A comprehensive evaluation of rodent malaria parasite genomes and gene expression
- Thomas D Otto1,
- Ulrike Böhme1,
- Andrew P Jackson2,
- Martin Hunt1,
- Blandine Franke-Fayard3,
- Wieteke A M Hoeijmakers4,
- Agnieszka A Religa5,
- Lauren Robertson1,
- Mandy Sanders1,
- Solabomi A Ogun6,
- Deirdre Cunningham6,
- Annette Erhart7,
- Oliver Billker1,
- Shahid M Khan3,
- Hendrik G Stunnenberg4,
- Jean Langhorne6,
- Anthony A Holder6,
- Andrew P Waters5,
- Chris I Newbold8, 9,
- Arnab Pain10,
- Matthew Berriman1Email author and
- Chris J Janse3Email author
© Otto et al.; licensee BioMed Central Ltd. 2014
Received: 28 July 2014
Accepted: 10 October 2014
Published: 30 October 2014
Rodent malaria parasites (RMP) are used extensively as models of human malaria. Draft RMP genomes have been published for Plasmodium yoelii, P. berghei ANKA (PbA) and P. chabaudi AS (PcAS). Although availability of these genomes made a significant impact on recent malaria research, these genomes were highly fragmented and were annotated with little manual curation. The fragmented nature of the genomes has hampered genome wide analysis of Plasmodium gene regulation and function.
We have greatly improved the genome assemblies of PbA and PcAS, newly sequenced the virulent parasite P. yoelii YM genome, sequenced additional RMP isolates/lines and have characterized genotypic diversity within RMP species. We have produced RNA-seq data and utilised it to improve gene-model prediction and to provide quantitative, genome-wide, data on gene expression. Comparison of the RMP genomes with the genome of the human malaria parasite P. falciparum and RNA-seq mapping permitted gene annotation at base-pair resolution. Full-length chromosomal annotation permitted a comprehensive classification of all subtelomeric multigene families including the `Plasmodium interspersed repeat genes' (pir). Phylogenetic classification of the pir family, combined with pir expression patterns, indicates functional diversification within this family.
Complete RMP genomes, RNA-seq and genotypic diversity data are excellent and important resources for gene-function and post-genomic analyses and to better interrogate Plasmodium biology. Genotypic diversity between P. chabaudi isolates makes this species an excellent parasite to study genotype-phenotype relationships. The improved classification of multigene families will enhance studies on the role of (variant) exported proteins in virulence and immune evasion/modulation.
KeywordsPlasmodium chabaudi Plasmodium berghei Plasmodium yoelii Genomes RNA-seq Genotypic diversity Multigene families pirs Phylogeny
Rodent malaria parasites (RMP) are used extensively as models of human malaria ,. Four different species that infect African rodents have been adapted for laboratory use: Plasmodium berghei, P. yoelii, P. chabaudi and P. vinckei. Small differences exist in the biology of the different RMP in laboratory mice and this makes them particularly attractive models to investigate different aspects of human malaria. Specifically, P. chabaudi is a model to investigate mechanisms of drug resistances and immune evasion, in particular antigenic variation ,. It invades normocytes and reticulocytes and mostly produces chronic, non-lethal, infections. In contrast, P. berghei preferentially invades reticulocytes and usually produces infections in mice that induce severe pathology . In combination with different mouse strains it has been used as a model to study immunopathology, experimental cerebral malaria, pregnancy-associated malaria and lung pathology . P. yoelii is widely used in studies on the biology of liver stages and on innate and acquired immunity against liver stages ,. Blood stage P. yoelii parasites of some lines are restricted to reticulocytes whereas others can invade all red blood cells and have been used to study receptors for erythrocyte binding ,. The availability of efficient reverse genetics technologies for P. berghei and P. yoelii - and the ability to analyse these parasites throughout the complete life cycle have made these two species the preferred models for analysis of Plasmodium gene function -. For these two species more than 600 different genetically modified mutants have been reported .
