The Position of Repetitive DNA Sequence in the Southern Cattle Tick Genome Permits Chromosome Identification

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, restriction 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. Following excised from the gel and the DNA in these bands was incubation, slides were washed with 1× PBS, 0.2 M purified using the Qiagen QIAEX II gel extraction kit MgCl2, serially dehydrated in 70%, 90%, and 100% (Qiagen). The DNA was then subjected to PCR to add ethanol and then air-dried. 3′ A-overhangs and cloned into the TOPO TA PCR 2.1 cloning vector (Invitrogen, Carlsbad, CA, USA). Fluorescence in-situ hybridization (FISH) These DNA fragments were then subjected to PCR- based sequencing reactions containing M13 forward, DNA probes were prepared by nick translation using M13 reverse, or T7 primers, and BigDye version 3.1 1 µg of DNA and either biotin- or digoxigenin- (Applied Biosystems, Foster City, CA, USA) using conjugated dUTP (Roche) according to the manufac- the following PCR conditions: initial denaturation at turer’s recommendations. In-situ hybridization was 96°C for 3 min followed by 96°C for 10 s, 50°C for performed with 40–100 ng of denatured probe DNA 5 s, and 60°C for 4 min for 30 cycles. The DNA in 10 µl of hybridization solution (10% dextran sequence was subsequently determined using an ABI solution, 2× SSC, 40% formamide, and 20 µg of 3730XL capillary DNA analyzer (Applied Biosys- herring sperm DNA) at 37°C for 12–15 h. Detection tems) by the PGCF. All DNA sequences associated was performed using Alexa Fluor 488-conjugated 80 C.A. Hill et al. anti-biotin (Molecular Probes, Eugene, OR, USA) and BAC 4H23 and when used as probe to screen the rhodamine-conjugated anti-digoxigenin (Molecular library it hybridized only with itself (data not shown). Probes). For multilayer detection of biotin conjugated This suggested that BAC 4G12 lacked repetitive probes, Alexa Fluor 488-streptavidin was used as the DNA and it was not analyzed further. In contrast, initial layer of immunodetection followed by a layer of when BAC 1D1 was used as probe it hybridized to biotin anti-streptavidin and a second layer of Alexa itself and 93 additional clones that had been negative Fluor 488-streptavidin. For multilayer detection of to gDNA, 1B14, and 4H23 (Fig. 1d). digoxigenin-conjugated probes, mouse anti-digoxigenin was used in the first layer of detection followed by a Isolation and cloning of repetitive DNA fragments layer of anti-mouse Alexa Fluor 568 (Molecular Probes). Digital images were collected under UVoptics After the series of library screens, BAC clones using an ORCA-ER (Hammamatsu, Iwata City, Japan) containing highly repetitive DNA could be assigned digital camera mounted on an Olympus BX51 micro- to three groups: (1) clones that were BAC 1B14 scope and MetaMorph (Universal Imaging Corp., positive; (2) clones that were BAC 4H23 positive; Downington, PA, USA) imaging software. and (3) clones that were 1D1 positive. Therefore, a series of restriction digests and gel electrophoresis experiments were performed with two or more clones Results isolated from each of these three groups. DNA fragments that appeared to be repetitive, based on BAC library screens their relative intensity of fluorescence after ethidium bromide staining, were subsequently cloned and To identify BAC clones containing highly repetitive sequenced (Fig. 2). R. microplus gDNA sequence, 4608 BAC clones Three clones positive for 1B14 hybridization were arrayed in twelve 384-well plates were screened with analyzed (1F18, 1O24, and 1B14 itself). Fragments 32P-labeled R. microplus gDNA. We observed 110 unique to each clone were observed, but intensely positive clones (2.4% of the total) on the filter staining fragments in all three clones appeared to have (Fig. 1a). To begin discriminating among these clones the same molecular weight. The corresponding MseI for different classes of repetitive DNA, a series of fragments of approximately 150 and 300 bp were hybridizations were performed on separate filters cloned and sequenced. This revealed a repetitive using four of the positive clones as probes. We first