Fine Mapping And Candidate Gene Analysis Of Powdery Mildew Resistance Gene Cmpmr2F In Melon

Melon(Cucumis melo L.)is a trailing plant of the Cucumis genus belong Cucurbitaceae with chromosome 2n=24,a worldwide fruit-based vegetable crop,with 358,900 ha planted in China and a total production of 12,788,200 tons(FAO,2018).The fruit is rich in soluble sugars,vitamins,minerals and other nutrients,and can be eaten fresh or processed,making it widely popular.Powdery mildew is one of the main fungal diseases affecting the yield and quality of melon production and occurs widely in open field and protected cultivation.Powdery mildew develops and spreads very quickly,with the leaves first producing white spots after infection,which turn yellow and wilt later in plant growth.The occurrence of powdery mildew weakens the growth of melons and affects the transition from nutritional to reproductive growth,eventually leading to plant death at a later stage of growth.The main bottleneck in breeding for powdery mildew resistance in melon is that there are few resistance sources available,and further research is needed in the areas of fine localization of powdery mildew resistance genes,functional analysis and molecular marker-assisted selection breeding.In this study,the powdery mildew pathogen infecting melon was identified.The process of powdery mildew infestation of melon leaves was observed microscopically to determine the critical period for the occurrence of resistance.We selected the susceptible melon line X055 as the mother and the resistance melon line PI 124112 containing a single resistance gene as the father,and formulated a six-generation population to analyze the inheritance law of melon powdery mildew resistance genes.QTL-seq analysis based on BSA technique was used to obtain chromosomal segments closely linked to powdery mildew resistance gene,and a second population was constructed with PI 124111 and M1-101 as parents to verify the initial location result.On this basis,genetic linkage analysis was carried out using CAPS(Cleave Amplified Polymorphic Sequence)molecular markers and the resistance gene was fine-mapped by screening recombinant monocultures.Identification of candidate genes through gene function annotation,sequence alignment and expression pattern analysis of genes in fine-mapped intervals,combining subcellular localization and bioinformatics analysis to preliminary identify candidate gene functions.Determination of the corresponding hormone levels and transcriptome sequencing analysis to predict the mechanisms of disease resistance regulation.To provide a theoretical basis for in-depth research on the molecular mechanism of powdery mildew resistance in melon,and to provide a genetic resource and theoretical basis for molecular marker-assisted breeding for powdery mildew resistance in melon.The main results of this study are as follows:By observing the microscopic features of powdery mildew conidia,haustorium and conidiophore as well as cloning the sequences of the ITS region of powdery mildew and performing Blastn analysis on NCBI and phylogenetic analysis with sequences of high homology,it was determined that the powdery mildew in this study belongs to Podosphaera xanthii.The results of a post-inoculation survey of 13 powder mildew identification hosts for resistance to susceptibility identified the physiological race as 2F.PI 124112 and X055 were stained with Thomas brilliant blue for 0h,24h,48h and 72h after inoculation with powdery mildew conidia.The resistance of PI 124112 to P.xanthii physiological race 2F was found to be characterized by the inability of the disease to spread,and the critical period for resistance development was 48h after inoculation with powdery mildew conidia.A six-generation population was constructed using X055 and PI 124112 as parents.By planting the six-generation population and investigating the ratio of disease-resistant plants to disease-susceptible plants in each population,it was found that F₁ was susceptible and the F₂segregating population had a 3:1 segregating ratio of susceptible plants:disease-resistant plants,while BC₁P₂ had a 1:1 segregating ratio of susceptible plants:disease-resistant plants,indicating that PI 124112 was resistant to P.xanthii physiological race 2F is controlled by a pair of recessive alleles,which were named Cmpmr2F.In the F2 population,20 powdery mildew-resistant and powdery mildew-susceptible plants were selected,and DNA was extracted and mixed in equal amounts to construct gene pools,named R-pool and S-pool respectively,and the sequenced data were re-sequenced by PI 124112,X055,R-pool and S-pool.QTL-seq analysis