Brad A. Freking

A second-generation linkage map of the sheep genome.

A genetic map of Ovis aries (haploid n = 27) was developed with 519 markers (504 microsatellites) spanning ∼3063 cM in 26 autosomal linkage groups and 127 cM (female specific) of the X Chromosome (Chr). Genotypic data were merged from the IMF flock (Crawford et al., Genetics 140, 703, 1995) and the USDA mapping flock. Seventy-three percent (370/504) of the microsatellite markers on the map are common to the USDA-ARS MARC cattle linkage map, with 27 of the common markers derived from sheep. The number of common markers per homologous linkage group ranges from 5 to 22 and spans a total of 2866 cM (sex average) in sheep and 2817 cM in cattle. Marker order within a linkage group was consistent between the two species with limited exceptions. The reported translocation between the telomeric end of bovine Chr 9 (BTA 9) and BTA 14 to form ovine Chr 9 is represented by a 15-cM region containing 5 common markers. The significant genomic conservation of marker order will allow use of linkage maps in both species to facilitate the search for quantitative trait loci (QTLs) in cattle and sheep.

Prion gene sequence variation within diverse groups of U.S. sheep, beef cattle, and deer

Prions are proteins that play a central role in transmissible spongiform encephalopathies in a variety of mammals. Among the most notable prion disorders in ungulates are scrapie in sheep, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer. Single nucleotide polymorphisms in the sheep prion gene (PRNP) have been correlated with susceptibility to natural scrapie in some populations. Similar correlations have not been reported in cattle or deer; however, characterization of PRNP nucleotide diversity in those species is incomplete. This report describes nucleotide sequence variation and frequency estimates for the PRNP locus within diverse groups of U.S. sheep, U.S. beef cattle, and free-ranging deer (Odocoileusvirginianus and O. hemionus from Wyoming). DNA segments corresponding to the complete prion coding sequence and a 596-bp portion of the PRNP promoter region were amplified and sequenced from DNA panels with 90 sheep, 96 cattle, and 94 deer. Each panel was designed to contain the most diverse germplasm available from their respective populations to facilitate polymorphism detection. Sequence comparisons identified a total of 86 polymorphisms. Previously unreported polymorphisms were identified in sheep (9), cattle (13), and deer (32). The number of individuals sampled within each population was sufficient to detect more than 95% of all alleles present at a frequency greater than 0.02. The estimation of PRNP allele and genotype frequencies within these diverse groups of sheep, cattle, and deer provides a framework for designing accurate genotype assays for use in genetic epidemiology, allele management, and disease control.

Porcine gene discovery by normalized cDNA-library sequencing and EST cluster assembly.

Genetic and environmental factors affect the efficiency of pork production by influencing gene expression during porcine reproduction, tissue development, and growth. The identification and functional analysis of gene products important to these processes would be greatly enhanced by the development of a database of expressed porcine gene sequence. Two normalized porcine cDNA libraries (MARC 1PIG and MARC 2PIG), derived respectively from embryonic and reproductive tissues, were constructed, sequenced, and analyzed. A total of 66,245 clones from these two libraries were 5?-end sequenced and deposited in GenBank. Cluster analysis revealed that within-library redundancy is low, and comparison of all porcine ESTs with the human database suggests that the sequences from these two libraries represent portions of a significant number of independent pig genes. A Porcine Gene Index (PGI), comprising 15,616 tentative consensus sequences and 31,466 singletons, includes all sequences in public repositories and has been developed to facilitate further comparative map development and characterization of porcine genes (http://www.tigr.org/tdb/ssgi/). The clones and sequences from these libraries provide a catalog of expressed porcine genes and a resource for development of high-density hybridization arrays for transcriptional profiling of porcine tissues. In addition, comparison of porcine ESTs with sequences from other species serves as a valuable resource for comparative map development. Both arrayed cDNA libraries are available for unrestricted public use.

