The Gene Mapping Of Resistance To Cucumber Green Mottle Mosaic Virus And Its Mechanism In Watermelon

Cucumber green mottle mosaic virus（CGMMV）is a damaging viral disease of several cucurbit crops,particularly watermelon（Citrullus lanatus）,causing mosaic,mottle and bobbling symptoms on leaves and“blood flesh”in the fruits of watermelon,resulting in considerable loss of fruit quality and economic losses to the watermelon industry.CGMMV has been listed as a quarantine disease by many countries,including China.To date,the resistance genes and the molecular mechanism of resistance to CGMMV have not been dlucidated.In this work,a CGMMV-resistant line（PI595203,C.mucosospermus）was then crossed with a CGMMV-susceptible line（M1511-3,C.lanatus）to generate a segregating population,based on BSA-seq and KASP genotyping assay,a candidate gene was mapped.The function of the candidate gene was revealed,and according to the SNP in the candidate gene,we developed a KASP marker for screening CGMMV resistance germplasm.The main results are follows:1)WPRb was mapped as resistance to CGMMV in watermelon.Based on BSA-seq,ten non-synonymous SNPs in the target region were selected to design Kompetitive allele-specific PCR（KASP）markers for the association analysis of candidate genes with CGMMV resistance in watermelon accessions.A non-synonymous SNP（G1282A）of Cl CG04G006480 was confirmed between the resistant and susceptible lines.The BC₁ population verified this molecular marker with 86%concordance.Cl CG04G006480 was identified as WPRb by phylogenetic tree.2)Function analysis of the WPRb gene.The relative expression of WPRb was upregulated in the susceptible line and downregulated in the resistant line after CGMMV infection.When challenged with CGMMV,the wild type of Nicotiana benthamiana plants showed obvious disease symptoms,while,the nbwprb did not show any symptoms.However,a mild symptom was observed in nbwprb plants during the late period of after inoculation.Replication of CP-CGMMV was similar in the WT and nbwprb plants.In addition,WPRb targeted plasmodesmata and the nucleus.These results provided strong evidence that WPRb plays an essential role in the viral movement,but not in replication.3)The molecular mechanism of resistance to CGMMV.Cl WPRb can interact with the MP of CGMMV,as determined by bimolecular fluorescence complementation（Bi FC）and coimmunoprecipitation（Co-IP）assays.Compared to WT plants,callose accumulation was increased and the PD permeability was restricted in nbwprb plants after 13 dpi of CGMMV inoculation,and the expressions of Cal S genes were up-regulated and BG gene was down-regulated in nbwprb plants.In a conclusion,WPRb interacting with MP confers resistance to CGMMV by interfering with the viral movement via affecting callose deposition at the PD.4)CGMMV resistance selection using a WPRb-based SNP marker in watermelon.Based on the CGMMV-SNP marker,21 CGMMV-resistance germplasms were screened among 601 watermelon germplasm bank.Meanwhile,the RIL population of the resistant line and the susceptible line in this study were obtained by self-crossing for several generations.The CGMMV-SNP marker in this study and white/red flesh SNP marker were used to assist breed some varieties with good taste,different colors and resistance to CGMMV.In conclusion,we found that WPRb,is recessively associated with CGMMV resistance in watermelon.Editing of WPRb conferred greater tolerance to CGMMV.We found WPRb targets to the plasmodesmata（PD）and biochemically interacts with the CGMMV movement protein,facilitating viral intercellular movement by affecting the permeability of PD.We developed a reproducible marker based on a single non-synonymous substitution（G1282A）in WPRb,which can be used for marker-assisted selection for CGMMV resistance in watermelon.