MBE Advance Access originally published online on February 28, 2006
Molecular Biology and Evolution 2006 23(5):1016-1018; doi:10.1093/molbev/msj116
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Letter |
Microsatellite Variation, Repeat Array Length, and Population History of Plasmodium vivax
* Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, Texas; Malaria Research Group, International Centre for Medical Research and Training, Cali, Colombia; The Institute for Genomic Research, Rockville, Maryland; and || Centre for Tropical Medicine and Vaccinology, Churchill Hospital, Oxford, United Kingdom
E-mail: tanderso{at}darwin.sfbr.org.
Key Words: microsatellite • array length • heterozygosity • selection • bottleneck
A recent paper (Leclerc et al. 2004
) described limited variation in dinucleotide microsatellites from Plasmodium vivax, suggesting very recent bottlenecks or genome-wide selective events. We describe patterns of variation in 11 dinucleotide microsatellites in P. vivax populations from Colombia, India, and Thailand. We find abundant variation with heterozygosity of 0.64, 0.76, and 0.77, respectively, in the three countries. The discrepancy between these two studies results is simply explained by the differences in the size of repeat arrays. The microsatellites studied by Leclerc et al. (2004) have very few repeats (median 5.5, range 4–13) and so would not be expected to be variable. Plasmodium vivax microsatellites show comparable levels of variation to those in Plasmodium falciparum when repeat array length is taken into account and provide no support for recent bottlenecks or widespread selective purging of variation from the genome of P. vivax.
The unusual patterns of variation in the P. falciparum genome have generated a lively debate about parasite origins and evolutionary history (Su, Mu, and Joy 2003; Hartl 2004). Recent studies have also revealed conflicting views on the ancestry of the related parasite Plasmodium vivax. Sequencing studies of both mitochondrial DNA and nuclear genes suggest a most recent common ancestor between 200,000 and 314,000 years ago in P. vivax (Feng et al. 2003; Escalante et al. 2005; Jongwutiwes et al. 2005). However, patterns of microsatellite variation muddy the picture. Leclerc et al. (2004) isolated 13 microsatellite sequences and found that 9/12 were monomorphic in eight populations examined, while of the remaining four loci only one showed extensive polymorphism. Because microsatellite repeats characteristically show high mutation rates relative to single nucleotide polymorphisms (Ellegren 2004), these data might suggest either expansion from a recent bottleneck (<10,000 years ago) and/or the recent removal of variation as a consequence of multiple selective events. However, such recent events are also expected to remove sequence variation, which clearly has not occurred. It therefore seems likely that there is an alternative explanation for the meager variation observed in the microsatellite data of Leclerc et al. (2004).
To further evaluate microsatellite variation in P. vivax, we screened the unpublished genome sequence data generated by The Institute for Genomic Research (TIGR) (http://www.tigr.org) for repeats using TANDEM REPEAT FINDER (Benson 1999) and designed oligos to amplify 16 dinucleotide microsatellite sequences. Five microsatellites amplified poorly or were not interpretable and were discarded. The remaining markers were assigned to chromosomes by comparison with the draft genome sequence for P. vivax. Nine of the markers were each found on different chromosomes, while two were situated on short contigs that have not yet been assigned to chromosomes (table 1). We measured length variation in these 11 markers in P. vivax populations from Thailand (n = 28), India (n = 27), and Colombia (n = 27). Genotyping was performed on an ABI 3100 capillary sequencer using GENESCAN and GENOTYPER software, and products were sized by comparison to LIZ-500 size standards (table 1). The samples from Thailand were collected from patients visiting the hospital from tropical diseases in Bangkok, Indian samples were collected from symptomatic patients at Calcutta School of Tropical Medicine, while Colombian parasites were collected from five different locations (Quibdo, Buenaventura, Guapi, and Tumaco on the coast west of the Andes and Amazonas state to the east of the Andes). All samples were collected with ethical permission from review boards in Thailand, India, and Colombia and from the Institutional Review Board of the University of Texas at San Antonio. We measured expected heterozygosity (He) at each locus using the formula 
where p is the frequency of ith allele and n is the number of alleles sampled. Where multiple alleles were observed within an infection, suggesting that >1 clone is present, we used only the predominant allele for calculation of He. All markers examined were polymorphic with 7–18 alleles per locus and mean expected heterozygosity (He) ± standard deviation (SD) of 0.64 ± 0.25, 0.76 ± 0.15, and 0.77 ± 0.18 in Colombia, India, and Thailand, respectively. These data and the high diversity observed at a single microsatellite sequence by Gomez et al. (2003) demonstrate that many microsatellites show high levels of variation in P. vivax.
