Next Generation Semiconductor Based-Sequencing of a Nutrigenetics Target Gene (GPR120) and Association with Growth Rate in Italian Large White Pigs

Abstract

The GPR120 gene (also known as FFAR4 or O3FAR1) encodes for a functional omega-3 fatty acid receptor/sensor that mediates potent insulin sensitizing effects by repressing macrophage-induced tissue inflammation. For its functional role, GPR120 could be considered a potential target gene in animal nutrigenetics. In this work we resequenced the porcine GPR120 gene by high throughput Ion Torrent semiconductor sequencing of amplified fragments obtained from 8 DNA pools derived, on the whole, from 153 pigs of different breeds/populations (two Italian Large White pools, Italian Duroc, Italian Landrace, Casertana, Pietrain, Meishan, and wild boars). Three single nucleotide polymorphisms (SNPs), two synonymous substitutions and one in the putative 3′-untranslated region (g.114765469C > T), were identified and their allele frequencies were estimated by sequencing reads count. The g.114765469C > T SNP was also genotyped by PCR-RFLP confirming estimated frequency in Italian Large White pools. Then, this SNP was analyzed in two Italian Large White cohorts using a selective genotyping approach based on extreme and divergent pigs for back fat thickness (BFT) estimated breeding value (EBV) and average daily gain (ADG) EBV. Significant differences of allele and genotype frequencies distribution was observed between the extreme ADG-EBV groups (P < 0.001) whereas this marker was not associated with BFT-EBV.

Keywords:

• Association study
• GPR120
• Heavy pigs
• Ion Torrent semiconductor sequencing
• SNP

ACKNOWLEDGMENTS

We thank ANAS for providing data and samples, Sara De Fanti (University of Bologna) for technical assistance, and Rita Casadio and Pier Luigi Martelli (Biocomputing Group, University of Bologna) for their advice on data analysis.

Notes

^a PCR profile for primer pairs Ex1, Ex2, and Ex3 was the following : 98°C for 1 min, followed by 35 cycles with 98°C for 10 sec, the appropriate annealing temperature for 30 sec, 72°C for 40 sec, the final elongation step was at 72°C for 8 min. PCR Profile for primer pair Ex3SNP was the following: 95°C for 5 min, then 35 cycles at 95°C for 30 sec, the reported annealing temperature for 30 sec, 72°C for 30 sec, the final elongation step was for 5 min at 72°C.

^a BFT = back fat thickness estimated breeding value (EBV); ADG = average daily gain EBV.

^b The two extreme and divergent tails for BFT EBV and ADG EBV.

^c P value of the chi square test for allele frequency difference between the negative and positive tails.

^d P value of the chi square test for genotype frequency difference between the negative and positive tails.

^e P value of the Cochran-Armitage trend test for genotype frequency differences between the negative and positive tails.