A high-resolution comparative map of porcine chromosome 4 (SSC4).

We used the IMNpRH2(12,000-rad) RH and IMpRH(7,000-rad) panels to integrate 2019 transcriptome (RNA-seq)-generated contigs with markers from the porcine genetic and radiation hybrid (RH) maps and bacterial artificial chromosome finger-printed contigs, into 1) parallel framework maps (LOD ≥ 10) on both panels for swine chromosome (SSC) 4, and 2) a high-resolution comparative map of SSC4, thus and human chromosomes (HSA) 1 and 8. A total of 573 loci were anchored and ordered on SSC4 closing gaps identified in the porcine sequence assembly Sscrofa9. Alignment of the SSC4 RH with the genetic map identified five microsatellites incorrectly mapped around the centromeric region in the genetic map. Further alignment of the RH and comparative maps with the genome sequence identified four additional regions of discrepancy that are also suggestive of errors in assembly, three of which were resolved through conserved synteny with blocks on HSA1 and HSA8.

Assignment of 128 genes localized on human chromosome 14q to the IMpRH map.

To provide a gene-based comparative map and to examine a porcine genome assembly using bacterial artificial chromosome-based sequence, we have attempted to assign 128 genes localized on human chromosome 14q (HSA14q) to a porcine 7000-rad radiation hybrid (IMpRH) map. This study, together with earlier studies, has demonstrated the following. (i) 126 genes were incorporated into two SSC7 RH linkage groups by CarthaGene analysis. (ii) In the remaining two genes, TOX4 linked to TCRA located in SSC7 by two-point analysis, whereas SIP1 showed no significant linkage with any gene/marker registered in the IMpRH Web Server. (iii) In the two groups, the gene clusters located from 19.9 to 36.5 Mb on HSA14q11.2-q13.3 and from 64.0 to 104.3 Mb on HSA14q23-q32.33 respectively were assigned to SSC7q21-q26. (iv) Comparison of the gene order between the present RH map and the latest porcine sequence assembly revealed some inconsistencies, and a redundant arrangement of 16 genes in the sequence assembly.

High-resolution comprehensive radiation hybrid maps of the porcine chromosomes 2p and 9p compared with the human chromosome 11.

We are constructing high-resolution, chromosomal 'test' maps for the entire pig genome using a 12,000-rad WG-RH panel (IMNpRH2(12,000-rad))to provide a scaffold for the rapid assembly of the porcine genome sequence. Here we present an initial, comparative map of human chromosome (HSA) 11 with pig chromosomes (SSC) 2p and 9p. Two sets of RH mapping vectors were used to construct the RH framework (FW) maps for SSC2p and SSC9p. One set of 590 markers, including 131 microsatellites (MSs), 364 genes/ESTs, and 95 BAC end sequences (BESs) was typed on the IMNpRH2(12,000-rad) panel. A second set of 271 markers (28 MSs, 138 genes/ESTs, and 105 BESs) was typed on the IMpRH(7,000-rad) panel. The two data sets were merged into a single data-set of 655 markers of which 206 markers were typed on both panels. Two large linkage groups of 72 and 194 markers were assigned to SSC2p, and two linkage groups of 84 and 168 markers to SSC9p at a two-point LOD score of 10. A total of 126 and 114 FW markers were ordered with a likelihood ratio of 1000:1 to the SSC2p and SSC9p RH(12,000-rad) FW maps, respectively, with an accumulated map distance of 4046.5 cR(12,000 )and 1355.2 cR(7,000 )for SSC2p, and 4244.1 cR(12,000) and 1802.5 cR(7,000) for SSC9p. The kb/cR ratio in the IMNpRH2(12,000-rad) FW maps was 15.8 for SSC2p, and 15.4 for SSC9p, while the ratio in the IMpRH(7,000-rad) FW maps was 47.1 and 36.3, respectively, or an approximately 3.0-fold increase in map resolution in the IMNpRH(12,000-rad) panel over the IMpRH(7,000-rad) panel. The integrated IMNpRH(12,000-rad) andIMpRH(7,000-rad) maps as well as the genetic and BAC FPC maps provide an inclusive comparative map between SSC2p, SSC9p and HSA11 to close potential gaps between contigs prior to sequencing, and to identify regions where potential problems may arise in sequence assembly.

Assignment of 115 genes from HSA9 and HSA14 to SSC1q by RH mapping to generate a dense human-pig comparative map.

