Cloning Of TaGS5 And TaTAC1 Genes Related To Important Agronomic Traits And Their Association With Agronomic Traits In Chinese Bread Wheat(Triticum Aestivum L.)

Wheat is an important food crop in the world,and improvement of wheat yield has always been an essential breeding target for wheat breeders.As one of the three wheat yield elements,thousand-kernel weight(TKW)is considered to have an important influence on wheat yield and could be determined by the kernel size.Common wheat has a largerhuge genome size and abundant repeat sequences.Therefore,direct cloning of yield-related genes from hexaploid wheat without reference genes was difficult by map-based cloning.Up to date,numerous yield-related QTLs have been identified in bread wheat.However,studies on cloning,molecular mechanism and genetic basis of yield-related genesome important agronomic traits are relatively rare.In this study,a total of 363 wheat cultivars or advanced lines from wheat germplasm of Yellow and Huai wheat regions in China was used to clone the two yield-related genes based on in silico cloning.One was a TaGS5 gene related to kernel size and the other was TaTAC1 gene related to tiller angle in bread wheat.Studies on structure,molecular characterization and their association with agronomic traits of these genes could provide important information for the dissection of molecular mechanism,genetic basis of yield-related traits.The main results were as follows.1.TaGS5 genes were cloned from bread wheat and composed of ten exons and nine introns,and further were physically mapped on 3AS and 3DS.Sequencing results revealed that a SNP was found in the sixth exon of TaGS5-A1 gene.The SNP resulted in amino acid change from alanine to serine at the 303 bp position of TaGS5-A1.These two alleles were designated as TaGS5-A1a(alanine at the 303 bp position)and TaGS5-A1 b genes(serine at the 303-bp position).Based on this SNP,aCAPS marker was developed to distinguish between TaGS5-A1 a and TaGS5-A1 b genotypes.2.Analysis of association of TaGS5-A1 alleles with agronomic traits indicated that cultivars with TaGS5-A1 b possessed wider kernel width and higher thousand-kernel weight,as well as significantly lower plant height,spike length,and internode length below spike than those of cultivars with TaGS5-A1 a over 3 years.These trait differences between TaGS5-A1 a and TaGS5-A1 b genotypes were larger in landraces than in modern cultivars.This finding suggested that TaGS5 gene probably played an important role in modulating yield-related traits in the landraces,which possibly resulted from numerous superior genes gathering in modern cultivars after strong artificial selection.The preferred TaGS5-A1 b haplotype probably underwent very strong positive selection in Chinese modern wheat breeding,but not in Chinese landraces.3.Expression analysis of the TaGS5-A1 gene indicated that TaGS5-A1 b allele possessed significantly higher expression level than TaGS5-A1 b allele in differently developmental seeds.It suggested that TaGS5-A1 gene was positively associated with thousand-kernel weight.4.To further study the regulatory mechanism of TaGS5 gene in this study,the promoter sequence of TaGS5-A1 gene was isolated from bread wheat.Sequencing results revealed that a G insertion was found in position-1925 bp of TaGS5-A1 gene(Reference to ATG),which occurred in the Sp1 domain of the promoter sequence.Combined with the G to T substitution in TaGS5-A1 exon sequence and the G insertion in the promoter sequence of TaGS5-A1 gene,four genotypes were formed at the TaGS5-A1 locus and were designated as TaGS5-A1a-a,TaGS5-A1a-b,TaGS5-A1b-a and TaGS5-A1b-b,respectively.5.Analysis of association of the promoter sequence of TaGS5-A1 alleles with agronomic traits indicated that cultivars with TaGS5-A1a-b allele possessed higher thousand-kernel weight,lower plant height and wider kernel width than those of cultivars with TaGS5-A1a-a allele.Cultivars with TaGS5-A1b-b allele showed higher thousand kernel weight,higher plant height and narrower kernel width than those ofcultivars with TaGS5-A1b-a allele over three years.The differences of these traits between TaGS5-A1a-a and TaGS5-A1a-b alleles were larger than those of TaGS5-A1b-a and TaGS5-A1b-b alleles,suggesting that the G insertion in the TaGS5-A1 promoter sequence probably played the more important role in TaGS5-A1 a genotypes than in TaGS5-A1 b genotypes.6.q RT-PCR indicated that TaGS5-A1b-b possessed significantly higher expression level than TaGS5-A1b-a at different developmental stages of the seeds.Due to TaGS5-A1b-b genotypes showing higher thousand kernel weight,it possibly suggested that high expression of TaGS5-A1 was positively associated with thousand kernel weight in bread wheat.Moreover,we found that TaGS5-A1 gene could express in root,stem and leaf,but its relative expression level was significantly higher in stem and leaf than in root.7.In silico cloning,TaTAC1 genes were cloned from bread wheat and composed of four exons and three introns,further were physically mapped on 5AL,5DL and5 DL.In addition,a 242 bp fragmental insertion was found in-419 bp site of the promoter regions of TaTAC1-A1 gene,forming two genotypes,and further developing functional marker.Sequencing results suggested that some insertions,deletion and SNP allelic variation were identified in the promoter sequence of TaTAC1-B1 gene,and the TaTAC1-D1 promoter or coding region had no divergence.8.Analysis of association of TaTAC1 alleles with agronomic traits found that cultivars with TaTAC1-A1 a genotype possessed more tiller number,larger angle and open angle of flag leaf than those of cultivars with TaTAC1-A1 b genotype,and cultivars with TaTAC1-B1 a genotype also showed more tiller number,larger angle and open angle of flag leaf than those of cultivars with TaTAC1-B1 b genotype in 2014.Genotype interaction analysis between TaTAC1-A1 and TaTAC1-B1 showed additive effects between the favorable haplotypes.9.Studies on relative expression levels of TaTAC1 gene under abiotic stress indicated that expression levels of both TaTAC1-A1 gene and TaTAC1-B1 gene were increased under drought(PEG 6000)and salt(Na Cl)stress,while expression levels of TaTAC1-A1 gene and TaTAC1-B1 gene were firstly increased and then decreasedunder high temperature(42 ℃)and low temperature(Na Cl)stress.It suggested that TaTAC1 gene positively responded to drought and salt stress.10.Using TILLING technology,a series of silencing mutants were constructed to enable TaTAC1-A1 gene expression to terminate in advance.Phenotype analysis of wild types and mutants of TaTAC1-A1 gene found that tiller number,angle and open angle of flag leaf of wild types were significantly larger than those of mutants.The results were consistent with the association results of TaTAC1 alleles with agronomic traits.