J. Gellin

Construction of a whole-genome radiation hybrid panel for high-resolution gene mapping in pigs

We have developed a panel of 152 whole-genome radiation hybrids by fusing irradiated diploid pig lymphocytes or fibroblasts with recipient hamster permanent cells. The number and size of the porcine chromosome fragments retained in each hybrid clone were checked by fluorescence in situ hybridization with a SINE probe or by primed in situ labeling (PRINS) with SINE-specific primers. A strategy based on the interspersed repetitive sequence polymerase chain reaction (IRS-PCR) was developed for selected clones to determine if the large fragments painted by the SINE probe corresponded to one pig chromosome or to different fragments of several chromosomes. This strategy was buttressed by a double PRINS approach using primers specific for α-satellite sequences of two different groups of swine chromosomes. Genome retention frequency was estimated for each clone by PCR with 32 markers localized on different porcine chromosomes. Of the 152 hybrids produced, 126 were selected on the basis of cytogenetic content and chromosome retention frequency to construct a radiation hybrid map of swine chromosome 8. Our initial results for this chromosome indicate that the resolution of the radiation hybrid map is 18 times higher than that obtained by linkage analysis.

THE PIGMAP CONSORTIUM LINKAGE MAP OF THE PIG (SUS SCROFA).

A linkage map of the porcine genome has been developed by segregation analysis of 239 genetic markers. Eighty-one of these markers correspond to known genes. Linkage groups have been assigned to all 18 autosomes plus the X Chromosome (Chr). As 69 of the markers on the linkage map have also been mapped physically (by others), there is significant integration of linkage and physical map data. Six informative markers failed to show linkage to these maps. As in other species, the genetic map of the heterogametic sex (male) was significantly shorter (∼16.5 Morgans) than the genetic map of the homogametic sex (female) (∼21.5 Morgans). The sex-averaged genetic map of the pig was estimated to be ∼18 Morgans in length. Mapping information for 61 Type I loci (genes) enhances the contribution of the pig gene map to comparative gene mapping. Because the linkage map incorporates both highly polymorphic Type II loci, predominantly microsatellites, and Type I loci, it will be useful both for large experiments to map quantitative trait loci and for the subsequent isolation of trait genes following a comparative and candidate gene approach.

A somatic cell hybrid panel for pig regional gene mapping characterized by molecular cytogenetics.

A panel of 27 pig x rodent somatic cell hybrids was produced and characterized cytogenetically. The first step of this study consisted of hybridizing a SINE probe to GTG-banded metaphases of each hybrid clone in order to count and identify the normal pig chromosomes and to detect rearranged ones. The second step consisted of using the DNA of each clone as a probe after pIRS-PCR (porcine interspersed repetitive sequence-polymerase chain reaction) amplification to highly enrich it in pig sequences. These probes, hybridized to normal pig metaphase chromosomes, enabled the identification of the complete porcine complement in the hybrid lines. Whole chromosomes and fragments were characterized quickly and precisely, and results were compared. In addition to this cytogenetic characterization, molecular verification was also carried out by using primers specific to six microsatellites and to one gene previously mapped to pig chromosomes. The results obtained allow us to conclude that we have produced a panel that is informative for all porcine chromosomes. This panel constitutes a highly efficient tool to establish not only assignments of genes and markers but also regional localizations on pig chromosomes.

A first-generation porcine whole-genome radiation hybrid map.

Abstract. A whole-genome radiation hybrid (WG-RH) panel was used to generate a first-generation radiation map of the porcine (Sus scrofa) genome. Over 900 Type I and II markers were used to amplify the INRA-University of Minnesota porcine Radiation Hybrid panel (IMpRH) comprised of 118 hybrid clones. Average marker retention frequency of 29.3% was calculated with 757 scorable markers. The RHMAP program established 128 linkage groups covering each chromosome (n = 19) at a lod ≥ 4.8. Fewer than 10% of the markers (59) could not be placed within any linkage group at a lod score ≥4.8. Linkage group order for each chromosome was determined by incorporating linkage data from the swine genetic map as well as physical assignments. The current map has an estimated ratio of ∼70 kb/cR and a maximum theoretical resolution of 145 kb. This initial map forms a template for establishing accurate YAC and BAC contigs and eventual positional cloning of genes associated with complex traits.

Detection of quantitative trait loci for growth and fatness in pigs.

