Transcript levels of genes implicated in steroidogenesis in the testes and fat tissue in relation to androstenone accumulation in fat of pubertal pigs.

The present study was performed to measure messenger RNA levels of steroidogenic enzymes in testes and fat tissue and determine whether they are related to fat androstenone level. Real-time polymerase chain reaction experiments were performed on 26 testes and 12 adipose tissue samples from pubertal boars using 21 genes. The absence of significant correlations between fat androstenone and the transcriptional activity of the SRD5A2 and SRD5A3 genes but the high correlation coefficient with that of the SRD5A1 gene (r = 0.62, P < 0.05) suggests that the enzyme coded by SRD5A1 is mainly responsible for the last step of androstenone synthesis. The testicular transcriptional activities of CYP17, CYP11A1, CYP19A, AKR1C-pig6, SRD5A1, LHCGR, and AR were significantly correlated. Only transcriptional levels of CYP17, CYP11A1, CYP19A, SRD5A1, and AKR1C-pig6 were correlated with the fat concentration of androstenone (0.57 < r < 0.70, P < 0.05) confirming that the amount of androstenone stored in fat is related to the production in testes of androstenone and more generally to all sex steroids. Altogether, our data are in favor of a preponderant role of AKR1C-pig6 instead of HSD17B3 for testicular synthesis of steroids. Concerning fat tissue, our data do not support a significant de novo biosynthesis of steroids in porcine adipose tissues. The presence of transcripts coding for steroid enzymes, especially those of AKR1C-pig6, suggests that steroids can be transformed. None of transcript abundance was related to androstenone accumulation (P > 0.1). Therefore, steroids synthesized elsewhere can be transformed in fat tissue but synthesis of androstenone is unlikely.

Molecular characterization of the porcine TEAD3 (TEF-5) gene: examination of a promoter mutation as the causal mutation of a quantitative trait loci affecting the androstenone level in boar fat.

A quantitative trait loci (QTL) for accumulation of androstenone in fat has been identified in an Large White × Meishan cross in a region of SSC7-containing TEAD3. In humans, TEAD3 is a transcription activator, known to be able to regulate the transcription of HSD3B. This enzyme is involved in the degradation of androstenone in the liver. In this study, porcine transcripts of TEAD3 were characterized and compared with mammalian transcripts. The complete structure of porcine TEAD3 gene was characterized including two 5' non-coding exons and one exon 5 not used in porcine transcripts. Variations were screened in sequences related to TEAD3: in exons, in flanking sequences of exons and in the promoter region. A SNP characterized at 726 bp at 5' of the first exon was tested on several pig populations without coherent and convincing results concerning its association with androstenone levels. We showed that in the liver of adult boars, the transcripts levels of TEAD3 and HSD3B were correlated. As in humans, it is possible that HSD3B is a target gene of TEAD3 in porcine liver. Nevertheless, no expression variation was observed for TEAD3 or HSD3B in liver between animals with different genotypes at the SNP. We concluded that this SNP was not the causal mutation of this QTL.

Towards candidate genes affecting body fatness at the SSC7 QTL by expression analyses.

A quantitative trait locus (QTL) affecting fatness in a way opposite to expectations based on breed means was mapped to swine chromosome 7 (SSC7) using crosses between Large White (LW) and Meishan (MS) founders. Defining the molecular fatness trait more explicitly would allow deducing positional candidate genes, for which expression differences must be analysed in experimental populations. First, mRNA levels of genes representing sequential steps in adipogenesis or involved in lipid metabolism were studied in backfat of pigs having homozygous LW(QTL7)/LW(QTL7) or heterozygous LW(QTL7)/MS(QTL7) alleles and considered at two ages. mRNA level of DLK1 expressed in preadipocytes was greater in MS(QTL7)/LW(QTL7) pigs than in homozygous pigs at 28 days. Transcript abundances of CEBPA involved in differentiation, the prolipogenic FASN gene and the adipocyte-specific marker FABP4 were lower in MS(QTL7)/LW(QTL7) pigs compared with LW(QTL7)/LW(QTL7) pigs at 150 days. Because these results suggest a lag time in terminal differentiation associated with the MS allele, seven genes in the QTL interval were deduced as promising candidates for the QTL effect by bioinformatics analysis. Among them, PPARD and CDKN1A had lower expression levels in MS(QTL7)/LW(QTL7) pigs at both ages. Genotype-related differences were observed in mRNA levels of PPARD target genes involved in cell differentiation (FZD7) or fatty acid oxidation (ACADL and ACOX1) at 150 days. These results re-evaluate the potential of PPARD to explain part of variation in pig adiposity.

