Identification And Functional Validations Of A Candidate Gene For Flesh Firmness In Watermelon

Watermelon is a widely cultivated fruit crop for its quality properties of fruit flesh.Among the several quality factors,flesh firmness is attributed as an essential quality influencing fruit texture,commerciality,and shelf life.The significant factors affecting the firmness of watermelon were the fruit flesh cell architecture,cell wall composition,and hormones,which positively or negatively regulate the flesh firmness.Fruit flesh firmness plays a crucial role in the fruit quality of watermelon.Thus,the identification of key regulatory factors related to flesh firmness can provide a theoretical and technical platform for watermelon quality breeding.In the current study,we measured the flesh firmness of 198 watermelon accession,performed a genome-wide association study（GWAS）,and detected 34 SNPs significantly associated with flesh firmness.Furthermore,we used a watermelon line“Zhongguojingxin”（CL）with low flesh firmness and“PI595203”（CM）with high flesh firmness as parental lines.We obtained the F₁ and F₂ populations by hybridizations between“CL x CM”and subsequent self-pollination.To map the QTL related to the flesh firmness in watermelon.The flesh firmness of each F₂individual plant was recorded,and constructed two extreme pools for bulk-segregant RNA-Seq（BSR-Seq）analysis.The results revealed three QTLs related to the watermelon flesh firmness trait,two QTLs on chromosome 2,located between35.65 to 35.66,35.68 to 37.79 Mb,and one QTL on chromosome 6,from 10.266 to 22.864 Mb,with a total length of 0.010 Mb,2.11 Mb,and 12.60 Mb,respectively.The RNA-Seq analysis between parental lines CL and CM at 34 DAP in fruits identified the differentially expressed genes（DEGs）.Further,the annotations of DEGs resulted in the significant pathways associated with cell wall biogenesis,xyloglucan metabolic process,and plant hormone signal transduction associated with fruit development and ripening.For the identification of accurate genetic region associated with flesh firmness,we used an integrative approach of GWAS and BSR-seq.We identified an overlapping candidate region from12776310 to 12968331 bp on chromosome 6 underlying ten candidate genes.The expression analysis using RNA-Seq data revealed an auxin-responsive gene,Cla97C06G118630（Aux/IAA）expression was higher in hard-flesh（CM）than in soft-flesh（CL）fruits.These results were validated via RT-q PCR analysis in 3 soft-fleshed and 3 hard-fleshed elite watermelon cultivars.The sequence alignment revealed a single base mutation in the coding region of Aux/IAA.Furthermore,the concomitant kompetitive/competitive allele-specific PCR（KASP）,genotyping data sets of the F₂ population,and germplasm accessions suggested that the allelic variation in Aux/IAA was associated with the flesh firmness,indicating the Aux/IAA as the strong candidate gene associated with flesh firmness in watermelon.Candidate gene Aux/IAA was enriched in the plant hormone signal transduction pathway,involving cell enlargement and leading to low flesh firmness.We determined higher ABA content in low-flesh firmness fruits than in hard-flesh ones.Further,ABA content was increased,and flesh firmness was decreased at different developmental stages.Additionally,the allelic variation in Aux/IAA for soft flesh firmness was found to exist in Citrullus mucosospermus and gradually fixed into Citrullus lanatus during domestication,indicating that soft flesh firmness was a domesticated trait.The Aux/IAA family genes involve in intense plant development and ripening mechanisms.In this study,28 Aux/IAA genes were identified through the Genome-wide characterization of Aux/IAA family genes in watermelon.These genes were located in six sub-clusters based on the phylogenic analysis.The genes characteristics revealed the highly conserved domains I,II,III,and IV,and promotors analysis detected auxin and ABA-responsive cis-regulatory elements.Among 28 Aux/IAA genes,the RNA-Seq revealed the expression of the candidate gene Cla97C06G118630（Aux/IAA）expression was noticeable,expression in hard-flesh wild fruits was higher than in the soft-flesh cultivars.Furthermore,ABA content was higher in soft-flesh than in hard-flesh fruits.In the histology of fruit flesh cells,soft-flesh fruit cell size was larger compared to hard flesh fruits with smaller cell size and a compact cell arrangement.Meanwhile,the correlation analysis revealed the association between flesh firmness ABA contents and cell size.Further,the overexpression of Aux/IAA reduced the ABA content and cell sizes and induced higher cell density in watermelon true leaves.Moreover,overexpression of Aux/IAA induced higher flesh firmness and increased the number of cells while reducing ABA content and flesh cell sizes in the watermelon fruit flesh.The current findings may advance our understanding of watermelon fruit flesh firmness,consequently improved the watermelon fruit quality.