Computational protocol: The Microevolution of V1r Vomeronasal Receptor Genes in Mice

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Protocol publication

[…] Seven M. musculus and seven M. domesticus individuals were collected from Czech Republic and France, respectively. Although the mice from each species were sampled from restricted geographic areas (supplementary table 1, Supplementary Material online), it should not affect our results because mice have little geographic differentiation (). The identity of the mice was confirmed by sequencing a 683-nt segment of the mitochondrial cytochrome c oxidase subunit I (COXI) gene that is commonly used as a barcode for identifying animal species.The liver genomic DNAs of the mice were extracted using the PUREGENE genomic DNA purification kit (Gentra Systems, Minneapolis, MN), following the manufacturer's instruction. Gene-specific primers for amplifying 44 V1r genes were designed according to the Mus musculus reference sequence from GenBank (supplementary table 2, Supplementary Material online). The protein-coding region of each V1r gene studied has 870–1,104 nt, which were completely amplified in our experiments. Polymerase chain reactions (PCRs) were performed with GoTaq DNA Polymerase (Promega Corp, Madison, WI) under conditions recommended by the manufacturer. PCR products were examined on 1.5% agarose gel. Samples showing duplicated electropherograms due to insertions/deletions were cloned with TOPO PCR cloning kit (Invitrogen, Carlsbad, CA) and sequenced with universal T7 and M13 primers using the Sanger method on an automatic DNA sequencer. Otherwise, the PCR products were enzymatically processed using calf intestinal phosphatase and exonuclease I (Exo I) (New England Biolabs, Ipswich, MA) before being sequenced bidirectionally with the gene-specific primers. Sequencher (GeneCodes, Ann Arbor, MI) and MEGA4 () were used to edit and align the sequences. Twenty-five presumably neutral noncoding regions (supplementary table 3, Supplementary Material online), most with ∼1,000 nt, were also amplified and directly sequenced in the same 14 mice. Watterson's θ, nucleotide diversity π, Tajima's D, and Fu and Li's D* were computed using DnaSP (). Tajima's test () and Fu and Li's test () were conducted by 10,000 coalescent simulations in DnaSP. Hudson–Kreitman–Aguade (HKA) test () was conducted using a program written by J. Hey (∼heylab/). The sequences reported in this article have been submitted to GenBank (accession numbers JF782602–JF783819, JF782044–JF782601, and JF783820–JF783959). […]

Pipeline specifications

Software tools Sequencher, MEGA, DnaSP, HKA
Application Population genetic analysis
Organisms Mus musculus, Monodelphis domestica