Expression levels of 25 genes in liver and testis located in a QTL region for androstenone on SSC7q1.2.

A quantitative trait locus (QTL) for boar fat androstenone levels has been identified near the SSC7 centromere in a Large White × Meishan cross. Backcrosses were produced to isolate the Chinese haplotype in a European genetic background. The expression of 25 genes from the QTL region was studied in the testes and livers of 5-month-old backcross boars, with the aim of identifying the causal gene. Using Fluidigm, a new high-throughput technology, the expression of 25 genes was measured in a single real-time PCR experiment. This study found six significantly down-regulated genes (C6ORF106, C6ORF81, CLPS, SLC26A8, SRPK1 and MAPK14) in the testes of MS-LW backcross boars. However, according to current knowledge, none of the genes appear to be related to androstenone metabolism. In the livers, none of the genes were significantly up- or down-regulated, including TEAD3, which was previously designated as a possible candidate to explain this QTL.

Mapping of porcine ESTs obtained from the anterior pituitary.

We report the physical mapping of porcine expressed sequence tags (ESTs) from anterior pituitary clones isolated by differential display PCR in a study using lines selected for reproduction. These ESTs were mapped using a somatic cell hybrid panel (SCHP) and a radiation hybrid panel (IMpRH) as follows (SCHP position, nearest marker on the RH map): SPARCL1 (8q23-q27, SSP1); ATF4 (5p11-p15, AC02); MEF2C [2(1/2q21)-(1/2q22), SW2134]; FTH1 (2p14-p17, SWR783); FRAP1 (6q22-q23, SW1355); PBP (14, SW2508); LOC92004 [13q23-(1/2q41), CP]; and PGRMC1 [Xq22, SW1943]. All RH assignments were at LOD score >6.0 except for PGRMC1 at LOD score 5.4. ESTs TCP1 [12p11-(2/3p13)], SF3B1 (15q23-q26) and Clock (8q11-q12) were assigned using only the SCHP. The map position of SPARCL1 coincides with a quantitative trait loci (QTL) for age at puberty found in the University of Nebraska selection lines. Physical mapping of ESTs reported in the present study contributes to characterization of the transcriptome of anterior pituitary of pigs, adds new information to the public database of the porcine genome expression map, and further develops the porcine-human comparative map.

A new contribution to the integration of human and porcine genome maps: 623 new points of homology.

In this study we examined homologies between 1,735 porcine microsatellites and human sequence. For 1,710 microsatellites we directly used the sequence flanking the repeat available in GenBank. For a set of 305 microsatellites, a BAC library was screened and end-sequencing provided 461 additional sequences. Altogether 2,171 porcine sequences were tentatively aligned with the sequence of the human genome using the fasta program. Human homologies were observed for 652 microsatellite loci and porcine chromosome assignments available for 623 microsatellites provide useful links in the human and pig comparative map. Moreover for 92 STS, a significant sequence similarity was detected using at least two sequences and in all cases corresponding human locations were consistent. The present study allowed the integration of anonymous markers and the porcine linkage map into the framework of the comparative data between human and porcine genomes (http://w3.toulouse.inra.fr/lgc/pig/msat/). Moreover all conserved syntenic segments were defined on human chromosomes.

Generation of a 5.5-Mb BAC/PAC contig of pig chromosome 6q1.2 and its integration with existing RH, genetic and comparative maps.

We generated a sequence-ready BAC/PAC contig spanning approximately 5.5 Mb on porcine chromosome 6q1.2, which represents a very gene-rich genome region. STS content mapping was used as the main strategy for the assembly of the contig and a total of 6 microsatellite markers, 53 gene-related STS and 116 STS corresponding to BAC and PAC end sequences were analyzed. The contig comprises 316 BAC and PAC clones covering the region between the genes GPI and LIPE. The correct contig assembly was verified by RH-mapping of STS markers and comparative mapping of BAC/PAC end sequences using BLAST searches. The use of microsatellite primer pairs allowed the integration of the physical maps with the genetic map of this region. Comparative mapping of the porcine BAC/PAC contig with respect to the gene-rich region on the human chromosome 19q13.1 map revealed a completely conserved gene order of this segment, however, physical distances differ somewhat between HSA19q13.1 and SSC6q1.2. Three major differences in DNA content between human and pig are found in two large intergenic regions and in one region of a clustered gene family, respectively. While there is a complete conservation of gene order between pig and human, the comparative analysis with respect to the rodent species mouse and rat shows one breakpoint where a genome segment is inverted.

