| Pre-harvest sprouting (PHS) occurs frequently in China, particularly in the Middle and Lower Yangtze Valley Winter Wheat Region and Southwest Winter Wheat Region in China. Recently, PHS is getting more and more serious in Yellow and Huai Valleys Winter Wheat Region and the Northern Winter Wheat Region. PHS resistance has become an important trait in wheat breeding. The thousand grain weight (TGW) is also an important targeting trait in wheat breeding. In the present study, wheat TaSdr and TaGS genes, orthologs of OsSdr4and OsGS3conferring seed dormancy and grain size in rice, respectively, were cloned by a comparative genomics approach. Baesd on the sequence diversity, functional markers were developed for each gene. The main results obtained in this study are summarized below.1. Wheat TaSdr genes, orthologs of OsSdr4conferring seed dormancy in rice, were cloned by a comparative genomics approach. They were located on homoeologous group2chromosomes, and designated as TaSdr-Al, TaSdr-B1and TaSdr-Dl, respectively. The coding sequences (CDS) were993bp,981bp and987bp for TaSdr-A1, TaSdr-B1and TaSdr-Dl, respectively. The open reading frame (ORF) of TaSdr genes were slightly shorter than that of OsSdr4, and the similarity was-75%. Like OsSdr4there was no intron in the TaSdr genes. Sequence analysis of TaSdr-Al revealed a single nucleotide polymorphism (SNP) at the position643bp downstream of the initiation codon, with bases G and A in cultivars with low and high germination indices (GI), respectively. These two alleles were designated as TaSdr-A1a (GenBank accession KF021988) and TaSdr-Alb (GenBank accession KF021989), respectively. The SNP resulted in coding amino acid change from Leu to Pro. DNA sequencing of the TaSdr-B1locus revealed no diversity in the CDS, but a SNP was found at position-11in the promoter region, with A in genotypes with lower GI values and G in those with higher GI, and these two alleles were designated as TaSdr-B1a (GenBank accession KF021990) and TaSdr-B1b (GenBank accession KF021991), respectively. No sequence diversity was found in TaSdr-D1gene (GenBank accession KF021992).2. Based on the SNP at position643bp in TaSdr-A1gene, a cleaved amplified polymorphism sequence (CAPS) marker Sdr2A was developed. The specific1,140-bp and525-bp/650-bp bands could be used to distinguish TaSdr-Ala and TaSdr-Alb alleles. Forty-four Chinese wheat cultivars were used to validate the CAPS marker Sdr2A. Association analysis indicated no significant differences in GI between two genotypes. A CAPS marker Sdr2B was developed based on the SNP at the position-11upstream of the initiation codon. Three fragments of60,116and650bp were generated in genotype TaSdr-B1a, whereas two fragments of60and766bp were produced in TaSdr-B1b. The specific bands of650and766bp can be used to distinguish TaSdr-Bla and TaSdr-Blb. Linkage analysis showed that Sdr2B was closely linked to Xwmc477(3.2cM), an SSR marker located near the centromere of chromosome2B. A QTL for GI co-segregating with Sdr2B explained6.4,7.8and8.7%of the phenotypic variances in a RIL population derived from Yangxiaomai/Zhongyou9507grown in Shijiazhuang and Beijing, and the averaged data from those environments, respectively. Two sets of Chinese wheat cultivars were used for association mapping, and results indicated that TaSdr-B1was significantly associated with GI. Markers for the TaSdr-B1and TaVp-1B genes were used to test Set I and Set II cultivars and significant associations were identified between genotypes and phenotypes. Therefore, the combination of two sequence tagged site (STS) markers, Sdr2B and Vp1B3, could improve the accuracy of marker assisted breeding for improving PHS tolerance.3. Analysis of the allelic distribution at the TaSdr-B1locus showed that the frequencies of TaSdr-B1a were different in different countries and regions. The frequencies of the TaSdr-B1a allele in Japanese, Australian and Argentine cultivars were62.5,37.1and36.4%, respectively. No TaSdr-Bla genotype was found among cultivars from Germany, Romania, Russia, Ukraine and Serbia. In China, the proportions of TaSdr-B1a were higher in the Middle and Lower Yangtze Valley Winter Wheat Region (48.9%) and Southwest Winter Wheat Region (41.9%). The Xinjiang Winter-Spring Wheat Region had the lowest frequency of TaSdr-Bla (14.8%).4. Wheat TaGS gene on chromosome7DS in wheat, an ortholog of OsGS3in rice, was cloned by a comparative genomics approach, and it was designated as TaGS-D1. The TaGS-D1gene contained four exons and three introns. The cDNA of TaGS-D1was255bp, and it encoded85amino acids. The similarities in cDNA and deduced polypeptides of TaGS-Dl and OsGS3were75.5and72.2%, respectively. DNA sequencing of the TaGS-Dl locus revealed no diversity in the CDS, but there were a SNP in the first intron,30SNPs, a40-bp InDel and a3-bp InDel in the second intron between genotypes with higher and lower thousand grain weights. The allele with a40-bp insertion in the second intron was designated as TaGS-Dla (GenBank accession KF687956) and the other as TaGS-D1b (GenBank accession KF687957), respectively.5. Based on the40-bp InDel a co-dominant marker, designated GS7D, was developed to discriminate the two alleles, This marker amplified a562-bp fragment from the genotype TaGS-Dla, whereas a522-bp fragment was generated from TaGS-D1b. Linkage analysis with the RIL population of Doumai/Shi4185showed that GS7D was7.9cM distal to Xbarc184on chromosome7DS. QTL for thousand grain weight and grain length co-segregating with GS7D explained up to14.6and6.8%, respectively, of the phenotypic variances in a RIL population derived from Doumai/Shi4185grown in Shijiazhuang and Beijing, and the averaged data from those environments. One hundred and seventy five Chinese wheat cultivars were genotyped with GS7D, and association analysis revealed that TaGS-D1was significantly associated with grain weight. Combining the marker detections of TaGS-D1and TaCwi-A1, the relationship between genotypes and phenotypes became more significant. Therefore, the combination of two STS markers improved the ability of marker assisted breeding to increase TGW.6. The allelic distribution at the TaGS-D1locus showed that the proportions of TaGS-D1a allele were high in Serbia (90.0%), Japan (87.5%), Australia (87.1%) and Canada (83.3%). Low frequencies of the TaGS-Dla allele were found among cultivars from Germany (8.1%), and Norway (4.5%). TaGS-D1a was very frequent in Chinese wheat cultivars. Among the444Chinese cultivars,355(80.0%) carried TaGS-D1a. The frequencies of TaGS-Dla were higher in the Northeastern Spring Wheat Region (96.7%) and Northern Winter Wheat Region (87.7%). The Northwestern Spring Wheat Region had the lowest frequency of TaGS-D1a (64.3%).This study first reported the allelic variations of TaSdr and TaGS genes, and the functional markers Sdr2B and GS7D were of important value in wheat breeding. |
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