International Journal for Parasitology 41 (2011) 669–675

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International Journal for Parasitology

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Construction of a genetic map for Theileria parva: Identification of hotspots of recombination ⇑ Frank Katzer b,1, Regina Lizundia a,1, Daniel Ngugi a, Damer Blake a, Declan McKeever a, a Royal Veterinary College, University of London, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire AL9 7TA, UK b Moredun Research Institute, Pentlands Science Park, Penicuik, Midlothian EH26 0PZ, UK article info abstract

Article history: The tick-borne protozoan parasite Theileria parva is the causal agent of East Coast Fever (ECF), a severe Received 17 November 2010 lymphoproliferative disease of cattle in eastern, central and southern Africa. The life cycle of T. parva is Received in revised form 11 January 2011 predominantly haploid, with a brief diploid stage occurring in the tick vector that involves meiotic Accepted 12 January 2011 recombination. Resolved genetic studies of T. parva are currently constrained by the lack of a genome- Available online 23 February 2011 wide high-definition genetic map of the parasite. We undertook a genetic cross of two cloned isolates of T. parva to construct such a map from 35 recombinant progeny, using a genome-wide panel of 79 var- Keywords: iable number of tandem repeat markers. Progeny were established by in vitro cloning of cattle lympho- Theileria parva cytes after infection with sporozoites prepared from Rhipicephalus appendiculatus ticks fed on a calf Recombination Marikebuni undergoing a dual infection with the two clonal parental stocks. The genetic map was determined by Muguga assigning individual markers to the four genome, whose physical length is approximately Hotspot 8309 kilobasepairs (Kb). Segregation analysis of the markers among the progeny revealed a total genetic Genetic map size of 1683.8 centiMorgans (cM), covering a physical distance of 7737.62 Kb (93% of the genome). The average genome-wide recombination rate observed for T. parva was relatively high, at 0.22 cM KbÀ1 per meiotic generation. Recombination hot-spots and cold-spots were identified for each of the chromo- somes. A panel of 27 loci encoding determinants previously identified as immunorelevant or likely to be under selection were positioned on the linkage map. We believe this to be the first genetic linkage map for T. parva. This resource, with the availability of the genome sequence of T. parva, will promote improved understanding of the pathogen by facilitating the use of genetic analysis for identification of loci responsible for variable phenotypic traits exhibited by individual parasite stocks. Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction tle become infected by inoculation of sporozoite forms in tick saliva. Sporozoites invade host lymphocytes and differentiate to Theileria parva is a tick-borne parasite of cattle that infects and multinucleate schizont forms, which transform the infected cell transforms bovine lymphocytes, causing a severe lymphoprolifera- to a state of uncontrolled proliferation. The parasite replicates in tive disease known as East Coast Fever (ECF). Transmitted by the synchrony with the dividing lymphocyte and, by associating with brown ear tick Rhipicephalus appendiculatus, the parasite is preva- the mitotic spindle (Hulliger et al., 1964), ensures transmission of lent in eastern, central and southern Africa. The pathogenesis of infection to each daughter cell. With the progress of infection, ECF arises largely from the invasion of lymphoid and non-lym- the parasite ceases to divide in a proportion of infected cells and phoid tissues with proliferating infected lymphoblasts, and suscep- differentiates to uninucleate merozoites. These are released from tible animals normally die within 3 weeks of infection. With an the dying cell and invade erythrocytes, where they develop into estimated 24 million cattle at risk of infection (Mukhebi et al., tick-infective piroplasms. Cattle that recover from infection are al- 1992) the disease is a major constraint to livestock productivity most invariably long-term carriers of these forms. Upon ingestion in the region. The life cycle of T. parva is similar to that of other api- by a subsequent feeding tick, piroplams give rise to macro- and mi- complexa and involves obligate developmental stages in both cro-gametes, which undergo syngamy in the gut lumen to yield mammalian and vector hosts (Mehlhorn and Shein, 1984). It is pre- diploid zygotes. These invade gut epithelial cells and undergo fur- dominantly haploid, with only a brief diploid phase in the tick. Cat- ther differentiation, including meiotic division, to form motile kinetes, which migrate to the tick salivary gland. There they under- go further nuclear division, ultimately giving rise to cattle-infective ⇑ Corresponding author. Tel.: +44 0 1707 666572; fax: +44 0 1707 666298. E-mail address: [email protected] (D. McKeever). uninucleate sporozoites. We have recently provided evidence for 1 Both first authors contributed equally to this work. substantial genetic recombination between T. parva strains during

