R. T. Stone

Selection and use of SNP markers for animal identification and paternity analysis in U.S. beef cattle

Abstract. DNA marker technology represents a promising means for determining the genetic identity and kinship of an animal. Compared with other types of DNA markers, single nucleotide polymorphisms (SNPs) are attractive because they are abundant, genetically stable, and amenable to high-throughput automated analysis. In cattle, the challenge has been to identify a minimal set of SNPs with sufficient power for use in a variety of popular breeds and crossbred populations. This report describes a set of 32 highly informative SNP markers distributed among 18 autosomes and both sex chromosomes. Informativity of these SNPs in U.S. beef cattle populations was estimated from the distribution of allele and genotype frequencies in two panels: one consisting of 96 purebred sires representing 17 popular breeds, and another with 154 purebred American Angus from six herds in four Midwestern states. Based on frequency data from these panels, the estimated probability that two randomly selected, unrelated individuals will possess identical genotypes for all 32 loci was 2.0 × 10−13 for multi-breed composite populations and 1.9 × 10−10 for purebred Angus populations. The probability that a randomly chosen candidate sire will be excluded from paternity was estimated to be 99.9% and 99.4% for the same respective populations. The DNA immediately surrounding the 32 target SNPs was sequenced in the 96 sires of the multi-breed panel and found to contain an additional 183 polymorphic sites. Knowledge of these additional sites, together with the 32 target SNPs, allows the design of robust, accurate genotype assays on a variety of high-throughput SNP genotyping platforms.

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.

A small-insert bovine genomic library highly enriched for microsatellite repeat sequences

A bovine genomic phagemid library was constructed with randomly sheared DNA. Enrichment of this single-stranded DNA library with CA or GT primers resulted in 45% positive clones. The 14% of positive clones with (CA · GT)>12, and not containing flanking repetitive elements, were sequenced, and the efficiency of marker production was compared with random M13 bacteriophage libraries. Primer sequences and genotyping information are presented for 390 informative bovine microsatellite markers. The genomic frequency for 11 tri- and tetranucleotide repeats was estimated by hybridization to a lambda genomic library. Only GCT, GGT, and GGAT were estimated to have a frequency of >100 per genome. Enrichment of the phagemid library for these repeats failed to provide a viable source of microsatellite markers in the bovine. Comparison of map interval lengths between 100 markers from the enriched library prepared from randomly sheared DNA and M13 bacteriophage libraries prepared from Mbo1 restriction digests suggested no bias in skeletal genomic coverage based on source of small insert DNA. In conclusion, enrichment of the bovine phagemid library provides a sufficient source of microsatellites so that small repeat lengths and flanking repetitive sequences common in the bovine can be eliminated, resulting in a high percentage of informative markers.

Linkage mapping bovine EST-based SNP

BackgroundExisting linkage maps of the bovine genome primarily contain anonymous microsatellite markers. These maps have proved valuable for mapping quantitative trait loci (QTL) to broad regions of the genome, but more closely spaced markers are needed to fine-map QTL, and markers associated with genes and annotated sequence are needed to identify genes and sequence variation that may explain QTL.ResultsBovine expressed sequence tag (EST) and bacterial artificial chromosome (BAC)sequence data were used to develop 918 single nucleotide polymorphism (SNP) markers to map genes on the bovine linkage map. DNA of sires from the MARC reference population was used to detect SNPs, and progeny and mates of heterozygous sires were genotyped. Chromosome assignments for 861 SNPs were determined by twopoint analysis, and positions for 735 SNPs were established by multipoint analyses. Linkage maps of bovine autosomes with these SNPs represent 4585 markers in 2475 positions spanning 3058 cM . Markers include 3612 microsatellites, 913 SNPs and 60 other markers. Mean separation between marker positions is 1.2 cM. New SNP markers appear in 511 positions, with mean separation of 4.7 cM. Multi-allelic markers, mostly microsatellites, had a mean (maximum) of 216 (366) informative meioses, and a mean 3-lod confidence interval of 3.6 cM Bi-allelic markers, including SNP and other marker types, had a mean (maximum) of 55 (191) informative meioses, and were placed within a mean 8.5 cM 3-lod confidence interval. Homologous human sequences were identified for 1159 markers, including 582 newly developed and mapped SNP.ConclusionAddition of these EST- and BAC-based SNPs to the bovine linkage map not only increases marker density, but provides connections to gene-rich physical maps, including annotated human sequence. The map provides a resource for fine-mapping quantitative trait loci and identification of positional candidate genes, and can be integrated with other data to guide and refine assembly of bovine genome sequence. Even after the bovine genome is completely sequenced, the map will continue to be a useful tool to link observable phenotypes and animal genotypes to underlying genes and molecular mechanisms influencing economically important beef and dairy traits.

