Center for Integrated Animal Genomics Research Experience in Molecular Biotechnology & Genomics Summer 2007 ASSOCIATIONS OF TRANSCRIPTION FACTOR 7 LIKE 2 GENE AND FAT TRAITS IN PIGS Randy Amoako 1, Zhi-Qiang Du 2 and Max F. Rothschild 2 1.Department of Animal Science, North Carolina Agricultural and Technical State University, Greensboro, NC Department of Animal Science and Center for Integrated Animal Genomics, Iowa State University, 2255 Kildee Hall, Ames, IA Program supported by the National Science Foundation Research Experience for Undergraduates DBI ABSTRACT Obesity is associated with an increased risk of developing insulin resistance and Type 2 Diabetes (T2D) in humans. Transcription factor 7 like 2 (TCF7L2) is a gene that is located on human chromosome 10q25.2, and is associated with T2D. We identified a single nucleotide polymorphism (SNP) in the 3’UTR (untranslated) region in the TCF7L2 gene in pigs. A genetic test was used to genotype a reference family constructed by two pig breeds, Berkshire and Yorkshire (BY), and the TCF7L2 gene was genetically mapped to Sus scrofa chromosome 14. We found that TCF7L2 is significantly associated with various fat-related traits, average backfat (P<0.01), tenth rib backfat (P<0.01), last rib backfat (P<0.05), lumbar backfat (P<0.01), and also muscle fiber type II ratio (P<0.01) in the BY population. However, in a pig line selected for intramuscular fat (IMF), only two genotypes of this SNP exist, and there were no significant association results with various carcass traits and fat-related traits. Further investigation on other pig populations have yet to be done, so that we can explore the possibility of utilizing this genetic marker in pig breeding. BACKGROUND  Transcription Factor 7 Like 2 (TCF7L2) is a gene that is located on human chromosome 10q25.2 and is associated with Type 2 Diabetes (T2D).  Type 2 Diabetes is a disease affecting the metabolism of sugar in humans.  Scientists at the National Genome Research Network (NGFN) demonstrated a relationship between the TCF7L2 gene and body weight in humans.  The TCF7L2 gene is involved in regulating hormones that control the balance between the feeling of fullness and appetite (National Genome Research Network 2007).  Obesity is a major contribution to diabetes because it decreases the human body's ability to use insulin.  The main purpose of our research is to examine more closely the relationship between the TCF7L2 gene and growth rate, or adipogenesis in pigs. AKNOWLEGDEMENTS RA would like to thank Mr. Benny Motes for his statistical analyses, and other members from Dr. Rothschild’s laboratory for technical assistance. This research was supported in part by Hatch Funds, the Iowa State Experimental Station and the NSF-REU summer internship program. CONCLUSIONS Based on the results there is possible evidence that the Transcription Factor 7 like 2 gene is associated with obesity. The future possibilities of this connection could benefit the pork industry greatly. First, we may be able to use the gene to produce a leaner healthier pig. Since consumers are now buying leaner less caloric meats this could be a great benefit. Also producing a leaner pig would cut down some spending on pig feed because a leaner pig is more efficient. Producing leaner less caloric pork might also help lower obesity in meat consuming countries. MATERIALS AND METHODS Animals  This research was carried out using a three-generation resource family of a cross between Berkshire and Yorkshire (BY) pig breeds. In the BY family a total of 515 F2 animals were harvested in a commercial facility when they approached 115kg.  Another Duroc population of 760 animals selected for the intramuscular fat percentage trait (IMF) was also used. Traits measured  Traits measured on the BY resource family included four growth-related traits recorded on live animals, several body composition traits and a total of 28 meat quality traits. SNP identification and genotyping  We pooled PCR products from the two different pig breeds and they were sequenced. Sequences obtained were compared using Sequencher software version 3.0 (Gene Codes Ann Arbor, MI USA) to identify SNPs.  We set up a PCR-restriction fragment length polymorphism (RFLP) test using the restriction enzyme Bsr I, in order to distinguish the different alleles of the SNP in the two pig populations. Linkage mapping and association analysis  Genotype data of the TCF7L2-E8 SNP were used to map this gene in the BY population using CRIMAP 2.4.  Association analyses were performed using a mixed model procedure (SAS procedure MIXED, SAS Institute, Cary, NC, USA). Live weight at slaughter was treated as a covariate, litter as a random effect, and sex, year-season and marker genotype as fixed effects for the BY population. For the IMF population, line, sex, sire and marker genotype were treated as fixed effects. RESULTS AND DISCUSSION  We have obtained three sequences (accession numbers: CT826377, CT737264, CT737332), after blast against the pig HTGS database. Based on human gene structure and exon homology, CT was found to contain the promoter, exons 1, 2 and 3, introns 1, 2 and partial intron 3; CT was composed of exons 4, 5, 6, 7, 8, partial intron 3, introns 4, 5, 6, 7, and the 3’UTR; and CT was composed of exons 6, 7, 8, partial intron 5, introns 6, 7, and the 3’UTR (Figure 1).  We used the genotype data in our BY population to map this SNP to Sus scrofa chromosome (SSC) 14. After the two point analysis, high LOD-scores were found between this SNP and microsatellite markers SW55 (LOD=27.78), SW77 (LOD=48.17), respectively.  The marker order of the best sex-averaged map from the multi-point linkage analysis (with distance in Kosambi cM) was: SW CTB-2.8-SW MM14AC-9.3-SWC6-7.8-JNK-2.8-SWR S SW TCF7L SW SW SWC27.  Association analyses in the BY population revealed that different fat traits were highly associated with this SNP, including average backfat, last rib backfat, lumbar backfat, and tenth rib backfat traits. Interestingly, another trait, muscle fiber type II ratio, was also highly associated with TCF7L2 (Table 1 and Figure 2).  However, we found that only two genotypes of this SNP exist in the IMF population, and no significant association results with various carcass traits and fat-related traits were found (Table 1 and Figure 3). A/G C Figure 1. TCF7L2 gene structure in human and pig A. Human TCF7L2 has 8 exons, and exon size is shown as proportional to its real sequence length. B. Three pig genomic sequences have been retrieved, and horizontal arrows indicate they align in both forward and reverse positions to the human sequence orientation. The SNP (A/G) position in the 3’UTR is indicated by a vertical arrow. C. The chromatogram demonstrates the SNP position. Table 1. Association analysis result of TCF7L2-E8 Bsr I polymorphism in the BY and IMF population Figure 2. TCF7L2 least squares means for fat traits in BY population Figure 3. TCF7L2 least squares means for the loin muscle area in the IMF population Genotypes cm Genotypes cm 2 * AVBFAT=average backfat, CARCWT=carcass weight, LASTRIB=last rib backfat, LEA=loin eye muscle area, LUMBAR=lumbar backfat, TENTHRIB=tenth rib backfat, TOTLIPPR%=total lipid percentage, FTYPIIR=muscle fibre type II ratio, IMF%=intramuscular fat percentage, LLBF=last lumbar backfat, LRBF=last rib backfat, LMA=Loin muscle area, PFAT=percentage fat, SBF=scanned backfat, SLMA=scanned loin muscle area, DAYS=days to 70kg.