Knud Christensen

Characterization of 24 porcine (dA-dC)n-(dT-dG)n microsatellites: genotyping of unrelated animals from four breeds and linkage studies

Twenty-four PCR primer pairs were designed for the detection of porcine microsatellites. Polymorphism was investigated in 76 unrelated animals from four different breeds: Duroc, Landrace, Hampshire, and Yorkshire. Compared with human microsatellites, a general lower heterozygosity was detected; however, for each microsatellite a significant variation between breeds in number of alleles and heterozygosity was seen. Mean heterozygosity was found to be significantly higher (P<0.01%) in the Yorkshire breed than in the other three breeds. Linkage analyses with the CEPH linkage packet were performed in a backcross family comprising 45 animals, of which 43 had informative meioses. Ten of the microsatellites could be assigned to six different linkage groups, demonstrating that linkage mapping with microsatellites can be carried out with great efficiency in a relatively small number of animals. Four of the linkage groups represent Chromosomes (Chrs) 4, 6, 7, and 8 respectively, while two linkage groups are unassigned.

Assignment of the porcine calcium release channel gene, a candidate for the malignant hyperthermia locus, to the 6p11----q21 segment of chromosome 6.

Several studies point to the possibility that malignant hyperthermia (MH) in pigs is caused by a defect in the calcium release channel (CRC) of skeletal muscle sarcoplasmic reticulum. The locus for MH is closely linked to the glucosephosphate isomerase (GPI) locus, near the centromere of chromosome 6. We demonstrate synteny of the genes for CRC and GPI using somatic cell hybrid lines, and assign the CRC gene to chromosome 6p11----q21 by in situ hybridization.

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

Recent fusion events during evolution of pig chromosomes 3 and 6 identified by comparison with the babirusa karyotype

The chromosomes of the babirusa, a species considered to have diverged from an ancestor of the pig during the Miocene epoch, about 12-26 million years ago, were studied to determine the sites of recent rearrangements during evolution of the domestic pig. It is shown that there is a pericentric inversion of the entire short arm on pig chromosome 1, compared to its counterpart in the babirusa (chromosome 15). We also present evidence suggesting that pig chromosome 3 was derived by a telomere-centromere fusion of two ancestral chromosomes homoelogous to babirusa chromosomes 12 and 17. Likewise, we conclude that pig chromosome 6 was most likely derived by a telomere-telomere fusion of ancestral chromosomes homoelogous to babirusa chromosomes 6 and 14. The detection of interstitial hybridization signals from presumptive subteloemeric repeats in the same chromosome region as the evolutionary fusion points on pig chromosomes 3 and 6 indicates that the fusion sites may still contain elements that are otherwise restricted to the telomere regions of pig chromosomes.

Albinism in the American mink (Neovison vison) is associated with a tyrosinase nonsense mutation

Albino phenotypes are documented in various species including the American mink. In other species the albino phenotypes are associated with tyrosinase (TYR) gene mutations; therefore TYR was considered the candidate gene for albinism in mink. Four microsatellite markers were chosen in the predicted region of the TYR gene. Genotypes at the markers Mvi6025 and Mvi6034 were found to be associated with the albino phenotype within an extended half-sib family. A BAC clone containing Mvi6034 was mapped to chromosome 7q1.1-q1.3 by fluorescent in situ hybridization. Subsequent analysis of genomic TYR sequences from wild-type and albino mink samples identified a nonsense mutation in exon 1, which converts a TGT codon encoding cysteine to a TGA stop codon (c.138T>A, p.C46X; EU627590). The mutation truncates more than 90% of the normal gene product including the putative catalytic domains. The results indicate that the nonsense mutation is responsible for the albino phenotype in the American mink.

Cytogenetics of donkey chromosomes: nomenclature proposal based on GTG-banded chromosomes and depiction of NORs and telomeric sites.

