Discovery of genetic variants for fatty acid binding proteins of pig ( Brief Report )

Back fat thickness (BFT) and intramuscular fat (IMF) contents are known as major issues affecting meat performance. Several types of fatty acid-binding proteins (FABPs), which involve signal transduction pathways, are abundantly presented in tissues such as intestine, liver, kidney, mammary gland, heart, and red skeletal muscle (Nechtelberger et al. 2001). FABPs have been reported to be differentially expressed genes during porcine adipogenesis (Samulin et al. 2008) and related to fat deposition (Szczerbal et al. 2007). Accordingly FABPs may be candidate genes to explain variation of fat related traits in pigs. Therefore, it is an essential process to search genetic variants that may provide useful genetic information to study associations with quantitative loci (QTL).


Background
Back fat thickness (BFT) and intramuscular fat (IMF) contents are known as major issues affecting meat performance.Several types of fatty acid-binding proteins (FABPs), which involve signal transduction pathways, are abundantly presented in tissues such as intestine, liver, kidney, mammary gland, heart, and red skeletal muscle (Nechtelberger et al. 2001).FABPs have been reported to be differentially expressed genes during porcine adipogenesis (Samulin et al. 2008) and related to fat deposition (Szczerbal et al. 2007).Accordingly FABPs may be candidate genes to explain variation of fat related traits in pigs.Therefore, it is an essential process to search genetic variants that may provide useful genetic information to study associations with quantitative loci (QTL).

Procedure
Amplification primers for FABP1, 2, 4, 5, and 7 genes were designed based on sequences from the GenBank (Table 1).PCR mixtures used were made up of 2 µl of 10 X reaction buffer (10 mM Tris, pH 8.3, 50 mM KCl, 0.1 % Triton X-100, and 1.5 mM MgCl 2 ), 25 mM of dNTP, 10 pmol of each primer, 50 ng of genomic DNA, and 0.2 units of Taq DNA polymerase (Gibco BRL, Grand Island, NY) in a final volume of 20 µl.After heating at 95 °C for 2 min, a total of 35 cycles were adapted for denaturation at 94 °C/1 min, annealing at 51 to 59 °C/1 min, and polymerization at 72 °C/1.5 min.After cleaning up PCR products using a PCR clean up kit (Nucleogen, Korea), the direct sequencing was performed with Big-dye terminator version 3.1 and an ABI3730XL Genetic Analyzer.Each experiment was duplicated for PCR amplification and sequencing reactions to minimize base calling errors.After identification of genetic variants, SNPs were detected for 355 Yorkshire pigs using the sequenom mass array system.

Results
This study aimed to search single nucleotide polymorphisms (SNPs) in FABPs as molecular markers accounting for variation of fat mechanisms of pigs.As shown in Table 2, a total of 26 SNPs were identified using 355 Yorkshire pigs, and the sequences with the newly identified SNPs were submitted to GenBank with accession numbers (FABP1, FABP2, FABP4, FABP5,  The primer selection based on GenBank accession numbers, and the newly identified sequences for FABP1, FABP2, FABP3, FABP4, FABP5, and FABP7 genes were submitted into GenBank with accession numbers (GU189560, GU189561, FJ755468, GU189562, GU189563, and GU189564), respectively.The nucleotide positions based on the GenBank accession numbers that we have been submitted (FABP1, FABP2, FABP4, FABP5, and FABP7 for GU189560, GU189561, GU189562, GU189563, and GU189564, respectively), 2 Substitutions of nucleotides, 3 Substitutions of amino acids, 4 The rates of nucleotide substitution for 355 Yorkshire pigs and FABP7 for GU189560, GU189561, GU189562, GU189563, and GU189564, respectively).Substitutions of amino acids detected with 2 SNPs at positions 1,328 (R/H) and 2,045 (R/Q) in FABP4 and FABP5 gene, respectively, and some of the amino acids revealed conservative patterns showing that the polymorphisms have not been observed in other species using the blast search.We also found di-repeat sequences, showing polymorphisms (CA) 17-20 .The identified SNPs are the first report to help understanding of genetic structures for pig populations regarding fat related traits.
The existence of mutation sites in coding regions of FABP genes may give useful genetic information due to the relation to fat deposition (Szczerbal et al. 2007).In addition, as candidate genes associated with fatness (Estelle et al. 2009, Mercade et al. 2006), either genotypes or haplotypes of FABP genes in this analysis may help animal breeders when they select animals and change genetic structures in pig populations for commercial purposes.

Table 1
Primer sequences, PCR conditions, and size of segments for the swine fatty acid binding proteins Primer sequences 1

Table 2
Identification of single nucleotide polymorphisms in the swine fatty acid binding proteins