| Wheat stripe rust, as a leave fungal disease caused by Puccinia striiformis West.f.sp.tritici, is a worldwide wheat disease and seriously threats to wheat yield and quality, especially in China. Breeding and cultivating resistant varieties have become the most economical and effective method. However, with the continually appearance of new stripe rust physiological race, especially V26, most disease-resistance wheat varieties lose their resistance features. Exploring and precise positioning of the molecular marker linked to disease-resistance genes, not only lay a basis for disease-resistance gene cloning and molecular marker assistant breading, but also brings the theoretical and practical value to inheritance and breeding research of resistance to stripe rust disease. In addition,wheat defense response is a complicated progress regulated and controlled by multi-genes. It will provide the scientific basis for understanding the molecular mechanism of interacting between host and stripe rust pathogen in defense response through the relative regulated gene cloning, expression and function analysis.Chuannong 19 （CN19）, a disease-resistant wheat cultivar with stripe rust disease-resistant gene Yr41, was used as research materials. Mianyang 11（MY11）, a disease-susceptible wheat cultivar, was used as the control. CN19/MY11 and Myll/CN19 F1, F2 and F2:3 genetic populations and high generation recombinant inbred lines were fabricated. Disease-resistance identification and genetic analysis were carried out after CRY32 inoculation in the field and greenhouse. New EST-STS and SNP primers were developed with genome comparative analysis method and RNA-Seq sequencing technique. The precise genetic linkage map of Yr41 gene was drawn with those primers above which are newly desighed. Through realtime PCR（Q-RT-PCR） and virus induced genetic silence （VIGS） method, the gene expression characteristics and functions of two genes TaLHY and TaNIT induced by stripe rust pathogen were investigated. The main results are as follows:1. Genetic analysis and gene precise linkage map were conducted on Yr41 in the research..The strip rust disease-resistant genetic analysis was studied by the prepared research groups involving of hybridized generation material F1, F2 and F2:3 from CN19/MY11, further confirming that the gene Yr41 in wheat CN19 is a dominant stripe rust resistant gene.3212 F2 individuals was used as a fine genetic mapping population. A high generation recombinant inbred lines from CN19/MY11F2 were reproduced.127 pairs of EST-STS primers were designed through EST sequence published and located on 2BS in wheat. By using F2:3bulked segregation analysis grouping method （BSA）,8 pairs of EST-STS markers closely linking to Yr41 gene were obtained. The linkage relationship with target gene were calculated via Kosambi mapping function. The locus order was BE444541-BE474133-BE604911-Yr41-BE44606-BE446068-BE444659-BF4 78477-BI479116- BE496167, with genetic distance of 0.83、0.19、0.06、0.19、0.06、 0.44、0.19 and 0.32 cM, respectively. The result showed that the linkage marker genetic distance reduced from 8.9 cM to 0.25 cM and the smallest marker distance to Yr41 is 0.06 cM, which further locate Yr41 in 2BS3-0.84-1.00 region.2. The homologous relationship analysis of the 8 pairs of closely linked EST sequence with wheat genome sketch, B. sylvaticum Beauv genome, rice （Oryza sativa L.） genome, and corn （Zea mays L.） genome were operated by using Blast localized program. Basing on the collinearity segments,201 pairs of primers were designed. However, the selection results of acrylamide gel electrolyte show that these markers have no linkage relationship with Yr41. Using bioinformatics method to annotate the corresponding gene these 8 sequences, the results show that the annotation information of anchieutectic separation EST sequence BE604911 is THO compound, which is proved as a key disease-resistance protein in Arabidopsis. Thus, the THO compound genes sequence can be further investigated as the candidate gene of Yr41.3. BSA-RNA-Seq transcriptome sequencing for 50 （fully resistance） and 50 （fully susceptible） plants of recombinant inbred lines （RILs） generation F6 were studied and 57.14Gb Clean Data were obtained. Analyzed by bioinformatics method,15,000 new wheat genes were annotated approximately and 5919 differential expression genes between disease-resistant and disease-susceptible phenotype groups. SNP locus’ correlative positioning analysis for BSR colony finds out that 52 SNP locus appear in 2BS chromosome, among which 38 SNP locus have amplification refractory mutation system primers. PCR product analyzed by agarose gel electrophoresis shows that 4 pairs of SNP locus are closed linked with Yr41. The locus order was 3523042/2963-3523042/ 3033-5186704/3797-Yr41-5245446/8902, with genetic distance of 0.06,0.06,0.06 and 0.25 cM, respectively.4.The investigation result of the expression and function of TaLHY gene primarily preliminary proofs that TaLHY gene regulate, control and participate in wheat heading and defense response towards stripe rust disease resistance. And TaLHY is a gene that has circadian rhythm whose expression up-regulates at day time and down-regulates at night. Q-RT-PCR result show that TaLHY gene expresses mainly in the leaf, ear, and stem of wheat, while less in the root. Exogenous hormones SA can significantly induce the up-regulation of this gene. Using VIGS （virus-induced gene silencing） technology to disease-resistant wheat in the fourth leaf stage, plants with silenced TaLHY cannot complete their heading stage and it is susceptible in the compatible interaction with stripe rust pathogen physiological race 32 （IT=4）.5. VIGS method was used to study the silence effect to the cloned TaNIT gene. The results demonstrated that the growth of silence and non-silence plant have no significant change to the defense response of stripe rust pathogen. |
