The position of repetitive DNA sequence in the ... - Semantic Scholar

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) using meiotic chromosome preparations and highly repetitive DNA from the southern cattle tick, Rhipicephalus microplus, was undertaken to investigate genome organization. Several classes of highly repetitive DNA elements were identified by screening a R. microplus bacterial artificial chromosome (BAC) library. A repeat unit of approximately 149 bp, RMR1 was localized to the subtelomeric regions of R.

Responsible Editor: Mary Delany. Electronic supplementary material The online version of this article (doi:10.1007/s10577-008-9003-0) contains supplementary material, which is available to authorized users. C. A. Hill (*) : J. P. Van Zee : N. S. Geraci : J. J. Stuart Department of Entomology, Purdue University, West Lafayette, IN 47907, USA e-mail: [email protected] F. D. Guerrero USDA-ARS Knipling-Bushland U.S. Livestock Insects Research Laboratory, Kerrville, TX 78028, USA J. G. Walling Department of Horticulture, University of Wisconsin, Madison, WI 53706, USA Present address: N. S. Geraci Chicago Children’s Memorial Research Center, Chicago, IL 60614, USA

microplus autosomes 1–6 and 8–10. A second repeat unit, RMR-2 was localized to the subtelomeric regions of all autosomes and the X chromosome. RMR-2 was composed of three distinct repeat populations, RMR-2a, RMR-2b and RMR-2c of 178, 177 and 216 bp in length, respectively. Localization of an rDNA probe identified a single nucleolar organizing region on one autosome. Using a combination of labeled probes, we developed a preliminary karyotype for R. microplus. We present evidence that R. microplus has holocentric chromosomes and explore the implications of these findings for tick chromosome biology and genomic research. Keywords fluorescent in-situ hybridization . heterochromatin . holocentric chromosomes . repetitive DNA . Rhipicephalus microplus Abbreviations BAC C-banding FISH gDNA GTE NCBI NOR PGCF RMR

bacterial artificial chromosome centromere banding fluorescent in-situ hybridization genomic DNA glucose tris-EDTA National Center for Biotechnology Information nucleolar organizing region Purdue University Genomics Core Facility Rhipicephalus microplus repeat

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Introduction Ticks (subphylum Chelicerata; class Arachnida; subclass Acari; family Ixodidae) cause global veterinary and medical health problems by transmitting a wide variety of bacteria, viruses, and protozoa and inflicting direct damage to their host via attachment and feeding. The southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), is widely distributed throughout the tropics and subtropics and is the most economically significant Rhipicephalus species. It transmits the causative agents of bovine babesiosis (Texas cattle fever) and anaplasmosis, diseases that cause severe milk and beef production losses and high mortality rates in affected herds (Dietrich and Adams 2000). In many parts of the world, R. microplus control is complicated by development of acaricide resistance. Rhipicephalus microplus was eradicated from the United States in the 1940s, but the possibility of its re-introduction represents a serious economic threat to the US cattle industry. For this reason, the USDA-ARS maintains a 10-mile wide quarantine zone along the US-Mexico border and a mandatory acaricide treatment program for all cattle imported into the United States. Studies of the R. microplus genome are underway to learn more about the biology of this significant pest and identify new methods for its control (Guerrero et al. 2006). Currently, little is known about the nature and organization of the R. microplus genome or tick genomes in general. The haploid genome size of R. microplus is an estimated 7.1 Gbp (Ullmann et al. 2005) and is the largest of any pro- or metastriate tick species examined thus far (Palmer et al. 1994; Ullmann et al. 2005; Geraci et al. 2007). Ullmann et al. (2005) used reassociation kinetics to study the composition of the R. microplus genome and found that the highly repetitive DNA fraction, comprising tandem and dispersed repeats of low sequence complexity, accounted for approximately 40% of the R. microplus genome, while the moderately repetitive fraction, comprising transposable elements and multigene families, accounted for approximately 30%. These findings raise intriguing questions about the accumulation of repetitive material in R. microplus and the organization of this material with respect to coding sequence on the chromosomes. Techniques to identify specific chromosomes and to study chromosome biology are needed for R.