A large number of significant QTL for economically important traits including average daily gain have been located on SSC1q, which, as shown by chromosome painting, corresponds to four human chromosomes (HSA9, 14, 15 and 18). To provide a comprehensive comparative map for efficient selection of candidate genes, 81 and 34 genes localized on HSA9 and HSA14 respectively were mapped to SSC1q using a porcine 7000-rad radiation hybrid panel (IMpRH). This study, together with the cytogenetic map (http://www2.toulouse.inra.fr/lgc/pig/cyto/genmar/htm/1GM.HTM), demonstrates that SSC1q2.1-q2.13 corresponds to the region ranging from 44.6 to 63.2 Mb on HSA14q21.1-q23.1, the region from 86.5 to 86.8 Mb on HSA15q24-q25, the region from 0.9 to 27.2 Mb on HSA9p24.3-p21, the region from 35.1 to 38.0 Mb on HSA9p13, the region from 70.3 to 79.3 Mb on HSA9q13-q21 and the region from 96.4 to 140.0 Mb on HSA9q22.3-q34. The conserved synteny between HSA9 and SSC1q is interrupted by at least six sites, and the synteny between HSA14 and SSC1q is interrupted by at least one site.

Assignment of 117 genes from HSA5 to the porcine IMpRH map and generation of a dense human-pig comparative map.

Twenty-two and eight significant quantitative trait loci for economically important traits have been located on porcine chromosomes (SSC) 2q and SSC16 respectively, both of which have been shown to correspond to human chromosome 5 (HSA5) by chromosome painting. To provide a comprehensive comparative map for efficient selection of candidate genes, we assigned 117 genes from HSA5 using a porcine radiation hybrid (IMpRH) panel. Sixty-six genes were assigned to SSC2 and 48 to SSC16. One gene was suggested to link to SSC2 markers and another to SSC6. One gene did not link to any gene, expressed sequence tag or marker in the map, including those in the present investigation. This study demonstrated the following: (1) SSC2q21-q28 corresponds to the region ranging from 74.0 to 148.2 Mb on HSA5q13-q32 and the region from 176.0 to 179.3 Mb on HSA5q35; (2) SSC16 corresponds to the region from 1.4 to 68.7 Mb on HSA5p-q13 and to the region from 150.4 to 169.1 Mb on HSA5q32-q35 and (3) the conserved synteny between HSA5 and SSC2q21-q28 is interrupted by at least two sites and the synteny between HSA5 and SSC16 is also interrupted by at least two sites.

Assignment of 204 genes localized on HSA17 to a porcine RH (IMpRH) map to generate a dense comparative map between pig and human/mouse.

Bi- and uni-directional chromosome painting (ZOO-FISH) and gene mapping have revealed correspondences between human chromosome (HSA) 17 and porcine chromosome (SSC) 12 harboring economically important quantitative trait loci. In the present study, we have assigned 204 genes localized on HSA17 to SSC12 to generate a comprehensive comparative map between HSA17 and SSC12. Two hundred fifty-five primer pairs were designed using porcine sequences orthologous with human genes. Of the 255 primer pairs, 208 (81.6%) were used to assign the corresponding genes to porcine chromosomes using the INRA-Minnesota 7000-rad porcine x Chinese hamster whole genome radiation hybrid (IMpRH) panel. Two hundred three genes were integrated into the SSC12 IMpRH linkage maps; and one gene, PPARBP, was found to link to THRA1 located in SSC12 but not incorporated into the linkage maps. Three genes (GIT1, SLC25A11, and HT008) were suggested to link to SSC12 markers, and the remaining gene (RPL26) did not link to any genes/expressed sequence tags/markers registered, including those in the present study. A comparison of the gene orders among SSC12, HSA17, and mouse chromosome 11 indicates that intra-chromosomal rearrangements occurred frequently in this ancestral mammalian chromosome during speciation.

Assignment of 101 genes localized in HSA10 to a swine RH (IMpRH) map to generate a dense human-swine comparative map.

Economically important traits such as growth and backfat in pigs have been shown to be influenced by genes in swine chromosome (SSC) 10q12-->qter corresponding to human chromosome (HSA) 10p. However, since gene information in the swine chromosomal region was limited, we attempted to generate a dense comparative map between SSC10 and HSA10 by mapping the 115 genes of HSA10 to a swine RH map (IMpRH map). In the mapping ten genes were assigned to SSC10, 88 to SSC14, and one to SSC3. One gene was suggested to link to SSC3, and another to SSC9. The correspondences between HSA10 and SSC10 and between HSA10 and SSC14 were essentially consistent with the observations obtained from bi/uni-directional chromosome painting or other results. This study further indicated that a large number of intrachromosomal rearrangements occurred in the synteny-conserved regions following species separation.