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/cR(12,000)) is two to three times more than that of IMpRH (35-37 kb/cR(7,000)). 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.

Comparative analysis of a BAC contig of the porcine RN region and the human transcript map: implications for the cloning of trait loci.

The poorly developed transcript maps and the limited resources for genome analysis hamper positional cloning of trait loci in farm animals. This study demonstrates that this will now be easier by the combined use of BAC contigs and the import of the near complete human transcript map. The conclusion was obtained by a comparative analysis of a 2.4-Mb BAC contig of the RN region in pigs. The contig was constructed as part of a successful positional cloning project, which identified PRKAG3 as the causative gene for the RN phenotype. A comparative map including the corresponding regions on human chromosome 2q35 and mouse chromosome 1 (region 36-44 cM) is reported. Sixteen coding sequences were mapped on the BAC contig. The majority of these were identified by BLAST searches of BAC end sequences and BAC shotgun sequences generated during the positional cloning project. Map data for the orthologues in humans were available for 12 of the 16 coding sequences, and all 12 have been assigned to 2q35. Furthermore, no evidence for any rearrangement in gene order was obtained. The extensive linkage conservation indicates that the near complete human transcript map will be an invaluable resource for positional cloning projects in pigs and other domestic animals.

Construction of a high-resolution RH map of the human 2q35 region on TNG panel and comparison with a physical map of the porcine homologous region 15q25.

This article describes the construction of a high-resolution radiation hybrid map of Hsap 2q35 by using the TNG RH panel generated by irradiation with 50,000 rads. We were able to build a framework map of 1300 cR(50,000) including 34 markers ordered with odds higher than 1000:1. The comprehensive map includes 77 loci and describes a region of 3 Mb around the SLC11A1 gene. Because of the very small size of the fragments retained and a reduced retention frequency, it was difficult to build a high-resolution multi-point map of this region by using the TNG RH panel. Nevertheless, this study confirmed the very high potential of this RH panel for constructing a human, high-resolution physical map (2.3 kb/cR(50,000)). Moreover, human ESTs from Hsap 2q35 were hybridized with porcine BAC contigs to establish a porcine transcript map of the homologous region Sscr 15q25 (greater than 2.5 Mb). We identified 17 new genes in this porcine chromosomal region. We were able to compare the location of 26 genes mapped in both species. The gene order was similar except for two possible minor discrepancies in the Desmin sub-region, suggesting the existence of a porcine micro-region between TNP1 and IL8RB with an unknown origin.

Comparative mapping between humans and pigs: localization of 58 anchorage markers (TOASTs) by use of porcine somatic cell and radiation hybrid panels.

To increase the number of Type I markers that are directly informative for comparative mapping, 58 anchorage markers, TOASTs (Traced Orthologous Amplified Sequence Tags), were mapped in pig. With specific consensus primers, 76 TOASTs were tested in pig: 50 were regionally localized in pig on a somatic cell hybrid panel (SCHP), and 51 were mapped on the whole genome, INRA/University of Minnesota porcine Radiation Hybrid panel (IMpRH). Comparison of marker positions on RH and cytogenetic maps indicated general concordance except for two chromosomal regions. For RH mapping, all markers, apart from one, were significantly linked (LOD > 4.8) to a marker of the first-generation radiation hybrid map. Localization of new markers on the initial map is necessary for drawing a framework map as shown for Chromosome Sscr 14. The addition of four TOASTs has enabled us to propose an improved map, using a threshold likelihood ratio of 1000/1. At the whole-genome level, this work significantly increased (by 50%) the number of precisely mapped genes on the porcine RH map and confirmed that the IMpRH panel is a valuable tool for high-resolution gene mapping in pig. Porcine PCR products were sequenced and compared with human sequences to verify their identity. Most of the localizations made it possible to either confirm or refine the previous comparative data between humans and pigs obtained through heterologous chromosomal painting or gene mapping. Moreover, the use of TOASTs in mapping studies appears to be a complement to other strategies using CATS, human ESTs, or heterologous FISH with BACs which had already been applied to improve the gene density of comparative genomic maps for mammals.

