Screening And Cloning Resistance Related Genes To Powdery Mildew In The Haynaldla. Villosa Using Barley Genechip

Wheat (T.aestivum L) is one of the most important cereal crops in the world. However, the production of wheat is adversely affected by a number of biotic and abiotic stresses. Wheat powdery mildew disease, which is caused by Erysiphegraminis DC, is one of the most serious wheat diseases in China and many other countries of the world. Identifying more resistant genes to powdery mildew disease and studying the resistance mechanism become more and more important. Pm21 gene, which is located on the short arm of chromosome 6V of H. villosa, is a broad-spectrum and powerful resistant gene to the powdery mildew disease. So, on the one hand, studying the transcription factors, signal transduction pathway and important defense genes involved in the resistance progress will be valuable to investigate the mechanism of the broad-spectrum resistance; on the other hand, isolating the resistance gene will facilitate improving the level of resistance to powdery mildew in wheat through genetic engeneering.Wheat-H.villosa translocation line (6VS/6AL) has been widely used in wheat breeding project as genetic material and some varieties containing the 6VS/6AL chromosome are now playing an important role in the wheat production. Identification the status of 6VS/6AL chromosome is tightly associated with the efficiency of the marker assisted selection. Because the marker published in the past years are all dominant markers, it is helpful to develop codominant PCR marker linked with Pm21 for the wheat breeding. Owing to the advantages of H.villosa, many genetic materials have been developed through molecular cytogenetic engineering. If a PCR marker is developed which can be used to identify the chromotin of H.villosa in the common wheat backgroud, the efficiency of screening will be improved dramaticly.1. Identification of Genes Involved in the Resistance to the Powdery Mildew and Investigating the Resistance Mechanism of Pm21 in Haynaldia villosa Profiling Using Barley GenechipThe inoculated resistant and susceptible mutant of H.villosa and the uninoculated resistant H. villosa were used to study the resistance mechanism of Pm21 to the powdery mildew by microarray analysis using the Barley1 genechip. About 5471 probes could hybridize with Haynaldia villosa genes in each split. Comparison of transcript profiling of inoculated sample with that of the uninoculated sample of resistant H.villosa, genes induced by Erysiphegraminis DC were identified, including pathogen related proteins, defense genes, transcription factors, signal transduction components and resistance gene analogs. Transcript profiling of the inoculated sample of the resistant H.villosa was also compared with the inoculated sample of the susceptible H.villosa mutant, the differentially expressed genes were screeded. The information of these genes indicated that salicytic acid and ethylene signal transduction pathway involved in the resistance defense pathway in resistant H.villosa, while jasmonic acid signal transduction pathway was induced instead of the salicytic acid signal transduction pathway in the susceptible H.villosa mutant. Salicytic acid signal transduction pathway may be the most effective in the powdery mildew resistance pathway of H. villosa.2. Cloning of resistance related geneAccording to the information of the upregulated genes, a few probes were analysed in detail in the following study. The expression level of the probe Contig17515 in the inoculated resistant H.villosa was higher than that in the uninoculated resistant H.villosa and than in the inoculated susceptible H.villosa. A pare of primers were designed based on the sequence of the Contig17515 and a putative serine/threonine kinase gene, named as Hv-S/TPK (cDNA), was cloned by RT-PCR. The complete sequence of Hv-S/TPK (cDNA), was obtained by RACE protocal and this gene was located on the short arm of the 6V chromosome. Hv-S/TPK (cDNA) contains 1376 nucleotides and the putative protein contains two conserved domain of serine/threonine kinase gene. The gene, named as Hv-S/TPK (gDNA), was cloned from the genomic DNA of H. villosa using the same primers. The results of southern blotting and the sequence comparison indicated that the Hv-S/TPK (gDNA) from the 6VS of H. villosa and Ta-A-S/TPK (gDNA), Ta-B-S/TPK (gDNA), Ta-D-S/TPK(gDNA) from