| Common wheat is a major crop in China and increasing the yield has been pursued by breeders. Wheat yield is determined by three main components:Head Number (HN), Kernel Numbers PerEar (KN) and Thousand Kernel Weight (TKW). Increasing the TKW is one of the major strategies to elevate yield in the past seven decades. TKW was a quantitative trait that determinated by many major and minor genes. Understanding their chromosomal location, evolution, the haplotypes and genetic effects of genes controlling grain weight will promote wheat improvement. Because of the higher statistical power and dissection efficiency than QTL mapping, marker/trait association analysis and haplotype analysis of candidate gene have been widely adopted in dissecting complex traits and related genes’cloning recently.Based on the target chromosome region associated with TKW, cfd78 and cfd67were used to screen the BAC library of Aegilops tauschii (D). Two BAC clones were obtained and fully sequenced. Combined with the genome scaffolds of Ae. tauschii (D), five functional genes were predicted by FGENESH. After analysis of RT-PCR and bioinformatics, TaWOX2 and TaB3 were chosen as the candidate genes directly or indirectly influencing TKW. Marker/trait association showed that TaWOX2 and TaB3 were significantly associated with agronomic traits. Geographical distribution and frequency chang eof their haplotypes showed that they were underwent strong selection in global wheat breeding.1. Two positive clones, C20 and H8 were obtained after screening the Aegilopps tauschii BAC library. They were sequenced, assembled into 14 and 11 contigs respectively. Three high similar scaffolds were obtained after blasting BAC-C20 and H8 contigs in Ae. Tauschii draft genome sequence database. Similarity between BACs and scaffolds reached as high as 98%. Five functional genes were predicated. Four genes, C20-4 (ion exchange protein), C20-8 (WOX2),5178-8(B3) and H8-12 (CBS), were transcription factors and C20-5 was an unknown protein, In the four transcription factors, In rice, the homogenous gene of C20-8 is 0sWOX2, a gene involved in seed development. The 5178-8 possesses the conservative B3 domain. So these two candidate genes were named as TaWOX2 and TaB3. Homologous genes of TaWOX2 and TaB3 were located on chromosome 1 and chromosome 3 in rice, as well as chromosomes2 and 1 in Brachypodium.2. Based on sequence differences between genomes of the two genes in wheat, D genome-specific markers were developed and they were mapped on chromosome5D, so they were further named as TaWOX2-5D and TaB3-5D, respectively. The expression of TaWOX2-5D was constitutive.In developing seeds, expression of TaWOX2-5D in Shi4185 was significantly higher than that in Shijiazhuang8, especially at 5days after flowering.3. The length of coding sequences of TaWOX2-5D was 969bp, composed of 323aa, with two exons and one intron. Its variation mainly occurred at promoter and 3’-UTR region. Satelliterepeat variations (CT)n were detected at its propoter reion. And one SNP mutation was found at its 3’-UTR region, respectively. These variations were strongly associated with TKW, heading data, mature date and GN. TKW differences of these two alleles reached significant in all environments (P< 0.001). The cfd78 locus in promoter underwent strong selection in breeding process and the frequency of cfd78259 has been elevating but that of cfd78261 has been declining from 40 years to 90 years in last century. One dCAPs marker was developed according to the SNP mutation in 3’-UTR and was assigned as Hap-C and Hap-G for TaWOX2-5D. They were associated with plant height and TKW in 348 wheat modern varietiesin all environments (P<0.001). The Hap-G was the major haplotype (93.3%) in all ten wheat ecological zones in China. It also reached to 90.3% in 384 Europe varieties.4. The TaB3-5D was 597bp long with no intron. Four SNPs (-465bp,-888bp,237bp and 270bp) were found, two in promoter region and two in coding region. Four polymorphic dCAPs markers were developed. Six haplotypes were found in 348 Chinese modern varieties, named as Hap-Ⅰ, Hap-Ⅱ, Hap-Ⅲ, Hap-Ⅳ, Hap-Ⅴ, Hap-Ⅵ, but only five haplotypes were found in Chinese wheat mini core collection (MCC). Hap-Ⅰ and Hap-Ⅱ are the two major haplotypes. They are significantly assocated with heading date, maturing date and TKW in the landraces, with KN and maturing date in 348 Chinese modern varieties. The Hap-I of TaB3-5D mainly distributed in spring wheat zones in landraces of MCC. However, in winter wheat zones, its frequency has a significant increasing in modern varieties. The Hap-Ⅱ mainly distributed in winter wheat zones in landraces, andits frequency declined in modern varieties but its frequency is increasing in spring wheat zones. Therefore, we inferred the TaB3-5D might play an important role invernalization. Only one SNP polymorphism at -465bp was found between Shijiazhuang8 and Shi4185 in TaB3-5D. The Shijiazhuang8 type (G) has a positive effect on TKW, with increasing of 5.56 g (13.14%,2008 Zhaoxian),3.97 g (8.81%,2009 Beijing),4.7 5g (13.1%,2010 Shijiazhuang),5.39 g (13.1%,2010 Luoyang). TKW differences of these two haplotypes were at the significant level in all environments (P<0.001). Therefore, bothof the two genes underwent strong selection in global breeding. They are important target genes in breeding.5. Combining with the D draft genome sequence datebase and the BAC library of Aegilops tauschii (D), we isolated the genes of TaWOX2-5D and TaB3-5D with association mapping and comparative genome, which proved that association mapping was effective method for gene mining.6. In chromosome region of cfd67-cfd78, we isolated two genes, TaWOX2-5D and TaB3-5D, and they were underwent strong selection in breeding progress, which demonstrated there were more than two important genes in this region and they were selected simultaneously in the progress of domestication. |
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