The first draft RMP genome was published in 2002 for P. yoelii yoelii 17XNL . This was followed by publication of draft genomes of P. berghei ANKA (PbA) and P. chabaudi chabaudi AS (PcAS) in 2005 . Comparisons with the genome of the human parasite P. falciparum and other primate malaria species defined a large set of core genes that are shared between RMPs and primate malarias -. Although availability of draft RMP genomes made a significant impact in applying post-genomic technologies for understanding malaria biology  and were used in many follow-up functional genomics studies to analyse gene regulation and function ,, these RMP genomes were highly fragmented and were annotated with little or no manual curation. The fragmented nature of the genomes has hampered genome wide analysis of gene regulation and function, especially of the (subtelomeric) multigene families. To utilise RMP models to their full potential, we therefore undertook production of high quality reference genomes: for PbA and PcAS large-scale improvement of their existing genomes, with re-sequencing, re-analysis and manual re-annotation, and for P. y. yoelii a genome sequence was produced de novo from the virulent YM line using the latest sequencing technologies and computational algorithms. In addition, we have utilised comprehensive RNA-seq data derived from a number of life-cycle stages to both improve gene model prediction and to provide genome-wide, quantitative data on gene expression. By sequencing additional isolates/lines of P. berghei, P. yoelii and P. chabaudi (including the subspecies P. c. adami) we have documented genotypic diversity that exists within different RMP species. The availability of RMP reference genomes in combination with the RNA-seq and genotypic diversity data will serve as excellent resources for gene-function and post-genomic analyses and, therefore, better interrogation of Plasmodium biology and development of anti-malaria interventions.
The genomes of RMP contain a number of multigene families located in the subtelomeric chromosomal regions. These include a large family of so-called `Plasmodium interspersed repeat genes' (pir) , that are present also in other human/primate Plasmodium species -. Most of these gene families are expressed in blood stages and these proteins show features that have been reported to contribute to immune evasion through antigenic variation - and may play a role in the sequestration of infected red blood cells and virulence ,. As a result of the improved annotation, we have been able to define all multigene families in the RMP genomes. Comparative phylogenetic analyses of the pir genes and analyses of pir expression patterns in blood stages of P. berghei provide evidence of functional diversification within this gene family. The improved classification of multigene families will enhance studies on the role of (variant) exported proteins in virulence and evasion and modulation of the immune system.
Generation of high-quality RMP reference genomes
Features of the reference genomes of P. berghei ANKA, P. c. chabaudi AS and P. y. yoelii YM
P. berghei ANKA
P. c. chabaudi AS
P. y. yoelii YM
P. falciparum 3D7 a
Previous assembly [ ]
Previous assembly [ ]
Genome size (Mb)
G + C content (%)
Genes with functional annotationd
Novel genes (see Additional file 1)
Genome size (bp)
G + C content (%)
Number of genes
Genome size (bp)
G + C content (%)
Conserved chromosome organization and gene orthology between RMP and primate malaria parasites
Different (subtelomeric) multigene families in the RMP genomes
Gene family (new name)
Other (previous) names
Number of genes
pir, bir, cir, yir
Pb-fam-1; Pc-fam-1; fam-a; PYSTA
Early transcribed membrane protein
Reticulocyte binding protein, putative
P235; 235kDA protein
rhoptry protein, putative
RMP-erythrocyte membrane antigen (RMP-EMA1)
haloacid dehalogenase-like hydrolase, putative
`Other subtelomeric genes'
We compared all predicted RMP protein-coding genes with those of three primate malaria species, P. falciparum, P. knowlesi and P. vivax using OrthoMCL and divided the predicted RMP proteome into three different categories: (1) RMP proteins with orthologs in any of the primate malarias; (2) RMP-specific proteins with no orthologs in primate malarias; and (3) primate malaria-specific proteins with no orthologs in any of the RMP (see Additional file 5). Between the predicted RMP proteomes (15,793 proteins in total) and primate malaria proteomes (15,853 proteins in total), approximately 87% of the RMP proteins had detectable orthologs in at least one of the primate malarias and only 2,104 proteins (13.3%) were predicted to be RMP-specific. Of those 2,104 proteins, 1,854 (88.1%) are from gene families, as defined in Additional file 4. For 2,306 primate malaria proteins (14.6%) no orthologs have been detected in the RMP. Of these primate malaria specific genes, approximately 1,635 (70.9%) are subtelomeric genes or members of subtelomeric gene families (see Additional file 5).
