Kendlbacher Vincent, Krenn Yoko Philipina, Vijayan Thapasya, Rupprecht Christina, Spießberger Magdalena, Curto Manuel, Landler Lukas, Meimberg Harald
Department of Ecosystem Management, Climate and Biodiversity, Institute of Integrative Nature Conservation Research BOKU University Vienna Austria.
Department of Ecosystem Management, Climate and Biodiversity, Institute of Zoology BOKU University Vienna Austria.
Ecol Evol. 2025 Jul 8;15(7):e71652. doi: 10.1002/ece3.71652. eCollection 2025 Jul.
Microsatellites (SSRs) are reliable markers for population genetic analyses but often suffer from null alleles caused by mutations in primer binding sites. Exon-primed intron-crossing (EPIC) markers address these limitations and serve as a complementary tool. In this study, we developed SSR and EPIC markers for the green toad (). We demonstrated their effectiveness using genotyping by high-throughput amplicon sequencing (GBAS) across urban and rural populations. Marker development followed an established in-house protocol. We then tested their functionality in singleplex-PCRs before employing them in multiplex reactions and preparing them for sequencing. We analyzed the resulting data using several variability measures as well as individual-based clustering methods. From the initial set of 48 markers of each type, 35 SSR and 46 EPIC markers consistently amplified across samples both in singleplex and multiplex assays. Data analysis did not corroborate the expectation of a continuous reduction of diversity in urban populations compared to rural ones. Clustering methods using EPIC markers revealed ecologically coherent results, showing weaker genetic structure in rural environments, whereas the SSRs' signal reflected drift-induced patterns. Our findings suggest that the green toad exhibits a degree of resilience to urban environments. Furthermore, EPIC markers not only complement SSRs by having fewer null alleles but also provide greater robustness to random drift events. We recommend their combined use, especially in fragmented environments prone to genetic drift, such as urban areas.
微卫星（简单序列重复，SSRs）是群体遗传分析的可靠标记，但常因引物结合位点的突变而出现无效等位基因。外显子引物内含子交叉（EPIC）标记克服了这些局限性，可作为一种补充工具。在本研究中，我们开发了绿蟾蜍的SSR和EPIC标记。我们通过对城市和农村种群进行高通量扩增子测序基因分型（GBAS）来证明其有效性。标记开发遵循既定的内部方案。然后，我们在将它们用于多重反应并为测序做准备之前，先在单重PCR中测试其功能。我们使用多种变异性测量方法以及基于个体的聚类方法分析所得数据。在最初每组48个每种类型的标记中，有35个SSR和46个EPIC标记在单重和多重检测中均能在样本间持续扩增。数据分析并未证实与农村种群相比城市种群多样性持续降低的预期。使用EPIC标记的聚类方法显示出生态上连贯的结果，表明农村环境中的遗传结构较弱，而SSR的信号反映了由漂变引起的模式。我们的研究结果表明，绿蟾蜍对城市环境具有一定程度的适应能力。此外，EPIC标记不仅通过具有较少的无效等位基因来补充SSR，而且对随机漂变事件具有更高的稳健性。我们建议联合使用它们，特别是在容易发生遗传漂变的碎片化环境中，如城市地区。
