Abstract:Identification of super-male plants is the key to all-male breeding in asparagus, while traditional method based on analyzing the separation ratio of hybrid offspring remains time-consumption and cost. It is of practical value to design and develop sex-specific markers assisted for asparagus super-male plant selection. In this study, 968 selfing populations of andromonoecious plants were constructed. STS bimolecular markers YSM and XSM, which can amplify the Y chromosome specific region gene SOFF and the X chromosome specific region gene WIP2/NTT, respectively, were produced. Based on the genotyping result using STS marker, 58 super-male plants carrying a single 293 bp fragment, 550 female plants carrying a single 245 bp fragment, as well as 360 male plants with two fragments (245 bp and 293 bp) were identified. Sanger sequencing of the 293 bp and 245 bp fragments revealed a sequence identity to the reference. By making crosses using 418 male plants, 58 F1 offspring lines were all male, thus revealing a super-male status for their male parents. 360 F1 families contained male plants and female plants, proving that their male parents were male. This result is coincident with that of STS molecular marker genotyping. Collectively, the STS bimolecular functional marker can accurately classify the sex status (female, male and super-male plants) of asparagus, which has implication in the all-male molecular marker assisted breeding of asparagus.