With the expansion of comparative genome analysis across different mammals, there is an increasing need to have well-defined banded karyotypes for the species chosen for investigation. In this context, the steadily growing gene mapping data in the donkey urgently require a framework whereby alignment/comparison of genetic information can be readily made with equids and other mammalian species. Hence a GTG-banded karyotype of the donkey (Equus asinus; EAS) is presented, along with schematic drawings and nomenclature of the banded chromosomes. In addition, the most characteristic features of individual chromosomes are described and their relative size estimated. Using the FISH approach, the location of nucleolous organizer regions (NORs) and telomeric repeat sequences (TTAGGG) were detected. Where possible, information on asine chromosomes is supplemented with known/likely equine and human homologues. The study thus primarily aims to provide an appropriate cytogenetic basis for the donkey chromosomes, so that research focused on gene mapping and comparative genomics in this species can be reported under a common format.

Construction of an American mink Bacterial Artificial Chromosome (BAC) library and sequencing candidate genes important for the fur industry

BackgroundBacterial artificial chromosome (BAC) libraries continue to be invaluable tools for the genomic analysis of complex organisms. Complemented by the newly and fast growing deep sequencing technologies, they provide an excellent source of information in genomics projects.ResultsHere, we report the construction and characterization of the CHORI-231 BAC library constructed from a Danish-farmed, male American mink (Neovison vison). The library contains approximately 165,888 clones with an average insert size of 170 kb, representing approximately 10-fold coverage. High-density filters, each consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs), representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library. These included candidate genes for coat coloring, hair growth and length, coarseness, and some receptors potentially involved in viral diseases in mink. The extensive screening yielded positive results for 19 of these genes. Thirty-five clones corresponding to 19 genes were sequenced using 454 Roche, and large contigs (184 kb in average) were assembled. Knowing the complete sequences of these candidate genes will enable confirmation of the association with a phenotype and the finding of causative mutations for the targeted phenotypes.Additionally, 1577 BAC clones were end sequenced; 2505 BAC end sequences (80% of BACs) were obtained. An excess of 2 Mb has been analyzed, thus giving a snapshot of the mink genome.ConclusionsThe availability of the CHORI-321 American mink BAC library will aid in identification of genes and genomic regions of interest. We have demonstrated how the library can be used to identify specific genes of interest, develop genetic markers, and for BAC end sequencing and deep sequencing of selected clones. To our knowledge, this is the first report of 454 sequencing of selected BAC clones in mammals and re-assures the suitability of this technique for obtaining the sequence information of genes of interest in small genomics projects. The BAC end sequences described in this paper have been deposited in the GenBank data library [HN339419-HN341884, HN604664-HN604702]. The 454 produced contigs derived from selected clones are deposited with reference numbers [GenBank: JF288166-JF288183 & JF310744].

Porcine 5S rRNA genes map to 14q23 revealing syntenic relation to human HSPA6- and 7

5S rRNA is one of the two small RNA molecules localized in the large ribosomal subunit and thereby is involved in protein synthesis. 5S rRNAs from many different organisms have been sequenced, and more than 700 5S rRNA transcripts and 5S rRNA gene sequences have been compiled (Specht et al. 1991). The 5S rRNA genes have been studied in a large number of organisms (reviewed in Korn 1982; Geiduscheck and Tocchini-Valentini 1988; Willis 1993). Compared with the lower eukaryotes, thorough knowledge of the 5S rRNA genes in mammals is scarce. The

Assignment of a porcine male-specific DNA repeat to Y-chromosomal heterochromatin

Primers were designed to amplify by PCR a 509-bp genomic fragment from male pig DNA, using the porcine male-specific repeat sequence described by McGraw et al. (1988). This PCR product showed male-specific hybridization in Southern blots. Nonradioactive in situ hybridization localized it to the entire length of the heterochromatic portion of Yq. The assignment was confirmed using the PCR primer pDYZ1-S for primed in situ labeling.

Assignment of the porcine growth hormone gene to chromosome 12

The porcine growth hormone gene was mapped to the p11→qter region of chromosome 12 with a porcine growth hormone DNA sequence using in situ hybridization and using Southern blot analysis and cytogenetic characterization of a pig × rodent hybrid cell line panel.