Genotypic diversity within RMP isolates: P. chabaudiisolates exhibit high level polymorphism amongst their genes
Sequence diversity and number of members of multigene families from different RMP isolates/lines
Genes with SNPs b
Assembly size (Mb)
pir genes c
fam-a genes c
fam-b genes c
fam-c genes c
fam-d genes c
P. berghei ANKA a
P. berghei NK65 E
P. berghei NK65NY
P. berghei SP11 A
P. berghei SP11 RLL A
P. berghei K173cl1
P. y. yoelii YM a
P. y. yoelii 17X
P. c. chabaudi AS a
P. c. chabaudi CB
P. c. chabaudi AJ
P. c. adami DS
P. c. adami DK
In contrast to the P. berghei isolates, the P. chabaudi isolates and subspecies have much higher SNP densities with 4,300 to 4,500 (out of 4,576) non-subtelomeric genes having at least one SNP (Table 3, Additional file 8). The high genotypic diversity is not only evident between the subspecies P. c. chabaudi and P. c. adami, but also between isolates of the same subspecies. For example, we found 94,668 and 71,074 unique SNPs (in 3,978 and 4,166 genes) in the P. c adami DK and DS isolates, respectively. Between different P. chabaudi isolates differences exist in virulence- and invasion phenotypes of blood stage infections (see Additional file 6). We detected multiple SNPs in P. chabaudi genes involved in binding to red blood cells (RBC) such as the Duffy-binding protein and reticulocyte binding proteins (see Additional file 8), genes that are associated with differences in virulence between P. y. yoelii lines. Isolate-specific protective immunity between P. c. chabaudi isolates has been linked to the merozoite surface protein 1 (MSP1; PCHAS_083130) ,. Our analyses revealed an excess of non-synonymous substitutions (reflected in high Ka/Ks values) in msp1 of all P. chabaudi isolates (see Additional file 8).
High resolution, genome-wide expression data from different RMP life cycle stages
To further improve the reference genomes we mapped the RNA-seq data onto the RMP genomes and visually inspected the alignments using the Artemis Comparison Tool (ACT), a genome viewing tool . A comparative analysis with the P. falciparum 3D7 genome allowed us to determine gene structure at base-pair (bp) resolution for at least 89% of the genes. Of the 896 newly annotated protein-coding genes that were absent in the previous genome assemblies, 70% have primate malaria orthologs, 83% have expression evidence (RNA-seq FPKM values >21) and we could ascribe functions to 75% (see Additional file 1). The different RNA-seq data sets have also been used to confirm splice sites and to identify putative alternative splice sites (see Additional file 11). This analysis resulted in the identification of 839 alternative splicing events in a total of 567 RMP genes.
Characterization of RMP multigene families
As a result of having dramatically improved the annotation of the subtelomeric regions we were able to accurately define the RMP multigene families that are located there (Table 2, Additional file 4). For proteins of nearly all of these families experimental evidence exists that they are exported into the host RBC in the absence of a PEXEL motif . The pir family is the most abundant multigene family (see next section) encoding exported proteins that lack a canonical PEXEL motif. The second largest gene family is the fam-a gene family, formerly identified as the pyst-a family in P. yoelii 17XNL and named as Pb-fam-1, Pc-fam-1 or fam-a ,. PbA fam-a proteins are exported into the host RBC and can be transported to the RBC surface membrane  but lack a PEXEL-motif. Single copy orthologs have been defined in all primate malarias and the expansion of this family is RMP-specific. Most members have a subtelomeric location (see Additional files 12, 13 and 14), but all three RMP have at least one internally located copy that is positionally conserved with the primate malaria orthologs and, therefore, likely to represent the ancestral copy of this family. In order to standardise the naming of orthologous multigene families in different RMP, we have renamed the two multigene families, pyst-b/pb-fam-3 and pyst-c genes ,, as fam-b and fam-c, respectively (Table 2; Additional file 4). The fam-b family is exclusively subtelomeric and is characterized by the presence of the pyst-b domain. Most members contain a transmembrane domain (58%), a signal peptide (75%) and PEXEL-motif (76%) (see Additional files 12, 13 and 14). PbA fam-b proteins are exported into the host RBC . The fam-c is also exclusively found in the subtelomeric regions and is characterized by the presence of a pyst-c1 and/or pyst-c2 domain . Most members have a transmembrane domain (60%) and a signal peptide (92%) (see Additional files 12, 13 and 14) and only a small percentage (24%) contain a predicted PEXEL-motif.