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What might explain the difference between these studies and the meager variation observed in the data of Leclerc et al. (2004)
? Microsatellite variation is strongly dependent on the length of repeat arrays. Evidence for this comes from experimental studies in microorganisms (Wierdl, Dominska, and Petes 1997) and from observing mutation in pedigree studies (Brohede, Moller, and Ellegren 2004). Furthermore, descriptive studies of numerous organisms including Plasmodium (Anderson et al. 2000) invariably show higher levels of variation in loci with long repeat arrays than those with short repeat arrays (Ellegren 2004) and highlight the importance of standardizing measures of genetic variation by repeat array length (Petit et al. 2005). The relationship between array length and genetic variability is not linear. There is a lower threshold length below which slippage mutations are rare and an exponential increase in slippage with increasing repeat number (Lai and Sun 2003). There is a simple explanation for the minimal diversity observed in the data of Leclerc et al. (2004): the microsatellite sequences isolated by these authors have very short repeat arrays (median = 5.5, range 4–13) and so would not be expected to show high levels of variation. In contrast, we examined microsatellites with 12–18 repeats (median = 16) in the genome sequence strain (Salvador 1). Interestingly, the single locus showing elevated variation in the data of Leclerc et al. (2004) had 13 repeats in the sequenced clone (fig. 1).
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Our data from Colombia, India, and Thailand reveal comparable levels of variation with data previously collected from P. falciparum. For example, Nair et al. (2003)
sampled 58 P. falciparum dinucleotide microsatellite markers from Chr 1, 2, 3, and 12 in parasites from the Thailand-Burma border. Of these, 24 had between 12 and 18 pure AT repeats (median = 16) in the genome sequence and a mean He ± SD of 0.82 ± 0.08. Similarly, 20 dinucleotide microsatellites with 12–18 pure repeat arrays (median = 16) sampled from across the genome in 12 parasite isolates from worldwide locations had mean He ± SD of 0.81 ± 0.08 (Anderson et al. 2000). Hence, dinucleotide microsatellites from P. vivax show comparable variation to P. falciparum microsatellites with similar repeat array length and structure.
These data demonstrate the importance of accounting for repeat array length when interpreting microsatellite data. Plasmodium vivax microsatellite sequences show comparable levels of variation to those seen in P. falciparum when repeat array length is taken into account and provide little support for recent origins or multiple selective events in this species. Rather, microsatellite sequences with short repeat arrays such as those isolated by Leclerc et al. (2004) would be expected to have very low mutation rates. While microsatellites are considerably less common in the P. vivax genome than in the AT-rich P. falciparum genome and also tend to be shorter in length, these markers can still provide useful tools for assessing population structure and for searching for evidence of recent selection events associated with drug resistance.
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Acknowledgements |
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 TOP Acknowledgements References |
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Preliminary sequence data from which microsatellite primers were designed were obtained from TIGR (http://www.tigr.org). Funding for the P. vivax sequencing project came from the National Institutes for Allergy and Infectious Disease, the U.S. Department of Defense, and the Burroughs Wellcome Fund. Financial support was provided by a Wellcome Trust fellowship to M.I. and National Institutes of Health (NIH) grant RO1 AI48071 to T.J.C.A. N.J.W. and N.P.J.D. were supported by the Wellcome Trust of Great Britain. This investigation was conducted in facilities constructed with support from Research Facilities Improvement Program Grant Number C06 RR013556 from the National Center for Research Resources, NIH.
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Footnotes |
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Laura Katz, Associate Editor
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References |
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TOPAcknowledgementsReferences |
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