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microplus and other tick species. Cytogenetic investigations of R. microplus have been limited to the work published by Oliver and Bremner (1968), Newton et al. (1972), and Hilburn et al. (1989). Early tick cytogenetics is reviewed by Oliver (1977) who reported that R. microplus has an XX:XO sex determination system with 22 diploid chromosomes in females and 21 in males. Preliminary C-banding karyotypes have also been developed for R. microplus (Hilburn et al. 1989; Garcia et al. 2002). Unfortunately, these are of limited utility for chromosome identification due to uniformity of autosome length and morphology. Here we present the first report to identify a range of DNA sequence repeats from R. microplus genomic sequence and localize these repeats to R. microplus chromosomes by FISH mapping. Our work provides an approach to identifying R. microplus chromosomes and analyzing the organization of the genome. These results will support the anticipated R. microplus genome sequencing effort and enable advances in cytogenetics and population genetics research in this and other tick species.

Materials and methods Source of tick material Genomic DNA (gDNA) for BAC library production and chromosome preparations was obtained from the R. microplus Deutsch strain maintained as described by Davey et al. (1980) at the USDA-ARS Cattle Fever Tick Research Laboratory in Mission, Texas. This is an acaricide-susceptible strain that is used routinely in research applications. It has been maintained in continuous culture since it was established from ticks collected in Webb Co., Texas in 2001. R. microplus library screens Approximately 10% (4608 clones) of the R. microplus BAC library (approximately 1-fold genome coverage and average insert size of 118 kbp, BamHI cloning site; Amplicon Express, Pullman, WA, USA) was arrayed on a nylon filter membrane at the Purdue University Genomics Core Facility (PGCF) using the BioRobotics Total Array System. Filters were probed with R. microplus gDNA extracted from embryos of

Repetitive DNA organization in R. microplus

the Deutsch strain. BAC clones containing highly repetitive DNA were also used as probes in library screens. Probes were prepared by labeling 25 ng of DNA with 32P-ddATP in separate random priming reactions (Prime-It II, Stratagene, La Jolla, CA, USA). Filters were pre-hybridized with PerfectHyb Plus (Sigma, St. Louis, MO, USA). Denatured probe was added directly to the pre-hybridization solution and hybridization was performed overnight at 60°C. Filters were washed in successively diluted solutions of SSC (2× SSC, 1× SSC, and 0.5× SSC) and 0.1% SDS at 60°C and then exposed on a Fujifilm 2325 imaging plate. Images were developed using a Fuji FLA-5000 phosphorimaging system (Fujifilm, Tokyo, Japan). BAC DNA isolation and analysis BAC DNA was isolated for library screening, restriction digest, and probe production using an alkaline lysis procedure modified after Sambrook et al. (1989). BAC clones were separately cultured overnight at 37°C in 5 ml LB media containing chloramphenicol (0.04 mg/ml). Pelleted cells were re-suspended in GTE buffer (glucose, Tris, EDTA) containing 10 mg/ml lysozyme (Sigma) and 2 mg/ml RNase A (Qiagen, Valencia, CA, USA). Following lysis with a 0.2 N NaOH, 1% SDS solution, DNA was purified by phenol–chloroform–isoamyl alcohol (25:24:1) extraction, and re-suspended in nuclease-free water. BAC DNA was digested with HpaII, MseI, and RsaI in separate reactions and the resulting restriction fragments were separated by agarose gel electrophoresis on 0.8% TBE gels. Bands of interest were excised from the gel and the DNA in these bands was purified using the Qiagen QIAEX II gel extraction kit (Qiagen). The DNA was then subjected to PCR to add 3′ A-overhangs and cloned into the TOPO TA PCR 2.1 cloning vector (Invitrogen, Carlsbad, CA, USA). These DNA fragments were then subjected to PCRbased sequencing reactions containing M13 forward, M13 reverse, or T7 primers, and BigDye version 3.1 (Applied Biosystems, Foster City, CA, USA) using the following PCR conditions: initial denaturation at 96°C for 3 min followed by 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min for 30 cycles. The DNA sequence was subsequently determined using an ABI 3730XL capillary DNA analyzer (Applied Biosystems) by the PGCF. All DNA sequences associated

