Pitaya (Hylocereus spp.) is a tropical fruit grown in Okinawa prefecture, Japan. In this study, we developed simple sequence repeat (SSR) markers to assess the genetic diversity of pitaya accessions collected in Okinawa prefecture. SSR markers were developed with next-generation sequencing, using the Roche 454 GS FLX+ platform. Seventy primer sets were tested for the genetic identification of pitaya, including 50 AG motif and 20 AC motif SSRs. In total, 16 SSR markers successfully amplified fragments and exhibited polymorphism among the accessions. The SSR markers revealed the genetic diversity parameters including number of alleles per locus (ranging from 6 to 12), expected heterozygosity (ranging from 0.5 to 0.85), observed heterozygosity (ranging from 0.23 to 0.93), polymorphic information content (ranging from 0.47 to 0.83), fixation index value (0.013), and out-crossing rate (0.973). Using these SSR markers, genetic identification was performed for 32 pitaya accessions collected in Okinawa prefecture. These accessions could be distinguished genetically using three SSR markers, with the exception of accessions between ‘Uruma White’/‘Nago White’ and ‘Ohgimi Red 1’/‘Ohgimi Red 2’. Genetic diversity analyses suggested six groups, and grouping appeared to be based on differences in species. The obtained SSR markers and clarified genetic background information may facilitate future breeding of pitaya.

火龙果（Hylocereus spp。）是生长在日本冲绳县的一种热带水果。在这项研究中，我们开发了简单的序列重复（SSR）标记来评估在冲绳县收集的火龙果种的遗传多样性。使用Roche 454 GS FLX +平台，通过下一代测序开发了SSR标记。测试了70套引物对火龙果的遗传鉴定，包括50个AG主题和20个AC主题SSR。共有16个SSR标记成功地扩增了片段，并在这些种之间表现出多态性。SSR标记揭示了遗传多样性参数，包括每个基因座的等位基因数目（6至12），预期的杂合度（0.5至0.85），观察到的杂合度（0.23至0.93），多态信息含量（0.47至0.45）。 0.83），注视指数值（0.013），和交叉率（0.973）。使用这些SSR标记，对冲绳县收集的32种火龙果种进行了遗传鉴定。除了“ Uruma White” /“ Nago White”和“ Ohgimi Red 1” /“ Ohgimi Red 2”之间的种质外，可以使用三种SSR标记物对这些种质进行遗传区分。遗传多样性分析建议分为六类，而分组似乎是基于物种的差异。获得的SSR标记和明确的遗传背景信息可能会促进火龙果的未来繁殖。除了'Uruma White'/'Nago White'和'Ohgimi Red 1'/'Ohgimi Red 2'之间的种质外。遗传多样性分析建议分为六类，而分组似乎是基于物种的差异。获得的SSR标记和明确的遗传背景信息可能会促进火龙果的未来育种。除了'Uruma White'/'Nago White'和'Ohgimi Red 1'/'Ohgimi Red 2'之间的种质外。遗传多样性分析建议分为六类，而分组似乎是基于物种的差异。获得的SSR标记和明确的遗传背景信息可能会促进火龙果的未来育种。