Identification And Development Of A Functional Marker Of TaGW2 Associated With Grain Weight In Wheat (Triticum Aestivum L.)

China is the largest wheat producer and consumer in the world, and the production of wheat are important for our country's food security. In current national conditions, higher yield breeding is the fundamental way for enhancing wheat total production level in China. Thousand grain weight is one of the three yield components, and it was mainly determined by grain width (GW), grain length (GL) and grain thickness (GT). Larger grain weight directly correlation with yield. So, it is an important target trait during wheat breeding program. Cloning the genes associated with grain weight and its components, found the favored allele and developed a functional marker was helpful for wheat yield breeding. However, cloning genes directly from wheat by map-based cloning is very difficult and need a lot of time to perform due to its large hexaploid (2n= 6x= 42) genome and no whole genome sequence available. Based on the comparative genomics principle and methods to cloning homolog gene, which has been proved associating with grain yield in model plant, and through reverse genetics methods to study its function is a high efficient way in wheat.OsGW2 control grain width and weight in rice. In addition, this gene has the ability to accelerate the grain milk filling rate. Therefore, OsGW2 was selected as a candidate gene in this study. Its wheat homology gene, TaGW2 was cloned and characterized, the function of TaGW2 in wheat was studied, the superior alleles were detected, and functional marker for TaGW2 was developed. The main results are summarised as following.1. Among the grain weight components, there has the highest correlation between grain width and weight (r=0.822), and it has high correlation with grain thickness (r=0.703). The result indicated that through modifying the gene associated with grain width to enhance grain weight is a preferred strategy in wheat.2. The TaGW2 were isolated from A, B and D genomes in wheat, and TaGW2 were mapped on the homologous group 6 in wheat by CS nullisomic-tetrasomic lines. TaGW2 homologs to OsGW2 is >87% and >88% in DNA and AA level respectively, and the constructions of TaGW2 is same to OsGW2 which consists of 8 exons and 7 introns. TaGW2 encoding a protein with 424 amino acids, and have a C5HC2 RING motify shared with OsGW2.3. The sequences of TaGW2-6A, TaGW2-6B and TaGW2-6D in exon and intron regions were fully conserved between varieties with variable grain widths. These results implied that the grain width (weight) variation was not attributable to sequence differences in coding sequence, and that the mechanism affecting grain width and weight possibly differed from that in rice. However, relative expression levels of TaGW2 showed negative relationship with grain weight and width in immature grain. This indicated that the sequence differences in the promoter region of TaGW2 possibly associated with grain width and weight.4. About 1.2kb upstreams of coding sequences of TaGW2-6A, TaGW2-6B andTaGW2-6D were isolated from wheat. The promoter core Cis-element were predicted according TaGW2-6A sequence, the TATA box was identified at -173 and the start transcription site at -141 from the ATG initiation codon. No sequence difference was detected in the promoter region of TaGW2-B and TaGW2-D between varieties with different grain width and weight. However, In the TaGW2-6A promoter regions, the width grains varieties were -593 (A) and -739 (G), whereas those with slim grains were-593 (G) and -739 (A). The two SNPs formed a typical haplotype in the promoter region of TaGW2-6A, and, accordingly, the two alleles of TaGW2-6A were designated as Hap-6A-A and Hap-6A-G, respectively.5. The nucleotide diversities at TaGW2-6A produced a restriction enzyme TaqI recognition site (TCGA) in wide grain genotypes (Hap-6A-A) at SNP-593-A, but not in (Hap-6A-G) SNP-593G (TCGG) in the slim grain genotypes. And a cleaved amplified polymorphism (CAPS) marker was developed base on the SNP-593 to distinguish the TaGW-6A alleles.6. The function of TaGW2-6A controlling grain width and weight was identified by candidate gene association analysis. The results indicated that TaGW2, like OsGW2 in rice, was involved in grain development, mainly affecting GW and TKW. The average TGW was about 3.1g higher in varieties with the Hap-6A-A than those with Hap-6A-G, and the varieties with Hap-6A-A allele had earlier heading and maturity about 3-4 days and 2-3 days than Hap-6A-G varieties. Because Hap-6A-A had a significantly positive effect on grain size, it was considered a potentially superior allele for the improvement of grain yield in wheat. Therefore, the CAPS marker of TaGW2-6A can be used as a functional marker for grain width and weight. TaGW2-6A was located in the near centromere region on the 6A short arm in wheat, between the marker Xcfd80 and Xbarc146. Comparative localization of TaGW2-6A with other QTL which associated with grain width and weight further demonstrated that TaGW2 involved in grain width and weight development.7. Compare the frequency change between the landrace and the modern variety sub-group showed that the superior allele of TaGW2-6A has been positive selected in different wheat production regions in China, this indicated TaGW2 contributions to grain width and weight is not limited by environmental conditions. The superior allele Hap-6A-A had been a preponderant allele in Chinese wheat varieties. However, in the European varieties the Hap-6A-G was more frequent.8. In five and ten days post flowering immature seeds, the average expression level of TaGW2 was higher in the varieties with Hap-6A-G than in those with Hap-6A-A. These results further indicated that TaGW2 negatively regulated grain width and weight in wheat. The difference of Cis-element between TaGW2-6A alleles had been predicted, the Hap-6A-A with more indentified Cis-element than Hap-6A-G. Due to the SNP at the position of -2070, in the Hap-6A-A there is an Endosperm tissue-specific expression element GCN4_motif, but it is an ABA response element, ABRE in corresponding region of Hap-6A-G. However, the SNPs in promoter region causal for TaGW2 expression difference needs to be further studied in the future.