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Factors influencing genotyping success and genotyping error rate of Eurasian otter ( Lutra lutra ) faeces collected in temperate Central Europe
European Journal of Wildlife Research ( IF 1.8 ) Pub Date : 2020-12-09 , DOI: 10.1007/s10344-020-01444-4
Marcia Sittenthaler , Eva Maria Schöll , Christoph Leeb , Elisabeth Haring , Rosemarie Parz-Gollner , Klaus Hackländer

The use of non-invasively collected DNA source material for genetic and genomic applications is usually characterized by low target DNA concentration and quality, genotyping errors and cost-intensive lab procedures. However, for otters (Lutrinae) as elusive species of conservation concern, genetic non-invasive sampling has become an important tool to study their ecology and demography. To increase cost-efficiency of monitoring programmes and to promote the expansion of genomic approaches to non-invasive samples, we aimed to refine sample collection and preparation. Therefore, we examined the effects of intrinsic sample characteristics (including diet), environmental conditions in the field and sample treatment in the molecular laboratory on the success of genotyping and allelic dropout (ADO) rates using microsatellite markers in 1970 fresh Eurasian otter (Lutra lutra) scats. Using fresh samples only, we probably eliminated one of the most important impediments of genotyping DNA from otter faecal samples beforehand. But, we observed higher genotyping success and lower ADO rates for anal glad secretions and faecal samples containing high proportions of mucus. Moist conditions during sample collection may promote DNA degradation and PCR inhibition, leading to decreased genotyping success rates. ADO was further affected by the type of extraction kit. However, a high proportion of variance remaining unexplained by our models implied that additional parameters were acting (amount of PCR inhibitors, non-uniform distribution of intestinal cells, efficiency of PCRs, specific microclimate at marking sites). We summarized influential factors maximizing genotyping quality of otter scats and give recommendations for sample collection, storage and DNA extraction based on our results and current literature.



中文翻译:

影响中欧温带地区欧亚水獭粪便基因分型成功和基因分型错误率的因素

非侵入性收集的DNA来源材料在遗传和基因组学应用中的使用通常具有以下特点:目标DNA浓度和质量低,基因分型错误和成本高昂的实验室程序。但是,对于水獭(Lutrinae作为非自然保护物种,基因无创采样已成为研究其生态学和人口统计学的重要工具。为了提高监测计划的成本效益并促进基因组学方法向非侵入性样品的扩展,我们旨在完善样品的收集和制备。因此,我们使用1970年新鲜的欧亚水獭(Lutra lutra),使用微卫星标记检查了固有样本特征(包括饮食),现场环境条件和分子实验室中的样本处理对基因分型和等位基因缺失(ADO)率成功的影响。)粪便。仅使用新鲜样品,我们可能事先消除了水獭粪便样品中进行基因分型的最重要障碍之一。但是,我们观察到较高的基因分型成功率和较低的ADO率(对于肛门高兴的分泌物和含有高比例黏液的粪便样本)。样品收集过程中的潮湿条件可能会促进DNA降解和PCR抑制,从而导致基因分型成功率降低。ADO进一步受到提取工具包类型的影响。但是,我们的模型尚无法解释的很大一部分方差表明,正在发挥作用的其他参数(PCR抑制剂的量,肠细胞的不均匀分布,PCR的效率,标记位点的特定微气候)。

更新日期:2020-12-09
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