Assessing the suitability of mitochondrial and nuclear DNA genetic markers for molecular systematics and species identification of helminths

Abstract

Background 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. Methods 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. Results 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. Conclusion 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.

The potential use of mitochondrial ribosomal genes (12S and 16S) in DNA barcoding and phylogenetic analysis of trematodes

“…Our findings corroborate previous studies, as successful discrimination of nematode species within the Angiostrongylus cantonensis lineage was also attained using these two genetic markers [ 28 ]. More recently, the suitability of the mitochondrial 12S and 16S rRNA genes for molecular identification of parasitic trematodes, cestodes, and nematodes were ascertained through comparisons with other genetic markers [ 36 ]. Although the mitochondrial rRNA genes are generally the most conserved of the genes in the mitochondrial genome, here we provide evidence of the ability of the mitochondrial genes as alternative genetic markers for molecular identification of trematodes, instead of the commonly used mitochondrial protein-coding genes.…”

“…The NAD1 gene is popular among family Echinostomatidae, and successful molecular discrimination among the morphologically similar Echinostoma revolutum species complex have been achieved [ 43 – 47 ]. Additionally, the NAD1 gene showed higher levels of inter species variation compared to the COI gene within Echinostoma and among trematodes [ 36 , 43 ]. Similar to the COI gene, although they are useful for species discrimination, the high sequence variatblity can be a limiting factor hindering amplification of broad species range.…”

The first complete mitochondrial genome sequence of Cynopterus brachyotis (Chiroptera, Pteropodidae) from the Philippines

“…This study focused on the mitochondrial genome due to its utility in taxonomic resolution. Mitochondrial DNA evolves faster compared to nuclear genetic markers, generating a higher degree of sequence variation which makes it useful to resolve lower taxonomic levels for organisms ( Chan et al 2021 , Hassanin et al 2013 ). Furthermore, the relatively small size of the mitogenome (~ 16,000 base pairs long) makes it a practical alternative as a molecular marker.…”

“…In particular, single nucleotide polymorphisms (SNPs) and microsatellites exhibit high polymorphism amongst populations or individuals ( Galbusera et al 2000 , Jiang et al 2016 ). In addition, the highly-conserved nature of nuclear rRNA gene sequences makes it suitable for resolving higher taxonomic levels for organisms ( Chan et al 2021 ). Other studies have shown that combining mitochondrial and nuclear DNA analysis provided better taxonomic resolution ( Abreu et al 2020 , Lambret-Frotté et al 2012 ).…”

Diagnosis of Taenia solium infections based on “mail order” RNA-sequencing of single tapeworm egg isolates from stool samples

“…The easy readout of the entire mtDNA sequence by single egg RNA-sequencing provides valuable insight into the regional transmission and spread of T . solium infections, because the “barcodes” of mitochondrial genes can be easily used for taxonomic fine tuning [ 45 , 46 ] and thus for elucidation of chains of infection.…”