Primers and polymerase chain reaction conditions for DNA barcoding teleost fish based on the mitochondrial cytochrome b and nuclear rhodopsin genes

This report describes a set of 21 polymerase chain reaction primers and amplification conditions developed to barcode practically any teleost fish species according to their mitochondrial cytochrome b and nuclear rhodopsin gene sequences. The method was successfully tested in more than 200 marine fish species comprising the main Actinopterygii family groups. When used in phylogenetic analyses, its combination of two genes with different evolutionary rates serves to identify fish at the species level. We provide a flow diagram indicating our validated polymerase chain reaction amplification conditions for barcoding and species identification applications as well as population structure or haplotyping analyses, adaptable to high-throughput analyses. 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 ABSTRACT This report describes a set of 21 PCR primers and amplification conditions developed to barcode practically any teleost fish species according to their mitochondrial cytochrome b and nuclear rhodopsin gene sequences. The method was successfully tested in more than 200 marine fish species comprising the main Actinopterygii family groups. When used in phylogenetic analyses, its combination of two genes with different evolutionary rates serves to identify fish at the species level. We provide a flow diagram indicating our validated PCR amplification conditions for barcoding and species identification applications as well as population structure or haplotyping analyses, adaptable to high-throughput analyses. Teleosts account for > 95% of the estimated 30,000 fish species alive today (Miya et al. 2003; Nelson 2006). The unequivocal identification and classification of living organisms to the species level frequently relies on genetic evidence. Specific DNA sequences act as unrepeatable signatures and therefore constitute a unique DNA barcode for each species. Initiatives, such as The Barcode of Life Database (www.barcodinglife.org) including The Fish Barcode of Life ( 43 www.fishbol.org), use a DNA-based identification system based on a relatively small fragment of the mitochondrial cytochrome c oxidase subunit I (COI). This short DNA sequence provides sufficient identification labels in terms of nucleotide positions (Hebert et al. 2003) to discriminate even between congeneric fish species, despite only 2% sequence divergence found in 98% of these species (Ward et al. 2005). It is nevertheless clear that longer length DNA barcodes will provide more efficient identification labels. Barcode efficiency can be further improved by the simultaneous use of two genes showing different evolutionary rates and genomic positions. The mitochondrial cytochrome b gene (cytb) and the nuclear rhodopsin gene (rhod) fulfill these requirements. The cytb gene, whose 44 45 46 47 48 49 50 51 52