The molecular basis of color vision in colorful fish: Four Long Wave-Sensitive (LWS) opsins in guppies (Poecilia reticulata) are defined by amino acid substitutions at key functional sites

Additional file 1:

List of LWS primer names and sequences used for PCR, RT-PCR and qPCR. Primer names and numbers correspond to reaction conditions shown in Additional file 2. Primer numbers corresponds to amplicons shown in Additional file 3. Sequences are given in the 5'to 3' orientation. Primers were synthesized by Operon^® Biotechnologies, suspended in sterile buffered TE (pH 7.0) and stored for no longer than one year at -20°C.

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Additional file 2:

Sequence data and PCR conditions for specific primer combinations used for all six species of Poeciliidae. Primer names are given with corresponding primer number from Additional file 1. Amplicons are shown in Additional file 3 along with corresponding n value. 0.5 U iProof™ DNA polymerase (BioRad^®) was used for each reaction with 5× iProof™ HF Buffer, 10 mM dNTP mix, ~100 ng template DNA, 0.5 μM of both forward and reverse primers and dH₂O. An Eppendorf^® silver block thermal cycler was used for all PCR reactions. Each reaction included an initial denaturation of 94°C for 30 seconds and a final extension of 72°C for 600 seconds. If >1 bands were found after gel electrophoresis, expected sized amplicons were cut and purified using a QIAquick^® Gel Extraction Kit.

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Additional file 3:

Primer and sequence maps for LWS opsin loci in Poeciliidae. Large colored boxes and horizontal bars represent exons and introns, respectively. Black and solid horizontal lines represent genomic sequences amplified using various PCR primer combinations (see legend; see Additional files 1 and 2). Dotted horizontal lines represent mRNA transcripts acquired through RT-PCR from cDNA samples. "n" represents the number of times a given sequence of near perfect identity had been acquired. Key site amino acids are shown within their respective exons. Inter-species and individual sequence differences due to SNP's, small strings of nucleotides, or codon selection (see discussion) are omitted and a consensus map has been created. Yellow exons and introns are highly similar to the guppy LWS S180 locus. Blue coloration represents sequences of high identity to the guppy LWS P180 locus. This use of color is exploited to highlight the hybrid nature of the LWS A180 gene in the guppy. Finally, green coloration represents the diverged, single exon LWS S180r locus (originally detected by Weadick and Chang [24]).

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Additional file 4:

Exon alignment of LWS genes used in phylogenetic analysis. Common names are listed and acquisition numbers can be found in the methods section.

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