Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
Mathematical and Economic Modelling (MAEMOD), Mahidol Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
Parasit Vectors. 2021 May 1;14(1):233. doi: 10.1186/s13071-021-04737-y.
Genetic markers are employed widely in molecular studies, and their utility depends on the degree of sequence variation, which dictates the type of application for which they are suited. Consequently, the suitability of a genetic marker for any specific application is complicated by its properties and usage across studies. To provide a yardstick for future users, in this study we assess the suitability of genetic markers for molecular systematics and species identification in helminths and provide an estimate of the cut-off genetic distances per taxonomic level.
We assessed four classes of genetic markers, namely nuclear ribosomal internal transcribed spacers, nuclear rRNA, mitochondrial rRNA and mitochondrial protein-coding genes, based on certain properties that are important for species identification and molecular systematics. For molecular identification, these properties are inter-species sequence variation; length of reference sequences; easy alignment of sequences; and easy to design universal primers. For molecular systematics, the properties are: average genetic distance from order/suborder to species level; the number of monophyletic clades at the order/suborder level; length of reference sequences; easy alignment of sequences; easy to design universal primers; and absence of nucleotide substitution saturation. Estimation of the cut-off genetic distances was performed using the 'K-means' clustering algorithm.
The nuclear rRNA genes exhibited the lowest sequence variation, whereas the mitochondrial genes exhibited relatively higher variation across the three groups of helminths. Also, the nuclear and mitochondrial rRNA genes were the best possible genetic markers for helminth molecular systematics, whereas the mitochondrial protein-coding and rRNA genes were suitable for molecular identification. We also revealed that a general gauge of genetic distances might not be adequate, using evidence from the wide range of genetic distances among nematodes.
This study assessed the suitability of DNA genetic markers for application in molecular systematics and molecular identification of helminths. We provide a novel way of analyzing genetic distances to generate suitable cut-off values for each taxonomic level using the 'K-means' clustering algorithm. The estimated cut-off genetic distance values, together with the summary of the utility and limitations of each class of genetic markers, are useful information that can benefit researchers conducting molecular studies on helminths.
遗传标记广泛应用于分子研究，其适用性取决于序列变异程度，这决定了它们适合的应用类型。因此，遗传标记对于任何特定应用的适用性都因它们在研究中的特性和用途而变得复杂。为了为未来的用户提供一个衡量标准，在这项研究中，我们评估了遗传标记在寄生虫分子系统学和物种鉴定中的适用性，并估计了每个分类水平的遗传距离截止值。
我们根据对于物种鉴定和分子系统学很重要的某些特性，评估了四类遗传标记，即核核糖体内部转录间隔区、核 rRNA、线粒体 rRNA 和线粒体蛋白编码基因。对于分子鉴定，这些特性是种间序列变异、参考序列的长度、序列的易于比对以及通用引物的易于设计。对于分子系统学，这些特性是：从目/亚目到种水平的平均遗传距离、目/亚目水平的单系分支数量、参考序列的长度、序列的易于比对、通用引物的易于设计、以及核苷酸替换饱和的不存在。使用“K-均值”聚类算法估计了截止遗传距离。
核 rRNA 基因表现出最低的序列变异，而线粒体基因在这三组寄生虫中表现出相对较高的变异。此外，核和线粒体 rRNA 基因是寄生虫分子系统学的最佳遗传标记，而线粒体蛋白编码和 rRNA 基因适用于分子鉴定。我们还揭示了使用线虫中广泛的遗传距离证据，一般的遗传距离衡量标准可能不够充分。
本研究评估了 DNA 遗传标记在寄生虫分子系统学和分子鉴定中的适用性。我们提供了一种新的分析遗传距离的方法，使用“K-均值”聚类算法生成每个分类水平的合适截止值。估计的截止遗传距离值，以及每类遗传标记的用途和局限性总结，是对进行寄生虫分子研究的研究人员有用的信息。
