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Proc Natl Acad Sci U S A. Jul 8, 2003; 100(14): 8502–8507.
Published online Jun 10, 2003. doi:  10.1073/pnas.1232502100
PMCID: PMC166258
Microbiology

Single-nucleotide polymorphisms and genome diversity in Plasmodium vivax

Abstract

The study of genetic variation in malaria parasites has practical significance for developing strategies to control the disease. Vaccines based on highly polymorphic antigens may be confounded by allelic restriction of the host immune response. In response to drug pressure, a highly plastic genome may generate resistant mutants more easily than a monomorphic one. Additionally, the study of the distribution of genomic polymorphisms may provide information leading to the identification of genes associated with traits such as parasite development and drug resistance. Indeed, the age and diversity of the human malaria parasite Plasmodium falciparum has been the subject of recent debate, because an ancient parasite with a complex genome is expected to present greater challenges for drug and vaccine development. The genome diversity of the important human pathogen Plasmodium vivax, however, remains essentially unknown. Here we analyze an ≈100-kb contiguous chromosome segment from five isolates, revealing 191 single-nucleotide polymorphisms (SNPs) and 44 size polymorphisms. The SNPs are not evenly distributed across the segment with blocks of high and low diversity. Whereas the majority (≈63%) of the SNPs are in intergenic regions, introns contain significantly less SNPs than intergenic sequences. Polymorphic tandem repeats are abundant and are more uniformly distributed at a frequency of about one polymorphic tandem repeat per 3 kb. These data show that P. vivax has a highly diverse genome, and provide useful information for further understanding the genome diversity of the parasite.

Plasmodium vivax is the most widespread species of human malaria parasite and is found throughout Central and South America, the Middle East, Central, South, Northeast, and Southeast Asia, and parts of Africa. Although rarely fatal, P. vivax parasites cause debilitating disease that severely affects the quality of life and economic productivity of its victims (1). Compared with the more virulent parasite Plasmodium falciparum, much less is known concerning the basic biology and genome diversity of P. vivax, primarily because of the difficulty of working with a species for which a continuous in vitro culture system is not available. Currently, studies involving P. vivax use lines adapted to growth in New World monkeys (2), with the associated problems of cost and availability.

The P. vivax genome, like all Plasmodium genomes studied so far, appears to be distributed among 14 haploid chromosomes (3), but has a much lower AT content (≈55%; ref. 4) in comparison to the genome of P. falciparum (≈80%; ref. 5). Conservation of gene synteny is extensive among all of the Plasmodium species and declines as the evolutionary distance between the species increases (3, 6). Despite the limited availability of P. vivax biological material, recent research has led to the construction of a P. vivax whole-genome yeast artificial chromosome library (7), a collection of ≈11,000 annotated P. vivax gene sequence tags (4), the identification of a P. vivax multigene family with a possible role in antigenic variation (8), and the completion of a 200-kb contiguous DNA sequence from one P. vivax chromosome (9). A whole genome shotgun project to sequence and annotate the P. vivax genome is also underway (www.tigr.org/tdb/ezk1/pva1). However, only a few genome markers, mostly orthologs of previously identified P. falciparum antigen genes, have been used for population studies of P. vivax (10, 11), in contrast to ≈1,000 polymorphic microsatellites (12) and hundreds of restriction fragment length polymorphism and single-nucleotide polymorphism (SNP) markers (13, 14) described for P. falciparum. The lack of genetic markers for the P. vivax genome has severely hampered an in-depth analysis of the population structure and evolutionary history of the parasite and prevented efforts to map determinants contributing to important parasite phenotypes such as drug resistance and relapse patterns (1519).