Other subtelomeric multigene families include the `early transcribed family of proteins' (ETRAMPs) and `putative reticulocyte binding proteins' (Table 2, Additional files 4, 12, 13 and 14). ETRAMPS are small exported proteins with a predicted signal peptide and transmembrane domain but without a PEXEL-motif. These proteins are mainly located in the parasitophorous vacuole membrane ,. The genes encoding putative reticulocyte binding proteins (RBP), that were first described in P. yoelii as Py235 and are expressed in merozoites , are clear orthologs of the reticulocyte binding proteins of P. vivax  and the RH proteins of P. falciparum . These large proteins typically have a predicted signal sequence and at the C-terminus a transmembrane domain containing a rhomboid cleavage site and a cytoplasmic domain, although P. falciparum RH5 contains just the signal peptide and N-terminal ligand binding domain . The RMPs have genes encoding two short RBPs reminiscent of P. falciparum RH5 (typified by PYYM_0101400 and PYYM_0701100) and six or more full length proteins (Table 2). Compared with PyYM, Py17X contains an additional full length RBP.
In PcAS several other expanded gene families are present in the subtelomeric regions. These include `putative lysophospholipases', `erythrocyte membrane antigen 1' (EMA1), and `putative haloacid dehalogenase-like hydrolases' (Table 2, Additional files 4, 12, 13 and 14). The genes encoding lysophospholipases are characterized by the `pst-a' domain  and all RMP have two copies with an internal chromosomal location that are syntenic with orthologs of primate malarias. For two of the five PbA lysophospholipases evidence exists that they are exported into the RBC  and again they lack a PEXEL-motif. In PcAS this family has expanded into 28 copies (Table 2, Additional file 4). In the genome of PyYM and PbA only a single gene encoding EMA1 is present whereas PcAS ema1 has expanded to more than 10 copies in the subtelomeric regions (Table 2, Additional file 4). These PEXEL-negative proteins, first described in P. chabaudi  are associated with the RBC membrane. The gene encoding the putative haloacid dehalogenase-like hydrolase has expanded only in PcAS, with eight subtelomeric copies.
A number of other genes are interspersed within the subtelomeric regions of RMP chromosomes. Many of these `other subtelomeric genes' (46 to 67 genes; Table 2, Additional file 4) encode proteins that are RMP-specific and more than 96% of these proteins contain a predicted signal peptide, transmembrane domain or PEXEL-motif and for several proteins experimental evidence exists for their export into the host RBC cytoplasm. Combined, these observations indicate that most, if not all, RMP subtelomeric genes (apart from the RBP family) encode exported proteins and most lack a PEXEL-motif. The presence of large numbers of PEXEL-negative exported proteins in RMP indicates alternative export mechanisms possibly common to all Plasmodium species and investigations with highly tractable RMP species can, therefore, be used to understand these mechanisms better.
The RMP pir multigene family: phylogeny and expression
By extensive re-sequencing and annotation we have generated three high quality RMP reference genomes with nearly all core genes as complete gene models and a much improved and almost complete representation of chromosomal subtelomeric regions. These reference genomes will greatly enhance the use of RMP as model organisms in malaria research. We provide full-length gene models for more than 98% of predicted protein-coding genes. The approximately 60% of genes with functional annotation is comparable to the percentage of functionally annotated genes in the P. falciparum 3D7 reference genome and a high percentage (approximately 90%) of the predicted RMP proteins have orthologs in primate malaria species. It is this high level of orthology between RMP and primate malaria genomes that strongly supports RMPs as models in experimental approaches to characterize the Plasmodium gene function. Similarly, the genome-wide RNA-seq data from different RMP developmental stages is a valuable resource to further analyse Plasmodium gene function and the regulatory networks underlying the multiple differentiation pathways of Plasmodium. The RNA-seq studies presented here provide information on gene expression at an unprecedented depth and breadth of coverage of multiple blood stages and ookinetes. Previously, only a few large-scale transcriptome (microarray) analyses of P. berghei blood stages and ookinetes had been performed ,. These studies were based on a highly fragmented draft P. berghei genome and, therefore, expression data were only generated for about half of all P. berghei genes. In addition, important/valuable large scale transcriptome studies have been performed on RMP life-cycle stages, such as sporozoites and liver stages -. These life-cycle stages would also benefit from re-examination using the latest RMP genome assemblies we provide in this study.
