Using RAD-seq to develop sex-linked markers in a haplodiplontic alga

For many taxa, including isomorphic haplodiplontic macroalgae, determining sex and ploidy is challenging, thereby limiting the scope of some population demographic and genetic studies. Here, we used double-digest restriction site-associated DNA sequencing (ddRAD-seq) to identify sex-linked molecular markers in the widespread red alga Agarophyton vermiculophyllum. In the ddRAD-seq library, we included 10 female gametophytes, 10 male gametophytes, and 16 tetrasporophytes from one native and one non-native site (N = 40 gametophytes and N = 32 tetrasporophytes total). We identified seven putatively female-linked and 19 putatively male-linked sequences. Four female- and eight male-linked markers amplified in all three life cycle stages. Using one female- and one male-linked marker that were sex-specific, we developed a duplex PCR and tested the efficacy of this assay on a subset of thalli sampled at two sites in the non-native range. We confirmed ploidy based on the visual observation of reproductive structures and previous microsatellite genotyping at 10 polymorphic loci. For 32 vegetative thalli, we were able to assign sex and confirm ploidy in these previously genotyped thalli. These markers will be integral to ongoing studies of A. vermiculophyllum invasion. We discuss the utility of RAD-seq over other approaches previously used, such as RAPDs (random amplified polymorphic DNA), for future work designing sex-linked markers in other haplodiplontic macroalgae for which genomes are lacking.