title: Association mapping by pooled sequencing identifies TOLL 11 as a protective factor against Plasmodium falciparum in Anopheles gambiae.
abstract: BACKGROUND
  The genome-wide association study (GWAS) techniques that have been used for genetic mapping in other organisms have not been successfully applied to mosquitoes, which have genetic characteristics of high nucleotide diversity, low linkage disequilibrium, and complex population stratification that render population-based GWAS essentially unfeasible at realistic sample size and marker density.
  
  
  METHODS
  We designed a novel mapping strategy for the mosquito system that combines the power of linkage mapping with the resolution afforded by genetic association. We established founder colonies from West Africa, controlled for diversity, linkage disequilibrium and population stratification. Colonies were challenged by feeding on the infectious stage of the human malaria parasite, Plasmodium falciparum, mosquitoes were phenotyped for parasite load, and DNA pools for phenotypically similar mosquitoes were Illumina sequenced. Phenotype-genotype mapping was carried out in two stages, coarse and fine.
  
  
  RESULTS
  In the first mapping stage, pooled sequences were analysed genome-wide for intervals displaying relativereduction in diversity between phenotype pools, and candidate genomic loci were identified for influence upon parasite infection levels. In the second mapping stage, focused genotyping of SNPs from the first mapping stage was carried out in unpooled individual mosquitoes and replicates. The second stage confirmed significant SNPs in a locus encoding two Toll-family proteins. RNAi-mediated gene silencing and infection challenge revealed that TOLL 11 protects mosquitoes against P. falciparum infection.
  