Our studies reveal that large scale changes in gene expression occur in ookinetes between 16 and 24 hours after fertilization, possibly required for the differentiation of (retort-form) zygotes into the mature ookinetes. The strong up-regulation in mature ookinetes of transcripts involved in ribosome biogenesis and protein translation suggest that the mature ookinete generates transcripts for proteins required after the ookinete has traversed the mosquito midgut wall and starts its rapid transition into the oocysts, possibly using mechanisms of translational repression similar to those in gametocytes , and sporozoites ,. What these three stages have in common is that they are fully differentiated cells that will undergo rapid cellular differentiation and/or growth expansion upon entering a new environment. Whether mature ookinetes store repressed transcripts requires further investigation.
The additional sequence data from multiple RMP isolates will help to further unravel gene function and establish relationships between phenotypic traits and genotypic diversity. The near absence of polymorphisms within the genomes of P. berghei isolates was unexpected. Low sequence diversity of a limited number of genes of P. berghei isolates had been reported previously and it was proposed that this may result from cross-contamination of P. berghei isolates in the laboratory after isolation ,. However, this seems unlikely as one line would have needed to be mislabelled with the names of all other isolates, then all these mislabelled lines would have had to be sent to all the different laboratories worldwide replacing the `correct' isolates that may have existed in their recipient laboratories. However, sequencing of additional stocks from these isolates, which were frozen in different laboratories soon after isolation from the natural host, may reveal whether low sequence diversity is due to cross-contamination. The P. berghei isolates we have sequenced were obtained from other laboratories (SP11, NK65) and they also show a similar lack of sequence polymorphism. In contrast, the isolates of P. chabaudi exhibit considerable genotypic diversity. These P. chabaudi isolates exhibit differences in virulence, RBC invasion, growth rates and immunogenic profiles ,- and further studies, for example using linkage or quantitative trait loci (QTL) analyses ,-, will facilitate identification of genes associated with defined phenotypes. For RMP species there is evidence that differences in virulence are associated with differences in RBC invasion ,,,,. For example, P. yoelii virulence has been associated with mutations in proteins involved in RBC invasion ,,. Interestingly, we found extensive sequence polymorphism in P. chabaudi genes encoding such proteins. While much attention is given to the role of exported proteins of multigene families and virulence in both human and RMP, further analysis of RMP proteins that regulate invasion phenotypes may reveal novel mechanisms that underlie virulence.
The new sequence data allowed for a much improved annotation of chromosomal subtelomeric regions and to better define the different subtelomeric multigene families. In addition to the large pir gene family, all three RMP contain an expanded gene family encoding exported proteins, fam-a, with orthology to a single-copy gene in primate malarias, which contains a START-domain (steroidogenic acute regulatory-related lipid transfer domain; ). START-containing proteins of eukaryotes are involved in the transfer of phospholipids, ceramide or fatty acids between membranes . A START domain has also recently been identified in an exported, PEXEL-containing, P. falciparum protein that was shown to transfer phospholipids . The single-copy RMP orthologs of this gene (PF3D7_0104200) also contain a PEXEL-motif, indicating that phospholipid-transporting proteins are exported into the RBC in both primate malarias and RMP. P. chabaudi contains an additional, highly expanded, gene family that contains domains involved in phospholipid/fatty acid metabolism. These genes, encoding putative lysophospholipases, lack a PEXEL motif; however, for several P. berghei orthologs as well as lysophospholipases of P. falciparum there is evidence for their export into the host RBC ,. Combined, these observations indicate the importance of phospholipid/fatty acid metabolism/transport mediated by Plasmodium proteins exported into the RBC cytosol. Why such genes have been differentially expanded into multigene families in different species remains to be investigated.
