Linkage Mapping of the Angiotensin I Converting Enzyme Gene in Pig V.Q. Nguyen 1, K.L. Glenn 2, B.E. Mote 2, and M.F. Rothschild 2 1 Department of Biological Science, University of California, Irvine, Irvine, CA 92697, USA 2 Department of Animal Science and Center for Integrated Animal Genomics Iowa State University, Ames, Iowa 50011, USA Sow productive life plays an important role in the economic efficiency of pork production. Several genes have been isolated in model organisms and humans that are associated with lifespan. Our hypothesis is that these same genes or regulatory pathways are also important for sow productive life. Angiotensin I converting enzyme (ACE) has been identified as being key to several diseases known to shorten human lifespan. In human, the ACE gene is located on chromosome 17, but it has not been mapped in the pig. Three primer sets were designed to amplify the pig ACE sequence and are anchored in exons and spanned introns covering from exon 1- exon 2, exon 9 - exon 10, and exon 12 - exon 13 based on pig sequence and human intronic sizes based on Ensembl. Sequence results from the primer set designed to amplify sequence from exon 12- exon 13 showed an exonic C/T single nucleotide polymorphism located 95 bases from the start of the amplified fragment. The restriction enzyme AluI was used to perform PCR-RFLP test on this SNP in the Berkshire x Yorkshire Resource Family. The genotypes were used to linkage map this loci using CRIMAP. The results showed that ACE is located between microsatellite markers S0229 and SW874 on pig chromosome 12 (SSC12), as expected by comparative mapping with the human location. Radiation Hybrid (RH) mapping confirmed that ACE is located on pig chromosome 12. The association studies of this SNP with sow productive life are ongoing.  Primers were designed using pig sequence accession number NM_ from NCBI.  Preparation for polymerase chain reactions are summarized in Table 1.  Polymerase chain reactions (PCR) were set up under the following protocol: Initial denaturation temperature at 94˚C for 2 min with 36 cycles of 94˚C for 30 sec, 53˚C for 45 sec, 72˚C for 50 sec, and final extension at 72 ˚C for 5 min.  PCR products were sequenced and SNPs were identified.  A PCR-RFLP test was performed for the C/T SNP in exon 12 using the ISU Berkshire x Yorkshire Resource Family (Malek et al. 2001) under the above conditions with the restriction enzyme AluI.  ACE-I12F: 5’ tca tca tcc agt tcc agt tcc 3’  ACE-I12R: 5’ gtt cgg cgt cca gtt gta ct 3’  ACE-I1F: 5’ tca tcc tcc tcc tgc tct gc 3’  ACE-I1R: 5’ gcc gtg atg ttg gtg ttg ta 3’  ACE-I9F: 5’ ggg cca cat tca gta ttt ca 3’  ACE-I9R: 5’ tcc acc act cct ggt tgt ag 3’  Two point and multipoint linkage analysis of the genotypes were completed using CRI-MAP software (Green et al. 1990).  RH mapping was performed using the IMpRH panel (Milan et al. 2000)  ACE-RHF: 5’ gtc cac cct ctg gcc tac tt 3’  ACE-RHR: 5’ tta ctg agg ccc agc ttc at 3’ ABSTRACT MATERIAL AND METHODS FIGURE 2. ACE LINKAGE MAP FIGURE 1. CHROMATOGRAM REFERENCES  Green, P., Falls, K. and Crooks, S. (1990) Documentation for CRI-MAP, version 2.4 (St. Louis, MO: Washington University School of Medicine).  Malek, M., et al A molecular genome scan analysis to identify chromosomal region influencing economic traits in the pig. I. Growth and body composition. Mammalian Genome 12:  Milan D, et al. IMpRH server: an RH mapping server available on the Web. Bioinformatics 16 (2000): RESULTS AND DISCUSSION IngredientsAmountConcentration Buffer2 µln/a dNTPs0.5 µl10 mM Forward Primer 0.1 µl25 pM Reverse Primer 0.1 µl25 pM GoTaq0.05 µl.35 µM Water6.35 µln/a DNA1.0 µl12.5 ng  Sequencing of PCR products, using the designated primers, revealed 17 single nucleotide polymorphisms, of which five were verified by PCR-RFLP tests (Table 2).  An exonic C/T SNP in the amplified fragment from exon 12 - exon 13 was identified in the sequence chromatogram (Figure 1).  ACE mapped to SSC12 between microsatellites, S0029 and SW874, (Figure 2) on pig chromosome 12, which was expected when compared to human map.  RH mapping confirmed the location of ACE on pig chromosome 12 between markers RH and SW957. ACKNOWLEDGEMENTS This study was supported by the National Science Foundation Research Experiences for Undergraduate Program. We also thank members of Dr. Rothschild’s lab for technical support. TABLE 1. PCR PREPARATION SNPLocationEnzyme C/TExon 1Cac8I C/TExon 9ApeKI C/TExon 12EarI C/TExon 12BseRI C/TExon 12AluI TABLE 2. PCR-RFLP TESTS C/TC/T C T F2 GENERATION PIGS S0229 ACE SW874 S0090 S0147 SWC23 SW LOCI Pig Chromosome 12 cM