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.

中文翻译：

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使用 RAD-seq 在单倍体藻类中开发性别连锁标记


对于许多类群，包括同构单倍体大型藻类，确定性别和倍性具有挑战性，从而限制了一些种群人口统计和遗传学研究的范围。在这里，我们使用双消化限制性位点相关 DNA 测序 (ddRAD-seq) 来鉴定广泛分布的红藻Agarophyton vermiculophyllum 中的性别连锁分子标记。在 ddRAD-seq 文库中，我们包含了来自 1 个本地和 1 个非本地位点的 10 个雌配子体、10 个雄配子体和 16 个四孢子体（总共N = 40 个配子体和N = 32 个四孢子体）。我们鉴定了 7 个假定的女性连锁序列和 19 个假定的男性连锁序列。四个女性连锁标记和八个男性连锁标记在所有三个生命周期阶段均得到扩增。使用一种性别特异性的女性连锁标记和男性连锁标记，我们开发了一种双重 PCR 并测试了该测定对在非天然范围内两个位点采样的菌体子集的功效。我们根据生殖结构的目视观察和先前对 10 个多态性位点的微卫星基因分型确认了倍性。对于 32 个营养菌体，我们能够确定性别并确认这些先前基因分型的菌体的倍性。这些标记将成为正在进行的A. vermiculophyllum入侵研究的一部分。我们讨论了 RAD-seq 相对于之前使用的其他方法（例如 RAPD（随机扩增多态性 DNA））的实用性，以便未来在缺乏基因组的其他单倍体大型藻类中设计性别连锁标记。