Dataset Information

IMP PCR primers detect single nucleotide polymorphisms for Anopheles gambiae species identification, Mopti and Savanna rDNA types, and resistance to dieldrin in Anopheles arabiensis.

ABSTRACT: Polymerase chain reactions to distinguish single-nucleotide polymorphisms are commonly used for mosquito identification and identifying insecticide resistance alleles. However, the existing methods used for primer design often result in analyses that are not robust or require additional steps.Utilizing oligonucleotides that are unique in having an intentional mismatch to both templates three bases from the SNP at the 3-prime end, three new PCR assays that distinguish SNP targets using standard gel electrophoresis of undigested DNA fragments were developed and tested. These were applied to: (1) an alternative ribosomal DNA PCR assay to distinguish five members of the Anopheles gambiae complex; (2) detection of the Mopti and Savanna rDNA types; and (3) an assay to distinguish resistance to dieldrin (Rdl) alleles in Anopheles arabiensis.Reproducible specific amplification of the target alleles was observed in all three assays. The results were consistent with existing analyses but proved simpler and the results more distinct in our hands.The simplicity and effectiveness of the method should be utilized in these and other PCR analyses to increase their specificity and simplicity. These results have the potential to be extended not only to mosquito analyses but also to parasite and human polymorphisms.

SUBMITTER: Wilkins EE

PROVIDER: S-EPMC1769388 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature

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IMP PCR primers detect single nucleotide polymorphisms for Anopheles gambiae species identification, Mopti and Savanna rDNA types, and resistance to dieldrin in Anopheles arabiensis.

Wilkins Elien E EE Howell Paul I PI Benedict Mark Q MQ

Malaria journal 20061219

<h4>Background</h4>Polymerase chain reactions to distinguish single-nucleotide polymorphisms are commonly used for mosquito identification and identifying insecticide resistance alleles. However, the existing methods used for primer design often result in analyses that are not robust or require additional steps.<h4>Methods</h4>Utilizing oligonucleotides that are unique in having an intentional mismatch to both templates three bases from the SNP at the 3-prime end, three new PCR assays that disti ...[more]

PMID: 17177993

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Project description:BackgroundGenetic diversity is a key factor that enables adaptation and persistence of natural populations towards environmental conditions. It is influenced by the interaction of a natural population's dynamics and the environment it inhabits. Anopheles gambiae s.s. and Anopheles arabiensis are the two major and widespread malaria vectors in sub-Saharan Africa. Several studies have examined the ecology and population dynamics of these vectors. Ecological conditions along the Kilombero valley in Tanzania influence the distribution and population density of these two vector species. It remains unclear whether the ecological diversity within the Kilombero valley has affected the population structure of An. gambiae s.l. populations. The goal of this study was to characterise the genetic structure of sympatric An. gambiae s.s and An. arabiensis populations along the Kilombero valley.MethodologyMosquitoes were collected from seven locations in Tanzania: six from the Kilombero valley and one outside the valley (-700 km away) as an out-group. To archive a genome-wide coverage, 13 microsatellite markers from chromosomes X, 2 and 3 were used.ResultsHigh levels of genetic differentiation among An. arabiensis populations was observed, as opposed to An. gambiae s.s., which was genetically undifferentiated across the 6,650 km2 of the Kilombero valley landscape. It appears that genetic differentiation is not attributed to physical barriers or distance, but possibly by ecological diversification within the Kilombero valley. Genetic divergence among An. arabiensis populations (FST = 0.066) was higher than that of the well-known M and S forms of An. gambiae s. s. in West and Central Africa (FST = 0.035), suggesting that these populations are maintained by some level of reproductive isolation.ConclusionIt was hypothesized that ecological diversification across the valley may be a driving force for observed An. arabiensis genetic divergence. The impact of the observed An. arabiensis substructure to the prospects for new vector control approaches is discussed.

Project description:BACKGROUND: Immune responses to parasites, which start with pathogen recognition, play a decisive role in the control of the infection in mosquitoes. Peptidoglycan recognition proteins (PGRPs) are an important family of pattern recognition receptors that are involved in the activation of these immune reactions. Pathogen pressure can exert adaptive changes in host genes that are crucial components of the vector's defence. The aim of this study was to determine the molecular evolution of the three short PGRPs (PGRP-S1, PGRP-S2 and PGRP-S3) in the two main African malaria vectors - Anopheles gambiae and Anopheles arabiensis. RESULTS: Genetic diversity of An. gambiae and An. arabiensis PGRP-S1, PGRP-S2 and PGRP-S3 was investigated in samples collected from Mozambique and Tanzania. PGRP-S1 diversity was lower than for PGRP-S2 and PGRP-S3. PGRP-S1 was the only gene differentiated between the two species. All the comparisons made for PGRP-S1 showed significant P-values for Fst estimates and AMOVA confirming a clear separation between species. For PGRP-S2 and PGRP-S3 genes it was not possible to group populations either by species or by geographic region. Phylogenetic networks reinforced the results obtained by the AMOVA and Fst values. The ratio of nonsynonymous substitutions (Ka)/synonymous substitutions (Ks) for the duplicate pair PGRP-S2 and PGRP-S3 was very similar and lower than 1. The 3D model of the different proteins coded by these genes showed that amino acid substitutions were concentrated at the periphery of the protein rather than at the peptidoglycan recognition site. CONCLUSIONS: PGRP-S1 is less diverse and showed higher divergence between An. gambiae and An. arabiensis regardless of geographic location. This probably relates to its location in the chromosome-X, while PGRP-S2 and PGRP-S3, located in chromosome-2L, showed signs of autosomal introgression. The two short PGRP genes located in the chromosome-2L were under purifying selection, which suggests functional constraints. Different types of selection acting on PGRP-S1 and PGRP-S2 and S3 might be related to their different function and catalytic activity.

Dataset Information

IMP PCR primers detect single nucleotide polymorphisms for Anopheles gambiae species identification, Mopti and Savanna rDNA types, and resistance to dieldrin in Anopheles arabiensis.

Publications

IMP PCR primers detect single nucleotide polymorphisms for Anopheles gambiae species identification, Mopti and Savanna rDNA types, and resistance to dieldrin in Anopheles arabiensis.

Similar Datasets

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