The CRISPR/Cas9 genome-editing system serves as a pivotal tool for enhancing crop genetics. Within this system, single guide RNAs (sgRNAs) are instrumental in the precise cleavage of DNA double strands. However, the efficiency of gene editing varies among sgRNAs, emphasizing the need to meticulously select target sites, especially in the context of Citrullus lanatus, a species notorious for its challenging and inefficient generation of transgenic plants through stable transformation. This study employed an Agrobacterium rhizogenes-mediated hairy root method to assess effective target sites for gene editing of ClCIPK17 across various Citrullus species. Hairy roots were successfully induced in different plant tissues at diverse growth stages of Citrullus lanatus, Citrullus mucosospermus, and Citrullus amarus. Employing a vector with two sgRNAs (sgRNA1 and sgRNA5) positioned within conserved regions of exon 1 and exon 5 of ClCIPK17 in the CRISPR/Cas9 system, targeted mutations were detected in 90.9% of accessions across the four Citrullus species. Notably, 73.94% of all examined hairy roots exhibited mutations at the sgRNA1 site, while the sgRNA5 site showed no mutations. Among the 31 different mutation types identified at the sgRNA1 site, base deletion was the most prevalent. Using the sgRNA1 site of ClCIPK17, stable transgenic watermelon buds were obtained from explants, and the targeted mutations of the sgRNA1 site were confirmed. These findings underscore the viability of the hairy root transformation system in assessing the editing efficiency of sgRNA targets in diverse Citrullus species.

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 12776310 and 12968331 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 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.