A transcriptome‐based analytical workflow for identifying loci for species diagnosis: a case study with Bactrocera fruit flies (Diptera: Tephritidae)

Abstract

Development of novel molecular methods for accurate and economical identification of species has become critical both for pure biological research and for a wide range of applied areas. The most widely used current molecular diagnostic tool, the mitochondrial cytochrome c oxidase subunit 1 gene (COI), the so-called DNA barcode, has been highly criticised and is known to be ineffective at distinguishing species in many groups. Alternative markers are needed to circumvent these issues and provide diagnosticians with a greater range of tools for making accurate identifications. To address this, we describe here a novel analytical workflow for diagnostic marker development that utilises near-genomic-scale data to search for potential informative loci. The workflow takes advantage of orthologous gene databases, in combination with tests of phylogenetic resolution, and benchmarking of nucleotide variation against COI, to determine putative loci that might outperform COI. We use transcriptomes of 14 tephritid fruit flies and especially the taxonomically complex genus Bactrocera, as a case study. Of 1646 orthologues searched, our workflow retained a total of five loci following our conservative filtering strategy. One locus, POP4, had strong potential as a novel diagnostic marker for Bactrocera fruit flies. POP4 discriminates most species in the training set of taxa, but like COI fails to separate the sibling species Bactrocera tryoni and Bactrocera neohumeralis. Further validation of this potential new marker against a broader taxonomic sample is ongoing. We advocate that this simple and efficient workflow is, with minor modification, customisable for diagnostic development in almost any taxonomic group.

Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini)

“…To this end, tephritids have long been a focal group for DNA barcoding studies, and especially the Old World tropical tribe Dacini (Armstrong & Ball, ; van Houdt et al ., ; Virgilio et al ., ; Jiang et al ., ; Krosch et al ., ). Within this tribe, the three major genera Bactrocera , Dacus , and Zeugodacus are highly species‐rich and comprise several major economic pest species.…”

“…Accurate identification of insect species is fundamental to both basic biological research and applied fields such as quarantine, surveillance and biosecurity. It is widely recognized that traditional morphological approaches are not always sufficient in all taxa or in all life stages, and a multitude of approaches that harness the discriminating power of molecular data have been developed to overcome these issues (Krosch et al, 2017). The burgeoning field of DNA barcoding, in particular the use of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene as a standard molecular "barcode," has seen rapid cataloguing of molecular variation alongside morphological species identifications in many insect groups (Hebert et al, 2003).…”

“…Differentiating between species is thus critical for determining and maintaining "area freedom" (geographical regions declared free of a given pest species), ensuring market access and trade, and understanding local biodiversity and species evolutionary histories. To this end, tephritids have long been a focal group for DNA barcoding studies, and especially the Old World tropical tribe Dacini (Armstrong & Ball, 2005;van Houdt et al, 2010;Virgilio et al, 2012;Jiang et al, 2014;Krosch et al, 2017). Within this tribe, the three major genera Bactrocera, Dacus, and Zeugodacus are highly species-rich and comprise several major economic pest species.…”

“…Within this tribe, the three major genera Bactrocera, Dacus, and Zeugodacus are highly species-rich and comprise several major economic pest species. The majority of DNA barcoding studies in dacines have utilized the universal COI primers of Folmer et al (1994) and have focused predominately on pest species (Krosch et al, 2017); however, there are many more nonpest species that are morphologically similar to known pests and may partially overlap in geographical and/or host range. Such taxa can be rare in trapping surveys because they may be more geographically or host-restricted than their pest siblings, or may not respond as strongly to male-attracting lures that are commonly used (Royer, 2015).…”

The Complete Mitochondrial Genome of Bactrocera carambolae (Diptera: Tephritidae): Genome Description and Phylogenetic Implications

“…The species status of B. carambolae in relation to B. dorsalis has also been explored by many researchers using multiple approaches. Data on morphology/morphometrics [1,24], certain genetic markers [12,14,21,[25][26][27][28][29], mating behavior [11] and chemoecology [20,[30][31][32] supported the identity of B. carambolae as a separate biological species and provided some diagnostic features for species discrimination. On the other hand, identification of morphological hybrids [33] and data from nuclear protein coding genes [14] and microsatellite analysis [34] suggest naturally occurring hybridization and gene flow between the two taxa.…”