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Physical assignment of microsatellitecontaining BACs to bovine chromosomes M. De Donato,a, b D.S. Gallagher,a S.K. Davis,a Y. Ji,b J.D. Burzlaff,a D.M. Stelly,b J.E. Womack,c and J.F. Taylora a Laboratory
of Genetics, Department of Animal Science, of Plant Molecular Cytogenetics, Department of Soil and Crop Sciences, and c Department of Veterinary Pathobiology, Texas A&M University, College Station, TX (USA) b Laboratory
Abstract. Here we report the physical assignment of 40 microsatellite markers by fluorescence in situ hybridization to 13 different bovine chromosomes. This information will be valuable in providing physically anchored landmarks for the construction of contigs throughout the bovine genome. It also is
useful for the purpose of integrating the linkage maps of these chromosomes to their physical maps and determining the physical coverage of these linkage groups.
The development of high-resolution physical maps in livestock species will complement linkage map development as well as the strategic mapping of targeted regions of the genome for the purpose of cloning economically important genes. The Meat Animal Research Center (MARC) of the U.S. Department of Agriculture constructed a second-generation linkage map of the bovine genome using 1,258 genetic markers (mostly microsatellites) spanning 2,949 cM (Kappes et al., 1997; map available at http://sol.marc.usda.gov/genome/cattle/cattle. html). Of these 1,258 markers, 107 have been physically assigned (Ferretti et al., 1997; Kappes et al., 1997). A bovine bacterial artificial chromosome (BAC) library (Cai et al., 1995) constructed in our laboratory has been expanded and currently contains 81,000 clones, with an average insert size of 125 kb, which represents about 350 % coverage of the
bovine genome and a 96.5 % probability of containing a unique sequence. Currently, efforts to construct a genome-wide contig assembly in cattle are underway using this BAC library (Davis and Taylor, unpublished results). Physical assignment of BAC clones is required for the orientation of the contigs and their assignment to chromosome regions to expedite the process of contig assembly. Around 400 clones have been physically assigned by fluorescence in situ hybridization (FISH) (Gallagher et al. unpublished results). These clones contain genes, gene family members, and microsatellites, as well as anonymous sequences. This article represents the first report on the physical mapping of BAC clones to bovine chromosomes. Our primary objective was to assign microsatellite markers to chromosomes distributed throughout the bovine genome. Here we report the physical assignment of 40 microsatellite markers by FISH to 13 different bovine chromosomes.
J.F.T. and S.K.D. were partially supported by grants from the National Cattlemen’s Beef Association, the Texas Higher Education Competitive Grants Program (Grant No. 999902-088), and USDA NRICGP Grant Nos. 94-37205-1224 and 95-37205-2273. M.D.D. was partly supported by the Universidad de Oriente and is a Tom Slick Senior Graduate Fellow at Texas A&M University. M.D.D.’s present address is Departamento de Biologia, Universidad de Oriente, Cumana (Venezuela). Received 20 April 1999; revision accepted 27 July 1999. Request reprints from Dr. Jeremy F. Taylor, Laboratory of Genetics, Department of Animal Science, Texas A&M University, College Station, TX 77843 (USA); telephone: 409-845-2695; fax: 409-845-6970; e-mail:
[email protected].
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Materials and methods A PCR systematic screening strategy (Cai et al., 1995) was used to screen the bovine BAC library to isolate BACs containing microsatellite sequences that had been mapped by linkage analysis. Chromosome slides were prepared from bovine fibroblast lines with the addition of BrdU to promote differential incorporation in the G-bands. FISH was performed according to Gallagher et al. (1998). Biotinylated probes were detected with avidin-Cy3, and the chromosomes were stained with Hoechst 33258 to produce QFH bands. Chromosome-band designations followed the standard cattle nomenclature (ISCNDA, 1989) as modified by Popescu et al. (1996).
Accessible online at: www.karger.com/journals/ccg
Fig. 1. Physical mapping of some of the BAC clones containing microsatellite sequences by FISH. Biotinylated BAC DNA was hybridized to mitotic bovine chromosomes and detected with Cy3. QFH bands are shown on the left of each composite picture, and the signal is shown on the right. The assignments of (a) AGLA17 to BTA 1, (b) TGLA226 to BTA 2, (c) URB014 to BTA 1, and (d) BMS1095 to BTA 5 are shown, and the FISH signals are indicated by arrows.
Fig. 2. FISH assignment of BACs containing microsatellites to 13 bovine chromosomes. The MARC linkage maps for each chromosome (Kappes et al., 1997) are shown. The space between two bars on the left of the map represents 10 cM. All maps are drawn to the same scale. Markers not included in the MARC linkage maps (TEXAN23 on BTA 3, TEXAN25 on BTA 6, and SRCRSP5 on BTA 21) are positioned at their expected distance calculated from common markers and are indicated by dotted lines. Gray vertical bars and markers with an asterisk represent previous assignments. Italicized markers represent polymorphic markers at or near the coding regions.
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Results and discussion A total of 40 microsatellite-containing BAC clones were assigned to specific G-bands on 13 bovine chromosomes (Figs. 1 and 2). These represent 39 new physical assignments of microsatellite markers. Thirty-seven of these markers have been mapped on the MARC map (Kappes et al., 1997), serving to further anchor the linkage maps to specific chromosome regions and allow more precise estimation of the chromosomal coverage. The assignment of end markers to the pericentromeric and telomeric bands indicates that the cytogenetic coverage of bovine chromosome (BTA) 1 and BTA 5 by the linkage map is complete or nearly so (Fig. 2). The estimated physical sizes for BTA 1 and BTA 5 of 176 and 134 Mb, respectively (from Popescu et al., 1996, based on an estimated cattle genome size of 3,000 Mb), was divided by the length of their linkage maps of 142 and 133 cM (Kappes et al., 1997), respectively, to produce a physical/genetic distance relationship of 1.24 and 1.01 Mb/ cM, respectively. On BTA 9, the physical assignments of ETH225 (8.1 cM away from the most centromeric marker) and BMS2094 also indicate that coverage of this chromosome is complete or nearly so. The estimated physical/genetic relationship for BTA 9 is 1.06 Mb/cM (116 Mb estimated physical size and 109 cM genetic length).
Our assignments, as well as the previous assignments of microsatellite markers, have shown almost complete chromosome coverage for BTA 2, BTA 10, and BTA 28 (Solinas-Toldo et al., 1995; Heaton et al., 1997; Smith et al., 1997; Sonstegard et al., 1997). The estimated physical/genetic relationships for these chromosomes are 1.28, 1.09, and 0.99 Mb/cM, respectively (154, 110, and 52 Mb estimated physical sizes and 120, 101, and 52 cM genetic length, respectively). It is interesting that most of the bovine chromosomes appear to have physical/ genetic relationships close to one, except for BTA 1 and BTA 2, which have higher physical/genetic relationships. This could be related to the size of these chromosomes (i.e., the longest chromosomes have lower overall recombination rates) or it could be that the higher density of markers on BTA 1 and BTA 2 affects the estimation of the physical/genetic relationship. Only one end marker per chromosome was cytogenetically assigned for BTA 20 and BTA 23 (Fig. 2), showing almost complete coverage of these chromosomal ends. The assignment of INRA006, RM012, CSSM47, and RM033 represent either the most centromeric or telomeric markers physically mapped to date for BTA 3, BTA 7, BTA 8 and BTA 23, respectively.
Acknowledgements We acknowledge Elaine Owens for excellent technical support.
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