Fine Mapping Of The Powdery Mildew Resistance Gene Pm6 And Molecular Cloning Of Its Candidate Genes In Common Wheat

Powdery mildew, which is caused by Blumeria graminis f.sp. tritici (Bgt), is one of the most devastating diseases of wheat worldwide. This biotrophic fungus seriously affects grain yields in wheat production. Compared with the application of fungicides, using resistant cultivars is the most effective and environmentally safe approach to control the disease. To date,60 powdery mildew resistance genes/alleles have been reported in wheat, but only the Pm3 gene has been cloned. The cloning of more Pm genes will be helpful both for improving powdery mildew resistance by a transgenic approach and for understanding the mechanism of resistance at the molecular level. The tetraploid Triticum timopheevii (2n=4x=28, genome AAGG) is a valuable resource for powdery mildew resistance. Pm6 has been transferred from T. timopheevii into hexaploid common wheat, and successfully used in wheat breeding for powdery mildew resistance. Virulence matching the Pm6 gene has occurred in many regions; nevertheless, Pm6 is still effective at the adult stage in the field, especially when it is used in combination with other Pm genes such as Pm2. Breeders are now paying more attention to the adult-plant resistance due to its broad spectrum and durability. For the better utilization of the Pm6 gene in wheat breeding, in the present research, we will:[1] develope more molecular markers closely linked with Pm6 gene based on comparative analysis of Triticeae EST and the genomes of model crop, and fine map the Pm6 locus; [2] characterize the collinearity of the Pm6 region in wheat genome and those in the genomes of model crop; [3] identify candidate genes of Pm6 in the target region, and clone the candidate genes in common wheat. The main results obtained from this study are as follow:1. Collinearity-based marker mining for the fine mapping of Pm6The genome sequences of rice (Oiyza sativa L.) and Brachypodium distachyon and the comprehensive Triticeae EST (Expressed Sequence Tag) resources provide invaluable information for comparative genome analysis. Pm6 was previously mapped to the wheat chromosome bin of 2BL [fraction length (FL) 0.50-1.00] with limited DNA markers. In this study, we saturated the Pm6 locus in wheat using the collinearity-based markers by extensively exploiting these genomic resources. All wheat ESTs which located in the bin 2BL FL 0.50-1.00 and their corresponding orthologous genes located on rice chromosome 4 were firstly used to develop STS (Sequence Tagged Site) markers. Comparative mapping in the two F2 populations derived from crosses of IGVI-465 X Prins (including 1,816 individuals) and IGVI-466 X Prins (including 891 individuals) found that, eight STS markers (CINAU117, CINAU132, CINAU133, CINAU135, CINAU136, CINAU139, CINAU142 and CINAU144) were closely linked to Pm6. Two of them, CINAU117 and CINAU139, could flank the Pm6 locus in the IGVI-466/Prins population. These eight markers that linked to Pm6 were then used to identify the collinear regions in the genomes of rice and Brachypodium. Triticeae ESTs containing these orthologous genes in these collinear regions were further used to develop new conserved markers and COS (Conserved Orthologous Set) markers for the fine mapping of Pm6. Totally, we mapped 29 markers to the Pm6 locus. Among them,14 markers were co-segregated with Pm6 in the IGVI-466/Prins population. Comparative mapping in the two populations showed that two conserved markers CINAU123 and CINAU127 flanked the Pm6 locus.2. Collinearity analysis of the Pm6 region in wheat with those in rice and Brachypodium, and identification of the candidate genes of Pm6All the corresponding EST sequences of those identified Pm6-linked markers were used as queries to perform a BLAST search against the genome sequences of rice and Brachypodium,89.6% and 79.3% of these ESTs had their orthologs in chromosome 5L of Brachypodiun and chromosome 4L of rice. The collinear region of these 29 linked markers covers a～5.6-Mb region in chromosome 5L of Brachypodium (from 21,159,780 bp to 26,682,408 bp) and a-6.0-Mb region in chromosome 4L of rice (from 27,802,151 bp to 33,785,309 bp). The marker order is conserved between rice and Brachypodium, but re-arrangements are present in wheat. From the orthologous regions of Pm6 (flanked by two markers CINAU123 and CINAU127), an LRR-receptor-like protein kinase (LRR-RLK) cluster was identified. It was deduced that the identified LRR-RLK cluster in the orthologous regions of rice and Brachypodium might be the candidate orthologous genes of Pm6.3. Cloning of candidate genes of Pm6 in common wheat In order to clone the LRR-RLK gene in wheat