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.

Use of bovine EST data and human genomic sequences to map 100 gene-specific bovine markers

Abstract. A system to use bovine EST data in conjunction with human genomic sequence to improve the bovine linkage map over the entire genome or on specific chromosomes was evaluated. Bovine EST sequence was used to provide primer sequences corresponding to bovine genes, while human genomic sequence directed primer design to flank introns and produce amplicons of appropriate size for efficient direct sequencing. The sequence tagged sites (STS) produced in this way from the four sires of the MARC reference families were examined for single nucleotide polymorphisms (SNPs) that could be used to map the corresponding genes. With this approach, along with a primer/extension mass spectrometry SNP genotyping assay, 100 ESTs were placed on the bovine genetic linkage map. The first 70 were chosen at random from bovine EST–human genomic comparisons. An additional 30 ESTs were successfully mapped to bovine Chromosome 19 (BTA19), and comparison of the resulting BTA19 map to the position of the corresponding human orthologs on the HSA17 draft sequences revealed differences in the spacing and order of genes. Over 80% of successful amplicons contained SNPs, indicating that this is an efficient approach to generating EST-associated genetic markers. We have demonstrated the feasibility of constructing a linkage map based on SNPs associated with ESTs and the plausibility of utilizing EST, comparative mapping information, and human sequence data to target regions of the bovine genome for SNP marker development.

The bovine homolog of the obese gene maps to Chromosome 4

region of bovine Chr 6 (Fig. 1). The chromosomes were classified according to ISCNDA, 1989 [6]. Previously identified homologs: The OBS gene is located on mouse Chr 6 [7] and human Chr 7q31.3 [8]. Discussion: The chromosomal location of OBS in cattle is concordant with expectations based on chromosome painting experiments [Zoo-FISH; 9, 10]. The cloning and sequencing of the mouse obesity gene and its human homolog reported by Zhang and associates [7] is of great interest to researchers working with farm animal species. The mouse and human OBS gene product, secreted by adipose tissue, may be involved in appetite control/regulation, regulation of fuel storage, and energy expenditure [7]. The role of OBS in feed intake regulation and the regulation of energy balance in cattle is yet to be determined.

COLONY ISOLATION AND SECONDARY CULTURE OF FETAL PORCINE HEPATOCYTES ON STO FEEDER CELLS

SummaryThe secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.

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.

Porcine SINE-associated microsatellite markers: evidence for new artiodactyl SINEs

Approximately 24% (170/710) of porcine (dG-dT)n·(dC-dA)n microsatellites isolated in our laboratory are associated with a previously described porcine Short Interdispersed Element (SINE) termed PRE-1 SINE. Another 5.6% (40/710) of the microsatellites were adjacent to two previously unidentified SINE sequences, which we have designated ARE-1P (Artiodactyl Repetitive Element-1 Porcine) and ARE-2P. The ARE repeats were also found in bovine microsatellite and genomic sequences in the GenBank database. Genotypic information was obtained from 68.9% of primers where at least one primer sequence was obtained from the PRE-1 SINE and 66.6% of primer pairs designed from the ARE SINEs. The use of primers derived from SINEs significantly increases the number of primer pairs available for genetic linkage studies in swine.