0020-7519/$36.00 Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2011.01.001 670 F. Katzer et al. / International Journal for Parasitology 41 (2011) 669–675 passage through R. appendiculatus ticks (Katzer et al., 2006). Cattle clone on the basis of previous observations in vivo. The progress can be immunised against T. parva by simultaneous inoculation of of infection was monitored by daily evaluation of rectal tempera- live sporozoites and long-acting formulations of oxytetracycline, a ture from day 5, and microscopic examination of needle aspirates process known as ‘infection and treatment’ (Radley et al., 1975). of the draining lymph node for the presence of T. parva schizonts There is strong evidence that the resulting protection is mediated on alternate days after the first manifestation of fever. Levels of by parasite-specific major histocompatibility complex (MHC) class parasitaemia for each clone were monitored during infection by I-restricted cytotoxic T lymphocytes (CTLs), which eliminate schiz- PCR amplification of DNA extracted from whole blood, using dis- ont-infected lymphoblasts (McKeever et al., 1994). The response is criminatory satellite markers. Approximately 1000 R. appendicula- highly specific and therefore vulnerable to breakthrough by heter- tus nymphs enclosed in cloth bags were applied at regular intervals ologous strains. Importantly, because it does not engage until the to each ear of the calf from day 9 p.i. to feed during the piroplasm schizont parasitosis is established, the CTL response does not pre- parasitaemia. Upon detachment, engorged ticks were evaluated for vent infection, even after challenge with the immunising strain relative levels of infection with each parasite clone by satellite (Morrison et al., 1987). We have recently observed that, although marker analysis of a representative sample. Those that detached CTLs impose considerable numerical attrition on targeted parasi- on day 21 p.i. produced the most equivalent ratio for the two geno- tised lymphoblasts in vivo, a proportion of parasites nonetheless types, and were chosen for stabilate production. Nymphs were re- emerge as piroplasms in the erythrocyte compartment. Responses moved after engorgement and incubated for 4 weeks at 28 °C and are almost invariably restricted by one or other of the parental 85% relative humidity to promote moulting. Moulted infected adult MHC haplotypes and generally focus almost entirely on a single ticks were pre-fed on rabbits for 4 days to stimulate sporogony. antigenic determinant (MacHugh et al., 2009). In addition, the They were then removed and surface-sterilized by sequential specificity of the response varies with MHC haplotype, suggesting rinses in 5% chlorhexidine, 70% ethanol and antibiotics, and ana- that individual cattle in an outbred herd are likely to target distinct lysed for parasite infection. The stabilate (CTVM St102) was pre- parasite components. We have shown that T. parva CTL antigens pared by trituration of sterilized ticks essentially as described by reassort during recombination in the tick (Katzer et al., 2006) Brown (1987). All animal experiments were conducted with the and raised the possibility that this could result in evasion of such formal approval of the institutional animal ethics committees. a tightly focussed effector response. In the face of immune selec- Standards of care and maintenance for experimental animals were tion, such an eventuality might be anticipated to result in aug- in accordance with government and institutional guidelines. mented recombination of CTL antigenic loci over evolutionary time. In both yeast and human genomes, recombination occurs 2.3. In vitro infection and cloning predominantly at specific regions known as hot spots (Petes, 2001; McVean et al., 2004), which can vary considerably in size Peripheral blood mononuclear cells were isolated from defibrin- (from hundreds of bp to tens of megabases (Mb)). A segment of ated jugular venous bovine blood by flotation on Ficoll-Paque as DNA that undergoes recombination at the genome average rate described previously (Goddeeris and Morrison, 1988). Cells (107) can also be considered a hot spot if it is embedded in a very ‘‘cold’’ were resuspended in 1 ml of RPMI 1640 medium containing region of the genome. Although evidence for genetic crossover of T. 2 mM glutamine, 5 Â 10–5 M 2-mercaptoethanol, 100 IU/ml peni- parva in the tick has been obtained by satellite marker analysis cillin, 100 lg/ml streptomycin and 10% FCS (culture medium) (Katzer et al., 2006) and by tracking the segregation of several and mixed with an equal volume of CTVM St102 diluted in culture polymorphic sequences (Morzaria and Young, 1993), the extent medium, to obtain a multiplicity of infection of one tick equivalent/ of recombination and the location of hotspots across the genome ml. The suspension was incubated for 2 h at 37 °C with occasional remain to be determined. The recent availability of the T. parva agitation before the addition of 8 ml culture medium and centrifu- genome sequence (Gardner et al., 2005) facilitated the establish- gation at 200 g for 10 min. Infected cells were then resuspended in ment of a genome-wide panel of satellite markers for high-resolu- culture medium at a density of 2.5 Â 106 cells/ml, dispensed into a tion genotyping of parasite populations. We have exploited these 24-well plate in aliquots of 1 ml/well and incubated for 48 h at developments to undertake a broad genotypic analysis of a set of 37 °C in a humidified atmosphere of 5% CO2 and air. Cells were recombinant T. parva clones and their parents, with the aim of con- then harvested, assessed for viability by trypan blue exclusion structing, for the first time, a genetic map of the parasite. We draw and suspended in culture medium at a density of 105 viable on the information provided to evaluate the evidence for selection cells/ml. This suspension was used to seed 96-well plates in ali- arising from bovine immunity and from other sources. quots of 100 ll/well, with two plates each at 1 Â 104/well, 3 Â 103/well, 1 Â 103/well, 3 Â 102/well, and 1 Â 102/well. Each well then received 5 Â 104 irradiated (50 Gy) autologous periphe- 2. Materials and methods ral blood mononuclear filler cells in 100 ll culture medium supple- mented with 50% conditioned medium derived from established T. 2.1. Parasite populations parva-infected lymphoblast cultures. Plates were incubated for 2–