A dense comparative gene map between human chromosome 19q13.3-->q13.4 and a homologous segment of swine chromosome 6.

The human chromosome (HSA)19q region has been shown to correspond to swine chromosome (SSC) 6q11-->q21 by bi-directional chromosomal painting and gene mapping. However, since the precise correspondence has not been determined, 26 genes localized in HSA19q13.3-->q13.4 were assigned to the SSC6 region mainly by radiation hybrid (RH) mapping, and additionally, by somatic cell hybrid panel (SCHP) mapping, and fluorescent in situ hybridization (FISH). Out of the 26 genes, 24 were assigned to a swine RH map with LOD scores greater than 6 (threshold of significance). The most likely order of the 24 genes along SSC6 was calculated by CarthaGene, revealing that the order is essentially the same as that in HSA19q13.3-->q13.4. For AURKC and RPS5 giving LOD scores not greater than 6, SCHP mapping and FISH were additionally performed; SCHP mapping assigned AURKC and RPS5 to SSC6q22-->q23 and SSC6q21, respectively, which is consistent with the observation of FISH. Consequently, all the genes (26 genes) examined in the present study were shown to localize in SSC6q12-->q23, and the order of the genes along the chromosomes was shown to be essentially the same in swine and human, though several intrachromosomal rearrangements were observed between the species.

Elucidation of correspondence between swine chromosome 4 and human chromosome 1 by assigning 27 genes to the ImpRH map, and development of microsatellites in the proximity of 14 genes.

Loci affecting swine intramuscular fat content, backfat thickness, carcass weight, and daily weight gain were assigned to regions of swine chromosome (SSC) 4, which were shown to correspond to human chromosome (HSA) 1p22--> q25 by ZOO-FISH, bidirectional chromosome painting, as well as by the linkage map of genes. In order to select candidate genes responsible for the above traits from the human genome database, precise correspondence between SSC4 and HSA1 is a prerequisite. In the present study, 27 genes, PTGFR, GBP1, GBP2, GFI1, GCLM, ABCD3, EXTL2, KCNA3, ADORA3, KCND3, WNT2B, NRAS, SYCP1, PTGFRN, IGSF2, NOTCH2, S100A10, SHC1, SSR2, LMNA, CCT3, CD5L, PEA15, FCER1G, EAT2, DDR2, and LAMB3, located in the HSA1 region corresponding to SSC4 or possibly SSC4, were assigned to the IMpRH map. The alignment of genes from centromere to telomere in the SSC4 q arm is basically conserved in HSA1p22-->q25 with the direction from the q arm to the p arm, which is in good agreement with results from linkage mapping. In addition, the present study first demonstrated that WNT2B residing in the middle of the HSA1 region was assigned to SSC18 with a high lod score (> 5), and that at least three intrachromosomal rearrangements occurred in the region in the process of swine and human evolution. PTGFR, and LAMB3 localized at both ends of the HSA1 region were assigned to SSC6 and SSC9, respectively, which is consistent with regional correspondence reported earlier. In the course of the above analysis, microsatellite markers were developed in the proximity of eleven genes localized on SSC4, and three genes on other swine chromosomes.

Development of 50 gene-associated microsatellite markers using BAC clones and the construction of a linkage map of swine chromosome 4.

The development of informative polymorphic markers is essential for QTL mapping. We developed 50 microsatellite markers from BAC clones containing genes that were predicted to map swine chromosome 4 (SSC4) according to comparative analysis between human and swine chromosomes, and constructed a linkage map that consisted of 37 markers including 24 markers closely linked to genes in BAC clones. Microsatellite markers were developed by direct-sequencing of BAC clones and our results demonstrated that this method was effective for developing microsatellite markers in specific regions on chromosomes. Effective development of microsatellite markers closely linked to genes can further accelerate the comparative studies of chromosomes between different species.

Construction of a high-resolution comparative gene map between swine chromosome region 6q11-->q21 and human chromosome 19 q-arm by RH mapping of 51 genes.