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.

A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle.

A high proportion of purebred Hampshire pigs carries the dominant RN- mutation, which causes high glycogen content in skeletal muscle. The mutation has beneficial effects on meat content but detrimental effects on processing yield. Here, it is shown that the mutation is a nonconservative substitution (R200Q) in the PRKAG3 gene, which encodes a muscle-specific isoform of the regulatory gamma subunit of adenosine monophosphate-activated protein kinase (AMPK). Loss-of-function mutations in the homologous gene in yeast (SNF4) cause defects in glucose metabolism, including glycogen storage. Further analysis of the PRKAG3 signaling pathway may provide insights into muscle physiology as well as the pathogenesis of noninsulin-dependent diabetes mellitus in humans, a metabolic disorder associated with impaired glycogen synthesis.

Mapping of 14 expressed sequence tags (ESTs) from porcine skeletal muscle by somatic cell hybrid analysis.

Chromosomal assignments are reported for fourteen porcine expressed sequence tags (ESTs)--CALM1, CRYAB, MYH7, MYL1, PDK4, PGAM2, PYGM, REV3L, RFC1, SLN, SPTBN1, SRM160, TPM1 and YWHAG. The ESTs were derived from our porcine skeletal muscle cDNA library. The ESTs sequences selected for mapping included the presence of the 3'-untranslated region. The assignments were performed using two independent somatic cell hybrid panels providing the possibility of confirmation of the results obtained. The observed localizations are compared with the locations predicted from heterologous (human-pig, pig-human) chromosome painting data and knowledge of the map locations of the human homologues. These results add new information to the porcine genome transcript map.

A high-density linkage map of the RN region in pigs.

The porcine RN locus affects muscle glycogen content and meat quality. We previously mapped the RN locus to chromosome 15. This study describes the identification of polymorphisms for four class I and four class II markers located in the RN region. Resource families were genotyped with F-SSCP markers (fluorescent single strand conformation polymorphism) and microsatellite markers. Subsequent multipoint linkage analysis revealed the order FN1-IGFBP5-S1000-S1001-IL8RB-VIL1-RN-Sw936-Sw906. The gene order is identical to the previously reported porcine RH map of the same region. The described map will facilitate positional cloning of the RN gene.

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

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 approximately 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.

Isolation and characterization of the porcine c-myc proto-oncogene and chromosomal assignment to SSC 4p13.

The proto-oncogene c-myc codes for a nuclear phosphoprotein, a transcription factor composed of the typical basic/helix-loop-helix/leucine zipper domains. Its expression is coupled to a multitude of physiological processes and regulated by a variety of hormones, growth factors, cytokines, lymphokines and the nutritional status, development and differentiation. Its key roles have been characterized, e.g. in adipogenesis, myogenesis and folliculogenesis. We have isolated and sequenced a 6.4-kb genomic fragment encoding the porcine c-myc proto-oncogene. The gene shows the typical c-myc structure with three exons, three putative promoters and a deduced protein of 439 amino acids. The porcine c-myc was mapped to chromosome 4p13 by screening of a porcine-rodent hybrid cell panel.

A radiation hybrid map of the RN region in pigs demonstrates conserved gene order compared with the human and mouse genomes.

We recently constructed a 7000-rad porcine whole-genome radiation hybrid (RH) panel with the primary objective of integrating linkage maps of microsatellites with evolutionary conserved genes into one ordered map. In order to evaluate the resolution of this RH panel, we have now constructed a radiation hybrid map of the Chromosome (Chr) 15q2.3-q2.6 region containing the RN gene. This gene has large effects on glycogen content in muscle and meat quality. Ten microsatellites covering a region of 55 centiMorgans and eight genes (AE3, FN1, IGFBP5, INHA, IRS1, PAX3, TNP1, and VIL1) were placed on the Sscr15 RH map. All the genes, except IRS1, were mapped on the RH map between microsatellites located in 15q2.5. The relative order of AE3 and INHA was inverted on the porcine physical map in comparison with the mouse linkage map. The order of other genes already mapped in the mouse (FN1, IGFBP5, TNP1, VIL1, INHA/AE3, and PAX3) was identical in pigs. We found no clear difference between the gene order on pig Chr 15 and human Chr 2q.