Chestnut rose(Rosa roxburghii Tratt),belonging to Rosa genus of Rosaceae family,is a new promising fruit crop in China due to its fruits having high content of vitamin C and displaying high levels of superoxide disomutase (SOD) activity,which can delay senescence and prevent cancer.However,powdery mildew disease is common in the production area,especially when large areas of chestnut rose are cultivated.Damage caused by powdery mildews can be stunting and distort leaves,buds,growing tips,and fruit,the symptoms become more serious in areas with high humidity and hot weather.Powdery mildew resistance breeding becomes one of the most important goals for various economical crops within Rosaceae family; understanding the molecular mechanism underlying powdery mildew resistance in a relative wild crop would be beneficial to the molecular breeding of disease resistant cultivars of fruit and ornamental crops in Rosaceae family.Using the powdery mildew resistant and susceptible genotypes as materials,phytopathological,cytological,genetics,and genomics researches were carried out to understand the mechanism involved in powdery mildew resistance in chestnut rose.The main results are as following:1.The annual life-cycle of powdery mildew fungi and the shapes of fungi in different stages,the host-microbe interaction,and the expression of defense-related enzymes upon powdery mildew infection were studied.The experiments included the observation or determination of conidiophores,conidia,ascospore,hyphae and ascocarps.Moreover,H2O2 were observed accumulating near around the hyphae attacking sites,and the callose was detected by fluorescence analysis.Chitinase and glucanase were significantly responded to the innoculation of powdery mildew.The biological habits and morphological characterization of powdery mildew fungi in chestnut rose studied herein provided evidence for the correct classification of this fungus.2.One hundred and twenty six resistance gene analogs (RGAs) were cloned from chestnut rose genome.The RGA genes,clustered in the genome,are rapidly evolved,meiotically instable,and evolutionarily complex.The reasons for these characteristics and the generation of new resistance specificity could be the positive selection,balancing selection,recombination,point mutation,and even transposable elements.From chestnut rose,96 resistance gene aanalogues (RGAs) were cloned and characterized,of which 34 were derived from resistant parent,30 from susceptible parent,32 from F1 progeny.Comparison revealed that the nucleotide similarity between the resistant and susceptible parent is averaged at 54%,higher than that within resistant parent.Phylogenetic analysis divided these 96 genes into two groups; one group showed high homology with non-TIR resistance genes,the other is highly homologous to TIR resistance gene.Genetic mapping divided these 96 genes into 3 linkage groups:the biggest group contained 23 genes,designated as CR1; the second one contained 12 genes,all were from susceptible parent; the last group CR3 contained 6 genes.Comparing the positions of genetic map with phylogenetic tree,the RGA gene from a phylogenetic clade tended to cluster in genetic map,which was caused by tandem duplication and diversification.The other case is RGA genes from different clades clustered together in the genetic map and formed heterogeneous cluster,this might caused by ecotopic recombination.From the parents to F1 progeny,deletion of RGA gene’s fragment was observed,which may related with meiotic instability.Using TAIL-PCR strategy,a retrotransposon-like gene flanking RGA gene was isolated,Southern analysis revealed that the copy number of retrotransposon-like gene is relative large with great difference among different materials.Based on the published RosaceaeRGA genes from chestnut rose,apple,peach,pear,strawberry,apricot and plum,were comparatively analyzed on genus and species level.The synteny of RGA gene between different genus were discussed,and the 125 H site was identified to be under positive selection by evolutionary prediction analysis.This is the first report of using overlap extension strategy for target isolation of RGA genes in plants; this method can overcome the problem of bias amplification of degenerate PCR.Also this is the first report of meiotic instability for RGA gene.3.The downstream components of plant immunity system in chestnut rose,including the PTO-like protein kinase and defense-related genes,were investigated.The immunity related genes mostly exist in the genome as gene family.Among the members,single nucleotide polymorphisms (SNPs) are more prevalent than other sequence polymorphisms.Several members are obviously responded to the powdery mildew attack.From the resistant cultivar,30 defense-related genes (DR genes) were cloned,including 9 PTO-like kinase genes,21 pathogenesis-related genes (12 PR2 genes and 9 PR5 genes).The polymorphism of gene family was mainly composed of single nucleotide polymorphisms (SNPs).The average frequency for PR5 gene was one SNP per 59 bp,64 bp for PR2 genes.Based on the SNPs,SNAP markers were developed，23 primer pairs were designed and 17 markers were finally mapped in F1 population.Reverse Northern revealed that all PR2 genes were not induced significantly after innoculation,while one PR5 gene’s expression was significantly enhanced.The immunity related genes in chestnut rose genome from upstream to downstream,i.e.R gene→STK gene→PR2 gene,were predicted to be involved in a co-evolution system.4.Highly expressed genes activated by powdery mildew pathogen attack were cloned and characterized.Suppression subtraction hybridization (SSH) library which enriched powdery mildew responded genes was constructed,and reverse Northern technology was used to screen the clones from the library.Sequencing the differentially expressed clones revealed many genes highly homologous to resistance-related genes reported previously,such as PR10、P450、STK-like kinase gene,Cf-9-like,LRR receptor-like genes,transcription factor NAC gene,and even the transposon elements.The most noticeable genes appeared in the library is photorespiratory-related genes,such as ribulose bisphosphate carboxylase (Rubisco),Rubisco activase,glyoxylate aminotransferase,glutamine synthetase,glyceraldehyde dehydrogenase,ferredoxin,transport protein,etc.Real-Time PCR were used to verify the three most important photorespiratory genes,which revealed that expression levels of Rubisco activase,glyoxylate aminotransferase were significantly enhanced after innoculation.The enzymatic activities of these three photorespiratory genes were also verified to be enhanced significantly after innoculation by spectrophotometric analysis.For example,the transcription level of aminotransferase gene in powdery mildew infected material was as high as 40 folds of that in control material,and the enzymatic activity of aminotransferase in treated material was 25 folds higher than that in control material.Genetic mapping showed that the three photorespiratory genes exist in the chesnut rose genome as gene cluster.The full length cDNA of these three genes were further obtained by RACE strategy.Moreover,the DNA regions of these genes among the genomes of chestnut rose with different genotypes and rosaceae fruit crops were compared.Interestingly,the sequence of ChrRBCs in Guinong No.6,which is highly resistant to powdery mildew,is identical with that in Wuzi Cili,a genotype immune to powdery mildew.5.A new mechanism for powdery mildew disease resistance in plants,i.e.photorespiration,was proposed.Photorespiratory genes are not only highly responded to powdery mildew pathogen attack with the transcription and the enzyme activites highly induced; but also,we found that the transcriptions of photorespiratory genes are significantly induced by resistance signal salycilic acid,and the salycilic acid with another signal peroxide was accumulated significantly in the powdery mildew infected samples.Altogether,photorespiratory gene may be a kind of new resistance gene; therefore photorespiration maybe a new mechanism for plant to defense against pathogen attack were proposed. We are now carring out a further research on Arabidopsis to verify the new function of photorespiratory genes from chestnut rose,and to investigate the molecular mechanism of photorespiratory gene functioned in powdery mildew resistance.