SNPs have received considerable attention recently because of their potential as markers for genetic mapping and for studying molecular evolution and population dynamics (20, 21). In P. falciparum, a dense SNP map for chromosome 3 with hundreds of SNPs has been developed (14), and numerous polymorphic sites on chromosome 2 have also been identified by microarray hybridization (22). SNPs are relatively easy to assay and are often present at high frequency, making them ideal genetic markers (23). Identification and development of large numbers of genetic markers such as SNPs from P. vivax will provide a framework on which studies of molecular evolution and genetic mapping can be based. Here, we analyze an ≈100-kb DNA segment from five isolates collected from geographically diverse areas of the world. We have also screened the segments for repeat sequences and developed 33 PCR-based polymorphic markers that will be useful for genetic studies in the field. This study reveals P. vivax as a genetically diverse species with an abundance of SNPs and polymorphic tandem repeats (TRs) that are useful tools for further genetic characterization of this parasite.

Methods

Parasite Isolates and DNA Extraction. Four P. vivax laboratory-adapted lines isolated from different geographical regions were chosen for sequencing: (i) India VII, isolated from a patient from India (24); (ii) Sal I from El Salvador (25); (iii) Belem from Brazil (26); and (iv) Thai-NYU from Thailand (27). All lines have been adapted for growth in New World monkeys. Infected monkey blood was harvested, and monkey leukocytes were removed by filtration. DNA was extracted from the parasites by using a QIAamp kit (Qiagen, Valencia, CA).

Amplification of DNA Sequences from Isolates. To identify potential SNPs by using existing P. vivax sequence data, we sequenced 100,924 bases of DNA from a 200-kb contiguous DNA sequence (coordinates 46,279–147,203 of yeast artificial chromosome 1H14; ref. 9) of one P. vivax chromosome, a region that is syntenic to a section of chromosome 3 of P. falciparum. Oligonucleotide primers along the P. vivax sequence, spaced ≈400 bases apart and 16–20 bases in length with a minimum of secondary structure, were synthesized. DNA was amplified by PCR using the following conditions: 4 μl of DNA (≈5 ng), 0.5 μl (≈50 pM) of primer (Invitrogen), and 45 μl of PCR mix containing 5 μl of 10× PCR buffer, 1.0 μl of dNTPs (10 mM), and 0.1 μl (5 units/μl) of Taq polymerase (Invitrogen). All amplifications were performed with one cycling condition: 94°C for 2 min, 35 cycles of 94°C for 20 sec, 55°C for 10 sec, 50°C for 10 sec, and 65°C for 1–2 min, and a final extension step of 65°C for 5 min.

DNA Sequencing. Five microliters of the PCR products were run on a 1% agarose gel to check for quality of amplification. If single bands and no apparent “primer–dimer” were present, the PCR product was treated with 1 μl of ExoSAP-IT (United States Biochemical) at 37°C for 15 min and at 80°C for another 15 min. Each sequencing reaction used 2–5 μl of the PCR product and dichlororhodamine or BigDye terminator chemistry on an ABI377 or ABI3100 automatic DNA sequencer (Applied Biosystems). Sequence cycling was as follows: denaturing at 94°C for 2 min, amplification for 25–27 cycles at 94°C for 20 sec, 50°C for 5 sec, 48°C for 5 sec, and 60°C for 3 min, and a final extension at 60°C for 5 min. The majority of the sequences (≈93%) have multiple-read coverage from independent PCR amplifications, and all SNPs were validated by sequencing independent PCR products covering DNA segments with SNPs. Five gaps of 20–50 bases flanked by polyA and/or polyT stretches could not be sequenced.

Data Analysis. SNPs were identified after DNA sequence alignment using SEQUENCHER 4.0 (Gene Codes, Ann Arbor, MI). The quality of the DNA sequences and individual SNPs were assessed by visual inspection with reference to the chromatograms. Repetitive sequences were aligned by using SEQUENCHER 4.0 initially, then with visual assistance to minimize artifactual SNPs. Nucleotide diversity (π) was estimated from the number of segregating sites (28) as implemented in DNASP 3.99 (ref. 29; www.ub.es/dnasp/betas/DnaSP_Beta399.html). Codon biax index (30), effective number of codons (31), and other parameters were also calculated by using DNASP. The mean nonsynonymous substitutions per nonsynonymous site (dN) and the mean synonymous substitution per synonymous site (dS) were estimated by using a modified Nei–Gojobori distance method in MEGA2.1 (32). Standard errors for the dN and dS estimates were determined by using 1,000 bootstrap replicates.