sequence, referred to hereafter as R. microplus repeat discovered that a single arbitrarily selected BAC 1 (RMR-1; Table 1). RMR-1 had a 149 bp repeat at (1B14) hybridized with 100 of the clones (including its core, but 21 unique copies of RMR-1 ranging in BAC 1B14 itself) that were positive to gDNA length from 82 bp to 152 bp were identified by (Fig. 1b). This indicated that one or more major sequencing 14 independent clones of MseI fragments species of repetitive DNA was present in BAC 1B14. (see Supplementary Fig. S1). The minimum nucleo- One clone, BAC 3M21, was positive for 1B14 but tide identity between copies of the 149 bp core repeat failed to hybridize to gDNA. We assumed that this was 71.8% and the average GC content of these clone contained a minor repeat species and it was not sequences was 54.4%. investigated further. Nine BACs (0.2% of the total) Two clones positive for 4H23 hybridization (4L11 that had previously shown strong hybridization to and 4H23 itself) were analyzed (Fig. 2). Again, gDNA failed to hybridize to 1B14 DNA. Therefore, fragments unique to each clone were observed, but one of those clones was used as a probe in a the intensely staining fragments in both clones subsequent library screen. This clone (BAC 4H23) appeared to have the same molecular weight. Intensely hybridized only with clones that were both gDNA- staining MseI fragments of approximately 150, 200 and positive and BAC-1B14-negative (Fig. 1c). BAC 350 bp and RsaI fragments of approximately 375 and 1B14 and BAC 4H23 hybridization accounted for 450 bp were cloned and sequenced. These fragments all but two (4G12 and 1D1) of the 110 BAC clones contained three populations of repetitive sequence that had been positive to whole-genome screening. hereafter referred to as R. microplus repeats RMR-2a, BAC 4G12 showed only very weak hybridization to RMR-2b and RMR-2c (Table 1) or collectively as Repetitive DNA organization in R. microplus 81

Fig. 1 BAC library screens for highly repetitive DNA in the R. for 1B14 but negative for R. microplus gDNA. (c) A separate microplus genome. Each panel (a, b, c and d) displays rows A filter probed with BAC 4H23 (black box) clearly hybridized to to P and columns 11 to 24 of the same R. microplus BAC BACs 4H17, 3L21, and 4L11 (white boxes) but only weakly library. Black and white circles, boxes, and pentagons identify hybridized to BAC 4G12 (white circle). Although there was the same BAC clones in each panel. (a) A library screen using weak hybridization associated with a number of additional R. microplus gDNA as probe identified BACs putatively clones, BACs 1B14 (black circle) and 3M21 (pentagon) clearly containing highly repetitive DNA. (b) A separate filter probed failed to cross-hybridize to BAC 4H23 DNA. (d) A separate with BAC 1B14 DNA (black circle) failed to hybridize to filter probed with BAC 1D1 shows lack of hybridization to BACs 4G12 (white circle), 4H17, 3L21, 4L11 (white boxes), clones 1B14, 4G12, 4H17, 4H23, 3L21, 4L11, and 3M21 (note and 4H23 (black box), which previously hybridized to R. different hybridization pattern in white box and pentagon microplus gDNA. BAC 3M21 (pentagon) was clearly positive representing clones 4H17 and 3M21, respectively) repeat 2. RMR-2a, RMR-2b, and RMR-2c had repeats the consensus repeats of RMR-2a and RMR-2b (Fig. 3). of 178, 177, and 216 bp at their core, respectively. There Due to their limited sequence identity, these sequences was 81.5% nucleotide identity between 13 copies of are referred to as independent repeats but we recognize RMR-2a, 82.5% identity between 10 RMR-2b copies, that they are probably highly diverged copies of one and 88.5% identity between 14 RMR-2c copies (see another. No significant sequence similarity was iden- Supplementary Fig. S1). The %GC content of the RMR- tified between these repeats and RMR-2c. Restriction 2a, -2b, and -2c consensus sequences was 37.6%, fragment analysis showed that RMR-2a, RMR-2b, and 39.0%, and 43.8%, respectively. A forced manual RMR-2c are arranged in tandem within a 748 bp alignment revealed 52.3% nucleotide identity between stretch of DNA (Fig. 3). Five copies of the (TTAGG)n 82 C.A. Hill et al.