  
  CONCLUSIONS
  We present an efficient and cost-effective method for genetic mapping using natural variation segregating in defined recent Anopheles founder colonies, and demonstrate its applicability for mapping in a complex non-model genome. This approach is a practical and preferred alternative to population-based GWAS for first-pass mapping of phenotypes in Anopheles. This design should facilitate mapping of other traits involved in physiology, epidemiology, and behaviour.
authors.affiliation: Department of Parasites and Insect Vectors, Institut Pasteur, Unit of Insect Vector Genetics and Genomics, 28 rue du Docteur Roux, Paris, 75015, France. snr@sanger.ac.uk.|CNRS Unit of Hosts, Vectors and Pathogens, Paris, France (URA3012), 28 rue du Docteur Roux, Paris, 75015, France. snr@sanger.ac.uk.
authors.forename: Seth N
authors.initials: SN
authors.lastname: Redmond
authors.affiliation: Department of Parasites and Insect Vectors, Institut Pasteur, Unit of Insect Vector Genetics and Genomics, 28 rue du Docteur Roux, Paris, 75015, France. kei@pasteur.fr.|CNRS Unit of Hosts, Vectors and Pathogens, Paris, France (URA3012), 28 rue du Docteur Roux, Paris, 75015, France. kei@pasteur.fr.
authors.forename: Karin
authors.initials: K
authors.lastname: Eiglmeier
authors.affiliation: Department of Parasites and Insect Vectors, Institut Pasteur, Unit of Insect Vector Genetics and Genomics, 28 rue du Docteur Roux, Paris, 75015, France. cmitri@pasteur.fr.|CNRS Unit of Hosts, Vectors and Pathogens, Paris, France (URA3012), 28 rue du Docteur Roux, Paris, 75015, France. cmitri@pasteur.fr.
authors.forename: Christian
authors.initials: C
authors.lastname: Mitri
authors.affiliation: Program in Genomics, Boston Children's Hospital, Harvard Medical School, 3 Blackfan Street, Boston, MA, 02115, USA. kmarkianos@enders.tch.harvard.edu.
authors.forename: Kyriacos
authors.initials: K
authors.lastname: Markianos
authors.affiliation: Centre National de Recherche et de Formation sur le Paludisme, 1487 Avenue de l'Oubritenga, 01 BP 2208, Ouagadougou, Burkina Faso. guelbeogo.cnrfp@fasonet.bf.
authors.forename: Wamdaogo M
authors.initials: WM
authors.lastname: Guelbeogo
authors.affiliation: Centre National de Recherche et de Formation sur le Paludisme, 1487 Avenue de l'Oubritenga, 01 BP 2208, Ouagadougou, Burkina Faso. gplouise@yahoo.fr.
authors.forename: Awa
authors.initials: A
authors.lastname: Gneme
authors.affiliation: Department of Parasites and Insect Vectors, Institut Pasteur, Unit of Insect Vector Genetics and Genomics, 28 rue du Docteur Roux, Paris, 75015, France. Alison.Isaacs@lstmed.ac.uk.|CNRS Unit of Hosts, Vectors and Pathogens, Paris, France (URA3012), 28 rue du Docteur Roux, Paris, 75015, France. Alison.Isaacs@lstmed.ac.uk.
authors.forename: Alison T
authors.initials: AT
authors.lastname: Isaacs
authors.affiliation: Malaria Research and Training Centre, Faculty of Medicine and Dentistry, University of Mali, Point G, Bamako, Mali. bcoulibaly@mrtcbko.org.
authors.forename: Boubacar
authors.initials: B
authors.lastname: Coulibaly
authors.affiliation: Department of Parasites and Insect Vectors, Institut Pasteur, Unit of Insect Vector Genetics and Genomics, 28 rue du Docteur Roux, Paris, 75015, France. emma.brito-fravallo@pasteur.fr.|CNRS Unit of Hosts, Vectors and Pathogens, Paris, France (URA3012), 28 rue du Docteur Roux, Paris, 75015, France. emma.brito-fravallo@pasteur.fr.
authors.forename: Emma
authors.initials: E
authors.lastname: Brito-Fravallo
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK. gmaslen@ebi.ac.uk.|Wellcome Trust Centre for Human Genetics, Oxford, UK. gmaslen@ebi.ac.uk.
authors.forename: Gareth
authors.initials: G
authors.lastname: Maslen
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK. da2@sanger.ac.uk.|Wellcome Trust Centre for Human Genetics, Oxford, UK. da2@sanger.ac.uk.
authors.forename: Daniel
authors.initials: D
authors.lastname: Mead
authors.affiliation: Malaria Research and Training Centre, Faculty of Medicine and Dentistry, University of Mali, Point G, Bamako, Mali. niare@mrtcbko.org.
authors.forename: Oumou
authors.initials: O
authors.lastname: Niare
authors.affiliation: Malaria Research and Training Centre, Faculty of Medicine and Dentistry, University of Mali, Point G, Bamako, Mali. cheick@icermali.org.
authors.forename: Sekou F
authors.initials: SF
authors.lastname: Traore
authors.affiliation: Centre National de Recherche et de Formation sur le Paludisme, 1487 Avenue de l'Oubritenga, 01 BP 2208, Ouagadougou, Burkina Faso. n.fale.cnlp@fasonet.bf.
authors.forename: N'Fale
authors.initials: N
authors.lastname: Sagnon
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK. dominic@well.ox.ac.uk.|Wellcome Trust Centre for Human Genetics, Oxford, UK. dominic@well.ox.ac.uk.
authors.forename: Dominic
authors.initials: D
authors.lastname: Kwiatkowski
authors.affiliation: Department of Microbiology, University of Minnesota, 1500 Gortner Avenue, Saint Paul, MN 55108, USA. mriehle@umn.edu.
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Riehle
authors.affiliation: Department of Parasites and Insect Vectors, Institut Pasteur, Unit of Insect Vector Genetics and Genomics, 28 rue du Docteur Roux, Paris, 75015, France. kvernick@pasteur.fr.|CNRS Unit of Hosts, Vectors and Pathogens, Paris, France (URA3012), 28 rue du Docteur Roux, Paris, 75015, France. kvernick@pasteur.fr.|Malaria Research and Training Centre, Faculty of Medicine and Dentistry, University of Mali, Point G, Bamako, Mali. kvernick@pasteur.fr.
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Vernick
mesh_terms: D000818:Animals; D000852:Anopheles / Q000235:genetics* / Q000469:parasitology; D002874:Chromosome Mapping; D049750:Genome, Insect; D055106:Genome-Wide Association Study; D005838:Genotype; D006790:Host-Parasite Interactions / Q000235:genetics; D006801:Humans; D007303:Insect Vectors / Q000235:genetics; D016778:Malaria, Falciparum / Q000235:genetics* / Q000469:parasitology / Q000635:transmission; D010641:Phenotype; D010963:Plasmodium falciparum / Q000235:genetics* / Q000472:pathogenicity; D020641:Polymorphism, Single Nucleotide; D051193:Toll-Like Receptors / Q000235:genetics*
chemical_list: D051193:Toll-Like Receptors