based on BSA technology of the data obtained by sequencing tentatively localized the disease resistance gene Cmpmr2F between 22,282,737-24,906,174 bp on chromosome 12.The accuracy of the initial location results was verified by applying the same analysis to another set of population PI 124111×M1-101,where the disease resistance locus was located between 21,618,212-24,071,898 bp on chromosome 12.Genotyping of individual plants of the F₂ population grown in 2017 with 15 polymorphic CAPS markers within the initial locus interval,combined with phenotypic data for genetic linkage analysis,narrowed the locus interval to 333,464 bp between markers CHR12PM7-CHR12PM9.In2018,the resistance gene Cmpmr2F was fine-mapped to a range of 26,520 bp between markers CHR12PMA1-CHR12PMIndel1 by planting an F₂ population of 1200 plants,encrypting markers between markers CHR12PM7-CHR12PM9 and screening recombinant plants.Based on the reference genome annotation information,two encoded genes were found in the fine-mapped interval,with the MELO3C002403 gene functionally annotated as allantoate deiminase.MELO3C002403 has three non-synonymous SNPs between PI 124112 and X055.Comparison of PI 124112 with X055 reveals that in PI 124112,amino acid position 8 is changed from serine(S)to phenylalanine(F),amino acid position 287 is mutated from isoleucine(I)to methionine(M);amino acid position 321 is changed from aspartic acid(D)to glutamic acid(E),there is a 7 bp insertion in PI 124112 at 882-889 bp in the coding region of MELO3C002403,which leads to the premature termination of the translation of MELO3C002403 and prevents the translation of the complete protein sequence.Expression analysis showed that the relative expression of MELO3C002403 in PI 124112 increased significantly at 48h after inoculation and decreased slightly to 72h,but was significantly higher than that of MELO3C002403 in X055.In X055,MELO3C002403 gene expression showed an overall upward trend but was lower than PI124112 at each time point.The expression pattern of the MELO3C002403 coincides with the response of PI 124112 to powdery mildew infestation.The other gene in the locus,MELO3C002402,has no non-synonymous SNP between the two parents and no obvious pattern of expression.Allantoate deiminase MELO3C002403 identifited as the candidate gene of Cmpmr2F.Bioinformatics analysis of the candidate gene MELO3C002403,construction of a GFP fusion vector and subcellular localization showed that the protein encoded by MELO3C002403 is localized to the cytoplasm and cell membrane.The MELO3C002403 encodes a protein with a conserved M20 family metallo-hydrolase domain,whose function is to catalyse the hydrolysis of the amide bond in the target substrate,converting allantoic acid to(S)-ureidoethanolic acid and ammonia.It was found that allantoin and jasmonic acid showed the same trend,that the content of allantoin and jasmonic acid increased significantly at 48h of inoculation,while the content of salicylic acid was not related to inoculation or not.The transcriptome of melon leaves was sequenced at 0h,24h,48h and 72h after treatment with powdery mildew resistant line PI 124112,inoculated with powdery mildew as the treatment group and sprayed with sterile water as the control group,and the expression pattern of MELO3C002403 was consistent with the fluorescence quantitative PCR results.We conducted Weighted Correlation Network Analysis(WGCNA)with hormone content data to obtain differentially expressed genes significantly correlated with hormone content,and then used functional enrichment analysis to find differentially expressed genes related to plant-pathogen interactions.The expression of calcium-binding protein,calmodulin and WRKY transcription factor are involved in the regulatory pathway of PI 124112 in response to P.xanthii physiological race 2F invasion.In summary,in this study,a recessive resistance gene was identified in the melon powdery mildew resistance line PI 124112 for P.xanthii physiological race 2F and fine-mapped,identifying the candidate gene as MELO3C002403.Preliminary functional analysis of candidate genes and preliminary exploration of the powdery mildew resistance mechanism of PI 124112 by means of transcriptome sequencing.The results of the study provide a genetic resource and theoretical basis for the functional analysis of melon powdery mildew resistance genes and the analysis of disease resistance regulatory networks,provide molecular markers for the selection of new varieties of melon resistant to powdery mildew,and lay the foundation for future research on the mechanism of melon resistance to powdery mildew and signal transduction pathways.