which provided genetic basis underlying various phenotype among the Chinese mini-core collection.2.Population structure analysis:Phylogenetic tree,population structure analysis and population principal component analysis were used to analyze the genetic structure of the Chinese mini-core collection in detail.Population diversity analysis demonstrated that the overall genetic diversity of the modern Chinese cultivars(π=2.08e-5)was comparable to that of Chinese landraces(π=1.71e-5).The population differentiation coefficient also indicated that the population differentiation coefficient(Fst)among modern Chinese cultivars,Chinese landraces and introduced modern cultivars was generally low and comparable.Extensive germplasm exchanges among these groups were identified during the breeding process.3.Population selection analysis:A total of 975.4 Mb genomic regions including 7,298 genes were subjected to artificial selection during the improvement process from landraces to cultivars,accounting for 6.7%of the genome.Among them,subgenome A had the longest selection interval of 554.9 Mb,including 3,978 genes;subgenome B was the second at 351.0 Mb,including 2,525genes;subgenome D had the shortest,with only 69.5 Mb,including 795 genes.The selected intervals contained 175 reported QTLs and numerous important genes,such as GS5,GW7 and Ta Sus2-2A.Population selection interval analysis also reveals key improving segments of the genome during modern breeding.4.GWAS analysis:Genome-wide association analysis(GWAS)was performed to identify1,489 significant SNPs associated with 11 agronomic traits.According to r~2=0.2,the distance of linkage disequilibrium(LD)was determined to be 3 Mb,and the above loci were incorporated into 707 GWAS-derived QTLs,which provided valuable genetic loci for the dissection of complex traits in wheat.At the same time,21 high-confidence GWAS-derived QTLs that repeated at more than 5 environmental locations were identified,and the most likely candidate genes in these QTLs were provided based on tissue expression characteristics and known functional gene information,laying a solid foundation for further determination of the final candidate genes.5.Identification of a novel plant height locus and a candidate gene:A new locus impacting plant height was identified on chromosome 2A.This locus could not only affect plant height,but also affect heading date,grain number per spike and grain related trais,showing obvious pleiotropy.Its distribution frequency increased by 34%from landraces to cultivars,reaching 46%,indicating that it has been selected in breeding.The candidate gene was identified as Ta ARF12 by gene expression pattern analysis of candidate interval and RNAi experiment.In the Rht-B1b background,down-regulation of Ta ARF12 expression could further reduce plant height,and simultaneously exhibit large panicle and increasing grain number,indicating that this gene has important application potential.6.Identification of novel grain trait loci and candidate genes:Two grain thickness candidate loci(the most significant SNP positions:chr6＿2798923 and chr5A＿430246395)and their respective candidate genes Ta Skp1 and Ta DEP1 were identified on chromosomes 6A and 5A.Among them,the elite haplotype of Ta DEP1 not only increased grain thickness,grain length,grain width and 1000-grain weight,but also significantly reduced effective tillering and plant height,and its frequency increase by 47%from landraces to cultivars,showing clear pleiotropic role.The CRISPR/Cas9 edited lines of Ta DEP1 simultaneously affected grain length,grain width,grain thickness and thousand-grain weight,which further confirmed that Ta DEP1 was the best candidate gene for this locus.7.Analysis of the genetic basis of synergistic improvement for multiple traits:400 elite allelic variants that could cause phenotypic differences and were consistent with the direction of artificial improvement were identified among 1,489 significant SNPs loci.Their frequencies were significantly increased from landraces to cultivars,indicating that they were elite alleles.It was found that in the process of breeding selection,there were 141 loci to synergistically improve plant height and grain traits,124 loci for plant height and heading date,and 87 loci for heading date and grain traits.These loci would provide valuable molecular marker resources for synergistic pyramiding breeding of key traits in wheat.8.An important breeding strategy for wheat yield improvement:Through investigation of the synergistic effect of the known functional genes Rht-B1,Ta DEP1 and Ta ARF12,it was found that the effect of loci on plant height was dominantly additive,while the effect on 1000-grain weight was more complicated,and the epistasis effect was obvious.We proposed a gibberellin pathway centered model for wheat breeding.During this process,components of the G-protein pathway and the auxin pathway were added to further improve wheat plant height and 1000-grain weight.Such a strategy may lend a useful concept for future creation of breakthrough varieties in wheat. |
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