telomeric repeat were identified within this 750- nucleotide stretch and were associated specifically with RMR-2a and RMR-2b. Two intensely staining fragments of approximately 175 and 350 bp were identified by RsaI digestion of clone 1D1. Sequencing revealed two potential repeats of 177 and 356 bp, referred to hereafter as R. microplus repeats RMR-3 and RMR-4 (Table 1). These repeats are likely separate, presumably dispersed repeats because there is no evidence from BAC fragments that they occur as tandem repeats. 92.5% nucleotide identity was observed between one full-length copy of RMR-3 and a truncated 134 bp copy of this repeat (see Supplementary Fig. S1). The %GC content of the consensus repeat was 55.4%. Despite similarity in repeat length, no sequence similarity was identified between RMR-3 and either RMR-2a or RMR-2b. Only one RMR-4 clone was obtained and this had a GC content of 37.1%. BLASTn searches did not identify any similarity between RMR-1, RMR-2a, RMR-2b, RMR-2c, RMR-3, and RMR-4, and sequences in the NCBI nonredundant (nr) database. This search included over 19 million trace reads from the Ixodes scapularis (Lyme disease tick) genome sequencing project. These repeats maybe specific to R. microplus, but Fig. 2 Agarose gel electrophoresis of restriction endonuclease-digested BACs containing highly repetitive R. microplus sequencing of additional tick species is needed to DNA. Agarose gels containing digestions of BACs 1B14, confirm this hypothesis. BAC library screens with 1F18, 1O24, 4L11, 4H23, and 1D1 are shown. Black and white RMR-1 and RMR-2 sequence identified the same arrows indicate the fragments that were extracted from the gels, clones previously discovered by whole gDNA screen- cloned, and sequenced (H = HpaII; M = MseI, and R = RsaI) ing (data not shown).

Table 1 Rhipicephalus microplus repeat sequences

Repeat Size of core No. repeat copies Size range of repeat GC content of core Average nucleotide identity between repeat (bp) identified copies (bp) repeat (%) copies of core repeat (%)

Repeat family 1 RMR-1 149 21 82–152 54.4 71.8 Repeat family 2 RMR-2a 178 13 55–178 37.6 81.5 RMR-2b 177 10 175–177 39.0 82.5 RMR-2c 216 14 53–216 43.8 88.5 Repeat family 3 RMR-3 177 2 134–177 55.4 92.5 Repeat family 4 RMR-4 356 1 356 37.1 ND

ND, not determined. Repetitive DNA organization in R. microplus 83

Fig. 3 The R. microplus RMR-2 family of repeats. (a) Manual found in some but not all RMR-2 repeat copies. (c) Schematic alignment of RMR-2a and RMR-2b. Identical nucleotides are diagram showing how the RMR-2a (black shading), RMR-2b shaded. (b) Schematic diagram showing the arrangement of the (dark gray shading) and RMR-2c (light gray shading) repeats RMR-2a, RMR-2b and RMR-2c repeats within a 748 bp identified from the RsaI 375 bp and 450 bp, and the MseI repetitive DNA unit; M, MseI restriction site; R, RsaI restriction 150 bp, 200 bp and 350 bp restriction fragments were used to site ; *, location of TTAGG telomeric repeat; ‡, denotes a reconstruct the 748 bp repetitive DNA unit in (b). Text below truncated copy of RMR-2a. Numbers below the diagram refer lines refers to the five different fragments identified by MseI to the size of hypothetical restriction fragments in bp. Dotted and RsaI digest of BAC clones 4L11 and 4H23 shown in Fig. 2 lines indicate the location of restriction endonuclease sites

Cytology size and a single X chromosome (Fig. 4). In metaphase I, chiasmata were evident between homo- To determine the positions of the repetitive sequences logues in the ‘end-to-end’ bivalent configuration that in the R. microplus genome we performed fluorescent is typical of holocentric chromosomes and that is in-situ hybridization (FISH) experiments on male R. proposed to result from terminalization of the chias- microplus meiotic chromosome preparations. Nuclei mata (Dernburg 2001). Sister chromatids appeared to in all stages of meiosis were observed. Chromosome separate in an equational division in meiosis I, and the morphology and behavior during meiosis suggested homologues appeared to separate in a reductional that the chromosomes were holocentric rather than division during meiosis II (data not shown). In phase- metacentric as previously reported (Oliver 1977). contrast and DAPI-stained preparations, only the During meiosis I, we observed 10 bivalents of similar unpaired X chromosome could be distinguished from 84 C.A. Hill et al.