3 weeks at 37 °C in a humidified atmosphere of 5% CO2 in air and The study focused on two cloned stocks of T. parva, both of screened for the presence of single clones. One-third of each posi- which were generated as described by Morzaria et al. (1995). Clone tive well was then harvested for the identification of hybrid clones 3308 was derived from the Muguga (3087) stock of T. parva, while using 24 polymorphic markers for preliminary screening by PCR clone 4210 originated from the Marikebuni (3014) stock. The ori- analysis (Supplementary Table S1) and the residual culture was gins of these stocks have been described previously (Morzaria replenished with fresh medium. Unique clones were expanded et al., 1995). for full genotyping as required and stored as live stabilates under liquid nitrogen. 2.2. Animal infections 2.4. Polymorphic markers and genotyping A male Boran (Bos indicus) calf was infected by s.c. inoculation behind the left and right parotid lymph nodes with sub-lethal 2.4.1. Satellite markers quantities of sporozoites of both 3308 and 4210 clones. Doses were The satellite marker panel described by Katzer et al. (2010) calculated to provide similar piroplasm parasitaemias for each based on the original panel of Oura et al. (2003) was used, supple- F. Katzer et al. / International Journal for Parasitology 41 (2011) 669–675 671 mented with an additional 13 polymorphic markers that were pre- ical distance corresponding to 1 cM). Haldane, Kosambi and Mor- viously excluded due to cross-reaction with other Theileria spp. The gan map functions were tested on the data at the specified level additional markers were chosen on the basis that they discrimi- of significance for comparative linkage criteria. nated between the parental clones and provided additional cover for the analysis due to their positions on the four . 3. Results

