The Position of Repetitive DNA Sequence in the Southern Cattle Tick Genome Permits Chromosome Identification
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Chromosome Research (2009) 17:77–89 DOI 10.1007/s10577-008-9003-0 The position of repetitive DNA sequence in the southern cattle tick genome permits chromosome identification Catherine A. Hill & Felix D. Guerrero & Janice P. Van Zee & Nicholas S. Geraci & Jason G. Walling & Jeffrey J. Stuart Received: 15 July 2008 /Revised: 24 October 2008 /Accepted: 24 October 2008 / Published online: 17 February 2009 # Springer Science + Business Media B.V. 2009 Abstract Fluorescent in-situ hybridization (FISH) microplus autosomes 1–6 and 8–10. A second repeat using meiotic chromosome preparations and highly unit, RMR-2 was localized to the subtelomeric repetitive DNA from the southern cattle tick, Rhipi- regions of all autosomes and the X chromosome. cephalus microplus, was undertaken to investigate RMR-2 was composed of three distinct repeat genome organization. Several classes of highly populations, RMR-2a, RMR-2b and RMR-2c of repetitive DNA elements were identified by screening 178, 177 and 216 bp in length, respectively. Locali- a R. microplus bacterial artificial chromosome (BAC) zation of an rDNA probe identified a single nucleolar library. A repeat unit of approximately 149 bp, RMR- organizing region on one autosome. Using a combi- 1 was localized to the subtelomeric regions of R. nation of labeled probes, we developed a preliminary karyotype for R. microplus. We present evidence that R. microplus has holocentric chromosomes and Responsible Editor: Mary Delany. explore the implications of these findings for tick Electronic supplementary material The online version of this chromosome biology and genomic research. article (doi:10.1007/s10577-008-9003-0) contains supplementary material, which is available to authorized users. Keywords fluorescent in-situ hybridization . heterochromatin . holocentric chromosomes . repetitive C. A. Hill (*) : J. P. Van Zee : N. S. Geraci : J. J. Stuart Department of Entomology, Purdue University, DNA . Rhipicephalus microplus West Lafayette, IN 47907, USA e-mail: [email protected] Abbreviations BAC bacterial artificial chromosome F. D. Guerrero USDA-ARS Knipling-Bushland U.S. Livestock Insects C-banding centromere banding Research Laboratory, FISH fluorescent in-situ hybridization Kerrville, TX 78028, USA gDNA genomic DNA GTE glucose tris-EDTA J. G. Walling Department of Horticulture, University of Wisconsin, NCBI National Center for Biotechnology Madison, WI 53706, USA Information NOR nucleolar organizing region Present address: PGCF Purdue University Genomics Core N. S. Geraci Chicago Children’s Memorial Research Center, Facility Chicago, IL 60614, USA RMR Rhipicephalus microplus repeat 78 C.A. Hill et al. Introduction microplus and other tick species. Cytogenetic inves- tigations of R. microplus have been limited to the Ticks (subphylum Chelicerata; class Arachnida; sub- work published by Oliver and Bremner (1968), class Acari; family Ixodidae) cause global veterinary Newton et al. (1972), and Hilburn et al. (1989). Early and medical health problems by transmitting a wide tick cytogenetics is reviewed by Oliver (1977) who variety of bacteria, viruses, and protozoa and inflict- reported that R. microplus has an XX:XO sex ing direct damage to their host via attachment and determination system with 22 diploid chromosomes feeding. The southern cattle tick, Rhipicephalus in females and 21 in males. Preliminary C-banding (Boophilus) microplus (Canestrini), is widely distrib- karyotypes have also been developed for R. microplus uted throughout the tropics and subtropics and is the (Hilburn et al. 1989; Garcia et al. 2002). Unfortu- most economically significant Rhipicephalus species. nately, these are of limited utility for chromosome It transmits the causative agents of bovine babesiosis identification due to uniformity of autosome length (Texas cattle fever) and anaplasmosis, diseases that and morphology. cause severe milk and beef production losses and high Here we present the first report to identify a range mortality rates in affected herds (Dietrich and Adams of DNA sequence repeats from R. microplus genomic 2000). In many parts of the world, R. microplus sequence and localize these repeats to R. microplus control is complicated by development of acaricide chromosomes by FISH mapping. Our work provides resistance. Rhipicephalus microplus was eradicated an approach to identifying R. microplus chromosomes from the United States in the 1940s, but the and analyzing the organization of the genome. These possibility of its re-introduction represents a serious results will support the anticipated R. microplus economic threat to the US cattle industry. For this genome sequencing effort and enable advances in reason, the USDA-ARS maintains a 10-mile wide cytogenetics and population genetics research in this quarantine zone along the US-Mexico border and a and other tick species. mandatory acaricide treatment program for all cattle imported into the United States. Studies of the R. microplus genome are underway Materials and methods to learn more about the biology of this significant pest and identify new methods for its control (Guerrero et Source of tick material al. 2006). Currently, little is known about the nature and organization of the R. microplus genome or tick Genomic DNA (gDNA) for BAC library production genomes in general. The haploid genome size of R. and chromosome preparations was obtained from the microplus is an estimated 7.1 Gbp (Ullmann et al. R. microplus Deutsch strain maintained as described 2005) and is the largest of any pro- or metastriate tick by Davey et al. (1980) at the USDA-ARS Cattle species examined thus far (Palmer et al. 1994; Fever Tick Research Laboratory in Mission, Texas. Ullmann et al. 2005; Geraci et al. 2007). Ullmann et This is an acaricide-susceptible strain that is used al. (2005) used reassociation kinetics to study the routinely in research applications. It has been main- composition of the R. microplus genome and found tained in continuous culture since it was established that the highly repetitive DNA fraction, comprising from ticks collected in Webb Co., Texas in 2001. tandem and dispersed repeats of low sequence complexity, accounted for approximately 40% of the R. microplus library screens R. microplus genome, while the moderately repetitive fraction, comprising transposable elements and multi- Approximately 10% (4608 clones) of the R. microplus gene families, accounted for approximately 30%. BAC library (approximately 1-fold genome coverage These findings raise intriguing questions about the and average insert size of 118 kbp, BamHI cloning accumulation of repetitive material in R. microplus site; Amplicon Express, Pullman, WA, USA) was and the organization of this material with respect to arrayed on a nylon filter membrane at the Purdue coding sequence on the chromosomes. University Genomics Core Facility (PGCF) using the Techniques to identify specific chromosomes and BioRobotics Total Array System. Filters were probed to study chromosome biology are needed for R. with R. microplus gDNA extracted from embryos of Repetitive DNA organization in R. microplus 79 the Deutsch strain. BAC clones containing highly with this study are available at the National Center for repetitive DNA were also used as probes in library Biotechnology Information (NCBI) GenBank data- screens. Probes were prepared by labeling 25 ng of base (accession numbers FJ223571- FJ223604). DNA with 32P-ddATP in separate random priming reactions (Prime-It II, Stratagene, La Jolla, CA, USA). Isolation of the R. microplus rDNA repeat Filters were pre-hybridized with PerfectHyb Plus (Sigma, St. Louis, MO, USA). Denatured probe was rDNA primers (forward primer: 5′-CTC TTG TGG added directly to the pre-hybridization solution and TAG CCA AAT GC-3′; reverse primer: 5′-AAG CGA hybridization was performed overnight at 60°C. CGT CGC TAT GAA CG-3′)weredesignedbasedon Filters were washed in successively diluted solutions a R. microplus 28S rDNA sequence obtained from of SSC (2× SSC, 1× SSC, and 0.5× SSC) and 0.1% GenBank (accession number: AF200189). PCR was SDS at 60°C and then exposed on a Fujifilm 2325 performed using 1 µg R. microplus gDNA and the imaging plate. Images were developed using a Fuji following conditions: initial denaturation at 94°C for FLA-5000 phosphorimaging system (Fujifilm, Tokyo, 2 min; 94°C for 10 s, 53°C for 30 s, and 72°C for Japan). 1 min for 30 cycles; final extension at 72°C for 10 min. The resulting 749 bp amplicon was subcloned and BAC DNA isolation and analysis labeled as described above, and used to screen BAC library filters. BAC DNA was isolated for library screening, restric- tion digest, and probe production using an alkaline R. microplus meiotic chromosome preparations lysis procedure modified after Sambrook et al. (1989). BAC clones were separately cultured overnight at Meiotic chromosome spreads were prepared from the 37°C in 5 ml LB media containing chloramphenicol testes of 25 newly molted adult males from the (0.04 mg/ml). Pelleted cells were re-suspended in GTE Deutsch strain of R. microplus. Tissues were dissected buffer (glucose, Tris, EDTA) containing 10 mg/ml in 0.5× Ringer’s saline, transferred to a 3:1 ethanol– lysozyme (Sigma) and 2 mg/ml RNase A (Qiagen, glacial acetic acid solution for 5 min, and pelleted by Valencia, CA, USA). Following lysis with a 0.2 N centrifugation. Cells were re-suspended in 50% NaOH, 1% SDS solution, DNA was purified by glacial acetic acid and 5 µl drops were placed on phenol–chloroform–isoamyl alcohol (25:24:1) extrac- microscope slides and air dried. Excess cytoplasmic tion, and re-suspended in nuclease-free water. material was removed from the preparations by a BAC DNA was digested with HpaII, MseI, and series of washes in 200 µl 2× SSC and 0.5% RNase at RsaI in separate reactions and the resulting restriction 37°C , followed by treatment with pepsin (100 mg/ml; fragments were separated by agarose gel electropho- Sigma-Aldrich, Saint Louis, MO, USA) in 85 µl of resis on 0.8% TBE gels. Bands of interest were pre-warmed 0.01 M HCl at 37°C for 2 h.