Identification And Powdery Mildew Resistance Evaluation Of An LRR Gene Cluster In The Short ARM Of 6V Chromosome Of Haynaldia Villosa

Powdery mildew is a worldwide disease in wheat,which caused great harm to both yield and quality of wheat.Improving resistance to powdery mildew in wheat is a long focus of attention for domestic and foreign breeders and agricultural scientists.Previous studies showed that Haynaldia villosa,an alien species of wheat,conferred broad-spectrum resistance to powdery mildew,which is controlled by Pm21 located on short arm of 6V.Since the recombination between 6VS and the chromosomes of common wheat,it is difficult to fine map of Pm21.It is not clear whether the Pm21 is a single gene or a gene cluster taking part together in the resistance to powdery mildew,so Pm21 is considered as a locus.Cytogenetics Institute in Nanjing Agricultural University cloned a key member of Pm21 Stpk-V using cytogenetics and molecular genetics technonogy.In recent years,a series translocation and deletion lines involving different regions of 6VS have been created by radiation of pollen and female gametes,and these stocks have been characterized by using situ hybridization,powdery mildew resistance evaluation and molecular marker analysis.These special cytogenetic stocks,which showed resistance or susceptibility to powdery mildew,give us valuable resources to fine map and colne the important genes in the Pm21 locus.The result of resistance evaluation,FISH and molecular marker analysis of different translocations and deletion lines involving the 6VS chromoses arm made it easy to locate the Pm21 locus to a comparatively small region.Comparative genomics study indicated a high collinearity between the resistance region and the second chromosome of rice,and there is an LRR gene-cluster in the linear region.Is there a similar LRR gene-cluster on the Pm21 locus of H.villosa?If there is an LRR cluster in this region,does it have a relationship with resistance to powdery mildew?If the the LRR cluster has a close relashionship with the resistance,which gene in the cluster play a key role in the resisitance?Based on the obove findings and assumptions,the following three researches are conducted:1.Based on the comparative genomics approach,the study to find the LRR gene-cluster in Pm21 locus was conducted.Primers were designed according the sequence of LRR gene from rice Chr.2,and the DNAs of China Spring,92R137,wheat-H.villosa amphipliod and different cytogenetic stocks were used as templates for PCR amplification.The results showed some specifc polymorphic bands could be amplified from the Pm21 locus when using some pairs of primers in.The study indicated that there was an LRR gene cluster in Pm21 locus.2.LRR gene-cluster in Pm21 locus were silenced by virus induced gene silencing to study the relationship between LRR gene-cluster and powdery mildew resistance.H.villosa has has a closer evolutionary relashionship with barley than with the rice.In order to obtain the fragment of LRR gene-cluster in Pm21 locus,the primers for PCR analysis were designed according to the LRR gene sequence of barley 6HS.The amplified sequence were used to silence the LRR gene on Nannong 9918,who containing 6VS,by VIGS,to study whether the resistance to powdery mildew was suppressed.Q-PCR results showed that the expression levels of the LRR insert fragments on leaves of virus infection leavedwere significantly declined in different extent compared with the leaves treated with water,indicating that these LRR genes have been silenced suscessfully.Leaves of genes silenced were inoculated with mixed Bgt and the the secondary hyphae werestatisticed under the microscope when the susceptible leaves used as the control were coverd with hyphae 7 days after inoculation.The results indicated that secondary hyphae in the HvLRR-12 and HvLRR-13 silenced leaves significantly increasedcompared with the Nannong 9918 mock.It was also found that in some HvLRR-12 silenced leaves,there were much more secondary hyphae,and secondary hyphae,were full of sights under the microscope,and some conidiophores could be detected.These results indicate that LRR genes of Pm21 locus have a certain relationship with resistance to wheat powdery mildew.3.LRR genes of Pm21 locus were cloned by homologous cloning,and the putative mutation were searching in a susceptible mutant of Nannong 9918.In the previous study,a susceptible mutant of Nannong 9918,NM14,was obtained by EMS treatment.In this study,the LRR fragments of Pm21 locus was cloned to examined whether the mutation of the above LRR gene-cluster cause the change in resistant phenotype of Nannong 9918.The primers for LRR gene amplification were designed based on LRR gene located in the barley 6HS,and the homologus were cloned from the wild-type and mutant of Nannong 9918 at the same time.Sequence alignments showed that PCR products of primers AK362389-PD2-44 and AK362389-PD2-1136 displayed single nucleotide polymorphism at 328bp,581bp and 959bp between sequences of Nannong 9918 and NM14.AK354248-PD1-4 and AK354248-PD1-1975 displayed single nucleotide polymorphism at 524bp,1293bp between sequences of Nannong 9918 and NM14.The relationship of the SNP and the real mutation needs to be further studied by different populations and more sequences.