Genome-wide identification of the HMG family in cucumber and regulatory analysis of CsHMG9 in spine density

Fruit spines constitute a critical protective barrier, and their density serves as a key determinant of appearance quality, directly influencing market value in cucumber (Cucumis sativus L.). Prior studies demonstrated that CsHMG9 (FS2), a high-mobility group (HMG) protein, regulates fruit spine density in cucumber. To elucidate the roles of the CsHMG in fruit spine density, we conducted comprehensive analyses of CsHMG family regarding their chromosomal localization, phylogeny, gene structures, conserved motifs, cis-acting elements and organ-specific expression patterns. Genome-wide identification revealed 17 CsHMGs in cucumber, with phylogenetic analysis classifying them into five distinct subfamilies. The subcellular localization of proteins varies among different CsHMG subfamilies. Reverse transcription quantitative PCR (RT-qPCR) analysis demonstrated differential gene expressions of CsHMGs under diverse treatments, indicating potential involvement in multiple signaling pathways. Furthermore, a Tnt1 retrotransposon insertion mutant of CsHMG9 exhibited significantly reduced fruit spine density. Allelic validation via genetic crossing between fs2-Tnt1 and fs2-EMS confirmed that CsHMG9 is the key regulator of fruit spine density in cucumber. Building on this, integrated approaches, including yeast one-hybrid (Y1H), dual-luciferase assays (Dual-LUC), RT-qPCR and genetic analyses, demonstrated that CsHMG9 acts downstream of key trichome regulators (Tril, Mict and TOE3), while feedback-regulating Tril expression to modulate fruit spine density in cucumber. This study clarified the function of CsHMG9 in fruit spine density and provided a foundational insight into CsHMG biology.