Open Access Research article

Identification of a second gene associated with variation in vertebral number in domestic pigs

Satoshi Mikawa1*, Shuji Sato16, Masahiro Nii2, Takeya Morozumi3, Gou Yoshioka4, Noriaki Imaeda4, Tsuneko Yamaguchi5, Takeshi Hayashi17 and Takashi Awata1

Author Affiliations

1 Animal Genome Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan

2 Livestock Research Institute, Tokushima Agriculture, Forestry and Fisheries Technology Support Center, Kamiita, Tokushima 771-1310, Japan

3 STAFF Institute, Tsukuba, Ibaraki 305-0854, Japan

4 Gifu Prefecture Livestock Research Institute, Minokamo, Gifu 505-0037, Japan

5 Chiba Prefecture Livestock Research Center, Yachimata, Chiba 289-1113, Japan

6 National Livestock Breeding Center, Odakura, Nishigo, Fukushima 961-8511, Japan

7 National Agricultural Research Center, Tsukuba, Ibaraki 305-8666, Japan

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BMC Genetics 2011, 12:5  doi:10.1186/1471-2156-12-5

Published: 14 January 2011

Additional files

Additional file 1:

Figure S1: Development of microsatellite markers in the 95% confidence interval of the QTL on SSC7. A. A part of the gene map for the human chromosome 14 (from the human genome reference genome assembly of NCBI, Build 37.1). Human sequences in the region corresponding to the QTL on SSC7 were used to search for homologous swine sequences by BLAST analysis. PCR primers for STSs were designed in these swine sequences. B. Swine BAC clones screened with STSs. C. Swine microsatellite markers developed in this study. Microsatellite markers were isolated from the BAC clones by a direct sequencing method using two-nucleotide repeats such as (CA)10 for sequencing primers. D. A part of the SSC7 sequence map (from the swine genome draft sequence, Sscrofa9 assembly, published by the International Swine Genome Sequencing Consortium). The microsatellite markers developed in this study and those on a linkage map developed by Rohrer [28] were assigned to the Sscrofa9 assembly. The underlined markers have not yet been found in the Sscrofa9 assembly. Dotted lines indicate that markers were assigned to multiple positions. E. A part of the SSC7 linkage map for microsatellite markers, developed by Rohrer [28].

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Additional file 2:

Table S1: Development of swine STSs and microsatellite markers.

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Additional file 3:

Table S2: Information on microsatellite markers.

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Additional file 4:

Figure S2: Swine VRTN cDNA. A swine VRTN cDNA. (number-increase-type; AB550854) was cloned by RT-PCR with three primer pairs: sVRTN 1 (red underlines), sVRTN 2 (green), and sVRTN 3 (blue). Comparison with the swine genome draft sequences revealed that exon 1 extended from the 1st to the 152nd nucleotides and exon 2 ranged from the 153rd to the end. The coding region was 2,094 bp long (154-2,247) and predicted to encode a protein of 698 amino acids. The positions of SNPs in a Large White population (the AY population) are shown with blue background.

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Additional file 5:

Table S3: Locations of genes surrounding the QTL.

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Additional file 6:

Table S4: Polymorphic markers used in this study.

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Additional file 7:

Figure S3: Expression analysis of VRTN in swine embryos.

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Additional file 8:

Figure S4: Expression of the green fluorescent protein (GFP)-fused vertnin in cultured cells. The plasmid vector pcDNA-DEST47-VRTN, which encoded GFP-fused vertnin, was constructed from VRTN cDNA and pcDNA-DEST47 plasmid vector (Invitrogen). NIH-3T3 and HeLa cells (1 × 104 cells/chamber) were seeded on BioCoat Poly-D-Lysine 4-well Culture Slides (BD Biosciences), and then the plasmid vectors pcDNA-DEST47-VRTN and pcDNA/GW-47/CAT (Invitrogen), which encoded a GFP-fused CAT (chloramphenicol acetyltransferase) and was a control for cytoplasmic expression, were transfected into cells by using FuGENE 6 (Roche Diagnostics). Forty-eight hours after transfection, the cells were washed and the nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen). The cells were mounted with the anti-bleaching reagent DABCO (Invitrogen) and analyzed by fluorescence microscopy to examine green (GFP) and blue (DAPI) fluorescence. DAPI staining indicates the locations of nuclei, and GFP-fused vertnin has a similar expression pattern in both types of cell.

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Additional file 9:

Figure S5: Orthologs of swine vertnin protein. A. In the public database, protein sequences probably coded by orthologous genes of swine VRTN were found not only in mammals, including opossum, but also in a bird (zebra finch) and fish (zebrafish and tetraodon). The orthologous genes were conserved at the start and stop codons, so it seems that VRTN encodes a functional protein. Alignment was performed with ClustalW software. Underlines in the swine sequence indicate the homologous region to helix-turn-helix domain of Transposase IS3/IS911. B. The identities (%) of amino acid sequences to swine VRTN. C. Phylogenic tree of vertnin proteins constructed with ClustalW software.

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