Fig. 4 Fluorescence in-situ hybridization of R. microplus and that rDNA hybridization (arrowhead) distinguished one repetitive DNA to R. microplus testicular chromosome prepa- bivalent (6). (c–j) FISH using both RMR-1 and RMR-2 as rations. (a) RMR-1 probes 1B14 and 1F18 hybridizing to probe. (c) DAPI stained bivalent. (d) RMR-1 hybridization to metaphase I chromosomes. Note that the X chromosome (X) the DAPI-stained bivalent in panel (c). (e) RMR-2 hybridiza- and one bivalent (7) failed to show any hybridization signal. tion to the DAPI-stained bivalent in panel (c). (f) False-color RMR-1 was clearly evident near the telomeres of the remaining overlay of images (c–e). (g) DAPI-stained interphase primary bivalents, and its intense hybridization distinguished one spermatocyte showing the 21 intensely DAPI-positive foci bivalent (4) from the others. An arrow indicates the position typical of these nuclei. (h) RMR-1 hybridization to the nucleus of the chiasmata of one bivalent. (b) RMR-2 (red fluorescence) shown in panel (g). (i) RMR-2 hybridization to the nucleus and 28S rDNA hybridization (green fluorescence) near the shown in panel (g). (j) A false-color overlay of images (g–i). telomeres of metaphase I chromosomes. Note that the X Scale bars represent 10 µm chromosome and each bivalent show RMR-2 hybridization Repetitive DNA organization in R. microplus 85 the other chromosomes; the X lacked a homologue longest bivalents, each of which contained a relatively and a chiasma, was larger than the autosomes, and small amount of RMR-1 DNA. The second group was slightly more DAPI-positive. (Fig. 5, group b) was composed of the three RMR-1 hybridized to all but one bivalent and the chromosomes that contained the greatest relative X chromosome (Fig. 4a). FISH experiments using amount of RMR-1 DNA. Hybridization to one of each of the RMR-1-containing BAC clones (1B14, these chromosomes (bivalent 4 in Fig. 4a and Fig. 5) 1F18, and 1O24) as probe were also performed. suggested that it was largely composed of RMR-1 Results of these experiments were indistinguishable heterochromatin. Identification of this chromosome in from those using the RMR-1 DNA alone as probe meiotic I cells was nearly unambiguous. Hybridiza- (data not shown). The position of this hybridization tion of 28 S ribosomal DNA made a second bivalent appeared to correspond to subtelomeric heterochro- in this group recognizable in every nucleus (bivalent matin. An absence of RMR-1 hybridization permitted 6, Fig. 4b and Fig. 5). The third group (Fig. 5, group the unequivocal identification of the X and one c) was composed of the shortest three bivalents each bivalent (bivalent 7 in Figs. 4 and 5). In addition, of which contained a moderate quantity of RMR-1 the relative lengths of the remaining bivalents DNA. The relative amount of RMR-1 DNA on one of combined with the relative intensity of RMR-1 these bivalents (bivalent 8, Fig. 5) made its recogni- hybridization made it possible to suggest the corre- tion almost unequivocal. spondence between bivalents in various meiotic I RMR-2 hybridized to each of the bivalents and the nuclei (Fig. 5). Such an attempt resulted in the X chromosome (Fig. 4b).AswithRMR-1,the placement of the bivalents into three groups. The first position of RMR-2 hybridization was subtelomeric. group (Fig. 5, group a) was composed of the three It appeared, however, that RMR-2 heterochromatin

