L. J. Alexander

Analysis of copy number variations among diverse cattle breeds

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. Here, we describe the first systematic and genome-wide analysis of copy number variations (CNVs) in modern domesticated cattle using array comparative genomic hybridization (array CGH), quantitative PCR (qPCR), and fluorescent in situ hybridization (FISH). The array CGH panel included 90 animals from 11 Bos taurus, three Bos indicus, and three composite breeds for beef, dairy, or dual purpose. We identified over 200 candidate CNV regions (CNVRs) in total and 177 within known chromosomes, which harbor or are adjacent to gains or losses. These 177 high-confidence CNVRs cover 28.1 megabases or approximately 1.07% of the genome. Over 50% of the CNVRs (89/177) were found in multiple animals or breeds and analysis revealed breed-specific frequency differences and reflected aspects of the known ancestry of these cattle breeds. Selected CNVs were further validated by independent methods using qPCR and FISH. Approximately 67% of the CNVRs (119/177) completely or partially span cattle genes and 61% of the CNVRs (108/177) directly overlap with segmental duplications. The CNVRs span about 400 annotated cattle genes that are significantly enriched for specific biological functions, such as immunity, lactation, reproduction, and rumination. Multiple gene families, including ULBP, have gone through ruminant lineage-specific gene amplification. We detected and confirmed marked differences in their CNV frequencies across diverse breeds, indicating that some cattle CNVs are likely to arise independently in breeds and contribute to breed differences. Our results provide a valuable resource beyond microsatellites and single nucleotide polymorphisms to explore the full dimension of genetic variability for future cattle genomic research.

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.

Construction and characterization of a large insert bovine YAC library with five-fold genomic coverage

Genetic linkage maps of the bovine genome have recently become available (Bishop et al. 1994; Barendse et al. 1994) and are now being used to identify markers associated with traits of economic importance (ETL; Lander and Botstein 1989). Once mapped with available markers, fine mapping of these loci will be greatly facilitated by the availability of large insert libraries from which additional markers can be rapidly obtained. In the human and mouse systems, yeast artificial chromosome (YAC) vectors have been used with great success for the construction of contigs in the vicinity of target genes (Grootscholten et al. 1991). Contig construction has depended on the availability of multiple YAC libraries that provide depth of coverage to minimize the impact of chimeric and deleted clones inherent in these libraries. A single bovine genomic YAC library has been reported (Libert et al. 1993) but is commercially owned and not universally available. We report the construction of a bovine YAC library with approximately fivefold coverage of the genome and a low rate of chimerism, to provide a public resource for positional cloning. Preparation of genomic DNA, methylation, restriction, and ligation were performed essentially as described (Larin et al. 1991). Enrichment for large fragments, ligation to vector arms of plasmid pYAC4, and transformations of the S. cerevisiae strain AB1380 (Burke et al. 1987) were performed as described; the resulting clones were stored in 96-well format (Broom and Hill 1994). Clones were pooled with 1536 clones in each primary pool (total of 15 pools), and secondary pools (rows and columns) comprised of 128 or 192 clones. Currently, the bovine YAC library consists of 239 plates containing 22,944 individual clones. The overall library coverage was assessed by Southern analysis of insert size. Agarose plugs containing total yeast DNA from individual YAC clones were prepared and subjected to pulsed field gel electrophoresis (PFGE), transferred to nylon membranes, and probed with radiolabeled sheared total bovine DNA (Fig. 1). A histogram of the results of this analysis is shown in Fig. 2A. The mean insert size of 141 clones analyzed was 730 kb, with a median size of 600 kb. Thus, the predicted level of coverage is theoretically 4.6 (median) to 5.6 (mean) genome equivalents, assuming a 3 billion bp genome. The number of genome equivalents in the library was more rigorously defined by subjecting the 15 primary DNA pools to PCR analysis with 60 primer pairs representing markers from each of the 29 autosomal linkage groups and both X and Y Chromosomes (Chrs; Bishop et al. 1994; Stone et al. 1995). PCR reactions contained 300 ng of primary pool total yeast DNA, and conditions were as described (Bishop et al. 1994). The number of primary pools for which a specific size fragment was produced was assumed to be equivalent to the number of YAC clones containing the target sequence. A mean value of 4.9 positive pools per primer pair was observed, with a range of 1-13 (Fig. 2B). The mean represents a minimum estimate of the number of genome equivalents, since analysis of secondary pools for 10 of the primers revealed that some primary pools contain more than one positive clone; therefore, actual coverage is 5 genome equivalents or greater. This figure is within the theoretical range predicted from the average insert size of the clones (4.6-5.6). The level of chimerism was assessed by FISH analysis. YAC DNA was separated by PFGE, excised, biotinylated, and hybridized to bovine metaphase spreads as described (Lichter et al. 1990). In total, 22 clones were analyzed; 18 were observed to hybridize to single chromosomal locations, while 4 were chimeric (18%). Two of the 22 randomly chosen clones were greater than 1600 kb, and both were chimeric. Since clones of this size represent 11% of the library, the level of chimerism may be higher (21% if all are chimeric). Although this estimate is based on a small number of clones, the level of chimerism (18-21%) is consistent with the level observed in a sheep YAC library constructed with identical procedures (Broom and Hill 1994), and significantly lower than the reported chimerism in the previous bovine genomic YAC library (Libert et al. 1993). We have produced a bovine YAC library of sufficient redundancy and low level of chimerism to be useful for a variety of purposes. We have directly shown that the library has overall fivefold coverage of the genome and includes markers from all 29 autosomes and both sex chromosomes. This makes it the most extensively characterized bovine library available. The combined coverage of the two bovine YAC resources should be sufficient to