Results

SNPs from a 100-kb Chromosome Segment. To study the genome diversity of the P. vivax parasite, we sequenced ≈100 kb of DNA containing 26 predicted genes from four P. vivax isolates. The DNA sequences from all four isolates plus the reference sequence deposited in the GenBank database (9) were aligned to identify SNPs. One hundred and ninety-one SNPs, including 66 coding SNPs (cSNPs) from 18 of the 26 genes, excluding the repeat regions in the circumsporozoite surface protein (csp) gene because of uncertainty of the sequence alignments, were identified (Table 1, Fig. 1). This frequency of SNPs is higher than that obtained from 204 predicted genes on P. falciparum chromosome 3 (Table 2; ref. 14), which is most likely a reflection of the greater number of intergenic regions analyzed in this study and also indicative of a highly polymorphic P. vivax genome. Among the 66 cSNPs, 24 are sSNP and 42 are nsSNP, yielding a ratio of nsSNP to sSNP of 1.75, which is similar to that seen in P falciparum (2.34). This finding is in sharp contrast to what has been found in the human genome (0.89), where functional constraints on amino acid changes are thought to reduce the number of nsSNPs in coding regions (33, 34).

Fig. 1.
SNP distribution, nucleotide diversity, base composition, and SNP haplotypes of a 100-kb P. vivax chromosomal segment. (A) A physical map integrated with SNPs and polymorphic TRs. Vertical lines above the thick horizontal chromosome line represent ...
Table 1.
Comparison of nucleotide substitution and diversity in 26 orthologous genes from P. vivax and P. falciparum
Table 2.
Comparison of SNP frequency and distribution between P. vivax and P. falciparum

In P. falciparum, part of the bias toward nsSNP can be explained by a reduction in the number of synonymous sites due to the extremely AT rich genome and consequent codon bias (14). Interestingly, the number of synonymous sites in P. vivax (22.1%) is similar to that found in P. falciparum (19.1%), despite the much lower AT content of the P. vivax genome. However, there is no obvious correlation between the percentages of synonymous sites with the AT content, the codon biax index, or the effective number of codons (data not shown), suggesting that neither AT content nor codon usage contributes to the reduction of synonymous sites. Positive selection by drug and/or immune pressure on some of the genes and the paucity of synonymous sites may provide an explanation for the increased number of nsSNPs.

SNP Distribution, Neutrality Tests, and Evidence of Constraint on Introns. The SNPs identified from our alignment are not distributed evenly across the segment, with the majority (120, or ≈63%) found in intergenic sequence (Fig. 1 A and B), averaging 4.9 noncoding SNPs (ncSNPs) per intergenic sequence. The majority of the intergenic sequences are highly polymorphic except for three regions that do not have SNPs (Table 3). The high density of ncSNPs (one SNP per 405 bp) may reflect a reduced level of functional constraint on P. vivax intergenic regions. Synonymous changes in coding sequences are also frequent, with one sSNP per 448 synonymous sites. This frequency of sSNPs is higher than that seen in P. falciparum (one per 620 bp), although it is not as high as the frequency of ncSNPs in intergenic regions. Significantly, fewer SNPs were identified in introns (five SNPs total, one per 944 bp), a frequency similar to that of nonsynonymous changes in the coding regions (Table 2). Assuming that introns have the same substitution rate as intergenic sequences, we would expect to see 12 SNPs instead of the 5 observed. This is a significant reduction in the substitution rate in the introns (χ2 = 4.026; P < 0.05) and suggests that functional constraints are acting on intron sequences.

