Gene Mining And Molecular Maker Developing Of Rind Hardness And Cracking Resistance In Watermelon

Fruit cracking is a common phenomenon during the production and marketing of fresh fruit such as watermelon,which causes huge yield losses in fields and logistical chains annually.Therefore,accurate quantification of cracking resistance traits and precise mapping of the genes of cracking resistance will provide important theoretical support for the breeding of cracking resistance varieties.In this study,we found that rind hardness could be an effective index for evaluating watermelon cracking resistance.And we identified Cl ERF4 as the causative gene that enabling watermelon cracking resistance by combining the toolkits of genetic map,BSA,Kompetitive/competitive Allele Specific PCR（KASP）genotyping.Meanwhile,transcriptome datasets provided clues for understaning the molecular mechanism of watermelon cracking resistance.The main results are outlined as follows:（1）A high-density watermelon genetic map was constructed and the rind hardness was found to be an effective index to evaluate the watermelon cracking resistance.We constructed a high-density genetic map based on resequencing datas of a segration population.Moreover,we found that the QTL of cracking indicators（Rind Hardness（RH）,Cracking Work（CRW）,Cracking Time（CRT）and Cracking or Not（CRN））all pointed to a region located in chromosome 10 and RH has the highest LOD value.Altogether,we found that RH could be a reliable,practical and effective parameter for the evaluation of watermelon cracking resistance.（2）Cl ERF4 was mapped as an important regulatory gene of watermelon rind hardness and cracking resistance.By combing genetic map,BSA analysis and KASP genotyping,the watermelon cracking resistance gene was fine-mapped to a 9372bp region and an In Del of 11 bp and a neighbouring SNP in the CDS of Cl ERF4 gene,conferring cracking resistance in F₂ populations with variable rind hardness.Additionally,the KASP genotyping data sets of 104 germplasm accessions strongly supported candidate Cl ERF4 as a causative gene conferring fruit cracking resistance.Genotyping of common cultivars undoubtedly suggest that ecotype breeding of watermelon for different cultivation patterns imposes on-target or off-target selection of the rind hardness-related Cl ERF4 locus.（3）Cl ERF4 was supposed to affect watermelon rind hardness and crack resistance via interacting with genes involved in the synthesis and regulation of polysaccharides associated with cell wall synthesis.Transcriptome studies were performed on P-a and P-b fruits during the fruit development.By weighted gene co-expression network analysis（WGCNA）and expression pattern cluster analysis（MFUZZ）,we identified brown model（3644 genes）and cluster 7（1500 genes）whose expressions showed high correlation between expression pattern and rind hardness development.Functional enrichment anaylysis suggested that these two gene sets were enriched in polysaccharide metabolic pathways related to cell wall synthesis.Transcriptome data showed that the expression level of primary cell wall related CESA genes was generally higher than those related to secondary cell wall,and the expression level of primary cell wall related CESA genes was higher in P-a than P-b.These results suggest that Cl ERF4 may regulate watermelon rind hardness and crack resistance via interacting with genes related to cell wall synthesis.（4）A new watermelon germplasm with cracking resistance was found and its QTL region was preliminarily mapped.We identified a special germplasm（P-c）which show P-a genotypes but P-b phenotypes.Further study indicated that P-c displays a different rind hardness development trendency with both P-a and P-b,and finally results to a high elasticity rind that is also creaking-resistance.Then we constructed a F₂ population and preliminary mapped two rind elasticity QTLs on chromosome 3 and chromosome 9 via BSA analysis.