Identification and characterization of sex-specific markers in the milky mangrove Excoecaria agallocha using double digest restriction site-associated DNA sequencing

Sex-specific markers can be used to infer sex determination systems of dioecious plants and to study the evolution of plant sex determination. Meanwhile, sex identification of plants with agricultural or economic value using sex-specific markers can be beneficial for rapid breeding practice and better commercial harvest. Although there are more than 15,000 dioecious flowering plants, the sex determination systems of less than 40 species have been identified. A major barrier to studying plant sex determination and evolution is the lack of information on the types of sex determination mechanisms. In this study, we used double digest restriction site-associated DNA sequencing (ddRAD-seq) to identify sex-specific markers in a dioecious mangrove tree, Excoecaria agallocha. We performed ddRAD-seq for multiple females and males of E. agallocha in a natural population and identified six candidate male-specific loci among 177,071 catalogue loci for these samples. In contrast, no female-specific loci were found among these catalogue loci. We further validated two of the six male-specific loci using PCR on the individuals used for ddRAD-seq and additional individuals from another geographically distant population. Our results indicated that E. agallocha has a genetic sex determination system. We found that sex ratios in the seeds and one-month-old seedlings of E. agallocha didn't deviate from 1:1 by using PCR amplification of one male specific locus, suggesting post-young-seedling selection may cause skewed sex ratio towards adult males. Reliable sex identification of seedlings of E. agallocha with male-specific markers is valuable for rapid recovery of mangrove forests. The ddRAD method developed here can also be used to identify sex-specific markers in other dioecious plants, especially for trees in which experimental crosses are impractical.