Figure 1.
Deduced Organization of the cRMP sera Locus
(A) The combination of three P. berghei, six P. chabaudi, and two P. yoelii contigs (thick black lines) in a region of Pfchr2 containing eight sera copies demonstrates the strength of the “composite genome approach.” Syntenic genes (black, linked by dashed vertical lines; left, PFB0315w and PFB0320c; right, PFB0365w) flank the sera clusters and reveal the presence of five sera genes in the RMPs.
(B) Phylogenetic analysis revealed a close relation between pfsera8, pfsera7, and pfsera6 and their syntenic orthologs in the RMPs (shaded gray, linked by dashed vertical lines in [A]). Other sera copies (pfsera1–5, pbsera1–2, and pysera1–2) clustered in species-specific groups (linked by solid horizontal lines in [A]). Circles represent branch points with bootstrap values of 100% (white), 90%–99% (light gray), and 65%–89% (dark gray).
Table 1.
Summary of the Characteristics of the cRMP Contigs, Scaffolds, SBs, and SBPs
Figure 2.
A Whole-Genome Synteny Map of P. falciparum and Three RMPs
Synteny map of the core regions of all chromosomes of P. falciparum (left) and the RMPs (right), showing the 36 SBs, 22 SBPs, 14 CAT regions, P. falciparum-specific indels, and translocations in the RMP chromosomes. The 36 SBs, colored according to their chromosomal location in the cRMP genome, are named with a Roman and an Arabic number referring to the corresponding chromosome location in P. falciparum and the cRMP genome, respectively. Letters give the order in which the SBs are connected. Small arrows indicate the inverted orientation of a SB in P. falciparum relative to the cRMP genome. Indels containing P. falciparum-specific intrasyntenic genes are indicated through interruption of the colored SBs. P. falciparum telomeres are shown as white arrow heads (▹). SBs forming the cRMP chromosomes are linked by gray lines. In the cRMP genomes, the 23 coinciding subtelomeric linked ends are shown as white arrowheads (▹) and the five P. falciparum subtelomeric ends that are chromosomal internal in the cRMP chromosomes are indicated by small white arrowheads (▹). The 11 syntenic P. falciparum CAT regions [29] are shown as white circles (○), two inconsistent CAT regions as white circles with a cross (⊗), and three newly recognized CAT regions as white diamonds (⋄). Chromosome-internal var clusters are shown as white arrows (); stars and circles on sticks indicate rrna gene units (
) and tstk genes (
); black stars and circles represent nonsyntenic genes; while syntenic genes (three rrna gene units and one tstk gene) are colored according to their chromosomal location in the RMPs. Bars under the cRMP chromosomes represent the differences in the organization of the SBs of P. yoelii, P. chabaudi, and P. vinckei as a result of translocations. Colors indicate the cRMP chromosome with which recombination has taken place, while color gradients represent the ill-defined regions of the translocation breakpoints.
Figure 3.
Schematic Representation of the 15 Recombination Events
Schematic representation of the 15 recombination events that would permit the 36 SBs to be rearranged to generate the P. falciparum genome from the cRMP genome. See Figure 2 for the numbering of the SBs and the symbols used in this figure. Gray lines between SBs represent links as present in the cRMP genome; gray dashed lines indicate intermediate links, and black arrows show links corresponding to the P. falciparum genome. Five subtelomeric regions of the cRMP genome must become chromosome-internal in the P. falciparum genome (A), thereby generating five subtelomeric regions in P. falciparum that are linked to SBPs in the cRMP genome. SB “XIVc:13b” is inverted (B), and SBs “VIIc:14b” and “VIIIb:14c” are inserted between SBs “VIIb:12c” and “VIIIc:12d,” a process likely to involve chromosome-internal clusters of var and rif genes possibly mediated by vicar genes (C). Eight single crossover events generate the remaining links between the remaining SBs (D).
Table 2.
Summary of Inter- and Intrasyntenic Gene Content of P. falciparum and Comparison to Intersyntenic Gene Content of the RMPs
Figure 4.