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with this study are available at the National Center for Biotechnology Information (NCBI) GenBank database (accession numbers FJ223571- FJ223604). Isolation of the R. microplus rDNA repeat rDNA primers (forward primer: 5′-CTC TTG TGG TAG CCA AAT GC-3′; reverse primer: 5′-AAG CGA CGT CGC TAT GAA CG-3′) were designed based on a R. microplus 28S rDNA sequence obtained from GenBank (accession number: AF200189). PCR was performed using 1 µg R. microplus gDNA and the following conditions: initial denaturation at 94°C for 2 min; 94°C for 10 s, 53°C for 30 s, and 72°C for 1 min for 30 cycles; final extension at 72°C for 10 min. The resulting 749 bp amplicon was subcloned and labeled as described above, and used to screen BAC library filters. R. microplus meiotic chromosome preparations Meiotic chromosome spreads were prepared from the testes of 25 newly molted adult males from the Deutsch strain of R. microplus. Tissues were dissected in 0.5× Ringer’s saline, transferred to a 3:1 ethanol– glacial acetic acid solution for 5 min, and pelleted by centrifugation. Cells were re-suspended in 50% glacial acetic acid and 5 µl drops were placed on microscope slides and air dried. Excess cytoplasmic material was removed from the preparations by a series of washes in 200 µl 2× SSC and 0.5% RNase at 37°C , followed by treatment with pepsin (100 mg/ml; Sigma-Aldrich, Saint Louis, MO, USA) in 85 µl of pre-warmed 0.01 M HCl at 37°C for 2 h. Following incubation, slides were washed with 1× PBS, 0.2 M MgCl2, serially dehydrated in 70%, 90%, and 100% ethanol and then air-dried. Fluorescence in-situ hybridization (FISH) DNA probes were prepared by nick translation using 1 µg of DNA and either biotin- or digoxigeninconjugated dUTP (Roche) according to the manufacturer’s recommendations. In-situ hybridization was performed with 40–100 ng of denatured probe DNA in 10 µl of hybridization solution (10% dextran solution, 2× SSC, 40% formamide, and 20 µg of herring sperm DNA) at 37°C for 12–15 h. Detection was performed using Alexa Fluor 488-conjugated

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anti-biotin (Molecular Probes, Eugene, OR, USA) and rhodamine-conjugated anti-digoxigenin (Molecular Probes). For multilayer detection of biotin conjugated probes, Alexa Fluor 488-streptavidin was used as the initial layer of immunodetection followed by a layer of biotin anti-streptavidin and a second layer of Alexa Fluor 488-streptavidin. For multilayer detection of digoxigenin-conjugated probes, mouse anti-digoxigenin was used in the first layer of detection followed by a layer of anti-mouse Alexa Fluor 568 (Molecular Probes). Digital images were collected under UV optics using an ORCA-ER (Hammamatsu, Iwata City, Japan) digital camera mounted on an Olympus BX51 microscope and MetaMorph (Universal Imaging Corp., Downington, PA, USA) imaging software.

Results BAC library screens To identify BAC clones containing highly repetitive R. microplus gDNA sequence, 4608 BAC clones arrayed in twelve 384-well plates were screened with 32 P-labeled R. microplus gDNA. We observed 110 positive clones (2.4% of the total) on the filter (Fig. 1a). To begin discriminating among these clones for different classes of repetitive DNA, a series of hybridizations were performed on separate filters using four of the positive clones as probes. We first discovered that a single arbitrarily selected BAC (1B14) hybridized with 100 of the clones (including BAC 1B14 itself) that were positive to gDNA (Fig. 1b). This indicated that one or more major species of repetitive DNA was present in BAC 1B14. One clone, BAC 3M21, was positive for 1B14 but failed to hybridize to gDNA. We assumed that this clone contained a minor repeat species and it was not investigated further. Nine BACs (0.2% of the total) that had previously shown strong hybridization to gDNA failed to hybridize to 1B14 DNA. Therefore, one of those clones was used as a probe in a subsequent library screen. This clone (BAC 4H23) hybridized only with clones that were both gDNApositive and BAC-1B14-negative (Fig. 1c). BAC 1B14 and BAC 4H23 hybridization accounted for all but two (4G12 and 1D1) of the 110 BAC clones that had been positive to whole-genome screening. BAC 4G12 showed only very weak hybridization to

