Linkage assignment of eleven genes to the porcine genome Zhiliang Hu, 1, * Gary A. Rohrer, 1 Roger T. Stone, 1 Mark Rutherford, 2 Mark A. Osinski, 2 Mary S. Pampusch, 2 Michael P. Murtaugh, 2 David R. Brown, 2 Craig W. Beattie 1, ** 1 U.S. Department of Agriculture, ARS, U.S. Meat Animal Research Center (MARC), Spur 18 D, P.O. Box 166, Clay Center, Nebraska 68933-0166, USA 2 University of Minnesota, Department of Veterinary and PathoBiology, St. Paul, Minnesota 66108, USA Received: 15 December 1996 / Accepted: 31 March 1997 Abstract. We report comparative linkage mapping of eleven genes in the swine genome by RFLP analysis. These genes in- clude: Acid phosphatase type 5 (ACP5), Cholecystokinin Type B Receptor (CCKBR), Antibiotic Peptide (FALL39), Insulin-like Growth Factor 1 Receptor (IGF1R), Integrin Alpha M (ITGAM), Integrin Beta 2 (ITG2), Opioid Receptor Mu-1 (OPRM1), Pro- hormone Converter (PC1/3), Retinol Binding Protein 3 (RBP3), Ribosomal DNA (RNR1), and Zona Pellucida Glycoprotein 1 (ZP1). The CCKBR and ITG2 loci define the ends of the linkage groups on Chromosomes (Chro) (SSC) 9p and 13qter, respec- tively. Introduction Microsatellite (ms) markers and the development of comparative gene mapping have made possible the construction of a high- resolution genetic map for swine (Rohrer et al. 1996). Two classes of loci (Type I and Type II; O’Brien 1991) have been used to construct genetic maps in swine. Type I loci (O’Brien 1991) rep- resent evolutionarily conserved, but minimally polymorphic cod- ing sequences, while Type II loci describe highly polymorphic regions of repetitive DNA. Microsatellites (ms), single-locus Type II markers, have been used to merge current swine linkage maps (Ellegren et al. 1994; Archibald et al. 1995; Rohrer et al. 1994) into a relatively high resolution genetic map (Rohrer et al. 1996). How- ever, comparative mapping of Type I markers in swine is just beginning to be developed (Rettenberger et al. 1995, 1996; Fro- nicke et al. 1996; Yerle et al. 1995). Identification of regions of synteny conserved between species can enhance a search for the basis of an inherited phenotype by providing candidate (gene) regions for eventual positional or positional candidate cloning (Womack 1987; Montgomery et al. 1995). Comparative linkage mapping by restriction fragment length polymorphisms (RFLPs; Botstein et al. 1980) defined by cDNA probes provide an impor- tant source of polymorphism(s) to order genes within regions of conserved synteny. Comparative linkage mapping also offers the opportunity to improve resolution about a quantitative trait locus (QTL). Since sufficient Type I markers are not present on the swine linkage map to permit precise alignment of the porcine and human genomes, and gene order rearrangements occur (Johansson et al. 1995), we initiated an effort to increase the number of linked Type I loci to more effectively utilize the human gene map. We report mapping 11 genes in the swine genome by linkage: Acid phos- phatase type 5 (ACP5), Cholecystokinin Type B Receptor (CCKBR), Antibiotic Peptide (FALL39), Insulin-like Growth Fac- tor 1 Receptor (IGF1R), Integrin Alpha M (ITGAM), Integrin Beta 2(ITG2), Opioid Receptor Mu-1 (OPRM1), Pro-hormone Con- verter (PC1/3), Retinol Binding Protein 3 (RBP3), Ribosomal DNA (RNR1), and Zona Pellucida Glycoprotein 1 (ZP1). Materials and methods Eight two-generation intraspecific backcross families comprising the USDA-MARC Swine Reference Population were used in this study (Rohrer et al. 1994). cDNA probes. Procedures used in transformation, amplification, and iso- lation of cDNA probes were as described (Sambrook et al. 1989). All cDNA clones were labeled with [ 32 P]ATP to a specific activity of 1–2 × 10 9 cpm/g by random priming (Feinberg and Vogelstein 1983). Nine probes were derived from porcine cDNA libraries (Table 1). All cDNA probes were essentially full length. The 4.7-kb fragment from the 5' regu- latory region of the ribosomal gene cluster (RNR1) was used to prevent hybridization to the numerous copies of ribosomal RNA genes. Two clones (CCKBR, IGF1R) were of human origin. Restriction endonuclease-digested swine genomic DNA (10 g) was electrophoretically separated on 0.8% agarose gels, blotted to Zeta-probe membranes, and hybridized to the radiolabeled cDNA probes at 65°C in 0.5 M sodium phosphate, 1% BSA (bovine serum albumin), and 5% SDS (sodium dodecyl sulfate) overnight; washed twice for 20 min each at 65°C in 2 × SSC and 0.5% SDS, and exposed to film for 48 h. HindIII-digested lambda markers were used as size standards. At least six restriction en- zymes were evaluated for each probe on the parents of the reference popu- lation. All informative families were genotyped. Linkage analysis. All possible two-point linkages were computed be- tween new markers and over 1000 existing markers (Rohrer et al. 1996) with CRIMAP (version 2.4; Green et al. 1989). When more than one enzyme was used to genotype a given locus (described by a cDNA), the genotypic information was haplotyped utilizing the ‘‘hap sys0’’ statement in CRIMAP. Significant (LOD > 3.0) two-point linkages were used to assign each new locus to a chromosome. The locus was inserted and order established by multipoint linkage analysis (the ALL option of CRIMAP). The CHROMPIC option was used to identify potential genotypic errors. If detected, genotypes were re-scored and corrections made. All results were entered into the USDA, ARS, MARC database (Keele et al. 1994). Results and discussion Unique RFLPs were identified for each of the 11 genes. Band sizes (Table 1) characteristic of each polymorphism and the number of Correspondence to: G.A. Rohrer * Present address: Iowa State University, Dept. Animal Science, Ames, IA 50011-3150, USA ** Present address: University of Minnesota, Department of Veterinary and Pathobiology, St. Paul, MN 66108, USA Mammalian Genome 8, 559–563 (1997). © Springer-Verlag New York Inc. 1997