A quantitative trait locus (QTL) analysis of growth and fatness data from a three-generation experimental cross between Meishan (MS) and Large White (LW) pig breeds is presented. Six boars and 23 F1 sows, the progeny of six LW boars and six MS sows, produced 530 F2 males and 573 F2 females. Nine growth traits, i.e. body weight at birth and at 3, 10, 13, 17 and 22 weeks of age, average daily gain from birth to 3 weeks, from 3 to 10 weeks and from 10 to 22 weeks of age, as well as backfat thickness at 13, 17 and 22 weeks of age and at 40 and 60 kg live weight were analysed. Animals were typed for a total of 137 markers covering the entire porcine genome. Analyses were performed using two interval mapping methods: a line-cross (LC) regression method where founder lines were assumed to be fixed for different QTL alleles and a half-/full-sib (HFS) maximum likelihood method where allele substitution effects were estimated within each half-/full-sib family. Both methods revealed highly significant gene effects for growth on chromosomes 1, 4 and 7 and for backfat thickness on chromosomes 1, 4, 5, 7 and X, and significant gene effects on chromosome 6 for growth and backfat thickness. Suggestive QTLs were also revealed by both methods on chromosomes 2 and 3 for growth and 2 for backfat thickness. Significant gene effects were detected for growth on chromosomes 11, 13, 14, 16 and 18 and for backfat thickness on chromosome 8, 10, 13 and 14. LW alleles were associated with high growth rate and low backfat thickness, except for those of chromosome 7 and to a lesser extent early-growth alleles on chromosomes 1 and 2 and backfat thickness alleles on chromosome 6.

IMpRH server: an RH mapping server available on the Web.

SUMMARY The INRA-Minnesota Porcine Radiation Hybrid (IMpRH) Server provides both a mapping tool (IMpRH mapping tool) and a database (IMpRH database) of officially submitted results. The mapping tool permits the mapping of a new marker relatively to markers previously mapped on the IMpRH panel. The IMpRH database is the official database for submission of new results and queries. The database not only permits the sharing of public data but also semi-private and private data.

Localization of 113 anchor loci in pigs: improvement of the comparative map for humans, pigs, and goats.

Abstract. In total, 113 genes that have already been located in humans and goats were cytogenetically mapped in pigs. For this purpose, 165 gene-containing bacterial artificial chromosomes (BACs) isolated in goats were used in heterologous fluorescent in situ hybridization on porcine chromosomes. Among them, 113 (or 69%) gave clear and specific signals, and 52 did not work in heterologous conditions. These localizations are a significant contribution to development of the porcine gene map and also to the comparative map for humans and pigs. They allowed us to specify the information obtained by Zoo-FISH while taking the gene order into account; the number of conserved fragments detected for human and pig chromosomes reached 84. The average size of conserved fragments could be estimated at 33 cM. As these genes had already been mapped in goats, the comparison was extended to ruminants. The previous results obtained in this species, suggesting a correlation between human chromosome abnormalities and evolutionary breakpoints, were confirmed in pigs.

The PiGMaP consortium cytogenetic map of the domestic pig (Sus scrofa domestica)

llNRA, Laboratoire de Grnrtique Cellulaire, BP27, 31326 Castanet-Tolosan, France 2Department of Functional Morphology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands 3Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden 4Division of Anatomy, Department of Anatomy and Physiology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark 5Division of Animal Genetics, Department of Animal Science and Animal Health, The Royal Veterinary and Agricultural University, Copenhagen, Denmark 6Department of Clinical Genetics, University of Ulm, Ulm, Germany 7Laboratoire de Cytomrtrie, CEA, Fontenay-aux Roses, France

Porcine linkage and cytogenetic maps integrated by regional mapping of 100 microsatellites on somatic cell hybrid panel

Recently two main genetic maps [Rohrer et al. Genetics 136, 231 (1994); Archibald et al. Mamm. Genome 6, 157 (1995)] and a cytogenetic map [Yerle et al. Mamm. Genome 6,175 (1995)] for the porcine genome were reported. As only a very few microsatellites are located on the cytogenetic map, it appears to be important to increase the relationships between the genetic and cytogenetic maps. This document describes the regional mapping of 100 genetic markers with a somatic cell hybrid panel. Among the markers, 91 correspond to new localizations. Our study enabled the localization of 14 new markers found on both maps, of 54 found on the USDA map, and of 23 found on the PiGMaP map. Now 21% and 43% of the markers on the USDA and PiGMaP linkage maps respectively are physically mapped. This new cytogenetic information was then integrated within the framework of each genetic map. The cytogenetic orientation of the USDA linkage maps for Chromosomes (Chrs) 3, 8, 9, and 16 and of PiGMaP for Chr 8 was determined. USDA and PiGMaP linkage maps are now oriented for all chromosomes, except for Chrs 17 and 18. Moreover, the linkage group “R” from the USDA linkage map was assigned to Chr 6.

Accurate mapping of the “acid meat” RN gene on genetic and physical maps of pig Chromosome 15

It has been shown that a major gene, called RN, is responsible for the RTN technological yield, a meat quality porcine trait. Experimental families informative for the segregation of RN gene were constituted from animals belonging to the Laconie composite line. We have previously mapped the RN gene to Chromosome (Chr) 15 (Milan et al. Genet. Sel. Evol. 27, 195-199, 1995). A Chr 15 map was established with 16 markers. The RN gene was found to be located between markers Sw120 and Sw936, at 2 cM from Sw936 (LOD = 38.1). In addition, by localizing Sw936 at 15q21–22 using DISC-PCR, we also located RN on the physical map.