2.4.2. Size polymorphisms 3.1. Generation of a linkage map The satellite marker set was further enhanced by the inclusion of PCR primers that amplify polymorphic regions of four A total of 35 recombinant T. parva clones were identified by and/or open reading frames (ORFs) of T. parva and give rise to screening 560 clonal cultures arising from the cross. These 35 un- amplicon size polymorphisms that distinguish T. parva Muguga ique clones were chosen from a preliminary screen using 24 poly- clone 3308 and T. parva Marikebuni clone 4210 (Katzer et al., morphic markers (six markers per chromosome) (Supplementary 2006, 2007, 2010). Table S1). All clones were genotyped using all 79 VNTR markers, providing inheritance patterns for each parent-specific marker 2.4.3. PCR conditions and genotyping (Supplementary Fig. S1). A genetic linkage map was constructed The PCR conditions used in this study have been described pre- based on the segregation of each parent-specific marker within viously (Katzer et al., 2006) and are essentially as outlined by Oura the recombinant progeny using the Map Manager QTX software. et al. (2003), except that the number of cycles was increased to 40, Haldane, Kosambi and Morgan map functions were tested on the and Bioline (UK) Taq polymerase and a custom-made 10Â PCR buf- data, but the linkage map described herein was informed by the fer (4.5 mM Tris–HCl (pH 8.8), 11 mM (NH4) SO , 4.5 mM MgCl , 2 4 2 Haldane function, which was in closest agreement with the con- 0.113 mg/ml BSA, 4.4 lM EDTA, 1.0 mM each of dATP, dCTP, dGTP, sensus. Genetic distances, based on the number of recombination dTTP) purchased from ABgene (UK), were used. PCR products were units between each marker, were expressed in cMs, which equate separated on 2% Metasieve agarose (Flowgen, UK), visualised with to a recombination frequency of 1%. The markers extended across ethidium bromide and photographed using a UV light box (BioRad, the four chromosomes of the 8309 Kb T. parva genome at an aver- UK). The amplicon size at each marker locus was recorded and as- age interval of 104 Kb (r = 11.2) and therefore comprised an signed a serial alphabetical code on the basis of the order in which approximately uniform set. Linkage mapping generated four link- it was first observed during the course of the analysis (Supplemen- age groups and a total genetic size of 1683.8 cM, representing a tary Fig. S1). Because the marker panel was first evaluated on a physical distance of 7737.62 Kb (93% of the genome; Table 1; clone of the Muguga stock 273 of T. parva, alleles occurring at all Fig. 1) covered by the marker set. Comparison of marker mapping loci in the Muguga population were designated A. Alleles occurring among the recombinant progeny identified 434 crossover events, at loci in the Marikebuni population were designated B. The clone providing an average of 56 crossover events per Mb of mapped 407 for marker MS73 showed a novel amplicon product designated genome. Crossover frequency varied considerably between the C, which may have arisen through replication slippage or possibly a chromosomes, ranging from 49 in chromosome 4 to 151 in chro- recombination event. mosome 1. However, crossover frequency correlated with neither genetic nor physical mapped chromosome size. 2.5. Organisation of polymorphic markers into a genetic linkage map

The inheritance of 79 variable number of tandem repeat (VNTR) 3.2. Genome-wide recombination parameters markers found to be polymorphic between the two parental T. par- va cloned isolates was determined for all 35 independent recombi- The physical rate of recombination was calculated by compar- nant progeny clones. These VNTR markers were organised into ing the genetic and physical distance between each mapped mar- À1 linkage groups and assigned to the four T. parva chromosomes ker (cM Kb ). Accordingly, the average rate of recombination À1 using Map Manager QTX Software. For every marker, the parental was approximately 0.22 cM Kb , although there was considerable alleles identified in each of the progeny clones were entered into variation within and between chromosomes (Table 1). The highest an Excel spreadsheet. Data were then prepared for analysis with rate of recombination was observed for chromosome 1, at À1 the Map Manager QTX software (version b.20) according to the 4.61 cM Kb . The presence of hot spots and cold spots of recombi- instruction manual (Chmielewicz and Manly, 2002). Linkage nation, defined here as at least five times higher or lower than the 6 À1 groups were formed with a P-value of 0.001 using the ‘‘Make Link- genome-wide average (i.e. P1.05 and 0.04 cM Kb ) respectively, age Groups’’ command in Map Manager with the Haldane mapping was evaluated across the four chromosomes. A total of 10 hot spots function. P-values in Map Manager indicate the probability of a and 13 cold spots were identified (Table 1). Type 1 error; that is, the probability of a false positive linkage. The optimum marker order and genetic distance between markers 3.3. Candidate association with recombination rate in centiMorgans (cM) was calculated using the ‘‘Ripple’’ command. Physical map unit sizes were calculated by comparison of genomic A panel of 27 genetic loci that encode previously iden- marker location with the associated genetic distance (i.e. the phys- tified as immunorelevant or annotated as likely to be under selec-

Table 1 Summary statistics for the Theileria parva genetic map.