Table 3.
Nucleotide diversity in the intergenic regions

To investigate nucleotide substitution patterns in the coding regions, we calculated dN and dS for each individual gene and pairwise dN and dS for entire coding regions. Although no significant positive or purifying selection can be detected when all of the coding sequences were analyzed as a whole (P = 0.063–0.928; Table 4, which is published as supporting information on the PNAS web site, www.pnas.org), significant differences in dN and dS were detected in two of the 18 individual genes with SNPs (Table 1). At locus 14110w (a hypothetical protein), dN is significantly greater than dS (P < 0.034), suggesting that the nonsynonymous sites are under positive selection, possibly from host immune or drug selection pressure, although constraints on synonymous changes could also explain this pattern. For gene 14070c (N-ethylmaleimide-sensitive fusion protein NSF), dS is significantly greater than dN (P < 0.024), indicating possible purifying selection operating on this gene. Indeed, a close inspection of the 100-kb DNA fragment identified two blocks, from gene 14055c to gene 14075c (≈30 kb) and from gene 14130c to gene 14145c (≈15 kb), that carry few nsSNPs, suggesting functional constraint (Fig. 1A, Table 1). The first block contains five well conserved genes, including 14070c, arranged in the same orientation. All of the three nsSNPs found in this block (average dN = 0.0001 ± 0.00006 compared with 0.0005 ± 0.0003 for all of the genes) are in gene 14075c, which is located at one end of the block. Not only are there fewer nsSNPs in these segments, but shared haplotype blocks, mostly consisting of ncSNPs, can also be seen among the Belem, Thai-NYU, and Sal I isolates (Fig. 1D). The second block (14130c–14145c) has only one coding SNP. Again, shared haplotypes can be identified from this region (Fig. 1D, block 2; average dN = 0.00006 ± 0.00005). Two of the four genes in this block are 60S ribosomal protein L44 and glutaredoxin. The section between the two blocks (≈30 kb) shows a more even distribution of SNPs and has a mosaic haplotype pattern (Fig. 1 A and D). The haplotype patterns also suggest that Belem and Thai-NYU isolates are closely related, which is supported by a dendrogram constructed based on the SNPs among the five isolates (Fig. 2, which is published as supporting information on the PNAS web site).

Comparison of Nucleotide Substitutions in Orthologous Genes and Interspecific Divergence of P. vivax and P. falciparum. In a previous study of diversity in P. falciparum (14), we compared polymorphisms identified within 204 genes (or gene fragments) on chromosome 3 among five P. falciparum isolates. The region of P. vivax described in this report is syntenic to P. falciparum chromosome 3, and the 26 genes described here are orthologs in the two species. Comparison of dN and dS from the 26 orthologs shows different distributions of nonsynonymous and synonymous SNPs in the two species (Table 1); most differ in the dN and dS patterns or ratio, suggesting different selection pressures and/or evolutionary histories, although some differences may simply reflect stochastic effects. The substitution rates are quite different in the nonrepeating regions of the csp genes. Whereas 11 nsSNPs were found in the five P. falciparum csp genes studied, clear evidence for the gene being under positive selection (P = 0.002), only one nsSNP and one sSNP were identified in the five P. vivax csp genes (P = 0.465). Although the number of parasite isolates analyzed is small, and repeat regions were excluded because of alignment difficulties, this difference may indicate that the csp gene is under different immune pressure in the two species. Similarly, dS for the P. falciparum gene c0125w (an ABC transporter), but not its orthologous 14050w, is significantly greater than its dN, indicating potential purifying selection. In contrast, whereas the P. vivax 14070c and 14110w have significant P values, the two P. falciparum orthologs, c0140c and c0175w, do not. Orthologs 14155c and c0215c (a hypothetical protein), however, have five nsNSPs (dN > dS), suggesting similar positive selection pressure acting on the gene in both species, although the P value for 14155c is not significant. Furthermore, the two conserved segments (Fig. 1, blocks 1 and 2) also appear to be conserved in P. falciparum; only one nsSNP is found in the first segment (c0126c–c0155c, 12,529 bp; average dN = 0.00009) and none are found in the second segment (c0190w–c0205c, 3,182 bp).

