QTL mfh2.1 Integrates Phytohormone Dynamics to Mediate Carpel Separation and Cavity Formation in Cucumber Fruit (Cucumis sativus)

Hollowness of the cucumber fruit, caused by carpel separation during growth, severely impacts fruit quality. Several Sikkim cucumber accessions originating from the India–Pakistan region exhibit pronounced internal cavities. We previously identified the QTL mfh2.1 as a key contributor to this phenotype. In this study, we investigated the genetic and physiological basis of fruit hollowness in the Sikkim cucumber line WI7120 through an integrative analysis combining histological staining, HPLC for hormonal profiling, and fine mapping using a large F2 segregation population. Comparative analysis between the hollow-fruited WI7120 and the non-hollow line 9930 revealed distinct growth dynamics: WI7120 displayed accelerated radial expansion and aberrant cell patterning at carpel junctions. Histological examination using paraffin sectioning uncovered disorganized endocarp cell arrangements in WI7120 occurring as early as pre-anthesis (0 days post-pollination), with enlarged suture cells that likely facilitate tissue separation during fruit enlargement. Hormonal assays indicated elevated levels of gibberellin (GA) and zeatin (ZT), along with reduced indole-butyric acid (IBA) in WI7120, suggesting that a hormonal imbalance and mechanical stress contribute to compromised cell adhesion. By screening ~2000 F2 individuals with SSR and InDel markers, we refined the mfh2.1 locus to a 50.92 kb interval on chromosome 2, pinpointing CsRPT4Bb—encoding a 26S proteasome subunit—as the candidate gene. A non-synonymous SNP (I135V) in CsRPT4Bb was associated with tissue-specific expression patterns during cavity formation, implicating proteasome-mediated cellular remodeling in carpel cohesion. Spatial-temporal expression analysis further revealed upregulation of CsRPT4Bb in the WI7120 exocarp during fruit expansion, potentially influencing cell wall dynamics. This study demonstrates a coordinated interplay among genetic, hormonal, and mechanical factors underlying cucumber fruit hollowness, offering new avenues for breeding cultivars with improved fruit integrity and postharvest quality.