Chromosome No. markers Mapped size (Kb) Total cM Crossovers cM/Kb Hot spots Cold spots 1 24 2418.67 811.9 151 0.34 4 3 2 18 1814.33 401.7 92 0.22 3 3 3 21 1771.65 270.5 142 0.15 2 4 4 16 1732.98 199.7 49 0.12 1 3 Total 79 7737.62 1683.8 434 – 10 13 Average 20 1934.41 421.0 109 0.22 3 3

Kb, kilobasepairs; cM, centiMorgans. 672 F. Katzer et al. / International Journal for Parasitology 41 (2011) 669–675

Fig. 1. Plots indicating genetic distance as a function of physical distance for each chromosome of Theileria parva. X-axis, cumulative physical distance in kilobasepairs (Kb); y- axis, cumulative genetic distance in centimorgans (cM). Approximate positions of variable number of tandem repeat (VNTR) markers along the chromosomes are indicated on the bar above each graph. Intervals of high recombination rate (hot spots) are shown in red, while those of low recombination rate (cold spots) are shown in blue. Positions of loci encoding determinants previously identified as immunorelevant or likely to be under selection are indicated with black arrows. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

tion were aligned with the linkage map (Fig. 1, Table 2). Allocation the T. parva genome is larger than has been described for any other of the relevant rate of recombination to each locus position re- apicomplexan parasite, despite its relatively small physical size of vealed two loci, comprising the T. parva repeat (Tpr) locus on chro- 8.3 Mb. For example, the estimated genetic size of the 23.3 Mb P. mosome 3 and the TashAT homologue gene cluster on falciparum genome is 1556 cM, while T. gondii, at 63 Mb, has a pre- chromosome 1, to be located within or between regions of high dicted genetic size of only 592 cM. (current predicted genome sizes recombination activity (Table 2, Fig. 1). By contrast, the p67 locus from EuPathDB, http://eupathdb.org/eupathdb/)(Su et al., 1999; and the loci encoding the CTL determinants Tp5 and Tp7 were Khan et al., 2005). The average genome-wide recombination rate found to be closely associated with regions of low recombination for T. parva is therefore relatively high at 0.22 cM KbÀ1 per meiotic activity (cold spots) while the loci encoding Tp1, Tp2, Tp4, Tp8 generation (equivalent to an average map unit of 4.6 Kb cMÀ1), and Tp9 were found in regions of average recombination rates. indicating that analysis of relatively few progeny can provide high The SVSP loci 1–4, located in sub-telomeric regions of chromo- mapping resolution. Pairwise comparison of the recombination somes 2, 1, 4 and 3, respectively, lay at the periphery of each rate between genetic markers revealed a high level of variation respective linkage group, which limited our ability to assign across the genome, both within and between chromosomes. Of recombination rates. The SVSP1 locus was found to be located close the 10 recombination hot spots identified, only two were found (a 166 bp interval) to a region of high recombination activity, but to be closely associated with the end of a chromosome, although the remaining three SVSP loci were linked to regions with recom- map coverage did not extend to the absolute end of any chromo- bination parameters comparable to the genome average. some. Recent studies on the chromosome structure of P. falciparum have supported similar observations of variable recombination along a chromosome, but also reported elevated rates at 4. Discussion chromosome ends, in contrast to our findings (Mu et al., 2005). Chromosome breakage occurs frequently in sub-telomeric genome Genetic maps have been constructed for a number of apicom- regions and, in P. falciparum, genes encoding a number of plexan parasites, including Eimeria tenella (Shirley and Harvey, immunodominant parasite proteins are located in these fragile 2000), Eimeria maxima (Blake et al., 2011), Plasmodium chabaudi chromosomal segments (Hernandez-Rivas et al., 1996). The chabaudi (Martinelli et al., 2005), Plasmodium falciparum (Su sub-telomeric regions of P. falciparum contain arrays of repetitive et al., 1999) and Toxoplasma gondii (Sibley et al., 1992). We believe sequences extending over 80–100 Kb (de Bruin et al., 1994). By that the genetic map presented here is the first available for T. par- contrast, sub-telomeric chromosome structure is much less com- va, representing the first non-Plasmodium map for parasites within plex in the T. parva genome. Indeed, many T. parva surface anti- the class Aconoidasida. Using a panel of 79 VNTR markers with gen-encoding loci are relatively conserved at the genus level and known genomic locations, four linkage groups were constructed, can be found distributed across the chromosomes, in contrast to consistent with the four chromosome T. parva genomic karyotype. the largely species-specific, sub-telomeric Plasmodium immuno- At 1683.8 cM after a single meiotic opportunity, the genetic size of dominant antigens (Kuo and Kissinger, 2008). Genetic recombina- F. Katzer et al. / International Journal for Parasitology 41 (2011) 669–675 673

Table 2 Annotation of 27 genetic loci encoding Theileriaparva antigens previously identified as immuno-relevant (or as likely to be under selection) with the genetic map.