In addition to intraspecific polymorphism, we also examined the pattern of interspecific divergence between P. vivax and P. falciparum in the 100-kb syntenic region by using dN and dS as indicators for selective constraints. Because of the sensitivity of dN and dS estimations to the quality of sequence alignment, only nine orthologous genes that have sequence identity >55% (55–77%, mean 65.5%, except for the nonrepeating csp region, which has ≈40% nucleotide identity) were analyzed. For the nine genes, dN has a range of 0.027–0.428 (mean 0.249 ± 0.016); and the range of dS is 0.055–0.776 (mean 0.573 ± 0.032). These data suggest that orthologous genes evolve at different rates with different mutation patterns, and reject the hypothesis of neutral evolution (dN = dS) governing sequence divergence between the two species (P < 0.001 for all of the eight genes except the csp genes; Table 5, which is published as supporting information on the PNAS web site). The range and variation of the dN/dS ratio (0.035–6.33) also show signatures of different evolutionary forces acting on individual genes (35). Selective constraint appears to be acting on nonsynonymous nucleotide substitutions in seven genes (dN < dS), in particular, the orthologs 14065c and c0135c (annotated as “conserved protein” with both intraspecific dN = 0). In contrast, for the orthologs 14130c and c0190c, dN is greater than dS, indicating positive selection favoring amino acid replacement (dN/dS = 6.33), although the intraspecific dN and dS are zero for both species. The null hypothesis of dN = dS is not rejected for the csp genes (z test, P = 0.749), and this may largely reflect the significant divergence during the csp evolution between the two parasites. The possibility of selection cannot be ruled out (dN is highest for csp) because purifying selection that occurred in the interspecific branch may be masked by the strong positive selection and rapid evolution observed in the P. falciparum internal branch, resulting in a close estimate of dN and dS. As in previous analyses, the repeat regions of the genes were excluded in the analysis.

Microsatellites and TRs. In addition to identification of SNPs between the five isolates, a search of the 100-kb DNA sequences for the presence of repeats identified: 37 polyT repeats and 39 polyA repeats >15 units, seven of which were found to be polymorphic among the isolates; three polymorphic polyG or C repeats; one polymorphic dinucleotide repeat (TA)15; and three single-nucleotide indels (these indels may be due to sequencing errors; data not shown). This finding is in contrast to the P. falciparum genome, where highly polymorphic polyT, polyA, (TA)n, and other TA-rich repeats are abundant (36). The absence of such repeats is likely due to the lower AT content of the P. vivax genome. In contrast, polymorphic TRs with repeating units of five nucleotides or greater are present at a high frequency in the P. vivax genome: 33 TRs were identified among the five isolates (Table 6, which is published as supporting information on the PNAS web site), at a frequency of one per 3 kb on average. Amplification of the TRs identified them as easy to assay by conventional PCR and agarose gel electrophoresis methods (Fig. 3, which is published as supporting information on the PNAS web site). This collection of SNPs and TRs of P. vivax constitutes a high-resolution map covering ≈100 kb of one P. vivax chromosome segment consisting of one polymorphic site per ≈0.4 kb, and represents a valuable resource for P. vivax population and genetic studies.

Discussion

This study represents a large-scale, multigene survey of variation within the P. vivax genome. Our data show that P. vivax has a highly polymorphic genome that may present some challenges for drug and vaccine development. In fact, SNPs appear to be present at a higher frequency in the P. vivax genome than in P. falciparum genome. Twenty of the 26 genes in the DNA segment are polymorphic among the isolates studied, with two thirds of the coding SNPs identified as resulting in amino acid changes. In addition, size polymorphisms are abundant, with half of the 33 TRs identified as occurring in coding regions.

This study also shows that SNPs tend to cluster in intergenic regions and even in specific genes that may be under selection. One interesting observation is that very high frequencies of SNPs are found in noncoding regions flanked by well conserved genes (Fig. 1, block 1). One possible explanation could be that the DNA segment is located at a recombination hotspot (subtelomeric region) that is frequently involved in chromosomal end exchange. A high frequency of SNPs is often positively correlated with a high recombination frequency (37). In contrast, the coding regions may be conserved because the genes are crucial to parasite survival and under functional constraint.

