Recovery of the mitochondrial COI barcode region in diverse Hexapoda through tRNA-based primers

Abstract

BackgroundDNA barcoding uses a 650 bp segment of the mitochondrial cytochrome c oxidase I (COI) gene as the basis for an identification system for members of the animal kingdom and some other groups of eukaryotes. PCR amplification of the barcode region is a key step in the analytical chain, but it sometimes fails because of a lack of homology between the standard primer sets and target DNA.ResultsTwo forward PCR primers were developed following analysis of all known arthropod mitochondrial genome arrangements and sequence alignment of the tRNA-W gene which was usually located within 200 bp upstream of the COI gene. These two primers were combined with a standard reverse primer (LepR1) to produce a cocktail which generated a barcode amplicon from 125 of 141 species that included representatives of 121 different families of Hexapoda. High quality sequences were recovered from 79% of the species including groups, such as scale insects, that invariably fail to amplify with standard primers.ConclusionsA cocktail of two tRNA-W forward primers coupled with a standard reverse primer amplifies COI for most hexapods, allowing characterization of the standard barcode primer binding region in COI 5' as well as the barcode segment. The current results show that primers designed to bind to highly conserved gene regions upstream of COI will aid the amplification of this gene region in species where standard primers fail and provide valuable information to design a primer for problem groups.

Simultaneous assessment of the macrobiome and microbiome in a bulk sample of tropical arthropods through DNA metasystematics

“…Within each of these orders, the success rate for Sanger sequencing was between 0% and 36%. This result supports the conclusion of past research that universal primers are not as suitable for some arthropod orders as others (6,15,24,25).…”

A newly recognised Australian endemic species of Austrolecanium Gullan & Hodgson 1998 (Hemiptera: Coccidae) from Queensland

“…We followed the same PCR conditions for amplifying 18S, 28S, EF-1α and wingless as Lin et al (2013) (Table 2), and included a negative control for all reactions. The PCR program from Park et al (2010) was used for all amplifications of COI, but using two different primer pairs to try to amplify the COI barcode region (Table 2). Firstly, we used the primer pair PcoF1 and HCO.…”

Systematic review of the Australian ‘bush-coconut’ genus Cystococcus (Hemiptera: Eriococcidae) uncovers a new species from Queensland

“…Gene regions used for analysis were the 5 0 region of 18S (small subunit nuclear rDNA, SSU rDNA) and the 'DNA barcode' region of COI (mitochondrial cytochrome c oxidase 1). Park et al's (2010) scale insect COI primer combination (PCO_F1 (Park et al 2010) and HCO (Folmer et al 1994)) was effective for many specimens of C. pomiformis and C. echiniformis, but yielded only poorly amplified or no PCR product for most specimens of C. campanidorsalis, sp. nov., so new primers were designed as follows.…”