Characterization of Conserved and Nonconserved Imprinted Genes in Swine

To increase our understanding of imprinted genes in swine, we carried out a comprehensive analysis of this gene family using two complementary approaches: expression and phenotypic profiling of parthenogenetic fetuses, and analysis of imprinting by pyrosequencing. The parthenote placenta and fetus were smaller than those of controls but had no obvious morphological differences at Day 28 of gestation. By Day 30, however, the parthenote placentas had decreased chorioallantoic folding, decreased chorionic ruggae, and reduction of fetal-maternal interface surface in comparison with stage-matched control fetuses. Using Affymetrix Porcine GeneChip microarrays and/or semiquantitative PCR, brain, fibroblast, liver, and placenta of Day 30 fetuses were profiled, and 25 imprinted genes were identified as differentially expressed in at least one of the four tissue types: AMPD3, CDKN1C, COPG2, DHCR7, DIRAS3, IGF2 (isoform specific), IGF2AS, IGF2R, MEG3, MEST, NAP1L5, NDN, NNAT, OSBPL1A, PEG3, APEG3, PEG10, PLAGL1, PON2, PPP1R9A, SGCE, SLC38A4, SNORD107, SNRPN, and TFPI2. For DIRAS3, PLAGL1, SGCE, and SLC38A4, tissue-specific differences were detected. In addition, we examined the imprinting status of candidate genes by quantitative allelic pyrosequencing. Samples were collected from Day 30 pregnancies generated from reciprocal crosses of Meishan and White Composite breeds, and single-nucleotide polymorphisms were identified in candidate genes. Imprinting was confirmed for DIRAS3, DLK1, H19, IGF2AS, NNAT, MEST, PEG10, PHLDA2, PLAGL1, SGCE, and SNORD107. We also found no evidence of imprinting in ASB4, ASCL2, CD81, COMMD1, DCN, DLX5, and H13. Combined, these results represent the most comprehensive survey of imprinted genes in swine to date.

Microsatellite Evolution: Testing the Ascertainment Bias Hypothesis

Abstract. Previous studies suggest the median allele length of microsatellites is longest in the species from which the markers were derived, suggesting that an ascertainment bias was operating. We have examined whether the size distribution of microsatellite alleles between sheep and cattle is source dependent using a set of 472 microsatellites that can be amplified in both species. For those markers that were polymorphic in both species we report a significantly greater number of markers (P < 0.001) with longer median allele sizes in sheep, regardless of microsatellite origin. This finding suggests that any ascertainment bias operating during microsatellite selection is only a minor contributor to the variation observed.

A porcine BAC library with tenfold genome coverage: a resource for physical and genetic map integration

