Development and application of sex-specific indel markers for Hippophae salicifolia based on third-generation sequencing

Hippophae salicifolia, a dioecious shrub endemic to the Himalayan region, holds substantial ecological and economic value. The lack of pre-flowering morphological traits for sex identification has long impeded efficient germplasm management and breeding efforts. In this study, we utilized third-generation sequencing technology to conduct whole-genome comparative analysis of known-sex individuals, identifying insertion/deletion loci significantly associated with sex. Based on these loci, we designed a specific primer pair, Hsa09. PCR amplification results demonstrated 100% accuracy in sex differentiation, with female individuals showing a single band and male individuals exhibiting a double-band pattern. Cross-species validation revealed limited applicability of Hsa09 in closely related Hippophae species, suggesting notable divergence in sex determination mechanisms within the genus. This study establishes the first molecular tool for early sex identification in H. salicifolia, overcoming the dependence on reproductive organ development in traditional methods. It provides essential technical support for understanding the evolutionary pathways of sex chromosomes and advancing sex-regulated breeding strategies within the Hippophae genus.