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BAC 4H23 and when used as probe to screen the library it hybridized only with itself (data not shown). This suggested that BAC 4G12 lacked repetitive DNA and it was not analyzed further. In contrast, when BAC 1D1 was used as probe it hybridized to itself and 93 additional clones that had been negative to gDNA, 1B14, and 4H23 (Fig. 1d). Isolation and cloning of repetitive DNA fragments After the series of library screens, BAC clones containing highly repetitive DNA could be assigned to three groups: (1) clones that were BAC 1B14 positive; (2) clones that were BAC 4H23 positive; and (3) clones that were 1D1 positive. Therefore, a series of restriction digests and gel electrophoresis experiments were performed with two or more clones isolated from each of these three groups. DNA fragments that appeared to be repetitive, based on their relative intensity of fluorescence after ethidium bromide staining, were subsequently cloned and sequenced (Fig. 2). Three clones positive for 1B14 hybridization were analyzed (1F18, 1O24, and 1B14 itself). Fragments unique to each clone were observed, but intensely staining fragments in all three clones appeared to have the same molecular weight. The corresponding MseI fragments of approximately 150 and 300 bp were cloned and sequenced. This revealed a repetitive sequence, referred to hereafter as R. microplus repeat 1 (RMR-1; Table 1). RMR-1 had a 149 bp repeat at its core, but 21 unique copies of RMR-1 ranging in length from 82 bp to 152 bp were identified by sequencing 14 independent clones of MseI fragments (see Supplementary Fig. S1). The minimum nucleotide identity between copies of the 149 bp core repeat was 71.8% and the average GC content of these sequences was 54.4%. Two clones positive for 4H23 hybridization (4L11 and 4H23 itself) were analyzed (Fig. 2). Again, fragments unique to each clone were observed, but the intensely staining fragments in both clones appeared to have the same molecular weight. Intensely staining MseI fragments of approximately 150, 200 and 350 bp and RsaI fragments of approximately 375 and 450 bp were cloned and sequenced. These fragments contained three populations of repetitive sequence hereafter referred to as R. microplus repeats RMR-2a, RMR-2b and RMR-2c (Table 1) or collectively as


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Fig. 1 BAC library screens for highly repetitive DNA in the R. microplus genome. Each panel (a, b, c and d) displays rows A to P and columns 11 to 24 of the same R. microplus BAC library. Black and white circles, boxes, and pentagons identify the same BAC clones in each panel. (a) A library screen using R. microplus gDNA as probe identified BACs putatively containing highly repetitive DNA. (b) A separate filter probed with BAC 1B14 DNA (black circle) failed to hybridize to BACs 4G12 (white circle), 4H17, 3L21, 4L11 (white boxes), and 4H23 (black box), which previously hybridized to R. microplus gDNA. BAC 3M21 (pentagon) was clearly positive

for 1B14 but negative for R. microplus gDNA. (c) A separate filter probed with BAC 4H23 (black box) clearly hybridized to BACs 4H17, 3L21, and 4L11 (white boxes) but only weakly hybridized to BAC 4G12 (white circle). Although there was weak hybridization associated with a number of additional clones, BACs 1B14 (black circle) and 3M21 (pentagon) clearly failed to cross-hybridize to BAC 4H23 DNA. (d) A separate filter probed with BAC 1D1 shows lack of hybridization to clones 1B14, 4G12, 4H17, 4H23, 3L21, 4L11, and 3M21 (note different hybridization pattern in white box and pentagon representing clones 4H17 and 3M21, respectively)

repeat 2. RMR-2a, RMR-2b, and RMR-2c had repeats of 178, 177, and 216 bp at their core, respectively. There was 81.5% nucleotide identity between 13 copies of RMR-2a, 82.5% identity between 10 RMR-2b copies, and 88.5% identity between 14 RMR-2c copies (see Supplementary Fig. S1). The %GC content of the RMR2a, -2b, and -2c consensus sequences was 37.6%, 39.0%, and 43.8%, respectively. A forced manual alignment revealed 52.3% nucleotide identity between

the consensus repeats of RMR-2a and RMR-2b (Fig. 3). Due to their limited sequence identity, these sequences are referred to as independent repeats but we recognize that they are probably highly diverged copies of one another. No significant sequence similarity was identified between these repeats and RMR-2c. Restriction fragment analysis showed that RMR-2a, RMR-2b, and RMR-2c are arranged in tandem within a 748 bp stretch of DNA (Fig. 3). Five copies of the (TTAGG)n

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Fig. 2 Agarose gel electrophoresis of restriction endonuclease-digested BACs containing highly repetitive R. microplus DNA. Agarose gels containing digestions of BACs 1B14, 1F18, 1O24, 4L11, 4H23, and 1D1 are shown. Black and white arrows indicate the fragments that were extracted from the gels, cloned, and sequenced (H = HpaII; M = MseI, and R = RsaI)

telomeric repeat were identified within this 750nucleotide 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 sequencing of additional tick species is needed to confirm this hypothesis. BAC library screens with RMR-1 and RMR-2 sequence identified the same clones previously discovered by whole gDNA screening (data not shown).

Table 1 Rhipicephalus microplus repeat sequences Repeat

Size of core repeat (bp)

Repeat family 1 RMR-1 149 Repeat family 2 RMR-2a 178 RMR-2b 177 RMR-2c 216 Repeat family 3 RMR-3 177 Repeat family 4 RMR-4 356 ND, not determined.

