Dissection Of A Haplotype Block On 6A Associating With Development And Yield Traits In Common Wheat

Bread wheat (Triticum aestivum. L) is an allohexaploid species with extremely large and complex genome (16,000 Mbp). Grain yield is such a complex quantitative trait, controlled by multi genes and influenced by the environments. All these enhance the difficult for gene isolation. However, there is an increasing need to clone yield-related genes, exploit favorable alleles and develop functional markers for marker-assisted selection (MAS). Thus, the dissecting of molecular mechanisms of yield-related is of great interest to plant biologists and breeders. In this study, based on random association, genomic region flanked by Xgpw7592-Xcfe273 on 6A was strongly associated with yield traits. SSR markers developed based on the synteny region between rice and Brachypodium were employed for association to narrow the interval of functional segment. Several important candidate genes such as CCT domain genes, auxin response factor (ARF), transcript elongation factor (TEF), Myb transcript factor etc were detected in the two sub-regions. Expression analysis, subcellular localization and overexpression in Arabidopsis were carried out to elucidate the biological functions of the candidate genes in wheat. Moreover, functional markers were developed based on the polymorphism to distinguish different haplotypes. The main results are summarized as follows:1. The cfe273 and gpw7592 were used to screen BAC libraries of Xiaoyan54 and Chinese spring. Two clones were identified and isolated, with insert size of 160kb and 20kb respectively. According to the annotated genes on tht two positive clones, we have identified its orthologous segments in rice (Os2g04750-Os2g06640) and Brachypodium (Bd3g03467-Bd3g04730) and 124 orthologous genes were found between Brachypodium and rice. Comparative studies have confirmed the collinear relationships of orthologous segments between barley, rice and Brachypodium. Synteny analysis suggested a rearrangement between barley, rice and Brachypodium in this orthologous segment2. Twenty five SSR markers were developed based on the colinear relationship among Poaceae species and the sequenced genome of Triticum urartu and Aegilops tauschii. Two sub-regions strongly swept by selection in domestication and breeding were detected after scanning the Chinese wheat mini core collection and 348 modern cultivars with 27 markers. Haplotype/trait association analysis revealed markers were significantly associated with heading date, mature date, tiller number and 1000-kernel weight. The results of association analysis, genetic diversity analysis and LD analysis suggested that two blocks, HapB-6A-1 and HapB-6A-2 significantly associated with wheat development and grain traits, respectively. They probably contain the genes causing selection sweep.3. A cross genome map-based dissection approach and annotated by WEGO、 Blast2go and KEGG were performed to identify two CCT domain genes as a major candidate for HapB-6A-1, and the auxin response factor (Os02g04810), transcription elongation factor (Os02g04160) and MYB transcription factor (Os02g04640) appeared as the major candidate gene for the HapB-6A-2 driving growth and development traits.4. TaCCT-6A flanking sequences and ORF were isolated. No diversity was found in the coding region, but 3 single nucleotide polymorphisms and 1 InDel were detected in the promoter regions among the 20 accessions. Marker was developed based on sequence variations in the promoter regions, and two haplotypes were identified based on this. TaCCT-6A was located on chromosome 6A and flanked by markers PSP3071 and GWM570. Haplotype/trait association analysis of the Chinese wheat mini core collection revealed that TaCCT-6A was significantly associated with grain number per spike, heading date and tiller number. Overexpression of TaCCT-6A in Arabidopsis thaliana produced effects in heading date and vegetative development. Phenotyping of NILs confirmed that Hap-6A-2 potentially increases grain yield and yield-related traits. Subcellular localization revealed that TaCCT-6A protein was localized in the nucleus and its expression pattern was detected in light-responsive tissues. Moreover, apparent rhythmicity of their transcripts were observed under long-day and short-day conditions. The favored haplotype frequency showed a positive correlation with yield in a global set of breeding lines. These results suggest that TaCCT-6A is a functional regulatory factor for yield traits and provide useful information for marker-assisted selection.5. In this chapter, TaCCT-7A, a CCT domain genes member in wheat, and its flanking sequences were isolated and located on homoeologous group 7. It was presumed that recombination have occurred going with the evolution of wheat by comparing with TaCCT-6A. Sequence analysis indicated that gDNA and ORF were 6 kb and 1.2 kb respectively. Subcellular localization revealed that TaCCT-7A protein was localized in the nucleus and its expression pattern was detected in a range of tissues. This gene expressed not in all organs, but with highest expression in seeding leaf and young spikes. In addition, daily oscillation mRNA abundances of this gene was regulated by long-day and short-day. Overexpression of TaCCT-7A in Arabidopsis thaliana caused heading date four days in advance, and enhanced vegetative development significantly in wild type comparisons. These further confirmed that TaCCT-7A potentially plays an important role in determination of yield in wheat.6. TaARF was chosen as the candidate gene of HapB-6A-2, gDNA and ORF sequence were obtained by a BLAST search against the Triticum urartu draft genome sequence. TaARF was located on homoeologous group 7 and sequence analysis indicated three ORF were found between TaARF homoeologous genes mainly produced by alternative splicing mechanism. The ORF of TaARF1 was longest with 3408 bp, which contains four conserved domains. Two 9 bp InDel were found in TaARF2, which not caused frame-shift in protein translation. Compare to TaARF1 and TaARF1, a 710 bp deletion was observed in TaARF3 at the position 1004. Further analyze indicated that deletion would result in premature termination of translation and loss conserved domains Ⅳ. Subcellular localization revealed that TaARF1 protein was localized in the nucleus. This gene expressed in all organs, but with highest expression in young spikes and developing seeds overexpression of TaARF in Arabidopsis produced pleiotropic effects in vegetative development, indicating that TaARF may have effects on development of wheat.7. In this study, TaTEF-7A, a TaTEF member in wheat, and its flanking sequences were isolated. TaTEF-7A was located on chromosome 7A and flanked by markers Xwmc83 and XP3156.3. Subcellular localization revealed that TaTEF-7A protein was localized in the nucleus and its expression pattern was detected in a range of tissues. This gene expressed in all organs, but with highest expression in young spikes and developing seeds. Overexpression of TaTEF-7A in Arabidopsis thaliana produced pleiotropic effects in vegetative and reproductive development that enhanced grain length, silique number and silique length. No diversity was found in the coding region of TaTEF-7A, but 16 single nucleotide polymorphisms and InDels were detected in the promoter regions of different cultivars. Markers were developed based on sequence variations in the promoter regions (InDel-629 and InDel-604), and three haplotypes were identified based on these markers. Haplotype/trait association analysis of the Chinese wheat mini core collection revealed that TaTEF-7A was significantly associated with grain number per spike. Phenotyping of NILs confirmed that TaTEF-7A potentialy increases grain yield and yield-related traits and Hap-7A-3, the favored haplotype frequency showed a positive correlation with yield in a set of breeding lines. These results suggest that TaTEF-7A is a functional regulatory factor for GN and provide useful information for marker-assisted selection.