The pir family is the largest RMP multigene family and is shared with human and non-human primate species P. vivax, P. knowlesi and P. cynomolgi -. PIR proteins are exported into the RBC in the absence of a PEXEL-motif, and there is evidence that they are located on, or close to, the RBC surface or dispersed in the RBC cytoplasm -,,,. The function of pirs is unknown and no functional domains have been identified so far. Recently, it has been shown that in P. chabaudi a change in virulence was associated with differential expression of members of the pir multi-gene family . It has been suggested that PIRs are transported to the surface of infected RBC and play a role in RBC sequestration comparable to the role of the Pfemp1 gene family of virulence factors in P. falciparum. However, for several P. berghei PIRs a direct role in RBC sequestration is unlikely since no evidence was found for their location on the RBC surface although they were exported into the RBC cytoplasm of both sequestering asexual blood stages and non-sequestering gametocytes . For P. vivax PIRs it has been shown that different members have distinct subcellular locations in the infected RBC . These observations indicate that functional differences may exist between members of the PIR family. Phylogenetic analyses support the possibility of functional differences between the PIRs. A recent phylogenetic analysis of the newly annotated PcAS pirs identified two distinct pir sub-families (A and B), which contain distinct amino acid sequence motifs . Our phylogenetic analyses included pirs from all three RMP species and resulted in the identification of a number of different clades. The presence of clearly distinguishable clades indicates that structural differentiation exists among pirs and that this evolved prior to the separation of the RMP species. Our observations of the stage-specific up- or down-regulation of expression of clusters of structurally different pirs in different blood stages supports the hypothesis that there is functional diversification within the pir family and that purifying selection plays a role in shaping this family. By including multiple species in the pir phylogeny it is clear that this gene family is subject to rapid turnover, that is, gene gain and loss, indicating the absence of strong selective forces that would result in distinct orthologous groups/clades that are shared and maintained in different species for functional reasons. Gain of pir genes in different species is evident in the multiple species-specific expansions of clades. Assuming that the common ancestor had a pir family equal in abundance and diversity, the relatively limited instances of orthology (12 clades) indicates significant losses of ancestral sequence types. A plausible explanation for both the abundance of species-specific sequences and the paucity of ancestral sequences is a continual process of gene turnover driven by gene conversion, a mechanism that has been proposed for pirs of P. chabaudi  and which was evident in each of the clades revealed in this study (data not shown). The effect of frequent gene conversion is the replacement of ancestral sequence types with species-specific sequences, which results in distinct species-specific clades without orthology. Loss of orthology is only resisted when selective forces maintain structurally distinct pirs, which we propose, explains the presence of the (limited) orthology between pir clades of the different RMP species. The improved annotation and phylogeny demonstrating clusters of structurally different pirs in all RMP combined with expression profiles are powerful data that can help to further delineate function, the relationship of expression with virulence and how the (species-specific) expansion of the pirs is related to distinct selective pressures.
To maximise the utility of RMP we have greatly improved the genome assemblies of P. berghei and P. chabaudi, comprehensively sequenced the P. yoelii YM genome, sequenced multiple RMP isolates and generated in-depth expression data from multiple RMP life-cycle stages. Comparison of the RMP and P. falciparum genomes and RNA-seq mapping permitted gene annotation at base-pair resolution and has defined the level of orthology between RMP and human parasite genomes. The very high level orthology between RMP and human malarias (both in genome structure and gene content) supports the use of highly tractable RMPs as experimental models to characterize the function of the very many Plasmodium genes that remain uncharacterised.
Only a few large-scale transcriptome (microarray) analyses of different P. berghei life-cycle stages had previously been performed. Moreover, these studies were based on highly fragmented draft RMP genomes and consequently, for example, for one of the most well studied RMP, P. berghei, gene expression data was only mapped to about half of all P. berghei genes that have now been characterised. The RNA-seq studies we present provide information on gene expression, at an unprecedented depth and breadth of coverage, of multiple life cycle stages and provide the foundational data needed for the performance of large-scale analyses of gene regulatory networks that underlie cellular differentiation.
We show that extensive genotypic diversity exists between P. chabaudi isolates making this species an excellent organism to study genotype-phenotype relationships. Differences in virulence red blood cell (RBC) invasion, growth rates and immunogenic profiles exist between parasites of these isolates. Therefore, studies, such as linkage or quantitative trait loci analysis, are now possible to help identify genes associated with these defined phenotypes. For RMP species there is evidence that differences in virulence are associated with differences in RBC invasion, and, indeed, we find extensive sequence polymorphism in P. chabaudi genes encoding proteins involved in RBC invasion. Much attention is given to the role of exported proteins of multigene families and virulence in both human and RMP (for example, var, pirs), and analysis of differences between RMP proteins, that regulate invasion phenotypes, may reveal novel mechanisms that underlie virulence.
Full-length chromosomal annotation has permitted a comprehensive classification of all RMP subtelomeric multigene families. Our analyses indicate that most, if not all, RMP subtelomeric genes (apart from the RBP family) encode proteins exported out of the parasite; however, most lack a canonical PEXEL-motif. The presence of large numbers of PEXEL-negative exported proteins indicates alternative export mechanisms possibly common to all Plasmodium species. Investigations with highly tractable RMP species can therefore be used to understand these mechanisms better.