Chromosome Locus Chromosomal Context Inter-species Intra-species Reference RRa location Polymorphism Polymorphism (bp) for T. parva

b 1 Sfi ST-c1a 3039–17,805 Sub-telomeric High dN/dS No data Weir et al. (2010) – 1 SVSP-c1a 18,028–30,312 Sub-telomeric High dN/dS No data Weir et al. (2010) –b 1 Tp2 122,521–123,539 Internal Highly polymorphic Polymorphic between Graham et al. –b stocks (2006) MacHugh et al. (2009) 1 TashAThomologues 1270,087–1274,623 Internal High dN/dS No data Weir et al. (2010) h 1 Tpms 2180,721–2181,765 Sub-telomeric High dN/dS Polymorphic between Weir et al. (2010) – stocks 1 Sfi ST-c1b 2514,060–2529,823 Sub-telomeric High dN/dS No data Weir et al. (2010) –b 1 SVSP-c1b 2531,301–2533,997 Sub-telomeric High dN/dS No data Weir et al. (2010) –b 2 Sfi ST-c2a 3020–7096 Sub-telomeric High dN/dS No data Weir et al. (2010) Cb 2 SVSP-c2a 7952–21,726 Sub-telomeric High dN/dS No data Weir et al. (2010) Cb 2 Tp8 285,551–287,131 Sub-telomeric Highly conserved No data Graham et al. – (2006) 2 Tp7 496,298–499,293 Internal Conserved gene No data Graham et al. C family (2006) 2 Tp5 1541,091–1541,855 Internal Highly conserved No data Graham et al. C (2006) 2 SVSP-c2b (Tp9) 1955,382–1969,335 Sub-telomeric High dN/dS No data Weir et al. (2010) –b 3 SVSP-c3a + Sfi ST- 2862–617,912 Subtelomeric + Internal High dN/dS No data Weir et al. (2010) –b c3a 3 Tp4 425,934–428,528 Internal Highly conserved No data Graham et al. – (2006) 3 Tp1 471,175–472,945 Internal Highly divergent Polymorphic between Graham et al. stocks (2006) MacHugh et al. – (2009) 3 p67 588,213–590,371 Internal Polymorphic Polymorphic between Sibeko et al. (2010) –b stocks 3 Tpr (at least 28 1300,000– Internal High dN/dS Highly polymorphic, Weir et al. (2010) H genes) 1400,000 Gardner et al. (2005) 3 p150 1808,704–1813,769 Sub-telomeric Polymorphic Polymorphic between Skilton et al. (1998) –b stocks 3 Sfi ST-c3b 1835,122– Sub-telomeric High dN/dS No data Weir et al. (2010) –b 1836,330c 3 SVSP-c3b 1836,591– Sub-telomeric High dN/dS No data Weir et al. (2010) –b 1885,398c 4 SVSP-c4a + 2938–174,574 Sub-telomeric High dN/dS No data Weir et al. (2010) –b 4 SfiST-c4a 796,343–797,246 Internal High dN/dS No data Weir et al. (2010) –b 4 PIM 85,086–86,644 Sub-telomeric High dN/dS Highly polymorphic Toye et al. (1995) –b 4 SVSP-c4b 1808,265–1821,765 Sub-telomeric High dN/dS No data Weir et al. (2010) cb 4 Sfi ST-c4b 1823,082–1832,984 Sub-telomeric High dN/dS No data Weir et al. (2010) cb

a Recombination rate annotation: H = in a hot spot, h = close to a hot spot, C = in a cold spot, c = close to a cold spot, – = no significant variation detected. b Low density marker coverage may obscure selection. c Based upon the sizes of the chromosome 3 contigs NW_876245 and NW_876244 plus the estimated missing sequence (Gardner et al., 2005). tion can play a key role in the development of allelic diversity, phic than previously thought (Sibeko et al., 2010), the p67 locus through mechanisms such as gene duplication followed by rapid was found to be located in a region of relatively neutral recombina- sequence divergence, and exon shuffling (Kuo and Kissinger, tion activity. Theileria parva antigens with high inter-specific non-

