| Pre-harvest sprouting(PHS)occurs frequently in Chinese major wheat regions,and has also become serious in the Yellow and Huai Valleys region and Middle and Lower Yangtze River Valley region due to frequent rainfall and high humidity during harvest,which influences grain yield and quality.Therefore,improvement of PHS tolerance has become an important objective in wheat breeding programs.PHS tolerance is controlled by multiple genes,however,its molecular mechanism remains unclear.To understand the genetics basis of PHS tolerance and accelerate the process of breeding,in this study,association analysis and linkage analysis were used together to identify genes or major loci underlying pre-harvest sprouting resistance,and then bulked segregant RNA sequencing(BSR-Seq)method was applied for exploring candidate genes underlying the major regions.The main results are as follows:1.Association analysis was performed in 260 wheat varieties differing in PHS tolerance.Combined with the germination index(GI)and field sprouting(FS)collected from different environments,a total of 47 loci were identified significantly(P<0.01)associated with PHS tolerance,distributed on all 21 chromosomes.Major loci were mapped on chromosome 2AL,3AS,3BL,5AL and 5BL and the locus(Xwmc658)on 2AL was considered as a novel QTL(Qsd.ahau-2AL).2.Two linkage populations were constructed based on Wanxianbaimaizi and Suiningtuotuomai carrying the favorable alleles of Qsd.ahau-2AL.The results showed that the Qsd.ahau-2AL expalined 9.10-38.80%of the phenotypic variation in the two populations.A total of 201 CMCC accessions was used to validate the association of the marker CAPS-2AL with PHS tolerance.The results indicated that the CAPS-2AL was tightly(P<0.01)associated with PHS tolerance,explaining 4.20-9.00%of the phenotypic variation,and the allele CAPS-2AL-b was significantly associated with lower GI.3.In the RIL population from the cross of Jing411/Hongmangchun21,TaCNGC8-A1 was cloned as a candidate gene for PHS tolerance on chromosome 2A using BSR-Seq approach,containing 7 exons and 6 introns with 6883 bp in length,and encoding 716 amino acids.4.Sequence analysis of TaCNGC8-A1 revealed there are 18 variations between wheat varieties differing in PHS tolerance.Of these,a SNP(T/A)in the promoter regions,a 39bp InDel mutation in the fourth intron,and a SNP(G/A)in the sixth exon,were all significantly associated with PHS tolerance,and a gene-specific marker CNG2AL was developed.The CNG2AL was significantly(P<0.01)associated with PHS tolerance,and the allele CNG2AL-b was significantly(P<0.01)associated with lower GI compared with CNG2AL-a.In Jing411/Hongmangchun21 population,the TaCNGC8-Al co-located a QTL for PHS tolerance within Xgwm356-Xwmc170 on 2AL chromosome,explaining 8.82-12.29%of phenotypic variation.5.Phylogenetic analysis showed that the CNGC gene derived from the monocotyledons and dicotyledons was located in the different phylogenetic branchs with far genetic relationship,and the TaCNGC8-A1 in Triticum aestivum had a close relationship with TuCNGC8 in T.urartu and AetCNGC8 in Ae.tauschii.Moreover,the TaCNGC8-A1 was distributed in the same evolutionary branch with OsCNGC6 in rice and AtCNGC6,AtCNGC7,AtCNGC8,and AtCNGC9 in Arabidopsis thaliana.6.The TaCNGC8-Al gene expressed in root,stem and leaf at the jointing stage and had a highest expression level in root.It also expressed in grain at different growth period after anthesis and different after-ripening period,the relative expression level of TaCNGC8-A1 decreased from 27 days post anthsis to 7 days post harvest,and then increased from 7 days post harvest to 21 days post harvest.Among them,the relative expression level of the TaCNGC8-A1 gene in Jing411 with low PHS resistance was significantly higher than that in Hongmangchun21 with high PHS resistance.Moreover,the expression level of the TaCNGC8-Al gene in seeds imbibied in water for 14h was higher than that in dry seed.The results indicated that the TaCNGC8-Al gene negatively regulated seed dormancy and PHS resistance. |
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