Transcriptome based co-expression network analysis coupled with genomic analysis insights candidate genes regulate sugar-acid ratio in watermelon fruit

Sugar-acid ratio (SAR) is a key determinant of flavor and consumer preference in watermelon ( Citrullus lanatus ), yet its genetic basis is largely unexplored. In this study, an integrated approach of transcriptomic and genomic analyses was employed across five fruit developmental stages, 8, 14, 20, 26, and 32 days after pollination, to elucidate the potential regulatory mechanisms underlying SAR in two contrasting watermelon accessions: improved ‘Xichunxuan’ (Xi) with high SAR and landrace ‘Sashenganiyougua’ (Sa) with low SAR. ‘Xi’ accumulated higher soluble solid contents (SSC), titratable acidity (TA), fructose, sucrose, glucose, malic, citric, and oxalic acids, resulting in superior SAR. Weighted gene co-expression network analysis identified four key modules correlated with SAR, from which seven hub genes were prioritized. Pectin lyase-like superfamily protein ( PLLSP ) might contribute to SAR by facilitating cell wall disassembly and carbon partitioning toward sugar and organic acid synthesis, while polyadenylate-binding protein-interacting protein 4-like ( PbPIP4L ) might coordinate SAR through post-transcriptional regulation of mRNA stability. Both genes showed the strongest positive correlations with SAR ( r = 0.85*** and r = 0.73**, respectively), and exhibited higher expression in ‘Xi’, consistent with the pattern of SAR accumulation. Comparative genomics revealed a 61-bp deletion in the first exon of PLLSP in ‘Xi’ and a C/G polymorphism in the promoter of PbPIP4L , generating a GTGANTG10 transcription factor binding site. Validation across 16 accessions showed the InDel deletion was present in all high-SAR improved accessions but absent in low-SAR landraces, suggesting an association with SAR of the limited germplasm examined in this study. Phylogenetic and multiple sequence alignment analyses demonstrated that both genes are evolutionarily conserved across diverse fruit species, including melon, tomato, citrus, grape, apple, and peach. In summary, this finding elucidated natural variation in promising key candidate genes associated with SAR, and provided valuable molecular targets for breeding strategies aimed at enhancing watermelon fruit flavor.