the 6AS, 6BS and 6DS of common wheat were ortholog genes. Hv-S/TPK (gDNA) could express in both in the H. villosa and in the translocation line 6VS/6AL, more over the Ta-B-S/TPK (gDNA) and Ta-D-S/TPK(gDNA) could also espress in translocation line. According to the protein sequence of the gene Hv-S/TPK (cDNA),Ta-B-S/TPK(cDNA) and Ta-D-S/TPK(cDNA), four differences were detected one of which involed the difference between the hydrophobic amino acid and the polar amino acid. When the PCR was conducted using the genomic DNA of F₂ population derived from Yang5×92R137 as templates, it was found that the plants resistant to powdery mildew disease had the 902bp amplicon specific to H.villosa, while the susceptible plants didn't. Further analysis indicated that the resistant plants of F₂ population could be divided into two types: one type of plants containing one 6VS/6AL chromosome and these plant could amplify the 984bp fragment from the 6AS; another type of plants containing two 6VS/6AL chromosomes and these plants couldn't amplify the 984bp fragment. So, a codominant PCR marker Nau/xibao15₉₀₂ was developed in this study. Since only fragments belonging to the group 6 chromosomes coule be amplified using 17515F and 17515R as primers, PCRs could be conducted using this pare of primers to detect whether the chromosomes added or translocatcd into other different species backgroud belong to the 6 homoeologous group.The probe Contig3156 was mostly upregulated by the inoculation with Erysiphegraminis DC in the resistant and susceptible H.villosa, and its expression level in resistant 92R137 was also higher than that in the susceptible Yangmai5. Contig3156 was a putative OxOLP gene (Oxalate Oxydase Like Protein, OxOLP). The result of RT-PCR indicated that the OxOLP was induced by the Erysiphegraminis DC in all four plant matierials, but the expression level of OxOLP was higher or the OxOLP was induced earlier in the resistant plant than in the susceptible plant. The complete sequence of the Hv-OxOLP in the H.villosa was cloned by TAIL-PCR. The whole gene contained one intron and two extrons, and the ORF contained 690 nucleotides. In the promoter of Hv-OxOLP, there was a TATA box and two W-box. Following the stop code, there was a polyA signal motif. Because the gene OxOLP was upregulated by Erysiphegraminis DC both in the resistant and susceptible plant, the gene may express during the defense response after the plant recognized the general elicite of the Erysiphegraminis DC. Owing to the character of the promoter of Hv-OxOLP, it may be used in the improving the resistance to diseases of crop through genetic engineering.3. Studying of the wheat evolution based on the sequence of the ortholog genesBecause there were differences among the sequences of the gene Hv-S/TPK (gDNA) of H.villosa and its orthologous genes in the different genomes of wheat, it was possible to study the evolution of Triticeae by analysing SNPs exsiting in the sequences from different species. The othologous genes of Hv-S/TPK (gDNA) from the different genomes of T.aestivum, T.turgidum, T.urartu, T.monococcum, Ae.speltoides, Ae.longissima, Ae.searsii, Ae.sharonensis, T.timopheevii, T.araraticum, A. tauschii were cloned and compared to study the relationship of these sequences and to investigate the evolution of the species in the Triticeae. The result indicated that the sequences of T. timopheevii and T. araraticum could be divided into one class, while the sequences of T.turgidum and T.aestivum could be divided into another. The A genome of tetraploid wheat T.timopheevii may originated from the T.monococcum, while the A genome of tetraploid wheat T.turgidum may originated from the T. urartu. The result of comparison among the sequences from the S, B, and G genomes indicated that the sequences from the Ae.searsii, Ae. longissima and Ae. sharonensis could be divided into one class, and the sequences from the G, B and the Ae.speltoides could be divided into another. T. timopheevii and T. araraticum came from a same donor and the B genome of the T. turgidum and T.aestivum came from another one. The G and B genome both may come from the S genome of Ae.speltoides. The sequence from the D genome of the T. aestivum shared high similarity with that from the D genome of the Ae. tauschii, so the above result supplied a new proof to the conclusion that the Ae. tauschii was the donor of the D genome of the T. aestivum.