Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics

Abstract

Mitochondrial genome sequences are important markers for phylogenetics but taxon sampling remains sporadic because of the great effort and cost required to acquire full-length sequences. Here, we demonstrate a simple, cost-effective way to sequence the full complement of protein coding mitochondrial genes from pooled samples using the 454/Roche platform. Multiplexing was achieved without the need for expensive indexing tags (‘barcodes’). The method was trialled with a set of long-range polymerase chain reaction (PCR) fragments from 30 species of Coleoptera (beetles) sequenced in a 1/16th sector of a sequencing plate. Long contigs were produced from the pooled sequences with sequencing depths ranging from ∼10 to 100× per contig. Species identity of individual contigs was established via three ‘bait’ sequences matching disparate parts of the mitochondrial genome obtained by conventional PCR and Sanger sequencing. This proved that assembly of contigs from the sequencing pool was correct. Our study produced sequences for 21 nearly complete and seven partial sets of protein coding mitochondrial genes. Combined with existing sequences for 25 taxa, an improved estimate of basal relationships in Coleoptera was obtained. The procedure could be employed routinely for mitochondrial genome sequencing at the species level, to provide improved species ‘barcodes’ that currently use the cox1 gene only.

The complete mitochondrial genome sequence of the western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae) contains triplicate putative control regions

“…An intermediate gene arrangement between the ancestral pattern and the arrangement found in T. imaginis and F. occidentalis would provide insight into the evolution of the CR and the gene arrangement of the mt genome of Thysanoptera. In addition, a study of the complete mt genomes of additional Thysanoptera species will provide more detailed information of the interordinal phylogeny of the hemipteroid assemblage and will help reconstruct the Thysanoptera phylogenetic relationships such as has been done for Hymenoptera (Castro and Dowton, 2007;Dowton et al, 2009), Diptera (Cameron et al, 2007), Orthoptera (Fenn et al, 2008), Coleoptera (Pons et al, 2010;Timmermans et al, 2010), and Phasmatodean (Kômoto et al, 2011).…”

Complete mitochondrial genome sequence of Cheirotonus jansoni (Coleoptera: Scarabaeidae)

“…To sequence the entire mitogenome of C. jansoni, a range of Coleoptera mitochondrial primers from Timmermans et al (2010) and a pair of universal insect mitochondrial primers from Simon et al (1994) were used. Species-specific primers were then designed on the basis of sequenced fragments to bridge the gaps.…”

“…Besides, it contains a 2590-bp long non-coding AT-rich region that is the second longest one (shorter than that of Rhopaea magnicornis, 2827 bp) among all the coleopteran mitogenomes (Sheffield et al, 2009). The gene arrangement and order is the same as that of most insect mitogenomes (Stewart and Beckenbach, 2003;Friedrich and Muqim, 2003;Bae et al, 2004;Sheffield et al, 2008Sheffield et al, , 2009Timmermans et al, 2010;Song et al, 2010;Wan et al, 2012;Timmermans and Vogler, 2012), and shares the proposed Pancrustacea ancestral organization (Boore et al, 1998). The A+T content of the genome (65.7%) was found to fall within the range observed so far for insects, from the lowest (64.8%) in Japyx solifugus (Diplura; Carapelli et al, 2005) to the highest (86.7%) in Melipona bicolor (Hymenoptera; Silvestre D and Arias MC, unpublished data from NCBI).…”

Morphological and Molecular Perspectives on the Phylogeny, Evolution, and Classification of Weevils (Coleoptera: Curculionoidea): Proceedings from the 2016 International Weevil Meeting

“…Julien Haran reported on his collaborative works that used mitochondrial genome (mitogenome) sequences in reconstructing the phylogeny of weevils. The development of high-throughput sequencing of full mitochondrial genomes in beetles [97] has made possible the exploration of weevil phylogenetic relationships based on this relatively large and easy-to-handle sequence dataset (12 protein coding genes, approximately 10,000 base pairs). Phylogenetic analyses were first conducted on 27 taxa, including major basal groups and subfamilies, representing the main lineages of Curculionidae [43].…”