No. repeat copies identified

Size range of repeat copies (bp)

GC content of core repeat (%)

Average nucleotide identity between copies of core repeat (%)

21

82–152

54.4

71.8

13 10 14

55–178 175–177 53–216

37.6 39.0 43.8

81.5 82.5 88.5

2

134–177

55.4

92.5

1

356

37.1

ND


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Fig. 3 The R. microplus RMR-2 family of repeats. (a) Manual alignment of RMR-2a and RMR-2b. Identical nucleotides are shaded. (b) Schematic diagram showing the arrangement of the RMR-2a, RMR-2b and RMR-2c repeats within a 748 bp repetitive DNA unit; M, MseI restriction site; R, RsaI restriction site ; *, location of TTAGG telomeric repeat; ‡, denotes a truncated copy of RMR-2a. Numbers below the diagram refer to the size of hypothetical restriction fragments in bp. Dotted lines indicate the location of restriction endonuclease sites

found in some but not all RMR-2 repeat copies. (c) Schematic diagram showing how the RMR-2a (black shading), RMR-2b (dark gray shading) and RMR-2c (light gray shading) repeats identified from the RsaI 375 bp and 450 bp, and the MseI 150 bp, 200 bp and 350 bp restriction fragments were used to reconstruct the 748 bp repetitive DNA unit in (b). Text below lines refers to the five different fragments identified by MseI and RsaI digest of BAC clones 4L11 and 4H23 shown in Fig. 2

Cytology

size and a single X chromosome (Fig. 4). In metaphase I, chiasmata were evident between homologues in the ‘end-to-end’ bivalent configuration that is typical of holocentric chromosomes and that is proposed to result from terminalization of the chiasmata (Dernburg 2001). Sister chromatids appeared to separate in an equational division in meiosis I, and the homologues appeared to separate in a reductional division during meiosis II (data not shown). In phasecontrast and DAPI-stained preparations, only the unpaired X chromosome could be distinguished from

To determine the positions of the repetitive sequences in the R. microplus genome we performed fluorescent in-situ hybridization (FISH) experiments on male R. microplus meiotic chromosome preparations. Nuclei in all stages of meiosis were observed. Chromosome morphology and behavior during meiosis suggested that the chromosomes were holocentric rather than metacentric as previously reported (Oliver 1977). During meiosis I, we observed 10 bivalents of similar

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Fig. 4 Fluorescence in-situ hybridization of R. microplus repetitive DNA to R. microplus testicular chromosome preparations. (a) RMR-1 probes 1B14 and 1F18 hybridizing to metaphase I chromosomes. Note that the X chromosome (X) and one bivalent (7) failed to show any hybridization signal. RMR-1 was clearly evident near the telomeres of the remaining bivalents, and its intense hybridization distinguished one bivalent (4) from the others. An arrow indicates the position of the chiasmata of one bivalent. (b) RMR-2 (red fluorescence) and 28S rDNA hybridization (green fluorescence) near the telomeres of metaphase I chromosomes. Note that the X chromosome and each bivalent show RMR-2 hybridization

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and that rDNA hybridization (arrowhead) distinguished one bivalent (6). (c–j) FISH using both RMR-1 and RMR-2 as probe. (c) DAPI stained bivalent. (d) RMR-1 hybridization to the DAPI-stained bivalent in panel (c). (e) RMR-2 hybridization to the DAPI-stained bivalent in panel (c). (f) False-color overlay of images (c–e). (g) DAPI-stained interphase primary spermatocyte showing the 21 intensely DAPI-positive foci typical of these nuclei. (h) RMR-1 hybridization to the nucleus shown in panel (g). (i) RMR-2 hybridization to the nucleus shown in panel (g). (j) A false-color overlay of images (g–i). Scale bars represent 10 µm


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the other chromosomes; the X lacked a homologue and a chiasma, was larger than the autosomes, and was slightly more DAPI-positive. RMR-1 hybridized to all but one bivalent and the X chromosome (Fig. 4a). FISH experiments using each of the RMR-1-containing BAC clones (1B14, 1F18, and 1O24) as probe were also performed. Results of these experiments were indistinguishable from those using the RMR-1 DNA alone as probe (data not shown). The position of this hybridization appeared to correspond to subtelomeric heterochromatin. An absence of RMR-1 hybridization permitted the unequivocal identification of the X and one bivalent (bivalent 7 in Figs. 4 and 5). In addition, the relative lengths of the remaining bivalents combined with the relative intensity of RMR-1 hybridization made it possible to suggest the correspondence between bivalents in various meiotic I nuclei (Fig. 5). Such an attempt resulted in the placement of the bivalents into three groups. The first group (Fig. 5, group a) was composed of the three

