| TaGW2-6A,cloned in earlier research,strongly influences kernel width and thousand kernel weight in common wheat,and a functional marker TaGW2-6A-CAPS associated with grain weight was developed.In this study,further research focused on sequencing and nucleotide diversity of the promoter regions of TaGW2-6B and TaGW2-6D,functional marker development,and an expression pattern comparison of the three homoeologous TaGW2 loci.Meanwhile,we compared gene diversity(π),genetic differentiation of TaGW2s between the promoter and coding regions in diploids,tetraploids,and hexaploids of bread wheat during the plolyploidization process and wheat improvement.It was expected that the study would identify important genes and functional markers for wheat yield improvement.This research also provides a valuable case for understanding interaction of genes that control complex traits in polyploid species.The main results are summarized as follows:1.About 2.9 kb of the promoter sequences of TaGW2-6B and TaGW2-6D were cloned from 34 bread wheat cultivars.Eleven SNPs were detected in the promoter region of TaGW2-6B,forming 4 haplotypes.No divergence was detected in the TaGW2-6D promoter or coding region.Three molecular markers including TaGW2-6B-CAPS,TaGW2-6B-dCAPS and TaGW2-6B-ACAS,were developed to discriminate the TaGW2-6B haplotypes.Haplotype association analysis indicated that TaGW2-6B has a stronger influence than TaGW2-6A on TKW,and Hap-6B-1&-2 were favored haplotypes increasing grain width and weight that had undergone strong positive selection in wheat breeding.2.We used wheat accessions worldwide including North America,Europe,Australia,Russia,Mexico and China,to analyze the selection intensity and geographical distribution of TaGW2s’ haplotypes in different wheat ecological regions.At TaGW2-6B,the favorable haplotype Hap-6B-1&-2 were more frequent in all regions.However,clear geographic distribution differences for TaGW2-6A haplotypes were found;Hap-6A-A was favored in Chinese,Australian and Russian cultivars,whereas Hap-6A-G was preferred in European,American and CIMMYT cultivars.3.The average relative expression analysis of TaGW2 three homologous genes in 22 wheat varieties revealed that TaGW2-6A was highest,TaGW2-6B followed and TaGW2-6D was lowest at different grain development stages.The average relative expression level of TaGW2s in varieties with low TKW was higher than that with high TKW,conversely,the average relative expression level of high TKW was lower,and the expression of three genes was consistent;in addition,the average relative expression level of favored haplotypes Hap-6A-A(TaGW2-6A)and Hap-B-1&-2(TaGW2-6B)were lower than other haplotypes on the same locus.These revealed that TaGW2 negatively regulated kernel width and kernel weitht in wheat and TaGW2-6B had a stronger effect.4.We analyze the interactions through haplotype combination among the three TaGW2 loci and phenotypic variation(R2)for grain traits.Haplotype interaction analysis between TaGW2-6A and TaGW2-6B showed additive effects between the favored haplotypes.Hap-6A-A/Hap-6B-1 was the best combination to increase TKW.R2 for grain traits in TaGW2-6B was higher than that in TaGW2-6A,and the value of the combination of TaGW2-6A/TaGW2-6B was higher than that of either TaGW2-6A or TaGW2-6B alone.5.One hundred and sixty-four collections of wheat and its relative species were used to analyze nucleotide polymorphisms of TaGW2s promoter and coding regions.In the process of wheat polyploidization,gene diversity of TaGW2s significantly decreased from diploids,tetraploids to hexaploids.Among different ploidy wheat,diversity of the promoter region was significantly higher than that of the coding region in TaGW2s.In both promoter and coding regions,TaGW2-6B had the highest diversity,following TaGW2-6A,and TaGW2-6D was the lowest.6.In the process of wheat polyploidization,genetic differentiation of TaGW2s’promoter and coding regions from diploids to tetraploids was larger than that from tetraploids to hexaplois.Based on sequence ploymorphisms of TaGW2s,phylogenic analysis showed that diploids was subgroup alone,and it was mixed for tetraploids and hexaploids,indicating a more variances in diploids.T.urartu and Ae.speltoides were more closer to the A and B genomes of common wheat respectively than other diploid species.7.According to Tajima’s D calculations,strongest selections mainly occurred on the promoter regions in both TaGW2-6A and TaGW2-6B.Fst values between Triticum species and related Aegilops species also showed that stronger differentiations occurred at the promoter region in TaGW2-6A and TaGW2-6B.8.The haplotype number was dramatically decreased from diploids,tetraploid to hexaploids in the promoter regions of TaGW2-6A and TaGW2-6B.There existed a clear relatedness between haplotypes in tetraploids and hexaploids,while diploids were classified into one cluster.Using SNPs from hexaploids,favored haplotypes at TaGW2-6A and TaGW2-6B were detected in tetraploid wheats,i.e.Hap-6A-A in T.durum,Hap-6B-1 in T.dicoccum and Hap-6B-2 in T.dicoccoides,T.dicoccum,T.durum and T.turgidum.Importantly,SNPs discovered in hexaploids were almost all monomorphic in diploids,but polymorphic in tetraploids.9.In wheat polyploidization,kernel width and weight increased from diploids,tetraploids to hexaploids,while relative expression levels of TaGW2s declined significantly.This further demonstrated negative regulatory role of TaGW2s to grain size,and they experienced strong seclection during wheat evolution. |