Our analyses of the phylogeny and expression of the largest RMP multi-gene family, the pirs, indicates functional diversification between members of the pir multigene family (this gene family is conserved between human/primate and RMP malaria species). Our new pir annotation and phylogeny demonstrates that clusters of structurally different pirs are differentially expressed. This is powerful data that can help to better understand their function, the relationship of pir expression with virulence and how the (species specific) pirs expansion is related to different selective pressures.
Animal experiments and parasites
All animal experiments performed in the Leiden malaria Research Group were approved by the Animal Experiments Committee of the Leiden University Medical Center (DEC 07171, DEC 10099). The Ethics Statement for P. yoelii YM and P. yoelii 17X: all animal work protocols were reviewed and approved by the Ethical Review Panel of the MRC National Institute for Medical Research and approved and licensed by the UK Home Office as governed by law under the Animals (Scientific Procedures) Act 1986 (Project license 80/1832, Malaria parasite- host interactions). Animals were handled in strict accordance with the `Code of Practice Part 1 for the housing and care of animals (21/03/05)' available at . The numbers of animals used was the minimum consistent with obtaining scientifically valid data. The experimental procedures were designed to minimize the extent and duration of any harm and included predefined clinical and parasitological endpoints to avoid unnecessary suffering. The study of P. chabaudi DNA and RNA was carried out in strict accordance with the UK Animals (Scientific Procedures) Act 1986 and was approved by the Ethical Committee of the MRC National Institute for Medical Research, and the British Home Office (PPL: 80/2538).
For sequencing of the RMP reference genomes the following were used: for PbA the cloned reference line cl15cy1 of the ANKA isolate of P. berghei ; for PcAS the 2722 clone of the AS isolate of P. chabaudi chabaudi (cloned after mosquito-transmission in 1978 and obtained from D. Walliker, University of Edinburgh, Edinburgh, UK); for PyYM the cloned 17XYM line of the YM line of P. yoelii yoelii . In Additional files 6 and 7 details are provided of the other RMP isolates/lines that have been sequenced.
Sequencing, assembly and annotation
Sequencing was performed using Sanger capillary, Illumina and 454 sequencing. Sequence assemblies were performed using different assemblers ,, which were improved automatically using a number of configuration tools - and manual inspection. First pass annotation was performed through a combination of ab initio gene finding via Augustus  and transfer of annotation through orthology using RATT . Gene models of the three reference genomes were corrected manually using RNA-Seq and orthologous information. Details of the assemblies and annotation are provided in Additional file 6. To define the orthologous and paralogous relationships between the predicted RMP proteins and those of human/primate malaria species OrthoMCL  was used. The presence of a PEXEL-motif was determined using the updated HMM algorithm ExportPred v2.0 with a cutoff value of 1.5 . Classification of the RMP multigene families was done through manual inspection of conserved domains (Interpro) and gene structure. SNPs in the genomes of these parasites were called by mapping the reads against their respective reference genomes, ignoring low complexity and repetitive regions. From the SNPs the Ka/Ks ratio was calculated for the P. chabaudi isolates with the Bio::Align::DNAStatistics Perl module.
RNA was collected from multiple synchronized blood stages  and purified gametocytes and ookinetes  of PbA, from PcAS blood stages (late trophozoites), isolated from different mice as described  and from PyYM late blood stages of two parasite lines (the cloned YM line and mutant PY01365-KO) . RNA was sequenced as described ,,,. To correct gene models and to compare the expression between samples, each sample was first mapped against its reference genome using TopHat  (version v2.0.6, parameter -g ). For the resulting output a custom Perl script was written to detect errors in the annotation and to find new or alternative splice sites. To determine transcript abundance FPKM values were calculated for all genes (FPKM: fragments per kilo base of exon per million fragments mapped) using Cufflinks . Accepting 10% of the intron as real signal, a cut-off FPKM value of 21 over all RNA-seq samples was determined. See also Additional file 6 for a detailed description of the generation and analysis of the RNA-seq data.
Heatmaps were generated with FPKM values of each gene and condition, using the heatmap.2 function of the gplots package. Correlation plots were done in R (Foundation for Statistical Computing; ) and generated with the corrplot function of the corrplot R library. Only genes were included that had one to one orthology in the three rodent species. For differential expression we used cuffdiff  (v2.0.2, with parameters -u -q) to compensate for GC variation and repetitive regions. GO enrichment of differentially expressed genes was performed in R, using TopGO. As a GO-database the predicted GO-terms from the reference RMP genomes were used.