longest bivalents, each of which contained a relatively small amount of RMR-1 DNA. The second group (Fig. 5, group b) was composed of the three chromosomes that contained the greatest relative amount of RMR-1 DNA. Hybridization to one of these chromosomes (bivalent 4 in Fig. 4a and Fig. 5) suggested that it was largely composed of RMR-1 heterochromatin. Identification of this chromosome in meiotic I cells was nearly unambiguous. Hybridization of 28 S ribosomal DNA made a second bivalent in this group recognizable in every nucleus (bivalent 6, Fig. 4b and Fig. 5). The third group (Fig. 5, group c) was composed of the shortest three bivalents each of which contained a moderate quantity of RMR-1 DNA. The relative amount of RMR-1 DNA on one of these bivalents (bivalent 8, Fig. 5) made its recognition almost unequivocal. RMR-2 hybridized to each of the bivalents and the X chromosome (Fig. 4b). As with RMR-1, the position of RMR-2 hybridization was subtelomeric. It appeared, however, that RMR-2 heterochromatin

Fig. 5 R. microplus bivalent identification using RMR-1 and rDNA FISH. The X chromosome and 10 bivalents from three meiotic I nuclei are shown. The bivalents from each nucleus were ordered from 1 to 10 by descending relative length. They are assigned to three groups (a, b, and c) according to their

relative lengths and relative quantity of RMR-1 DNA (red fluorescence). An absence of RMR-1 DNA on the X chromosome and bivalent 7 makes identification of these chromosomes unequivocal. Hybridization with rDNA (green fluorescence) also makes the identification of bivalent 6 unmistakable

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was more closely associated with the telomere than RMR-1 DNA (Fig. 4c–j). RMR-2 DNA appeared to be closely associated with brightly staining foci at the telomere of each bivalent, whereas RMR-1 hybridized at a position that was slightly closer to the chiasma (Fig. 4c). Interestingly, similar foci were evident as 21 DAPI-positive dots in primary spermatocytes (Fig. 4g). FISH experiments clearly indicated that these foci were also tightly associated with RMR-2 DNA and much less closely associated with RMR-1 heterochromatin (Fig. 4g–j). FISH experiments using RMR-3 and RMR-4 as probe failed to localize these repeats to a specific chromosomal location. Instead, the RMR-3 and RMR-4 signal was evenly distributed along each R. microplus chromosome (data not shown), which further suggests that these are dispersed repeats. BLASTn searches did not identify any similarity between RMR-3 and -4 sequences and retro-element sequences in the NCBI nr database.

Discussion Our finding of 10 paired autosomes and an unpaired X chromosome in R. microplus males agrees with earlier cytogenetic work of Oliver and Bremner (1968), who reported 21 diploid chromosomes and an XX:XO sex determination system in male R. microplus. These results are also consistent with chromosome numbers in Mexican (Newton et al. 1972; Hilburn et al. 1989) and Brazilian (Garcia et al. 2002) R. microplus populations. Although R. microplus karyotypes have been developed using traditional C-banding techniques (Hilburn et al. 1989; Garcia et al. 2002), chromosome identification is complicated by the fact that the autosomes are similar in size, lack distinguishing morphological traits, and possess relatively uniform C-banding patterns. Our DAPI staining results suggest that significant amounts of heterochromatin are associated with the termini of chromosomes, but the distribution was too uniform to permit identification of individual chromosomes. The X chromosome was easily identified in metaphase I as the only unpaired chromosome. It was also possible to identify the X chromosome based on its size and the intensity of DAPI staining, suggesting that the X chromosome may have a higher heterochromatin content compared to the autosomes. Given the