Fig. 5 R. microplus bivalent identification using RMR-1 and relative lengths and relative quantity of RMR-1 DNA (red rDNA FISH. The X chromosome and 10 bivalents from three fluorescence). An absence of RMR-1 DNA on the X chromo- meiotic I nuclei are shown. The bivalents from each nucleus some and bivalent 7 makes identification of these chromosomes were ordered from 1 to 10 by descending relative length. They unequivocal. Hybridization with rDNA (green fluorescence) are assigned to three groups (a, b, and c) according to their also makes the identification of bivalent 6 unmistakable 86 C.A. Hill et al. was more closely associated with the telomere than limitations associated with traditional staining techni- RMR-1 DNA (Fig. 4c–j). RMR-2 DNA appeared to ques, FISH mapping was pursued in this study as an be closely associated with brightly staining foci at the approach for identification of R. microplus chromo- telomere of each bivalent, whereas RMR-1 hybridized somes and analysis of DNA organization on the at a position that was slightly closer to the chiasma chromosomes. (Fig. 4c). Interestingly, similar foci were evident as 21 FISH mapping enabled an investigation of R. DAPI-positive dots in primary spermatocytes microplus chromosome morphology and behavior (Fig. 4g). FISH experiments clearly indicated that during meiosis. Previous studies have reported acro- these foci were also tightly associated with RMR-2 centric chromosomes in R. microplus (Oliver and DNA and much less closely associated with RMR-1 Bremner 1968; Hilburn et al. 1989; Garcia et al. heterochromatin (Fig. 4g–j). 2002) with the presumed centromere occurring at the FISH experiments using RMR-3 and RMR-4 as tip of the chromosome and the ‘short’ arm being probe failed to localize these repeats to a specific indistinguishable (Hilburn et al. 1989). Using FISH chromosomal location. Instead, the RMR-3 and and high-resolution digital images, we identified RMR-4 signal was evenly distributed along each R. several lines of evidence that suggest that R. micro- microplus chromosome (data not shown), which plus chromosomes are actually holocentric. Holocen- further suggests that these are dispersed repeats. tric chromosomes have been described in a number of BLASTn searches did not identify any similarity organisms, including the round worm Caenorhabdtis between RMR-3 and -4 sequences and retro-element elegans and heteropteran insects (Bongiorni et al. sequences in the NCBI nr database. 2004), and are characterized by the lack of a localized centromere, and consequently of a localized kinetic activity. During mitotic metaphase, diffuse kineto- Discussion chores are thought to form along the entire length of the chromosome and the sister chromatids separate in Our finding of 10 paired autosomes and an unpaired parallel. Rhipicephalus microplus chromosomes X chromosome in R. microplus males agrees with lacked a clearly defined centromere in all spreads earlier cytogenetic work of Oliver and Bremner examined. Garcia et al. (2002) also failed to identify a (1968), who reported 21 diploid chromosomes and clearly defined centromere in R. microplus chromo- an XX:XO sex determination system in male R. somes using conventional acetic orcein staining. In microplus. These results are also consistent with addition, we observed apparent end-to-end pairing of chromosome numbers in Mexican (Newton et al. homologous R. microplus chromosomes in meiosis I 1972; Hilburn et al. 1989) and Brazilian (Garcia et al. and sister chromatids in meiosis II (data not shown), 2002) R. microplus populations. Although R. micro- which is consistent with other holocentric arthropods plus karyotypes have been developed using traditional (Pérez et al. 1997, 2000; Mandrioli 2002, 1999; C-banding techniques (Hilburn et al. 1989; Garcia et Bongiorni et al. 2004). The studies of Hilburn et al. al. 2002), chromosome identification is complicated (1989) and Garcia et al. (2002) concluded that R. by the fact that the autosomes are similar in size, lack microplus constitutive heterochromatin bands were distinguishing morphological traits, and possess rela- centromeric, where as our observations suggest that