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

Abstract

Background: DNA 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.

Results: Two 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.

Conclusions: A 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.

Figure 1

Alignment of tRNA-W genes from varied Hexapoda. All 126 Hexapod tRNA-W genes retrieved from GenBank were aligned and manually compared, revealing two groups with high internal sequence homogeneity. Sequence variation within representative species in each group is presented.

Figure 2

Amplification success for CO1 using standard primers (LepF1/LepR1) and two new forward primers positioned in the tRNA-W gene in combination with a standard reverse primer (LepR1) both separately and as a cocktail. TW-J1301 is previously reported tRNA-W primer [9].

Figure 3

Success in recovering high quality sequence records from members of 8 insect orders represented by 6 or more different families. The number of families examined varied among orders: Coleoptera (23), Diptera (25), Ephemeroptera (6), Hemiptera (11), Hymenoptera (17), Lepidoptera (12), Plecoptera (6) and Trichoptera (6).

Figure 4

Sequence characteristics for the 5' region of COI and cases of amino acid deletion. a) Sequence attributes for 11 species that failed to amplify with the standard barcode primers, but that were recovered with the tRNA-W primer cocktail. Sequences are shown from the presumed initiation codon. b) Amino acid deletions in three species of Pseudococcidae and a species of Tenthredinidae. The numbers are the amino acid position from the proposed translation initiation codon.

Figure 5

Gel images of PCR amplicons for 28 scale insect species. The samples included 9 Pseudococcidae (1-9), 9 Diaspididae (10-18), 6 Coccidae (19-24), 1 Ortheziidae, 1 Eriococcidae, 1 Margarodidae and 1 Conchaspididae.