Data from: Two highly informative dinucleotide SSR multiplexes for the conifer Larix decidua (European larch)

We have designed two highly polymorphic microsatellite multiplexes for Larix decidua Mill. (European larch), a coniferous tree species with a fragmented distribution across Europe. The multiplexes combine microsatellites previously designed for the sister species L. kaempferi and newly identified microsatellites obtained by pyrosequencing of an enriched microsatellite library and subsequent marker candidate selection. As we wanted to target highly polymorphic markers, only microsatellite motifs with a high number of repeats (≥12) were selected. An important proportion of the marker candidates presented multiple bands, bad amplification or insufficient polymorphism. Such difficulties were expected owing to the large genome size of the studied species. Our strategy for marker validation followed most recent recommendations for microsatellite development, e.g. verifying marker quality in terms of polymorphism and accurate allele binning before multiplexing. The most promising loci were combined in two multiplexes, a 7-plex and a 6-plex. These were tested on a sample of 413 individuals from 18 populations distributed across the natural range. The 13 loci had from 9 to 36 alleles. Markers were successfully tested in another laboratory, confirming robustness of the marker protocols. We also tested transferability on six other larch species from Asia and North America. Overall, this study shows that, even in species with large genome size and relatively low overall polymorphism, microsatellites can be successfully developed using next-generation sequencing technologies provided that some additional precautions are taken compared to species lacking these characteristics.

我们为欧洲落叶松（*Larix decidua* Mill.）设计了两套高多态性微卫星（microsatellite）多重扩增组合。欧洲落叶松是一种在欧洲呈碎片化间断分布的针叶树种。该两套组合整合了此前为其姊妹物种日本落叶松（*L. kaempferi*）开发的微卫星标记，以及通过富集微卫星文库焦磷酸测序（pyrosequencing）并筛选候选标记后新获得的微卫星位点。由于本研究旨在筛选高多态性标记，仅选取了重复单元数≥12的微卫星基序。但有相当比例的候选标记出现了多条带扩增、扩增效率不佳或多态性不足的问题。鉴于该研究物种的基因组规模较大，这类困难本就在预期之中。本研究的标记验证流程遵循了当前微卫星开发的最新规范，例如在构建多重扩增组合前，先从多态性表现与等位基因精准分箱两个维度验证标记质量。最终将最具应用潜力的13个位点整合为两套多重扩增组合，分别为7重与6重扩增体系。我们在覆盖其自然分布范围的18个种群共413个个体的样本中对该体系进行了测试。这13个位点的等位基因数量介于9至36之间。该标记体系已在其他实验室成功完成验证，证实了其实验方案的稳定性与可靠性。我们还针对来自亚洲和北美洲的另外6种落叶松开展了标记跨物种转移性测试。综上，本研究证实：即便针对基因组规模较大、整体多态性相对偏低的物种，只要相较于常规物种采取额外的优化措施，即可利用下一代测序（next-generation sequencing）技术成功开发微卫星标记。