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limitations associated with traditional staining techniques, FISH mapping was pursued in this study as an approach for identification of R. microplus chromosomes and analysis of DNA organization on the chromosomes. FISH mapping enabled an investigation of R. microplus chromosome morphology and behavior during meiosis. Previous studies have reported acrocentric chromosomes in R. microplus (Oliver and Bremner 1968; Hilburn et al. 1989; Garcia et al. 2002) with the presumed centromere occurring at the tip of the chromosome and the ‘short’ arm being indistinguishable (Hilburn et al. 1989). Using FISH and high-resolution digital images, we identified several lines of evidence that suggest that R. microplus chromosomes are actually holocentric. Holocentric chromosomes have been described in a number of organisms, including the round worm Caenorhabdtis elegans and heteropteran insects (Bongiorni et al. 2004), and are characterized by the lack of a localized centromere, and consequently of a localized kinetic activity. During mitotic metaphase, diffuse kinetochores are thought to form along the entire length of the chromosome and the sister chromatids separate in parallel. Rhipicephalus microplus chromosomes lacked a clearly defined centromere in all spreads examined. Garcia et al. (2002) also failed to identify a clearly defined centromere in R. microplus chromosomes using conventional acetic orcein staining. In addition, we observed apparent end-to-end pairing of homologous R. microplus chromosomes in meiosis I and sister chromatids in meiosis II (data not shown), which is consistent with other holocentric arthropods (Pérez et al. 1997, 2000; Mandrioli 2002, 1999; Bongiorni et al. 2004). The studies of Hilburn et al. (1989) and Garcia et al. (2002) concluded that R. microplus constitutive heterochromatin bands were centromeric, where as our observations suggest that the heterochromatin actually occurs in the R. microplus telomeres. The localization of repetitive DNA in telomeric regions is a common feature of holocentric chromosomes (Schweizer and Loidl 1987). In meiosis, the behavior of holocentric chromosomes is strikingly different in that the kinetic activity of autosomes and sex chromosomes is restricted to either of the two chromatid ends during the first and second meiotic divisions (Pérez et al. 2000). In many holocentric organisms such as coccids, aphids, and mites, it is common for the meiotic divisions to be


inverted relative to mitosis such that the first division is equational (sister chromatids separate) and the second reductional (homologous chromosomes separate) (Wrensch et al. 1993; Dernburg 2001; Bongiorni et al. 2004). The segregational behavior of meiotic R. microplus chromosomes was similar to one of three alternative patterns documented for autosomes of the holocentric heteropteran, Triatoma infestans (Pérez et al. 1997, 2000). Kinetic activity appeared to be restricted to the heterochromatic termini of the R. microplus chromosomes at first metaphase with division being reductional. At second metaphase, the kinetic activity appeared to shift to the euchromatic termini such that division was equational. In T. infestans, both inverted and normal meiotic behavior is possible within an individual, although the ‘inverted’ sequence (Pérez et al. 1997, 2000) is more common. The mechanisms that determine the selection of a particular segregational type are currently unknown. The suggestion of holocentric chromosomes in R. microplus is not without precedent. Both monocentric and holocentric chromosomes have been reported in the Acari but the distribution of chromosome types among taxa has not been widely studied (Oliver 1977). Evidence suggests that the two-spotted spider mite, Tetranychus urticae, the water mites, Eylais setosa and Hydrodroma despiciens, and the grass mite Siteroptes graminum (reviewed by Oliver 1977) have holocentric chromosomes. Little is currently known about the segregational behavior of tick and mite chromosomes during meiosis. The suggestion of holocentric chromosomes in R. microplus raises interesting questions about the mechanisms that determine kinetic termini during meiosis and the possible role of heterochromatin in these processes. Other approaches such as silver and immunohistochemical staining, and atomic force microscopy (Mandrioli and Manicardi 2003) could lend weight to our hypothesis of holocentric chromosomes. If true, this finding would have important implications for the study of chromosome biology in R. microplus and other members of the Ixodidae. Four families of repetitive DNA were identified from R. microplus BACs that appear to be species specific tandem repeats based on searches of the NCBI nr database. The R. microplus family 1 repeat (RMR-1) localized to telomeric regions of 9 of the 10 autosomes (autosomes 1–6, 8–10) and the family 2

