Genetic And Molecular Analyses Of The Resistance To Sclerotinia Sclerotiorum In Brassica Napus

Sclerotinia stem rot（SSR）caused by necrotrophic fungus Sclerotinia sclerotiorum is a major disease of rapeseed（Brassica napus）in China,causing 10%-20%of yield losses every year.Breeding and cultivation of resistant varieties are the most efficient and economic approach to the control of SSR in rapeseed.Unfortunately,no immune or highly resistant germplasm in B.napus and its close relatives has been identified thus far.Quantitative resistance is the most important form for Sclerotinia-resistance breeding in B.napus.Here,we performed QTL mapping,genome-wide association study and transcriptomic analysis of resistant and susceptible lines to systematically investigate the genetic and molecular basis of the resistance to S.sclerotiorum in B.napus.The main results of this study are as follows:1.Identification of QTLs for resistance to Sclerotinia stem rot in B.napusThe resistance performance of a doubled haploid population（HJ-DH）was assayed by detached leaf inoculation and stem inoculation in multiple years/environments.The heritability of the leaf resistance（LR）and stem resistance（SR）was as high as 61.01%and 68.31%,respectively.A total of 13 QTLs for LR and SR were ident ified.Among the QTLs,3 for LR were mapped on LG A3,A9 and C5,and 10 for SR mapped on 9 LGs（A1,A2,A3,A6,A8,A9,C6,C7 and C8）,respectively.Notably,a major QTL LRA9 for LR was identified in both growing seasons,which accounted for 8.54%-15.86%of the trait variation.A major QTL,SRC6,which explained 29.01%-32.61%of the phenotypic variation for SR in the three environments,was detected in LG C6.To the best of our knowledge,SRC6 is the most stable QTL with the largest genetic effect for resistance to S.sclerotiorum in B.napus reported to date.2.Comparative mapping and functional genomes were used to predict the candidate genes for the major QTL SRC6Comparative mapping of C6 linkage group with Arabidopsis based on B.oleracea genome sequences and data mining from previous gene profiling experiments anchored a candidate gene Bna C.IGMT5.a for SRC6.Molecular cloning,genetic mapping and expression analysis were performed to validate the candidate gene.Bna C.IGMT5a was located in the confidence interval of SRC6,and could be detected as an integrated gene in Hua 5（the parent with resistance allele）.The expression of the resistant Bna C.IGMT5a allele in Hua 5 increases significantly at 24 hpi（hour post-inoculation）compared with mock-inoculated control,and continues to increase from 24 to 96 hpi.3.Fine mapping of SRC6The near isogenic lines SRC6 were constructed by continuous backcross,and used for fine mapping.The genetic effect of SRC6 was confirmed both in BC₃F₂ and BC₃F_2:3populations.A 2.36 cm difference of lesion length on stem at 7 dpi between the AA and aa genotypes（based on marker at the peak）was observed.At last,we narrowed the SRC6interval to 3.7 c M（about 700 kb）by QTL mapping.4.Genome-wide association study identifies loci for resistance to SSR in B.napusThe resistance of a panel of 448 inbred lines of B.napus was assayed by detached stem inoculation in two consecutive years.The population showed a larger segregation,and the heritability of the stem resistance was as high as 61.7%.A total of 26 highly resistant lines were idetified in the population.After the 448 inbred lines were genotyped using a 60K SNP array,genome-wide association study was performed.Three significant associated loci DSRC4,DSRC6 and DSRC8 located in the C-subgenome were identified for SR,which explained 6.11%,5.61%and 5.19%of the phenotypic variation,respectively.Moreover,six potential associated loci p DSRA2,p DSRA3,p DSRA10,p DSRC5,p DSRC9a and p DSRC9b were identified,which explained 3.57%-5.04%of the phenotypic variation.This is the first report of genome-wide association study for resistance to SSR in B.napus.5.Integration analysis of QTLs for resistance to S.sclerotiorumIntegration analysis of QTLs identified in the present and previous studies were performed based on the recent release of the B.napus genome sequences.We found that93%of the QTLs could be mapped to the physical map,of which,62%were mapped to C subgenomes.Five resistance QTL hotspots were identified,including C2（0-6.7 Mb）,C6（23.2-36.6 Mb）,A9（22.1-29.4 Mb）,A2（0-7.7 Mb）and A3（0.8-6.8 Mb）.6.Transcriptomic analysis uncovers the molecular mechanisms of the interactions between B.napus and S.sclerotiorumWe performed an in-depth transcriptomic analysis to understand the differential defense responses to S.sclerotiorum in a resistance line（J964,R-line）and a susceptible line（J902,S-line）of B.napus at 0,24,48 and 96 hpi.Both the numbers of and fold changes in differentially expressed genes in the R-line were larger than those in the S-line at 48 hpi.To better understand these genes with different expression patterns or fold changes between the R-and S-lines,we introduced the concept of relative differentially expressed genes（RDEGs）.In total,5,910 up-regulated RDEGs and 3,091 down-regulated RDEGs in the R-line were identified.After monitoring the RDEGs,we found that a set of genes involved in immune responses were induced more intensely in the R-line,including pathogen recognition,MAPK signaling cascades,WRKY transcription regulation,jasmonic acid/ethylene signaling pathways,defense-related protein（PR2,PR3,PR4,PR5-like,PDF1.2b,chitinase,lectin,β-1,3-glucanase and PGIP）production and indolic glucosinolate biosynthesis.Our data also reveal that the degree of differential expression tends to become larger between the R-and S-lines along with the defensive signal transduction chains.The change of the chitinase,β-1,3 glucanase,PGIP activity and the glucosinolates content in the R-and S-lines after S.sclerotiorum inoculation were measured.We found that the activities of chitinase,β-1,3 glucanase,PGIP were indeed higher in the R-line than that in the S-line after inoculation,and indole glucosinolate content in the R-line were dramatically increased after inoculation.These results were consistent with the ones obtained by transcriptomic analysis.Thus,the differences between R-and S-line were associated with the intensity of defense responses.Interestingly,we found that the difference of defense responses between R-and S-lines at the early time of infection（before 24 hpi）may be associated with their physical barriers.The stem of R-line developed a more compact vascular and higher content of lignin in the pith,which contributes to limiting the invasion and expansion of S.sclerotiorum at the early time of infection.Moreover,we found that the endo-polygalacturonase gene（SS1G＿10167）,cellobiohydrolase gene（SS1G＿09020）and oxaloacetate acetylhydrolase gene（SS1G＿08218）and eight candidate effectors might play important roles in S.sclerotiorum pathogenicity by monitoring the transcriptomic changes in S.sclerotiorum.Thus,above results provide novel information for understanding the complex molecular mechanism of the B.napus-S.sclerotiorum interactions.