Phylogenetic analyses of pirs
All full-length RMP pir coding sequences, including predicted pseudogenes, were used. Translated nucleotide sequences for 1,160 genes were aligned in ClustalW ; all multiple alignments were manually edited to resolve all frame-shifts. Non-homologous positions at the N-terminus were removed by curtailing the alignment to the N-terminal-most conserved cysteine position. Non-homologous repetitive motifs were removed from `long-form' PIRs (that is, 188 proteins >1,200 amino acids in length). The resultant 1,266-character alignment constitutes the conserved core of all PIRs and almost the complete repertoire of `short-forms' (that is, <1,200 amino acids in length and 972/1,160 genes). A Maximum Likelihood phylogeny was estimated from the nucleotide sequence alignment using RAxML v7.0.4  using a GTR + G model. Node support was assessed using 100 non-parametric bootstrap replicates . A Bayesian phylogeny was estimated using MrBayes v3.2.1  with a GTR + G model for a subsample of pir nucleotide sequences (MCMC settings: Nruns = 4, Ngen = 1,000,000, sample burnin = 1,000, and default prior distribution). See also Additional file 6 for a detailed description of the phylogenetic analyses.
All the raw data used in the assemblies of the genome and the RNA-seq data have been deposited with accession numbers shown in Additional file 17. The reference genomes have the following accession numbers: P. chabaudi chromosomes: LK022878-LK022893 and scaffolds: LK022855-LK022877, P. berghei chromosomes: LK023116-LK023131 and scaffolds: LK022894-LK022977; P. yoelii YM chromosomes: LK934629-LK934644 and scaffolds: LK023132-LK023312 and P. yoelii 17X chromosomes: LM993655-LM993670 and scaffolds: LK022978-LK023115.
TDO, UB, MH, LR, SAO, CIN, AP, MB contributed to the genome assembly, annotation and analysis. TDO, BFF, WAMH, AAR, OB, SMK, HGS, APW, CJJ generated and analysed RNA-seq data of P. berghei. DC, JL, AAH provided materials for genome sequences and/or RNA-seq data of different isolates/lines of P. yoelii and P. chabaudi. AE provided materials for genomes of isolates of P. berghei. APJ performed the phylogenetic analysis. TDO, UB, APJ, BFF, WAMH, SMK, AAH, CIN, AP, MB, CJJ conceived the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.
The work was funded by the Wellcome Trust (grant WT 098051). C. Newbold was supported by a Wellcome Trust program grant (082130), B. Franke-Fayard and W.A.M. Hoeijmakers by grants from The Netherlands Organization for Scientific Research (ZonMW TOP Grant No. 9120_6135; NWO Toptalent 021.001.011), T.D. Otto, M. Hunt, H.G. Stunnenberg, and C.J. Janse by a grant from the European Community's Seventh Framework Program (FP7/2007-2013; Grant Agreement No. 242095) and A.P. Water and A.A. Religa were supported by the Wellcome Trust (Ref. 083811/Z/07/Z). Work in the Holder and Langhorne labs is funded by the MRC (U117532067 and U117584248 respectively).
We thank the following individuals: Martine Zilversmit for producing an Augustus training set; Jai Ramesar (LUMC, Leiden) and C. van Overmeir (ITM, Antwerp) for technical support; The European Malaria Reagents Repository  as the source for P. chabaudi isolates; Sandra Cheesman for DNA preparation of the P. chabaudi isolates; Richard Carter for helpful discussion and providing information for the P. chabaudi isolates; R. Menard for providing P. berghei NK65 NY; Ph. van den Steen for P. berghei NK65 E; S. J. Boddey and T. Sargeant for assisting in the PEXEL analysis; R. Davies and Q. Lin for help with data release; L. Robertson, D. Harris, K. Segar, A. Babbage, H. Beasley, L. Clark, J. Harley, P. Heath, P. Howden, G. Kerry, S. Pelan, D. Saunders and J.Wood for manual improvement of the P. chabaudi AS assembly; R. Rance, M. Quail and members of DNA Pipelines for sequencing libraries at WTSI; J. Keane, M. Aslett, N de Silva for database support.
We acknowledge Roche (Branford, USA) for the generation of 454 20 kb libraries. The authors have no competing financial interests.
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