Abstract

Distant hybridization is vital for enhancing cultivated cotton by introgressing beneficial genes from wild relatives, yet existing identification methods for hybrids face limitations. Simple sequence repeat (SSR) markers are DNA quality-dependent and inefficient, while morphological traits are time-consuming and unreliable. Here, we developed a structural variation (SV)-based molecular approach to efficiently identify hybrids between Gossypium anomalum and Gossypium davidsonii . Genome comparison revealed 924 SV loci, from which 598 primer pairs were designed. A high-specificity primer pair was selected, yielding distinct electrophoretic bands: each parent exhibited a unique band, while reciprocal F 1 hybrids ( G. anomalum  ×  G. davidsonii and G. davidsonii  ×  G. anomalum ) displayed combined parental bands, confirming hybridity. This SV-based method is rapid, reliable, and reproducible, with clear band resolution and minimal technical complexity. It enables precise identification of distant hybrids using a single primer pair, overcoming key drawbacks of traditional techniques. Our findings provide a robust framework for hybrid validation in cotton and other crops, advancing germplasm innovation and breeding efficiency.