Computational protocol: Divergent Evolution among Teleost V1r Receptor Genes

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[…] The following ENSEMBL databases were searched using the BLASTN and TBLASTN algorithms: Dr Zv6 (August 2006 release, 7×coverage), Ga BROAD S1 (June 2006 release, 11×coverage), Ol MEDAKA1 (May 2006 release, 6.7×coverage), Tn Tetraodon 7 (September 2004 release, 8.3×coverage), and Tr Fugu 4.0 (May 2005 release, 90% coverage). Database mined sequences from Dr, Ol and Ga were PCR amplified from genomic DNA and sequenced for confirmation. Da, Dc, Dd, Dk, Dky, Dm, Dn, Dp and Dy V1r1 and V1r2 were PCR amplified from genomic DNA using the following primers: TTC CCG GTA ACA CCG CTG TCA TCT G, GAA GAA GAG CCG GAG GTC AGT GAT CAG, TAT GGA CCT GTG TGT CAC, CAG CCT TTA TGA AAC ACA TTC AC, TGG ACC TGT GTG TCA CCA TCA AAA GG, TCA GTT CTT GCC GCT GGA GTT CTT GCC, and sequenced. The Dm intergenic sequence was amplified from genomic DNA and sequenced. Conditions for degenerate PCRs performed on fugu and tetraodon genomic DNA were: 4 min. at 95°C followed by 48 cycles of 45 sec. at 95°C, 4 min. at 50°C and 3 min at 72°C, followed by a final extension time of 10 min at 72°C, using the following primers: TCT GSM TCA CCT KCA TKC TGA GYG YST WCC A and GGG CTG AGV GMK GCR TAS MAY GAG GAR AAA AA. Low stringency amplifications were performed with an annealing temperature of 52°C. Sequences were deposited in GenBank with the following accession numbers: DQ887609, DQ887610, DQ887610, DQ887612, DQ887613, AY279523, Q887614, DQ887615, DQ887616, 880989, 884592, 884596, 884602, 884604, 884606, 881043, 881043, 881049, 881053, 884622, and 884628. [...] The 75 base pair sequences corresponding to the V1r1 5′UTR homology region of Dr, Dm, Ol, Ga, Tr, and Tn, initially identified using the Pipmaker analysis tool available at http://pipmaker.bx.psu.edu/pipmaker, were aligned. A logo was generated using the sequence logo software available at http://weblogo.berkeley.edu/logo.cgi. [...] Nucleotide sequences of 24 teleost V1r sequences and mouse V1rb2, V1re4 and V1rf3 were aligned using ClustalX and manually rearranged using the Bioedit alignment software (v. 7.0.5.3).The MODELTEST v3.7 program was used to determine the model of DNA sequence evolution that fit our data best. The best fit model was the General Time Reversible (GTR) with a gamma shape distribution of evolutionary rates (α = 1.18; 8 categories of sites). Phylogenetic tree reconstruction based on DNA sequences was performed using the Maximum Likelihood (ML) method as implemented in the program Phyml and using NNI and SPR branch swapping methods, or imposing different starting topologies to avoid local optima. The amino acid tree reconstruction was performed using the JTT model of amino acid changes and a gamma shape distribution of evolutionary rates (α = 2.15; 4 categories of sites). The same topology was retrieved when using two different starting trees: the Bionj Tree or the best topology found using the DNA alignment. For DNA and amino acid analyses, support for branches was assessed by bootstrap analyses of 500 replicates. [...] Differences in the evolutionary rates of the two paralogous V1r copies were statistically tested using the RRtree program . Selective pressures acting on the V1r receptor genes were assessed using different models as implemented in the PAML software v.3.15 . Couples of nested models were compared using Likelihood Ratio Tests (LRTs) to statistically determine the best model. Twice the difference of the likelihood values of the tree obtained under each model approximately follows a chi-square distribution and, together with the number of degrees of freedom (df) separating the two models, allows the calculation of the associated p-value.To evaluate a possible unequal ratio of non-synonymous vs. synonymous substitutions (ω) in the two paralogous V1r copies, we performed an LRT comparing the two-ratio model (one ω per clade) with the one-ratio model (M0) where ω is constant. Varying selective pressure along the genes was assessed by a LRT test comparing the site-specific model M3 (discrete) with 2 or 3 classes of sites displaying different ω, against the M0 model (one-ratio). The branch-site model D , an extension of the model M3 which allows selective pressure at one class of sites to be different in two parts of the phylogeny, was used to test for divergent selective pressure between the two paralogous V1r genes, by applying an LRT on models D vs. M3. […]

Pipeline specifications

Software tools PipMaker, WebLogo, Clustal W, BioEdit, ModelTest-NG, PhyML, PAML
Applications Phylogenetics, Genome data visualization
Organisms Danio rerio, Oryzias latipes