The sex-determining region and early-stage gender identification in the dioecious industrial plant Herpetospermum pedunculosum

Herpetospermum pedunculosum ( Cucurbitaceae ) is a dioecious species whose seeds from female individuals are highly valued for their hepatoprotective properties. However, the industrial cultivation is severely constrained by the low natural feminization rate and the absence of reliable sex-determination markers at the seedling stage. Here, through ND-FISH, multi-omics and PCR technique, we find that: (i) H. pedunculosum is diploid ( 2n = 20) and probably has a heterogametic male system (XX/XY); (ii)A 241-kb sex-determining region (SDR01) harbors several candidate sex-determining loci, located at the end of pseudochromosome Chr01, including the MADS-box transcription factor HpAP3 and genes involved in phytohormone metabolism and signaling; (iii) The auxin-responsive gene HpSAUR50 in SDR01 is strictly present only in female individuals. Our results indicate that phytohormones, particularly ethylene and auxin, are integral to sex determination in H.pedunculosum , and that HpSAUR50 functions as a reliable molecular marker for the early sex detecting. • The cytological karyotype evidence reveals that Herpetospermum pedunculosum , an important medicinal plant, is diploid (2 n = 20) and harbors a male-specific dwarf "Y-like" chromosome, suggesting the existence of an XX/XY sex system in this species. • Phytohormone-related differentially expressed genes (DEGs) are mainly involved in the biosynthesis pathways of ethylene and auxin, indicating that ethylene and auxin play a pivotal role in sex determination of H. pedunculosum. • The putative sex-determining region (SDR01) on Chr01 harbors the key sex-related genes HpAP3 and HpSAUR50 . The auxin-related gene HpSAUR50 , specifically detected in females, serves as a reliable marker for early sex identification of H. pedunculosum. • PCR detections of HpSAUR50 (as a marker) are consistent with cytological observations, further confirming the XX/XY sex determination system in this species.