Although noncoding regions, including introns, are generally considered to be neutral, we found some of these regions to be conserved in our study. Recent studies indicate that some introns play a significant role in gene expression and gene regulation and may therefore not be as neutral as previously thought (3840). Purifying selection acting on exons, or selective sweeps resulting from directional positive selection acting on exons, is expected to reduce nucleotide diversity in “linked” introns. The data from this study indicate that some introns in P. vivax may be under functional constraint: A total of five SNPs were found in 25 introns from 10 genes (total 4,719 bp), averaging one SNP per ≈1 kb. This SNP frequency in introns is similar to that found in 25 introns of 10 housekeeping genes in P. falciparum (41). In contrast, a much higher frequency of SNPs is found in intergenic sequences. The uneven distribution of SNPs and variety of selection pressures acting on different genes argue for the analysis of a large number of genes and more parasite isolates, including other Plasmodium species, to chart the parasite's evolutionary history more precisely.

Analysis of allele frequency distributions can identify genetic loci under most intense selection and provide information for the identification of new drug or immune targets. High nucleotide substitution rates have been found in genes encoding proteins associated with the cell membrane in P. falciparum; most of these encode proteins of immune targets (22). Indeed, all of the currently known malaria vaccine candidates, such as pfMSP-1 (42, 43), are highly polymorphic, with the majority of substitutions leading to amino acid changes. Although none of the genes (csp is a possible exception because many SNPs in the repeating regions are not analyzed) from this chromosomal fragment has the high substitution rates seen in the known vaccine candidate genes, some of them clearly show signs of selection (such as 14110w, and possibly 14155c). A genomewide search for nucleotide substitution patterns may provide valuable information for the identification of new vaccine targets or genes involved in drug resistance.

Microsatellites are abundant in the highly AT-rich P. falciparum genome, especially (TA) microsatellite repeats (36). The P. vivax genome exhibits a more balanced nucleotide composition; hence, the paucity of such dinucleotide repeats in the region analyzed in this study is not surprising. The predominant repeat type (CA)n found in other genomes such as human and mouse is also rare in the P. vivax genome. In contrast, polymorphic TRs appear to be quite abundant, with a frequency of about one polymorphic TR per 3 kb. Based on this frequency, we can expect to see >10,000 polymorphic TRs in the ≈30-Mb genome, rendering the construction of a high-resolution genetic map a realistic option. Additionally, the TRs appear to be distributed evenly between coding and noncoding regions. Half of the 100-kb sequence analyzed is noncoding; and 17 of the 34 TRs are in noncoding regions, including three TRs in intron sequences. This even distribution of TRs suggests that these repeats may not be under strong selection, making them potentially useful markers for population studies.

In P. falciparum, microsatellites are highly polymorphic and have a high mutation rate (44, 45). Although these highly polymorphic repeats are informative and useful for studying closely related populations, the high mutation rate limits the usefulness of microsatellites in comparison of distantly related populations. The P. vivax TRs identified here have repeat units that are longer than microsatellites, but they are not typical minisatellites that usually have repeating motifs spanning several kilobases (46). Instead, most of the TRs consist of two to three imperfect repeats that appear to be less polymorphic than microsatellites or minisatellites. TRs found in the P. vivax genome may therefore prove useful in estimating recombination frequency and linkage disequilibrium, and in identifying isolate haplotypes in the field. Understanding these processes is crucial, because conventional genetic cross studies and linkage analysis to identify loci involved in significant phenotypic traits, such as drug resistance, are not feasible in P. vivax. In addition, these polymorphic repeats are easy to amplify and can be detected on an agarose gel, making the markers useful for laboratories in areas where P. vivax is endemic.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Jonathan Baker for help in the design of oligonucleotide primers; Marina Tchavtchitch and Allan Saul for access to sequence data before release; Steven Sullivan and Joana Silva for critical reading of the manuscript; and Brenda Marshall for editorial assistance.

Notes

Abbreviations: SNP, single-nucleotide polymorphism; sSNP, synonymous SNP; nsSNP, nonsynonymous SNP; TR, tandem repeat.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AY216936–AY216939).

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