Identification fi cation of an Aux/IAA regulator for fl esh fi rmness using combined GWAS and bulked segregant RNA-Seq analysis in watermelon

Watermelon is a highly cultivated fruit crop renowned for its quality properties of fruit flesh. Among various quality factors, fruit flesh firmness is a crucial quality parameter influencing the fruit texture, shelf life and its commercial value. The auxin/indole-3-acetic acid (Aux/ IAA) plays a significant role in fruit development and ripening of non-climacteric fruits. However, the regulatory mechanism of Aux/IAA in controlling fruit flesh firmness and ripening in watermelon remains unknown. In this study, we employed an integrative approach combining genome-wide association study (GWAS) and bulked segregant RNA-Seq analysis (BSR-Seq) to identify an overlapping candidate region between 12 776 310 and 12 968 331 bp on chromosome 6, underlying an auxin-responsive gene (Aux/IAA) associated with flesh firmness in watermelon. Transcriptome analysis, followed by real-time quantitative reverse transcription PCR (qRT-PCR), confirmed that the expression of Aux/IAA was consistently higher in fruits with high flesh firmness. 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 for F2 2 population and germplasm accessions identified Aux/IAA as a strong candidate gene associated with flesh firmness. Aux/IAA was enriched in the plant hormone signal transduction pathway, involving cell enlargement and leading to low flesh firmness. We determined the higher accumulation of abscisic acid (ABA) in fruits with low flesh firmness than hard flesh. Moreover, overexpression of Aux/IAA induced higher flesh firmness with an increased number of fruit flesh cells while reducing ABA content and flesh cell sizes. 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. These findings significantly enhanced our understanding of watermelon fruit flesh firmness and consequently the watermelon fruit quality.