QTL Mapping Of Leaf Rust Resistance And Functional Characterization Of Genes Involved In Defense Response In Poplar

Poplar is the major timber tree species in China,whose production is seriously restricted by the occurrence of diseases.In this study,we aimed to identify QTLs for leaf rust resistance and characterize genes involved in defense response in poplar.In the QTL mapping study,a high-density genetic map for I-69×Xiaoyeyang was constructed by high throughput GBS?Genotyping By Sequencing?technology.The symptom of leaf rust diseased for this F₁ population was invested in 3 different environments.Combing the genotypic and phenotypic data,QTL mapping for leaf rust resistance in the F₁ population was conducted and 2 major QTLs were detected.In the functional characterization of disease resistance related genes,eight genes were identified as candidate gene.Transgenic Arabidopsis and poplar were generated for these genes and phenotyping of diseased resistance was also performed for these newly generated transgenic lines.The main findings in this study are listed as follows:1.Previously,an F₁ population,I-69×Xiaoyeyang,was constructed.A total of92,097 high-quality SNP markers were developed by stringent filtering and identification.In total,889 and 1650 SNPs formed the female and male genetic maps,respectively.To test the application of the genetic maps,QTL mapping of leaf shape was conducted for this F₁ population.A total of 9 parameters were scored for leaf shape variation in three different environments and the results showed high quality of this genetic map.In2017-2018,the symptom of leaf rust was scored for this F₁ population in 3 different environments.Combining genetic maps and measurements of leaf rust score,we mapped a total of 11 significant QTLs.The highest LOD score of all QTLs was 8.61,and that QTL explained the most?12.84%?trait variation.A total of two QTLs could be detected in two environments,and they were located in 2 genomic regions.Within one of the 2QTL regions,a R gene in Xiaoyeyang genome at the peak position of QTL,which has601 bp fragment deletion polymorphism,were found through functional annotation.According to the information of this polymorphism,we were able to design PCR primers and successfully obtain a co-dominant molecular marker closely linked to the QTL of poplar leaf rust resistance.2.Through integrated transcriptome analyses of gene expression data on jasmonic acid?JA?/salicylic acid?SA?treatments and Melampsora larici-populinainfection,943 genes in total were identified as common responsive genes?CRG?.Among the CRG,six disease resistance related genes were selected according to certain thresholds.These 6genes are Potri.005G257900,Potri.003G166500,Potri.007G099400,Potri.004G158200,Potri.006G055600,and Potri.004G161500,and they were named as XW1,XW2,XW4,bZIP44,ZI,GATA6 gene respectively in this study.In addition,two disease resistance related genes,Potri.007G123700?YH3?gene and Potri.011G070100?YH9?,were also studied as candidate disease resistance genes.We constructed a vectors of GFP?green fluorescent protein?fusion with candidate transcription factors,and carried out subcellular localization in tobacco epidermal cells.It was found that these transcription factors were localized in the nucleus.3.We constructed the overexpression vectors of the above 8 candidate genes and reduced expression vector for the bZIP44 gene.The overexpression vectors of the bZIP44and the YH9 were transferred into wild-type Arabidopsis by inflorescence infection.Meanwhile,the 8 overexpression vectors were transferred into Nanlin 895 poplar by agrobacterium-medicated transformation.After carrying out positive identification by three levels,DNA,RNA and protein assay,the positive transformation lines were successfully obtained.Additionally,two T₃ transgeeic lines of OE-bZIP44 and OE-YH9were also successfully obtained.4.Two pathogens,Botrytis cinerea and Pseudomonas syringae pstDC3000,were inoculated in Arabidopsis for disease resistance assay.The results showed overexpression of YH9 in Arabidopsis enhanced its resistance to B.cinerea,while there was no significant change for resistance to pstDC3000;overexpression bZIP44 in Arabidopsis also enhanced resistance to pstDC3000 and decreased its resistance to B.cinerea.5.Two pathogens,Botryosphaeria dothidea?stain 3C?and Alternaria alternata?strain 3E?,were used for disease resistance assay for transgenic poplar.Compared to WT,transgenic poplars with overexpression of YH9,ZI,XW4 were highly susceptible to 3C,while other tested transgenic lines did not showed significantly different to strain 3C.Meanwhile,transgenic poplars with overexpression of bZIP44,YH3 and YH9 were highly susceptible to strain 3E.Owing to limited time,there is insufficient material for some transgenic lines and we were not able to get knowledge for the resistance to 3C or 3E for all studied genes.With the conduct of this research,we successfully obtained the 2 major leaf rust resistance QTL in poplar.We also developed molecular markers closely linked to the resistance QTL region.Meanwhile,we were able to investigated the disease resistance related functions for 8 candidate genes.The results revealed some genes were involved in poplar biotic defense response.Based on this study,it deepened our understanding of poplar leaf rust resistance and the defense regulation processes.The development of molecular markers can guide molecular breeding program in future.At the same time,we also obtained some transgenic poplar material which can help us in future study.