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repeat (RMR-2) localized to telomeric regions of all autosomes and the unpaired X chromosome. RMR-2 appeared to map more closely to the telomeric regions of meiotic chromosomes than RMR-1. FISH mapping to interphase nuclei further supports a telomeric location for the R. microplus repeats. DAPI staining revealed 21 brightly stained, presumably AT-rich heterochromatic dots associated with diploid, interphase nuclei. Telomeres are known to be enriched in heterochromatin (Panzera et al. 1995; Bizzaro et al. 1996) and these dots are expected to correspond to the telomeres of each of the 21 chromosomes in diploid males. The localization signal of the family 2 repeat immediately overlaid the heterochromatic dots observed in R. microplus nuclei, while the family 1 repeat exhibited a more diffuse signal in regions adjacent to these dots. The RMR-2 repeat also contained five copies of the TTAGG penta-nucleotide repeat that has been associated with the telomeres of hymenopteran and lepidopteran insects (Okazaki et al. 1993; Kipling 1995; Meyne et al. 1995). In addition, the RMR-2 core repeats are relatively AT rich. Taken together, these results suggest that RMR-1 and -2 are located within subtelomeric, heterochromatic regions of R. microplus chromosomes, with family 2 having closer proximity to the R. microplus telomeres. Although the distribution of RMR-3 and RMR-4 could not be resolved by FISH, our results indicate that these repeats are distributed evenly along all R. microplus chromosomes, and are in fact, dispersed repeats. RMR-1 and RMR-2 repeats may also be distributed in low copy number in other regions of the R. microplus chromosomes. Sequencing of gDNA is needed to investigate the distribution of these repeats in euchromatic regions of the R. microplus genome. The functions of the R. microplus repeats identified in this study are not known. Repeats of similar size and unknown function have been identified in other invertebrate species (Spence et al. 1998; Mandrioli et al. 1999), but these have no sequence similarity to any of the R. microplus repeats. Blocks of repetitive sequence have been localized to heterochromatic subtelomeric and pericentromeric regions of chromosomes in a number of organisms including yeasts, nematodes, insects, higher plants, and mammals (Spence et al. 1998; Chen et al. 2004). Heterochromatin is believed to function in a range of important cellular processes such as chromosome pairing and segregation, and regulation of gene expression (Lohe

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and Hilliker 1995). Loidl (1990) proposed that telomeric and subtelomeric repeats may facilitate chromosome pairing in meiosis and this has recently been substantiated in various organisms (Scherthan et al. 1994, 1996; Bass et al. 1997). It is possible that the R. microplus repeats have such a function given their presumed association with heterochromatic termini. The potential role of these repeats in determination of kinetic activity and segregation of chromosomes requires further investigation. The fact that the RMR-1 does not occur on all chromosomes suggests that it does not function in chromosome capping. Tandem and dispersed DNA repeats can account for a significant proportion of the eukaryote genome (Lander et al. 2001; Adams et al. 2000; Nene et al. 2007). FISH results suggest that large blocks of RMR-1 and RMR-2 occur in heterochromatic, telomeric regions of the R. microplus chromosomes. These repeats obviously comprise a substantial proportion of the highly repetitive DNA fraction of the R. microplus genome, and as such, have important implications for genome sequencing efforts and genomic studies of this tick. Long, contiguous stretches of low-complexity sequence are difficult to assemble and, even with repeat masking, may result in fragmented assemblies. However, the fact that the R. microplus repeats are largely localized to chromosome termini suggests that the sequencing of CoT based libraries enriched for high complexity sequence could offer a feasible approach to get at the fraction of the genome comprising unique, presumably coding DNA. The lack of suitable chromosome markers has hindered the study of tick chromosome biology. The repeats identified in this study provide useful markers to identify individual R. microplus chromosomes. Using different combinations of probes, including a 28S rDNA probe, it was possible to identify three groups of autosomes (groups a, b, and c) and the X chromosome. We were often able to distinguish autosomes 4, 6, 7, and 8 based on signal intensity. Hybridization results were not always consistent, possibly due to differences in chromosome condensation and/or variation between slide preparations. We were able to improve probe visualization and overcome problems associated with cytoplasmic material and cell debris in some slide preparations by pretreatment of these slides with pepsin. FISH mapping revealed the presence of a single rRNA cistron on autosome 6,

C.A. Hill et al.

suggesting a single nucleolar organizing region (NOR) in the R. microplus Deutsch strain. This finding is in agreement with that of Hilburn et al. (1989), who also reported a single NOR in R. microplus. The number and chromosomal location of NORs varies greatly in the arthropods and between species of hard ticks. Single NORs have also been reported in other metastriates in the genus Amblyomma by Gunn and Hilburn (1995), while Chen et al. (1994) identified three NORs per nucleolus on autosomes 3, 7, and 10 from an immortalized cell line of the prostriate tick, Ixodes scapularis. This study is the first to demonstrate FISH mapping in an ixodid tick; this represents a powerful technique for positioning and ordering BACs and cDNA clones on R. microplus chromosomes. As such, the FISH mapping reported herein will be an important tool for assembly of the anticipated R. microplus genome sequence. The repeats identified in the present study also provide useful markers for further investigation of tick chromosome biology and the advancement of studies of tick population genetics.

Acknowledgements This work was supported by USDAARS cooperative agreement no. 58-6205-4-008 to C.A.H. and J.J.S. The authors thank S.A. Jackson and two anonymous reviewers for insightful review of this manuscript.

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