A comprehensive and comparative map was constructed for the porcine chromosome (SSC) 6q11-->q21 region, where the gene(s) responsible for the maldevelopment of embryos are localized using swine populations of the National Institute of Animal Industry, Japan (NIAI). Since the chromosomal region corresponds to a region of human chromosome (HSA) 19q13.1-->q13.3 based on bi-directional chromosome painting, primer pairs were designed from porcine cDNA sequences identified, on a sequence comparison basis, as being transcripts from genes orthologous to those in the HSA region. Fifty-one genes were successfully assigned to a swine radiation hybrid (RH) map with LOD scores greater than 6. ERF and PSMD8 genes were assigned to SSC4 and SSC1, respectively. The remaining 49 genes were assigned to SSC6, demonstrating that the synteny between the SSC6 and HSA19 chromosomal regions is essentially conserved, therefore confirming, the results of bi-directional chromosome painting. However, when examined precisely, rearrangements have apparently occurred within the region of conserved synteny. For the ERF and PSMD8 genes assigned to SSCs other than SSC6, additional mapping using somatic cell hybrid (SCH) panels was performed to confirm the results of RH-mapping.

Glucocorticoids activate transcription of the gene for the glucose-6-phosphate transporter, deficient in glycogen storage disease type 1b.

Deficiencies in the glucose-6-phosphate transporter (G6PT) cause glycogen storage disease type 1b (GSD-1b), a heritable metabolic disorder. The G6PT protein translocates glucose-6-phosphate from the cytoplasm to the lumen of the endoplasmic reticulum, where glucose-6-phosphatase metabolizes it to glucose and phosphate. Therefore, G6PT and glucose-6-phosphatase work in concert to maintain glucose homeostasis. To delineate the control of G6PT gene expression, we first demonstrated that transcription of the gene requires hepatocyte nuclear factor 1alpha. Consequently, hepatocyte nuclear factor 1alpha-null mice manifest a G6PT deficiency like that of GSD-1b patients. In this study, we delineated the role of glucocorticoids in the transcription of the G6PT gene. We showed that the basal G6PT promoter is contained within nucleotides -369 to -1 upstream of the translation start site, which contains three activation elements. Further, we demonstrated that glucocorticoids activate G6PT transcription and that glucocorticoid action is mediated through a glucocorticoid response element within activation element-2 of the promoter. Taken together, the results suggest that glucocorticoids play a pivotal role in regulating the G6PT gene.

Genomic organization and assignment of the interleukin 7 gene (IL7) to porcine chromosome 4q11-->q13 by FISH and by analysis of radiation hybrid panels.

Interleukin 7 (IL7) is a cytokine that has many immunological functions, including regulation of hematopoiesis and peripheral lymphocytes. cDNA and a genomic DNA segment containing the porcine IL7 gene were isolated and sequenced, showing that porcine IL7 consists of 176 amino acids and that its gene spans over about 13 kb of genomic DNA. Porcine IL7 has 85% and 73% homology with human IL7 in terms of the nucleotide and amino acid sequences, respectively. Whereas the murine IL7 gene does not have an exon corresponding to human exon 5 (Lupton et al., 1990), the porcine IL7 gene was found to contain the same exon-intron structure as the human gene. These findings, together with the upstream structure of the cDNA elucidated in the present study, indicate that the relationship between swine and human IL7 is closer than that between mouse and human IL7. The IL7 gene was mapped to swine chromosome 4q11-->q13 by fluorescence in situ hybridization and, using a radiation hybrid panel, was localized between microsatellite markers Sw1336 and Sw1073 on the same chromosome.

Assignment of T cell receptor (TCR) alpha-chain gene (A), beta-chain gene (B), gamma-chain gene (G), and delta-chain gene (D) loci on swine chromosomes by in situ hybridization and radiation hybrid mapping.

Using the cDNA sequence of porcine T-cell receptor (TCR) alpha-, beta-, gamma-, and delta-chain genes, we screened a porcine BAC library to isolate clones containing these genes. The isolated BAC clones were confirmed to carry these TCR genes by partial nucleotide sequencing. Among the clones obtained in the present screening, two clones carried both the TCR alpha-chain gene (TRA) and the TCR delta-chain gene (TRD) while one clone each carried only the sequence of either TRA or TRD. This observation demonstrated that TRA and TRD are localized in close proximity on a swine chromosome. Also two clones contained the sequence of the TCR beta-chain gene (TRB), and two clones contained the sequence of TCR gamma-chain gene (TRG). Fluorescence in situ hybridization using the above BAC clones as probes revealed that TRA and TRD, TRB, and TRG loci reside on swine chromosomes 7q15.3-->q21, 18q11.3-->q12, and 9q21-->q22, respectively. The chromosome positions of TRA and TRB are consistent with those determined by somatic cell hybrid analysis (Rettenberger et al., 1996). In addition, RH mapping of these genes was performed using the INRA-University of Minnesota RH panel DNA. The result confirmed the position of TRA and TRB reported earlier (http://imprh.toulouse.inra.fr/), and further demonstrated that TRG was located 11 cR away from genetic marker SW989 toward the marker S0019.