genome, coding sequences of the LRR-RLK cluster in rice and Brachypodium were compared with their orthologous Triticeae EST to identify conserved extron regions. Conserved primers were designed based on the identified conserved extrons, and were firstly used for amplification and cloning of the partial sequence of LRR-RLK in IGVI-465. Then, the partial sequence was used to designed primers. By RACE strategy, the full-length ORF of LRR-RLK gene was isolated from IGVI-465 and was designated as TaLRR-RLK. Sequence analysis indicated that its ORF was 3045 bp, coding a protein of 1014 amino acids. The primary structure of the putative protein TaLRR-RLK contained a signal peptide,11 leucine-rich repeats motifs in the extracellular domain, a transmembrane domain, and a predicted intracellular serine-threonine kinase domain. Subcellular localization assay showed that the fusion protein of TaLRR-RLK and the green fluorescence protein (GFP) were mainly targeted to the plasma membrane and nucleolus. Both semi-quantitative RT-PCR and Q-PCR analyses showed that, the transcript level of TaLRR-RLK increased in the leaves of IGVI-465 from the second leaf stage (susceptible stage) to the fourth leaf stage (resistant stage), whereas in the susceptible line Prins, the transcript level of TaLRR-RLK showed no significant difference between different growth stages. The expression pattern of TaLRR-RLK in the Pm6-carrier IGVI-465 was correlated with the Pm6-mediated resistant phenotype, and which indicating that TaLRR-RLK was related to the Pm6-resistance. Moreover, transcript levels of TaLRR-RLK increased in response to Bgt during the compatible and incompatible recognition. In addition, the expression of TaLRR-RLK was also up-regulated by salicylic acid (SA), suggesting that that TaLRR-RLK may be involved in defense response to Bgt via a SA signaling pathway.4. Cloning of a conserved receptor protein kinase (TaRPK) gene near the Pm6 locusIn previous studies, an RFLP probe BCD135 was found to be closely linked with Pm6, and the probe sequence was highly conserved among several species including barley, wheat, rye, and rice. Park et al. (2004) revealed the presence of two conserved genes at the BCD135 region of barley and rice. The first conserved gene was a putative unknown protein. Based on this gene sequence in barley, Ji et al. (2008) developed two STS markers which were closely linked to Pm6. The second conserved gene was a putative receptor protein kinase (RPK). RPK showed highly conserved between dicotyledon and monocotyledon species. In this study, the barley sequence of RPK (HvRPK) was used to design STS markers. These markers were used to amplify a series of wheat relative species and the alien addition lines of these species in the background of wheat variety’Chinese spring’. The results showed that these STS markers produced specific fragments in all the addition lines involving the groups 2 chromosomes, such as chromosome 2G of T. timopheevii,2R of Rye,2H of barley,2S of Ae. speltoides,2S1 of Ae. longissima,2Mg of Ae. geniculata,2SP and 2UP of Ae. peregrine, respectively. We also found the STS markers based on the RPK gene was closely linked to Pm6 in the two F2 populations, i.e. IGVI-465/Prins and IGVI-466/Prins. All these indicated that RPK gene should be a conserved marker gene for homoeologous group 2 chromosomes of wheat and its relative species.By screening the BAC library of wheat variety Xiaoyan 54 using the primer pair STSrpk-FI/R4, the full-length DNA sequence of RPK gene was firstly isolated and named as TaRPK. Base on the TaRPK sequence obtained from Xiaoyan 54, we further cloned the full-length of DNA and cDNA sequence of the gene in IGVI-465, and named as TaRPK1. Sequence analysis indicated that the ORF of DNA and cDNA of TaRPK1 was 1650 bp and 1275 bp, respectively. TaRPK1 contains 4 introns and 5 extrons, and codes a protein of 424 amino acids. The primary structure of the putative proteins TaRPK1 contained a signal peptide, a transmembrane domain but lack of classical extracellular structure, and a predicted intracellular serine-threonine kinase domain. There are three copies of TaRPK1 present in the introgression line IGVI-465, which are from chromosomes of 2A,2D and 2G. However, in IGVI-465 only the copy TaRPK1-2G from the introgressed chromosome segment of 2G (1650 bp) was actively transcriped. TaRPK1-2G was an alternative splicing gene, and produced two different transcripts through alternative splicing. Subcellular localization assay showed the fusion protein of TaRPK 1-2G TaLRR-RLK and the green fluorescence protein (GFP) were mainly targeted to the plasma membrane and nucleolus. Q-PCR showed that the transcript level of TaRPK1-2G was up-regulated by methyl jasmonate (MeJA).