Annie Robic

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.

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.

Generation and characterization of a 12,000-rad radiation hybrid panel for fine mapping in pig

We have constructed a 12,000-rad porcine whole-genome radiation hybrid panel to complement the first generation 7,000-rad panel (IMpRH) and allow higher resolution mapping studies both in specific areas of interest and on the whole genome. We analyzed 243 hybrid clones on the basis of their marker retention frequency to produce a final panel of 90 hybrid clones with an average retention frequency of 35.4%. The resolution of this 12,000-rad panel (IMNpRH2) was compared to the resolution of the 7,000-rad panel (IMpRH) by constructing framework maps in the 2.4-Mb region of porcine chromosome 15 containing the acid meat RN gene. In this region, two-point analysis was used to estimate RH distances and demonstrates their reliability with the estimation of physical distances. This study demonstrates that the 12,000-rad panel constitutes a powerful tool for constructing high-resolution maps. Indeed, the resolution of IMNpRH2 (12–14 kb/cR12,000) is two to three times more than that of IMpRH (35–37 kb/cR7,000). As expected, the increase in the radiation dose allows an increase of the mapping resolution in terms of kb/cR with the same suppleness of use for mapping experiments. In addition the RH map constructed in the region investigated proved to be more homogeneous on IMNpRH2 than on IMpRH.

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.

A successful strategy for comparative mapping with human ESTs: 65 new regional assignments in the pig

Abstract. Large-scale sequencing of cDNAs from numerous tissues is currently being performed within the framework of the Human Genome Project. These expressed sequence tags (ESTs) are then mapped on a radiation hybrid panel to produce a high-resolution map of human genes. In this report, we estimate the efficiency of mapping these ESTs in the pig. A total of 344 human ESTs from Généthon were selected for amplification in other species by Zoo-PCR: 186 of these could be reproducibly amplified by use of pig DNA and the corresponding human primer pairs. One-hundred seven of these were tested on a porcine–rodent somatic cell hybrid panel, permitting regional localizations of 65 ESTs with agarose or single-strand conformation polymorphism analysis gels. The corresponding pig PCR products were sequenced: 60 ESTs matched significantly with the expected human sequences. Fifty-one of these localizations in the pig are in agreement with the comparative mapping data between humans and pigs based on heterologous chromosome painting. Seven ESTs that were localized in an unexpected region may indicate new chromosomal correspondences. This work significantly increases the number of genes mapped on the pig genome and demonstrates that this approach can be successfully applied to improve the gene density of mammalian genomic maps in chromosomal regions of interest, such as those in which QTL (Quantative Trait Loci) have been identified.

Genetic and metabolic aspects of androstenone and skatole deposition in pig adipose tissue: A review

High levels of androstenone and skatole in fat tissues are considered the primary causes of boar taint, an unpleasant odour and flavour of the meat from non-castrated male pigs. The aim of this article is to review our current knowledge of the biology and genetic control of the accumulation of androstenone and skatole in fat tissue. Two QTL mapping studies have shown the complexity of the genetic control of these traits. During the last ten years, several authors have taken a more physiological approach to investigate the involvement of genes controlling the metabolism of androstenone and skatole. Although some authors have claimed the identification of candidate genes, it is more appropriate to talk about target genes. This suggests that genes affecting androstenone and skatole levels will have to be sought for among specific or non-specific transcription factors interacting with these target genes.

Prolactin receptor maps to pig Chromosome 16

Species:Mouse Locus name: methionine synthase or 5-methyltetrahydrofolatehomocysteine methyltransferase Locus symbol:Mtr Map position: proximal–D13Mit1–1.06 cM ± 1.06 SE– Mtr, D13Bir4, D13Bir6–1.06 ± 1.06–D13Abb1e–2.13 ± 1.49–D13Bir7–distal Method of mapping:Mtr was localized by RFLP analysis of 96 animals from an interspecific backcross panel ((C57BL/6JEi × SPRET/Ei)F1 × SPRET/Ei) provided by The Jackson Laboratory, Bar Harbor, Me. (BSS panel) [1]. Database deposit information: The data are available from the Mouse Genome Database, accession number MGD-JNUM-39061. Molecular reagents:A 1095-bp mouse cDNA was obtained by reverse transcription/PCR of mouse liver RNA, with degenerate oligonucleotides based on regions of homology within the methionine synthase sequences of lower organisms. The two primers (D1730 and D1733), as described by Leclerc et al. [2], were successful in amplifying both human and mouse cDNAs. The PCR products from both species were subcloned and sequenced; they showed 89% identity. The mouse cDNA was labeled by random priming and hybridized to Southern blots of EcoRI-digested mouse genomic DNA. Allele detection:Allele detection was performed by RFLP analysis of an EcoRI polymorphism. The C57BL/6J strain has alleles of approximately 13 kb, while theMus spretusstrain has alleles of approximately 9 kb and 4 kb. A constant band of approximately 0.5 kb was seen in both strains. Previously identified homologs: Human MTR has been mapped to chromosomal band 1q43 by fluorescence in situ hybridization [2–4]. Discussion: Methionine synthase (EC 2.1.1.13, 5-methyltetrahydrofolate-homocysteine methyltransferase) catalyzes homocysteine remethylation to methionine, with 5-methyltetrahydrofolate as the methyl donor and methylcobalamin as a cofactor. Nutritional deficiencies and genetic defects in homocysteine metabolism result in varying degrees of hyperhomocysteinemia. Dramatic elevations in plasma and urinary homocysteine levels are associated with the inborn error of metabolism, homocystinuria. Consequent to the recent isolation of the human cDNA for methionine synthase [2–4], two groups of investigators have identified mutations in methionine synthase in homocystinuric patients [2, 5]. Mild elevations in plasma homocysteine are thought to be a risk factor for both vascular disease and neural tube defects [6–8]. A genetic variant in methylenetetrahydrofolate reductase (MTHFR), the enzyme that synthesizes 5-methyltetrahydrofolate for the methioninesynthase reaction, is the most common genetic determinant of hyperhomocysteinemia identified thus far [9]. Mild defects in the methionine synthase reaction are also potential candidates for hyperhomocysteinemia and the associated multifactorial diseases. A common variant has been reported for the human methionine synthase gene, but its physiologic consequences have not yet been determined [2, 4]. The mapping of the human MTR gene to 1q43 and of the mouse gene to proximal Chromosome (Chr) 13 is consistent with previous findings of human/mouse homologies between these 2 chromosomal regions; the human and mouse nidogen genes have been mapped to 1q43 and proximal Chr 13, respectively [10]. Several genes have already been implicated in neural tube defects in mice [11]. Studies involving the mouse methionine synthase gene will be useful in assessing the role of this important enzyme in the development of birth defects and/or vascular disease.