Department of Integrative Biology, University of Colorado Denver, Denver, Colorado, United States of America.
Department of Biology, Linfield College, McMinnville, Oregon, United States of America.
PLoS One. 2018 Oct 18;13(10):e0205423. doi: 10.1371/journal.pone.0205423. eCollection 2018.
Pinus albicaulis (whitebark pine) is a widely-distributed but rapidly declining high elevation western North American tree and a candidate for listing under the U.S. Endangered Species Act. Our objectives were to develop reliable nuclear microsatellite markers that can be used to assess within-population genetic diversity as well as seed and pollen migration dynamics, and to validate markers using two geographically proximal P. albicaulis populations. We identified 1,667 microsatellite-containing sequences from shotgun DNA libraries of P. albicaulis. Primer pairs were designed for 308 unique microsatellite-containing loci, and these were evaluated for PCR amplification success and segregation in a panel of diploid needle tissue. DNA was extracted with an SDS protocol, and primers were screened through gel electrophoresis. Microsatellites were genotyped through fluorescent primer fragment analysis. Ten novel and 13 transferred loci were found to be reproducible in analyses based on 20 foliage samples from each of two locations: Henderson Mountain, Custer Gallatin National Forest, Montana, and Mt. Washburn, Yellowstone National Park, Wyoming (USA). Transferred loci had higher numbers of alleles and expected heterozygosities than novel loci, but also revealed evidence for a higher frequency of null alleles. Eight of the 13 transferred loci deviated significantly from Hardy-Weinberg Equilibrium, and showed large positive FIS values that were likely inflated by null alleles. Mantel's tests of transferred and novel markers showed no correlation between genetic and geographic distances within or among the two sampled populations. AMOVA suggests that 91% of genetic variability occurs within populations and 9% between the two populations. Studies assessing genetic diversity using these microsatellite loci can help guide future management and restoration activities for P. albicaulis.
白皮松（白皮松）是一种分布广泛但迅速减少的高海拔北美西部树种，也是美国濒危物种法案下的候选树种。我们的目标是开发可靠的核微卫星标记，用于评估种群内遗传多样性以及种子和花粉迁移动态，并使用两个地理上相近的白皮松种群验证标记。我们从白皮松的鸟枪法 DNA 文库中鉴定出 1667 个含有微卫星的序列。为 308 个独特的含有微卫星的基因座设计了引物对，并在一组二倍体针叶组织中评估了 PCR 扩增成功率和分离情况。用 SDS 方案提取 DNA，并用凝胶电泳筛选引物。通过荧光引物片段分析对微卫星进行基因分型。在基于蒙大拿州卡斯特-加廷国家森林亨德森山和怀俄明州黄石国家公园的 Mt. Washburn 的 20 个叶样的每个位置的 20 个样本的分析中，发现 10 个新的和 13 个转移的基因座是可重复的。转移的基因座比新的基因座具有更多的等位基因和预期杂合度，但也显示出更高的无效等位基因频率的证据。13 个转移的基因座中有 8 个偏离 Hardy-Weinberg 平衡，表现出较大的正 FIS 值，这很可能是由无效等位基因引起的。转移和新标记的 Mantel 检验表明，在两个抽样种群内或种群间，遗传和地理距离之间没有相关性。AMOVA 表明，91%的遗传变异发生在种群内，9%发生在两个种群之间。使用这些微卫星标记评估遗传多样性的研究可以帮助指导白皮松的未来管理和恢复活动。
