Analysis Of Genetic Variation And Powdery Mildew Resistance Of Brassica Carinata × Brassica Napus Hybrid Progeny

The damage of rapeseed powdery mildew is becoming more and more serious.Brassica napus is the main variety of rapeseed in China,but its resistance to powdery mildew is weak.A white flowered plant was found in SAO TOME of Brassica carinata,which is near immune to powdery mildew.A good powdery mildew resistance gene of Brassica carinata can be introduced into Brassica napus through distant hybridization.In this paper,the genetic variation and resistance to powdery mildew of Distant Hybridization Progenies of Brassica napus and Brassica carinata were studied by means of genetic polymorphism analysis,pathogen and leaf cytology observation,quantitative PCR detection of gene expression level and screening of transcriptome differentially expressed genes.The results suggested that:1.In order to promote the outcrossing of female parent Brassica carinata,the stamen abortion was induced by chemical hybridizing agent treatment,and the seeds of distant hybridization with the excellent cultivar‘ZS11’of Brassica napus were obtained.Five BC₁F₃ lines,named‘w7-1’,‘w7-4’,‘w7-6’,‘w8-1’and‘w8-3’,and one BC₂F₂line‘w3ps-1’were identified as resistant or moderately resistant to powdery mildew.The morphological traits（leaf shape,petal color,cuticular wax,pod length）and seed quality of most of the resistant moderately resistant progenies were similar to those of the parent‘ZS11’,indicating that the resistant genes had been successfully introduced into Brassica napus.2.The cytological process of rape leaves infected by powdery mildew was observed,and the physiological and biochemical characteristics of the leaves of resistant and susceptible lines of Distant Hybridization Progenies inoculated with powdery mildew were compared:（1）through the observation of cuticular wax and the extraction and weighing of wax,it was found that needle like wax and more wax content were helpful for the resistance of leaves to powdery mildew;（2）the paraffin section of leaf cross section was observed The results showed that the resistant materials had dense and regular palisade tissue and regular spongy tissue,which might be conducive to the resistance of leaves to powdery mildew infection;（3）the observation and determination of callose in leaves showed that the distribution of callose fluorescent spots in resistant leaves was lower than that in susceptible varieties at different stages after inoculation,and the distribution of callose fluorescent spots in resistant varieties was lower than that in susceptible varieties after inoculation for two weeks.The relative expression trends of cals5 and cals12 were different in susceptible and resistant materials.3.The information of Mildew Locus O in Bn Mlo gene family members that negatively regulate powdery mildew resistance was screened from the database of Brassica napus.The phylogenetic tree was constructed,and the structural patterns of Bnmlo genes and proteins were identified.The expression trends of Bnmlo2,Bnmlo6 and Bnmlo12 in resistant and susceptible Brassica napus were detected.4.A total of 465 differentially expressed genes（DETs）related to disease resistance were identified by comparing the folia transcriptomics of susceptible（Group S）and resistant（Group R）plants at the peak stage of powdery mildew infection.Among them,303DETs in Group R expressed more than those in group S,while 162 in Group R expressed less.These dets are mainly concentrated in the aspects of epidermal defense,lipid metabolism,expression of R protein,receptor protein kinase and regulatory factors,among which the high expression in leaves of Group R is mainly:genes encoding the proteins related to waxiness,chloroplast and cell wall metabolism,such as kcs6,csp41b,RWA,callose synthetase 3,pectinase 9,fructosidase 2,9s-lipoxygenase Lox2,etc.;Protein kinase including RKL,ERECTA,BAK1,BAM2,Lys M receptor like kinase,lipid transfer protein kinase ERl1 and ERl2,which recognize pathogens and trigger PTI immunity;and broad spectrum powdery mildew resistance proteins RPW8,calmodulin Mlo2,PMR5,MLP328,EDR2,RPS4 and RPS6,etc.In group S,pectinesterase,cytochrome CYP81f2,13s-lipoxygenase,cysteine rich receptor protein kinases and serine/threonine protein kinases such as mitogen activated protein kinase MEKK,RLK6,CRK45,APK1,BRl3,WAK1,WAK10,etc.,and TIR-NB-LRR receptor like proteins R1M1,DSC1,DSC2,course related protein RP-1,etc.were the most expressed genes.The expression of several copies of genes encoding protein families in group S was higher than that in resistant plants,such as Mlo2,EDR2,EDR4,RPW8 like protein HR3;the expression of E3 ubiquitin transferase,ATP hydrolase,RLP23,NPR3,NPR4,detoxification protein（EDS5）,WRKY transcription factor family such as WRKY53,WRKY70 in S group was higher than that in R group.Based on these evidences,the transcriptional regulation network of resistance response to Powdery Mildew in Brassica napus was constructed.The results of this study accumulated technical data for interspecific distant hybridization to improve the adaptability of rapeseed varieties,provided the basis for breeding of resistance to powdery mildew,and preliminarily revealed the genetic regulation mechanism of cell activity and defense response related to the invasion and expression of resistance to powdery mildew.