Construction and characterization of a large insert porcine YAC library

The recent construction of genetic linkage maps of the porcine genome (Rohrer et al. 1994, 1996; Ellegren et al. 1994; Archibald et al. 1995) allows the assignment of loci affecting heritable traits of economic importance (ETLs; Lander and Botstein 1989) to specific chromosomal segments. Markers can thus be identified that may be useful in marker-assisted selection (MAS) to increase the frequency of favorable allele(s) in resource populations (reviewed in Soller 1994). In addition, mapping of these loci creates the opportunity to identify gene(s) influencing a trait, through positional cloning or positional cnadidate gene approaches (Grootscholten et al. 1991). A positional cloning strategy requires the construction of contigs that physically span large sections of chromosomes. In the human and mouse systems, contig construction has depended on the availability of multiple YAC libraries that provide depth of coverage to minimize the impact of chimeric and deleted clones inherent in these libraries. A single porcine genomic YAC library has been reported (Leeb et al. 1995), but contains only one genome coverage, which limits the ability to make large contigs. We report the construction of a porcine YAC library, with approximately 5.5-fold coverage of the genome and a low rate of chimerism, that provides an additional resource for contig construction and positional cloning. Preparation of genomic DNA, methylation, restriction, and ligation were performed essentially as described (Latin et al. 1991). Enrichment for large fragments, ligation to vector arms of plasmid pYAC4, and transformations of the S. cerevisiae strain AB1380 (Burke et al. 1987) were performed as described (Broom and Hill 1994; Smith et al. 1996). Ura + colonies were passaged on UraTrp-, 20 p.g/ml adenine plates, and transferred to YPD media in 96-well plates (Falcon) in triplicate for storage (-80~ The library was gridded onto filters with a Biomek 1000 work station (Beckman) as described (Bentley et al. 1992; Broom and Hill 1994). Clones were pooled with 1440 clones in each primary pool (total of 23 pools), and secondary pools (rows and columns) of 120 or 180 clones. Currently, the porcine YAC library consists of 345 plates containing approximately 33,120 individual clones. Overall library coverage was assessed by Southern analysis of insert size. Agarose plugs containing total yeast DNA from individual YAC clones were prepared and subjected to pulsed field gel electrophoresis (PFGE), transferred to nylon membranes, and probed with radiolabeled sheared total porcine DNA (Sambrook et al. 1989). A histogram of the results of this analysis is shown in Fig. 1A. The mean insert size of 152 clones analyzed was 589 kb,

Effect of reduced heifer nutrition during in utero and post-weaning development on glucose and acetate kinetics.