tively uniform C-banding patterns. Our DAPI staining the heterochromatin actually occurs in the R. micro- results suggest that significant amounts of heterochro- plus telomeres. The localization of repetitive DNA in matin are associated with the termini of chromo- telomeric regions is a common feature of holocentric somes, but the distribution was too uniform to permit chromosomes (Schweizer and Loidl 1987). identification of individual chromosomes. The X In meiosis, the behavior of holocentric chromo- chromosome was easily identified in metaphase I as somes is strikingly different in that the kinetic activity the only unpaired chromosome. It was also possible to of autosomes and sex chromosomes is restricted to identify the X chromosome based on its size and the either of the two chromatid ends during the first and intensity of DAPI staining, suggesting that the X second meiotic divisions (Pérez et al. 2000). In many chromosome may have a higher heterochromatin holocentric organisms such as coccids, aphids, and content compared to the autosomes. Given the mites, it is common for the meiotic divisions to be Repetitive DNA organization in R. microplus 87 inverted relative to mitosis such that the first division repeat (RMR-2) localized to telomeric regions of all is equational (sister chromatids separate) and the autosomes and the unpaired X chromosome. RMR-2 second reductional (homologous chromosomes sepa- appeared to map more closely to the telomeric regions rate) (Wrensch et al. 1993; Dernburg 2001; Bongiorni of meiotic chromosomes than RMR-1. FISH mapping et al. 2004). The segregational behavior of meiotic R. to interphase nuclei further supports a telomeric microplus chromosomes was similar to one of three location for the R. microplus repeats. DAPI staining alternative patterns documented for autosomes of the revealed 21 brightly stained, presumably AT-rich holocentric heteropteran, Triatoma infestans (Pérez et heterochromatic dots associated with diploid, inter- al. 1997, 2000). Kinetic activity appeared to be phase nuclei. Telomeres are known to be enriched in restricted to the heterochromatic termini of the R. heterochromatin (Panzera et al. 1995; Bizzaro et al. microplus chromosomes at first metaphase with 1996) and these dots are expected to correspond to the division being reductional. At second metaphase, the telomeres of each of the 21 chromosomes in diploid kinetic activity appeared to shift to the euchromatic males. The localization signal of the family 2 repeat termini such that division was equational. In T. immediately overlaid the heterochromatic dots infestans, both inverted and normal meiotic behavior observed in R. microplus nuclei, while the family 1 is possible within an individual, although the repeat exhibited a more diffuse signal in regions ‘inverted’ sequence (Pérez et al. 1997, 2000) is more adjacent to these dots. The RMR-2 repeat also common. The mechanisms that determine the selec- contained five copies of the TTAGG penta-nucleotide tion of a particular segregational type are currently repeat that has been associated with the telomeres of unknown. hymenopteran and lepidopteran insects (Okazaki et al. The suggestion of holocentric chromosomes in R. 1993; Kipling 1995; Meyne et al. 1995). In addition, microplus is not without precedent. Both monocentric the RMR-2 core repeats are relatively AT rich. Taken and holocentric chromosomes have been reported in together, these results suggest that RMR-1 and -2 are the Acari but the distribution of chromosome types located within subtelomeric, heterochromatic regions among taxa has not been widely studied (Oliver of R. microplus chromosomes, with family 2 having 1977). Evidence suggests that the two-spotted spider closer proximity to the R. microplus telomeres. mite, Tetranychus urticae, the water mites, Eylais Although the distribution of RMR-3 and RMR-4 setosa and Hydrodroma despiciens, and the grass mite could not be resolved by FISH, our results indicate Siteroptes graminum (reviewed by Oliver 1977) have that these repeats are distributed evenly along all R. holocentric chromosomes. Little is currently known microplus chromosomes, and are in fact, dispersed about the segregational behavior of tick and mite repeats. RMR-1 and RMR-2 repeats may also be chromosomes during meiosis. The suggestion of distributed in low copy number in other regions of the holocentric chromosomes in R. microplus raises R. microplus chromosomes. Sequencing of gDNA is interesting questions about the mechanisms that needed to investigate the distribution of these repeats determine kinetic termini during meiosis and the in euchromatic regions of the R. microplus genome. possible role of heterochromatin in these processes. The functions of the R. microplus repeats identified Other approaches such as silver and immuno- in this study are not known. Repeats of similar size histochemical staining, and atomic force microscopy and unknown function have been identified in other (Mandrioli and Manicardi 2003) could lend weight to invertebrate species (Spence et al. 1998; Mandrioli et our hypothesis of holocentric chromosomes. If true, al. 1999), but these have no sequence similarity to any this finding would have important implications for the of the R. microplus repeats. Blocks of repetitive study of chromosome biology in R. microplus and sequence have been localized to heterochromatic other members of the Ixodidae. subtelomeric and pericentromeric regions of chromo- Four families of repetitive DNA were identified somes in a number of organisms including yeasts, from R. microplus BACs that appear to be species nematodes, insects, higher plants, and mammals specific tandem repeats based on searches of the (Spence et al. 1998; Chen et al. 2004). Heterochro- NCBI nr database. The R. microplus family 1 repeat matin is believed to function in a range of important (RMR-1) localized to telomeric regions of 9 of the 10 cellular processes such as chromosome pairing and autosomes (autosomes 1–6, 8–10) and the family 2 segregation, and regulation of gene expression (Lohe 88 C.A. Hill et al. and Hilliker 1995). Loidl (1990) proposed that suggesting a single nucleolar organizing region (NOR) telomeric and subtelomeric repeats may facilitate in the R. microplus Deutsch strain. This finding is in chromosome pairing in meiosis and this has recently agreement with that of Hilburn et al. (1989), who also been substantiated in various organisms (Scherthan et reported a single NOR in R. microplus. The number al. 1994, 1996; Bass et al. 1997). It is possible that the and chromosomal location of NORs varies greatly in R. microplus repeats have such a function given their the arthropods and between species of hard ticks. presumed association with heterochromatic termini. SingleNORshavealsobeenreportedinother The potential role of these repeats in determination of metastriates in the genus Amblyomma by Gunn and kinetic activity and segregation of chromosomes Hilburn (1995), while Chen et al. (1994)identified requires further investigation. The fact that the three NORs per nucleolus on autosomes 3, 7, and 10 RMR-1 does not occur on all chromosomes suggests from an immortalized cell line of the prostriate tick, that it does not function in chromosome capping. Ixodes scapularis. Tandem and dispersed DNA repeats can account This study is the first to demonstrate FISH mapping for a significant proportion of the eukaryote genome in an ixodid tick; this represents a powerful technique (Lander et al. 2001; Adams et al. 2000; Nene et al. for positioning and ordering BACs and cDNA clones 2007). FISH results suggest that large blocks of on R. microplus chromosomes. As such, the FISH RMR-1 and RMR-2 occur in heterochromatic, telo- mapping reported herein will be an important tool for meric regions of the R. microplus chromosomes. assembly of the anticipated R. microplus genome These repeats obviously comprise a substantial sequence. 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