Analyses of genetic variations at microsatellite loci present in-and-around the Pfcrt gene in Indian Plasmodium falciparum Kshipra Chauhan a , Veena Pande b , Aparup Das a,⇑ a Evolutionary Genomics and Bioinformatics Laboratory, Division of Genomics and Bioinformatics, National Institute of Malaria Research, Sector 8, Dwarka, New Delhi 110077, India b Department of Biotechnology, Kumaun University, Nainital 263001, Uttarakhand, India article info Article history: Received 5 August 2013 Received in revised form 10 October 2013 Accepted 10 October 2013 Available online 21 October 2013 Keywords: Malaria Plasmodium falciparum India Microsatellites Pfcrt Genetic diversity abstract Evolution and spread of chloroquine resistant (CQR) malaria parasite Plasmodium falciparum have posed great threat in malaria intervention across the globe. The occurrence of K76T mutation in the P. falcipa- rum chloroquine resistance transporter (pfcrt) gene has been widely attributed to CQR with four neigh- boring mutations providing compensatory fitness benefit to the parasite survival. Understanding evolutionary patterns of the pfcrt gene is of great relevance not only for devising new malaria control measures but also could serve as a model to understand evolution and spread of other human drug-resis- tant pathogens. Several studies, mainly based on differential patterns of diversities of the microsatellite loci placed in-and-around the pfcrt gene have indicated the role of positive natural selection under the ‘hitchhiking’ model of molecular evolution. However, the studies were restricted to limited number of microsatellite loci present inside the pfcrt gene. Moreover, comparatively higher level of diversities in microsatellite loci present inside the pfcrt gene than the loci flanking the pfcrt gene are hallmarks of Indian P. falciparum, presenting contrasting evolutionary models to global isolates. With a view to infer evolutionary patterns of the pfcrt gene in Indian P. falciparum, we have adopted a unique sampling scheme of two types of populations (cultured and field collected) and utilized 20 polymorphic microsat- ellite loci (16 located inside the pfcrt gene and four in the two flanking regions) to disentangle between genetic drift (inbred cultured isolates) and natural selection (field isolates). Data analyses employing dif- ferent population genetic tests could not straightforwardly explain either the model invoking ‘genetic hitchhiking’ or ‘genetic drift’. However, complex evolutionary models influenced by both demography and natural selection or an alternative model of natural selection (e.g. diversifying/balancing selection) might better explain the observed microsatellite variation in-and-around the pfcrt gene in Indian P. falciparum. Ó 2013 Elsevier B.V. All rights reserved. 1. Introduction Variations in microsatellite (functionally inert components of the genome) repeats are generally considered as neutral polymor- phisms that help in inferring population genetic structure of spe- cies populations (Anderson et al., 2000; Ellegren, 2004; Tyagi et al., 2011). Due to their high abundance in genomes and higher degree of genetic variations, microsatellites have been studied in many model as well as non-model organisms to successfully estab- lish history of species populations (Anderson et al., 2000; Dieringer et al., 2000; Serrano et al., 2009; Schultz et al., 2010; Wu et al., 2011). Since microsatellites are considered to be highly mutable regions of the genome, inferences of evolutionary history using these repeat polymorphisms are considered to be ‘recent’. Apart from inferring species evolutionary history, microsatellite repeats also offer great opportunities in establishing local signature of po- sitive natural selection under the hitchhiking (or selective sweep) model (Maynard Smith and Haigh, 1974) with the help of popula- tion genetic data analyses (Schlotterer, 2003, 2004). Therefore, whether any specific functional genetic region has been recently targeted by positive natural selection could be well visualized by analyzing patterns of microsatellite diversities in-and-around the concerned functional gene under population genetic framework (Nair et al., 2003; Stephan, 2010; Wootton et al., 2002). To this respect, malaria is a highly infectious disease in the trop- ical and sub-tropical regions of the globe causing high mortality and morbidity. As part of intervention measures; several antima- larial compounds were used in the past (Schlitzer, 2008). No other antimalarial than chloroquine (CQ) was that successful as malaria chemotherapy for a longer duration, and due to its high efficacy, low side effects and affordability by the community, CQ was once considered to be the ‘golden drug’ for malaria treatment. However, malaria parasite, Plasmodium falciparum quickly developed 1567-1348/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2013.10.010 ⇑ Corresponding author. Tel.: +91 011 25307322; fax: +91 011 25307377. E-mail address: aparup@mrcindia.org (A. Das). Infection, Genetics and Evolution 20 (2013) 476–487 Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid