Research On The Method And Its Application Of Identifying Cucumber Resistance To Fusarium Wilt By Fusaric Acid

Fusarium wilt caused by Fusarium oxysporum seriously affected the growth and development of cucumber and led to the decline of yield and quality.Fusaric acid（FA）is a non specialized toxin synthesized and secreted by all tested Fusarium wilt pathogens in cucurbits,and it is also a key factor in the pathogenesis of Fusarium wilt.At present,many studies have shown that the resistance of cucumber and other crops to FA is highly positively correlated with their resistance to Fusarium wilt.I In this study,a method to identify the resistance of cucumber to Fusarium wilt by FA was established,and the physiological indexes such as root growth,reactive oxygen species production,ion penetration and photosynthetic efficiency of Cucumber Seedlings under FA stress were detected;Cucumber inbred lines CsFA-R1（resistance）and CsFA-S1（sensitivity）with significant differences in resistance and susceptibility were used as male and female parents respectively.F₂ segregant population was constructed through artificial hybridization and selfing,and the genetic analysis and mapping of FA resistance gene in cucumber were carried out.BSA-seq was used to preliminarily locate the FA resistance gene CsFAR1 in cucumber,and then combined with transcriptome analysis to mine the candidate genes in the target interval.The expression profile of the candidate genes was verified quantitatively by real-time PCR,and the transient expression vector was constructed for preliminary functional analysis of the candidate genes.This study provides a new method for identification of Cucumber Fusarium wilt resistance,and provides a basis for breeding and basic research of Cucumber Fusarium wilt resistance.The main results are as follows:1.The method of using FA to identify and screen cucumber plants resistant to Fusarium wilt,namely leaf disc treatment method,was established.2.Treatment of Cucumber Seedlings with FA showed that FA could inhibition of Cucumber Root Growth,increase the permeability of cell membrane,accumulate reactive oxygen species,reduce the maximum photosynthetic efficiency（FV/FM）and the actual photosynthetic efficiency Y（II）.It was found that these measured physiological responses were significantly different in Cucumber materials CsFA-R1 and CsFA-S1 resistant to Fusarium wilt.3.The leaf disk method was used to identify the resistance of F2 isolated population,and the extreme resistant and susceptible individual plants were selected for BSA-seq analysis,Two potential cucumber FA resistance loci were identified by BSA-seq,which were located on chromosomes 3 and 5 respectively.TheΔSNP peak of the locus on chromosome 5 was the most obvious.There are 178 genes located on chromosome 5,ranging from 3.51 MB to 6.79 MB,with an interval size of 3.28 MB.4.Transcriptome sequencing was performed on the roots and leaves of cucumber materials treated with different concentrations of FA.The RNA of roots and leaves were taken for RNA-seq.20797 expressed genes were detected Through WGCNA analysis of leaf transcriptome,two modules of gene differential expression in resistant and susceptible materials,black module and red module,with 433 genes and 571 genes respectively,were obtained.KEGG analysis showed that the pathways with more enriched differential genes were plant hormone signal transduction and spliceosome,autophagy regulation and nitrogen metabolism.5.Through the gene intersection analysis in the differential expression module between the genes in the localization interval and the genes of resistant and susceptible materials,combined with the SNP information extracted by BSA-seq,16 candidate genes were discovered.Three candidate genes were preliminarily selected through gene function annotation for expression profile verification and transient expression subcellular localization analysis.The fluorescence quantitative results showed that;The expression of CsFAR1-3 in the root and leaf of the susceptible material was significantly higher than that of the resistant material,while the expression of CsFAR1-2 in the root and leaf of the resistant material was significantly higher than that of the susceptible material,while the expression of candidate gene CsFAR1-1 in the root resistant material was higher than that of the susceptible material,but the opposite was true in the leaf;The transient expression results showed that CsFAR1-1 and CsFAR1-2 were expressed in the nucleus and cytoplasm,and CsFAR1-3 was expressed in the cytoplasm.