Inter- and Intrasyntenic Indels Contain Clusters of P. falciparum-Specific Genes
(A) A detailed illustration of the SBP between the SBs “Xa:12a” and “Xb:5a” (Pfchr10) flanked by P. yoelii contigs MALPY00409 (gray) and MALPY00055 (purple). The last gene on MALPY00409 is located on Pfchr3 (“IIIc:12b”) and defines the SBP; MALPY00055 is the last syntenic contig flanking a subtelomeric region that contains a cRMP-specific gene encoding a hypothetical protein (red) and a nonsyntenic etramp (white with red outline). The P. falciparum intersyntenic region contains three annotated genes (white with red outline): gbp130, pftstk10a, and etramp; and three genes encoding hypothetical proteins (red). Interestingly, four of six genes encode putative secreted proteins with N-terminal transmembrane domains destined for the parasite surface or infected host cell membrane (asterisks; see Table S33).
(B) A detailed illustration of a ~22-kb indel within SB “Xb:5a” that contains P. falciparum-specific genes directly upstream of a region containing genes that are highly diverged in the RMPs. Only four of 12 genes annotated on MALPY00271 have a clear ortholog (purple) and the last gene (PY01020), which encodes a hypothetical protein, shows low similarity at the N-terminal end with PF10_0348 (horizontal purple lines). Comparison of the P. yoelii and P. falciparum annotations revealed the presence in both species of six genes with the same orientation and comparable size, including four genes that encode hypothetical proteins (black with purple outline) and two annotated genes (white with purple outline): the putative P. yoelii lsa1, and the putative msp paralog P. yoelii H103. MALPY01161 and MALPY00271 are physically linked as determined with Grouper software and are therefore between 500 and 2,000 bp apart, leaving no space for the remaining genes in the ~22 kb P. falciparum indel that include S-antigen, glurp, msp3, msp6, and H101 (all white with red outline) and one gene encoding a hypothetical protein (red). In the entire regions, 12 of 15 genes encode putative secreted proteins destined for the parasite surface or infected host cell membrane (asterisks; see Table S34).
Figure 5.
Origin and Putative Mechanism of Expansion of the tstk Family in P. falciparum
(A) Analysis of P. falciparum-specific genes at the SBPs revealed a gene family encoding receptor-associated protein kinases (TSTK). Maximum likelihood distances were calculated for the C-terminal 400 amino acids of all TSTKs, including those found for other Plasmodium species, Toxoplasma gondii, Cryptosporidium parvum, and C. hominis. The tree was rooted using the clade with the three non-Plasmodium sequences as the outgroup (shaded dark gray). The syntenic progenitor genes clearly form one clade (shaded light gray), while the clustering of the other 20 mainly subtelomeric pftstk is more ambiguous (the three non-subtelomeric copies are shown in bold and include pftstk7a, which appears most closely related to the clade of progenitor genes). Circles represent branch points with bootstrap values of 100% (white), 90%–99% (light gray) and 65%–89% (dark gray).
(B) See Figure 2 for the numbering of the SBs and the symbols used in this figure. Based on the 15 recombination events described in Figure 3 and the phylogenetic analysis of the tstk family, we suggest the origin and putative evolution of the pftstk family as shown here. Phylogenetic analysis suggests that the intersyntenic pftstk7a is most closely related to the progenitor founder gene, pftstk0. Interestingly, this gene is the first nonsyntenic gene upstream of SB “VIIe:2b.” This SB is linked in the cRMP genome to SB “I:2a” that in P. falciparum is also flanked by a member of the tstk family, the subtelomeric pftstk1. Based on these observations we suggest that the founder gene pftstk0 was duplicated after the split of P. falciparum from the other Plasmodium species but before SBs “VIIe:2b” and “I:2a” were separated (1). This gene was then directly involved in the breakage of this link, creating Pfchr1 (“I:2a”) and destroying the telomere of “VIId:6d” by addition of “VIIe:2b” (2). During this recombination process, the gene was duplicated and is now present not only as two chromosome-internal copies on “VIIIc:12d” (pftstk0) and between “VIId:6d” and “VIIe:2b” (pftstk7a) but also as a first telomeric copy on the newly formed telomere of Pfchr1 (pftstk1). From here the gene could expand to the other subtelomeric regions (3). Local gene duplications resulted in the generation of seven copies on Pfchr9 and two copies on Pfchr4. After a copy of pftstk ended up at the left-hand cRMP subtelomeric end of SB “Xb:5a,” the telomere conversion linked SB “Xa:12a” to SB “Xb:5a,” which turned this telomeric copy into an intersyntenic gene (pftstk10a). The last non-subtelomeric copy, pftstk13, most likely resulted from a different process of mobility of P. falciparum-specific elements creating the intrasyntenic genes.