Genetic Mapping And Transcriptome Analysis Of Gummy Stem Blight Resistance In Cucumis Sativus-hystrix Introgression Line IL77

Gummy stem blight(GSB)caused by fungus Didymella bryoniae is widely distributed in Cucurbit crops and infects stem and roots,leading to rotten leaves and dead vine.Cucumber(Cucumis sativus L.)is an important horticultural crop and very popular in China with a long history of cultivation.However,the stable resistance to GSB in cultivated cucumber is absent due to the narrow genetic base,which restricts the resistance breeding.Wild species of Cucumis exhibits good resistance to disease hence interspecific hybridization was adopted to explore and utilize these valuable genes for resistance improvement in cucumber.Cross and backcrosses between Chystrix and C.sativus were perfored and introgression lines were generated.By inoculation with D.bryoniae,the IL77 was selected for stable resistance to GSB.By constructing genetic linkage map,joint traditional QTL mapping and bulk segrant analysis was conducted and made it more quickly to discover resistance genes and promoted the breeding work.Transcriptome sequencing can be helpful in gene expression analysis in the whole genome level and screening differentially expressed genes(DEGs)for resistance in cucumber.Therefore,RNA-seq analysis will play an important role in clarifying the related resistance mechanism.In this study,traditional map-based cloning and RNA-seq were conducted to detect resistant genes to GSB and related mechanism in cucumber,results are as follows:1.Mapping of major QTL for GSB resistanceA linkage map containing SSR and Indel markers was constructed with IL77 and 8419 for parental lines and F2:6 population for the progeny.SSR primers were derived from the sequence information of ’9930’ and ’Gy14’ while Indel primers were from the resequencing of IL77 and 8419.The polymorphic analysis of 1335 pairs of SSR primers on 7 chromosomes and 31 pairs of Indel primers on Chrl was carried out in parental lines and F2:6 population respectively.A linkage map containing 137 SSR and 6 Indel primers was obtained.The longest linkage group is on Chr6 while the shortest on Chr1,the primer number of each linkage group is between 11～35.According to the phenotypic statistics in 2017 spring and autumn,1 major QTL was detected with LOD 2.0 as threshold,the QTL was between SSR22637 and UW083739 in Chrl with LOD 2.5 and 2.3 respectively,which could explain the phenotypic variation rate of 12.1%and 11.3%respectively.Genetic distance was 7cM and the QTL was between 22.8Mb-27.5Mb.Joint QTL-seq and traditional QTL analysis delimited resistant gene to Chr1 24.6-27.1Mb.2.Transcriptome studies on the related pathway of resistance to GSBInoculation experiment was performed in resistant line IL77 and susceptible line 8419 and samples were prepared after 0/2/4/6 days post inoculation respectively.By comparative analysis of R2 VS R0,R4 VS R0,R6 VS R0 in IL77 and S2 VS S0,S4 VS S0,S6 VS S0 in 8419,DEGs for up or down regulation all the time were selected and validated by qRT-PCR.Finally 23 genes were screened for the participation of quorum sensing,NOD-like receptor pathway or phenylpropanoid biosynthesis pathway in IL77 or 8419.Therefore,the 23 genes were chosen as the candidate genes for GSB resistance.3.Screen and identification of candidate genes for GSB resistanceWith traditional QTL mapping and BSA-seq conducted,a comparison with the reference genome 9930 in 24.6-27.1Mb in Chr1 was performed,a total of 15 non synonymous mutations in the exon region were detected and 13 genes were included.According to the Cucurbit Genomic Database annotation,Csa1G654870 caught our sight for encoding the Dicer-like protein,and the homologous gene AT3G03300 in Arabidopsis thaliana functions in the antiviral silencing response in turnip-crinkle virus-infected plants and substitutes for DCL4 to produce viral siRNA when DCL4 is missing or inhibited.Furthermore,the nucleotide sequence similarity is 55.7%while protein sequence similarity is 61.6%between Csa1G654870 and AT3G03300.Both the high sequence similarity and participation of encoding Dicer-like protein make Csa1G654870 a candidate gene for GSB resistance.By RNA-seq,23 genes were related to disease resistance for participation in quorum sensing,NOD-like receptor pathway or phenylpropanoid biosynthesis pathway in IL77 or 8419.Based on the research above,genes including Csa1G654870 and the others involved in resistance pathway were considered as candidate genes for GSB resistance in IL77.