Using Next Generation RAD Sequencing to Isolate Multispecies Microsatellites for <i>Pilosocereus</i> (Cactaceae)

Microsatellite markers (also known as SSRs, Simple Sequence Repeats) are widely used in plant science and are among the most informative molecular markers for population genetic investigations, but the development of such markers presents substantial challenges. In this report, we discuss how next generation sequencing can replace the cloning, Sanger sequencing, identification of polymorphic loci, and testing cross-amplification that were previously required to develop microsatellites. We report the development of a large set of microsatellite markers for five species of the Neotropical cactus genus Pilosocereus using a restriction-site-associated DNA sequencing (RAD-seq) on a Roche 454 platform. We identified an average of 165 microsatellites per individual, with the absolute numbers across individuals proportional to the sequence reads obtained per individual. Frequency distribution of the repeat units was similar in the five species, with shorter motifs such as di- and trinucleotide being the most abundant repeats. In addition, we provide 72 microsatellites that could be potentially amplified in the sampled species and 22 polymorphic microsatellites validated in two populations of the species Pilosocereus machrisii. Although low coverage sequencing among individuals was observed for most of the loci, which we suggest to be more related to the nature of the microsatellite markers and the possible bias inserted by the restriction enzymes than to the genome size, our work demonstrates that an NGS approach is an efficient method to isolate multispecies microsatellites even in non-model organisms.微卫星标记（Microsatellite markers，又称SSRs，简单序列重复Simple Sequence Repeats）在植物科学领域应用广泛，是群体遗传学研究中信息含量最高的分子标记之一，但这类标记的开发仍面临诸多实质性挑战。本研究探讨了下一代测序技术如何替代此前开发微卫星标记所需的克隆、桑格测序（Sanger sequencing）、多态性位点筛选及跨扩增验证等流程。本研究基于罗氏454平台的限制性位点相关DNA测序（RAD-seq）技术，为新热带仙人掌科毛花柱属（Pilosocereus）的5个物种开发了一套大规模微卫星标记。研究平均每个个体可鉴定出165个微卫星位点，个体间的绝对位点数量与每个个体获得的测序读段数呈正比。5个物种的重复单元频率分布特征相似，其中二核苷酸、三核苷酸等短基序的重复类型最为丰富。此外，本研究提供了72个可在采样物种中实现有效扩增的微卫星标记，以及22个在毛花柱属物种Pilosocereus machrisii的两个种群中验证的多态性微卫星标记。尽管多数位点在个体间存在测序覆盖度偏低的情况，我们认为该现象更可能与微卫星标记本身的特性及限制性酶引入的偏好性有关，而非基因组大小差异。本研究证实，即使在非模式生物中，下一代测序技术也是一种高效分离多物种微卫星标记的可行方案。