Recent advances in porcine genomics have identified quantitative trait loci (QTL) that influence pork production traits such as carcass traits, meat quality, and reproductive efficiency (Rohrer 2000; Cassady 1999). The low resolution to which most of these loci have been defined precludes the accurate application of markerassisted selection (MAS) strategies for increasing production efficiency. Large-insert genomic libraries are an excellent resource for marker development aimed at increasing the resolution of QTL and for the development of contiguous physical maps (contigs) of the chromosomal regions containing them. To facilitate applications requiring genomic clones, several porcine yeast artificial chromosome (YAC) libraries have been developed representing onefold (Leeb et al. 1995), threefold (Rogel-Gaillard et al. 1997), and 5.5-fold (Alexander et al. 1997) coverage of the pig genome. These YAC libraries have been valuable resource owing to their large insert size. However, marker isolation from YAC libraries is confounded by the equimolar representation of the complex yeast genome and YAC DNA, susceptibility to insert rearrangement, and a relatively high degree of chimerism. The development of bacterial artificial chromosome (BAC) libraries (Shizuya et al. 1992; Ioannou et al. 1994) represents a compromise between insert size, stability, and ease of clone DNA isolation. BACs are capable of stably maintaining insert sizes exceeding 200 kb and can be easily isolated by standard alkaline lysis from bacterial genomic DNA by virtue of their closed-circular conformation (Shizuya et al. 1992; Ioannou et al. 1994). The ease of BAC DNA isolation allows for efficient restriction analysis, subcloning, and direct BAC DNA sequencing, procedures invaluable for marker isolation and the development of contiguous physical maps. Recently BAC libraries representing livestock species, including cattle, sheep, and pigs (Cai et al. 1995; Warren et al. 2000; Vaiman et al. 1999; Suzuki et al. 2000; Rogel-Gaillard et al. 1999) have been developed. Among these are two porcine BAC libraries representing fourfold (Suzuki et al. 2000) and fivefold (RogelGaillard et al. 1999) coverage of the porcine genome. We undertook the development of a publicly available pig BAC library, RPCI-44, representing at least a tenfold coverage of the pig genome as a resource for physical map development and pig genome sequencing. High-molecular-weight genomic DNA from four crossbred male pigs (breed composition: 37.5% Yorkshire, 37.5% Landrace, and 25% Meishan) was isolated as previously described (Osoegawa et al. 1998), partially digested with EcoRI (New England Biolabs, Beverly, MA) and EcoRI methylase (New England Biolabs), and fractionated by clamped homogeneous electrical field (CHEF; Chu et al. 1986) electrophoresis (BioRad, Hercules, CA). Digested genomic DNA in the range of 150–200 kpb was gelisolated by electroelution and ligated to EcoRI-cut pTARBAC2 (Wang et al., unpublished; http://www.chori.org/bacpac/ vectorframe.htm). Ligated DNA was drop-dialyzed on floating membranes, first against sterile water, followed by 0.5 × TE containing 30% PEG8000, and introduced by electroporation into DH1OB electrocompetent cells (Life Technologies, Rockville, MD). Transformed bacterial cells were allowed to recover in SOC medium for 1 h at 37°C, and were spread on LB agar plates containing 20 mg/ml chloramphenicol and 5% sucrose. Individual colonies were picked with an automatic colony-picking robot (Qbot; Genetix, Stony Brook, NY) and arrayed into 528 individual 384-well microtiter dishes (Genetix) with LB medium containing 7.5% glycerol and 20 mg/ml chloramphenicol. The library was divided into two segments and gridded onto 11 high-density replica filters, each containing up to 18,432 independent clones that have been spotted in duplicate. To serve as anchor spots, an endsequenced BAC clone (1A1) from a Caenorhabditis brigsea BAC library (RPCI-94), containing DNA apparently unique to nematode genomes, was gridded on the corner of each of the six fields that comprise the high-density filters. The inclusion of a probe directed against the anchor spots can facilitate orientation of hybridized filters. The predicted coverage of RPCI-44 is a function of the total number of insert-containing clones and the size of the genomic DNA fragments they contain. From a total of 202,752 clones picked, 1,764 (segment 1, 1.9%) and 1,519 (segment 2, 1.4%) clones failed to grow, leaving a total of 199,469 clones. Non-recombinant clones containing the original vector were identified by high-density colony filter hybridization with the vector as a probe. The probing identifies two classes of clones with strong versus weak hybridization signals corresponding to the presence or absence of the original vector stuffer fragment (“pUClink”), which contains a high-copy-number replicon. Recombinant clones lack the stuffer fragment, resulting in much weaker hybridization signals. In total, 119 clones (0.06%) were identified as containing intact vector by this approach. The plate and well addresses of these are available at http://www.chori.org/framenonrec.htm. To evaluate the average insert size and insert size distribution in the library, randomly selected clones from each segment were subjected to restriction analysis with Not1, insert size determination by CHEF electrophoresis, and comparison with molecular weight standards (Osoegawa et al. 2000). Segment 1 was composed of 90,396 clones containing inserts averaging 157 kpb * Present address: University of Minnesota, Department of Animal Science, 495 An Sci/Vet Med, 1988 Fitch Ave., St. Paul, MN 55108, USA.

Abnormal postnatal maintenance of elevated DLK1 transcript levels in callipyge sheep

The underlying mechanism of the callipyge muscular hypertrophy phenotype in sheep (Ovis aries) is not presently understood. This phenotype, characterized by increased glycolytic type II muscle proportion and cell size accompanied by decreased adiposity, is not visibly detectable until approximately three to eight weeks after birth. The muscular hypertrophy results from a single nucleotide change located at the telomeric end of ovine Chromosome 18, in the region between the imprinted MATERNALLY EXPRESSED GENE 3 (MEG3) and DELTA, DROSOPHILA, HOMOLOG-LIKE 1 (DLK1) genes. The callipyge phenotype is evident only when the mutation is paternally inherited by a heterozygous individual. We have examined the pre- and postnatal expression of MEG3 and DLK1 in sheep of all four possible genotypes in affected and unaffected muscles as well as in liver. Here we show that the callipyge phenotype correlates with abnormally high DLK1 expression during the postnatal period in the affected sheep and that this elevation is specific to the hypertrophy-responsive fast-twitch muscles. These results are the first to show anomalous gene expression that coincides with both the temporal and spatial distribution of the callipyge phenotype. They suggest that the effect of the callipyge mutation is to interfere with the normal postnatal downregulation of DLK1 expression.