Energetic efficiency was evaluated in composite bred heifers born from dams receiving 1·8 or 1·2 kg/d winter supplementation for approximately 80 d before parturition. Heifers were then developed post-weaning and randomly assigned to heifer development treatments of either control (100 %; ad libitum; n 8/year) or restricted (80 %; fed 80 % of supplementation fed to controls adjusted to a common body weight: n 8/year) in a 2-year study. A glucose tolerance test (GTT) and acetate irreversible loss test (AILT) were administered to heifers at the termination of a 140 d development period when the heifers were approximately 403 d of age and consumed a silage-based diet, and again at 940 d of age when pregnant with their second calf and grazing dormant forage. No differences were measured (P>0·08) for dam winter nutrition or heifer development treatment for baseline serum metabolites or measures in either the GTT or the AILT. However, changes in baseline serum concentrations (P>0·05) were different between metabolic challenges, which occurred at different stages of development. No difference in acetate disappearance (P = 0·18) and half-life (P = 0·66) was measured between the two metabolic challenges. A trend for glucose half-life to be shorter in heifers born from dams receiving in utero winter treatments that supplied 1·2 kg/d of winter supplementation was observed (P = 0·083). Heifers developed with lower total DM intake during a 140 d development period had similar glucose and acetate incorporation rates as ad libitum-fed heifers when evaluated at two different production stages.

An unassigned porcine microsatellite linkage group maps to Chromosome 6

The construction of species-specific comprehensive genomic maps is facilitated when anonymous polymorphic markers have also been physically anchored. This provides marker assignment and orientation, and improves estimates of chromosomal coverage (Murray et al. 1994). Although major advances (Rohrer et al. 1994: Ellegren et al. 1994; Archibald et al. 1995) have been made in the development of a porcine microsatellite (ms) linkage map, the number of ms loci physically anchored to individual porcine chromosomes is much lower than in other species. To physically anchor an unassigned porcine ms linkage group, a cosmid containing an ms (Sw2415) known to be near one end of the group was assigned by fluorescence in situ hybridization (FISH; Lichter et al. 1990). The assignment of the linkage group was confirmed by assigning an additional ms (Sw2419) with FISH. Forward and reverse primer sequences for Sw2415 were 5-ATACGTCTAAGCCCCTTGGC-3 and 5-TAGAGAAACCCACCAGTGGG-Y, respectively. The forward primer for Sw2419 was 5-AGGGCGTGCTCTTCTAACTG-3; the reverse primer was 5-TGACTCAGCATCTCCTGCC-3. Annealing temperatures for Sw2415 and Sw2419 were 50~ and 58~ respectively. Sw2415 has six alleles that range from 118 to 144 bp; Sw2419 has eight alleles ranging from 115 to 135 bp. Metaphase spreads from male porcine peripheral lymphocytes were prepared by standard techniques. Cosmid DNA was labeled with biotinylated 14 dATP by nick translation (Gibco BRL, Gaithersburg, Md.). Labeled cosmids were purified over a G-50 Sephadex column prior to use in FISH. FISH were performed according to manufacturers instructions (Oncor. Gaithersburg, Md.). Slides were treated with 2x SSC for 30 vain at 37~ and dehydrated in 70%, 80%, and 95% ethanol for 2 min each. After air drying, slides were denatured two at a time in 70% formamide/2x SSC at 71~ for 2 rain, After denaturation, slides were dehydrated in an ascending series of cold ethanol washes for 2 rain each, air dried, and warmed to 37~ The labeled probe (100M 50 ng) was hybridized to metaphase chromosomes overnight in a humidified chamber at 37~ Slides were washed post hybridization in 50% formamide/ 2x SSC at 43~ for 15 rain, followed by 8 rain at 37~ in 2x SSC. Hybridization signals were detected by FITC-avidin and amplified with additional applications of biotinylated anti-avidin and FITC avidin. The chromosomes were counterstained with propidium iodide (25 pg/ml in 2x SSC) for 2 min and R-banded by mounting in alkaline p-phenylenediamine (PPD-11; Lemieux et al. 1992). FITC and propidium iodide were excited with a BP 450-490 filter, and chromosomes were photographed with Kodak Ektachrome 400 color slide film in a Leitz DM RX epifluorescent microscope. Sw2415 (Fig. 1) and Sw2419 (Fig. 2) mapped to the telomeric region of the long arm of Chromosome (Chr) 6. As can be seen in Figs. 1 and 2, the two ms are physically very near each other. The