2008). In addition, recombination can support rapid dissemination synonymous/synonymous substitution (dN/dS) ratios, indicative of of an advantageous allele within a population under selection. For diversifying selection, include the polymorphic immunodominant example, recombination could play a significant role in generating molecule (PIM) and p150 (Toye et al., 1995; Skilton et al., 1998). diversity at loci relevant to evasion of host immunity by Theileria. Although the PIM and p150 loci occur in sub-telomeric regions of The identification of variable rates of recombination across the T. the T. parva genome, both also mapped to regions of relatively neu- parva genome therefore prompted an assessment of the associa- tral recombination activity. Similarly, the T. parva merozoite sur- tion between genes previously identified as immunorelevant and face antigen 1 (Tpms1), homologous to the highly polymorphic the local rate of recombination. Candidate antigens included Tp1, Theileria annulata merozoite surface antigen 1 (Tams1) (Katzer Tp2, Tp4, Tp5, Tp7, Tp8 and Tp9, previously shown to be targeted et al., 1998), is encoded sub-telomerically, in a region with a rela- by the T. parva-specific CTL response (Graham et al., 2006; Katzer tive neutral recombination rate. Analysis of many Tams1 alleles et al., 2006). Whilst polymorphism has been reported for several from field isolates has provided evidence of high levels of intra- of the CTL-targeted antigens of T. parva, their coding sequences genic recombination within this gene (Gubbels et al., 2000). The were all located within genomic regions characterised by low or observation that Tpms1 locates to a region with a relatively neutral neutral levels of recombination (Table 2). Other immunorelevant recombination frequency may reflect differences between these antigens include p67, a sporozoite surface antigen and vaccine closely related parasites in recombination rates for this locus. candidate (Musoke et al., 1992; Nene et al., 1996). Despite recent However, this does not explain why other T. parva genes encoding reports that p67 in cattle-derived T. parva may be more polymor- proteins seen by the host immune response occur in regions with 674 F. Katzer et al. / International Journal for Parasitology 41 (2011) 669–675 similarly low recombination frequencies. It is possible that these sequence assembly. Development of additional markers targeting observations reflect a limited requirement for the parasite to gen- hot spots of genetic recombination will refine the map and support erate diversity in these regions. Indeed, the extent to which the bo- identification of genes with the greatest potential for diversifica- vine immune response imposes selection on T. parva has been tion. Progress towards the annotation of putative and hypothetical questioned previously, on the basis that protective cellular re- coding sequences residing within each recombination hot spot will sponses fail to prevent the emergence of tick-infective piroplasms inform development of novel anti-Theileria control strategies, (Katzer et al., 2007) and the observation that many of the identified while identification of regions within the T. parva genome with antigenic targets of the response are conserved (McKeever, 2009). the highest and lowest potential for genetic recombination will It has been argued that polymorphism observed in some immune help to maintain the long-term efficacy of these strategies. determinants of the parasite arises from its evolution in the buffalo prior to its establishment in cattle (McKeever, 2009). Comparison Acknowledgements of the genome sequences of T. parva and T. annulata has revealed strong conservation of synteny outside of sub-telomeric regions, We are indebted to the International Livestock Research Insti- with only a few inversions of small sequence blocks and no in- tute (ILRI), Kenya for provision of the parental clonal T. parva stocks ter-chromosomal rearrangements (Pain et al., 2005). Exceptions and to Stephen Mwaura of ILRI for co-ordinating the in vivo infec- corresponded to the insertion or deletion of genes and usually in- tion work. The work was entirely supported by project grants volved members of large gene families, most notably within the 062610 and 064654 to DM from the Wellcome Trust. Tpr locus. The location of the Tpr locus within a recombination hot spot in these studies is consistent with these findings and Appendix A. Supplementary data the notion that the complex Tpr domain structure appears likely to support the rapid generation of genetic diversity (Gardner Supplementary data associated with this article can be found, in et al., 2005). Other T. parva gene families associated with diversify- the online version, at doi:10.1016/j.ijpara.2011.01.001. ing selection based upon inter-species dN/dS values include the Ta- shAT/TashHN homologues, and the SVSP and Sfi sub-telomeric genes (Weir et al., 2010). Conservation of the T. annulata TashAT/ References TashHN gene family has recently been found at the intra-species Blake, D.P., Oakes, R., Smith, A.L., 2011. A genetic linkage map for the apicomplexan level, despite the observation of elevated inter-species diversity protozoan parasite Eimeria maxima and comparison with Eimeria tenella. Int. J. (Weir et al., 2010). 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