Comparative mapping of BTA15 and HSA11 including a region containing a QTL for meat tenderness

Abstract. The starting point of the present study was the reported identification of a chromosomal region on bovine Chromosome (Chr) 15 (BTA15) carrying loci affecting meat tenderness. A comparative linkage map of BTA15 and human Chr 11 (HSA11) was constructed to identify potential positional candidate genes and to provide a resource of genetic markers to support marker-assisted selection (MAS). Relative rearrangements between the bovine and human genomes for these chromosomes are the most complex observed in comparative mapping between the two species, with nine alternating blocks of conserved synteny between HSA11 and bovine Chrs 15 and 29. The results of this study were the addition of nine genes to the HSA11/BTA15 comparative linkage map, and development of five microsatellite markers within the quantitative trait locus (QTL) interval. One gene with known effects on muscle development (MYOD1) was mapped to the interval. A second gene (CALCA) involved in regulation of calcium levels, a key factor in postmortem tenderization, also mapped within the interval. Refinement of the comparative map and QTL position will reduce the interval on the human transcription map to be scanned in search of candidates, reducing the effort and resources required to identify the allelic variation responsible for the genetic effect.

Comparative mapping of the ovine clpg locus

Abstract. We used a comparative mapping approach to identify segments of conserved synteny between human Chromosome 14 (HSA14), bovine Chromosome 21 (BTA21), and the portion of ovine Chromosome 18 (OAR18) that contains the clpg locus. A bovine radiation hybrid map of the region was constructed with available Type II genetic markers and seven candidate genes to establish the comparative interval between BTA21 and HSA14. We developed polymorphic microsatellite and SNP markers associated with five candidate genes and placed them on the ovine and/or bovine genetic maps by multipoint linkage analysis. Three additional genes were mapped by virtue of their physical linkage to genetically mapped makers. Development of integrated linkage and physical maps facilitates the selection of positional candidate genes from the gene rich human map. The physically linked candidate genes PREF-1 and MEG3 map to the interval containing the clpg locus. Comparative biology suggests imprinting of MEG3 and/or the influences of PREF-1 on cellular differentiation, should be examined for their role in the parent-of-origin dependent influence of mutant clpg alleles on sheep muscle characteristics.

Evaluation of Dorset, Finnsheep, Romanov, Texel, and Montadale breeds of sheep: II. Reproduction of F 1 ewes in fall mating seasons

Objectives were to estimate effects of sire breed (Dorset, Finnsheep, Romanov, Texel, and Montadale), dam breed (Composite III and northwestern whiteface), mating season (August, October, and December), ewe age (1, 2, and 3 yr), and their interactions on reproductive traits of F1 ewes. A total of 1,799 F1 ewes produced 3,849 litters from 4,804 exposures to Suffolk rams during 35-d mating seasons over 3 yr. Ewes were weighed at breeding. Conception rate and ewe longevity (present or absent at 42 mo of age) were determined. Number born and litter birth weight were recorded, and number and weight at weaning and 20 wk of age were analyzed separately for dam- and nursery-reared litter mates. Total productivity through 3 yr of age for each ewe entering the breeding flock was calculated as the sum of 20-wk weights for dam- or nursery-reared lambs. Interactions of sire breed x mating season, sire breed x ewe age, and mating season x ewe age were generally significant, whereas interactions of sire breed, mating season, and ewe age x dam breed were seldom detected. Interactions of sire breed x mating season were often due to changes in rank as well as magnitude, indicating the importance of matching sire breed to a specific mating season. The number born to Dorset-, Texel-, and Montadale-sired ewes was not affected by dam breed; however, Finnsheep-sired ewes out of northwestern whiteface dams were more prolific than Finnsheep-sired ewes out of Composite III dams, and the opposite situation existed for Romanov-sired ewes. Least squares means of sire breeds (P < 0.001) for total productivity of dam-reared lambs were 98.5, 103.5, 106.9, 124.6, and 154.9 kg/ewe entering the breeding flock for Texel, Dorset, Montadale, Finnsheep, and Romanov, respectively. Superior reproduction of Romanov-sired ewes was due to greater conception rate and prolificacy for each mating season and ewe age, as well as greater ewe longevity. Total productivity of F1 ewes by Composite III dams (125.6 kg) was greater (P < 0.001) than for ewes born to northwestern whiteface dams (109.7 kg), and the effect of mating season increased (P < 0.001) from August to October to December. Litter weight at 20 wk of age of 2- and 3-yr-old ewes was similar but greater (P < 0.001) than for 1-yr-old ewes. Experimental results provide comprehensive information about the appropriate use of these breeds in crossbreeding systems to meet specific production-marketing objectives.