Highly specific PCR-RFLP assays for karyotyping the widespread 2Rb inversion in malaria vectors of the Anopheles gambiae complex

Autor: Maria Calzetta, Raquel Montanez-Gonzalez, Vincenzo Petrarca, Rachel R. Love, Alessandra della Torre, Lydia Schaecher, Verena Pichler, Alexandra C. Vallera, Nora J. Besansky, Marco Pombi, Beniamino Caputo
Jazyk: angličtina
Rok vydání: 2020
Předmět:
0106 biological sciences
0301 basic medicine
Chromosomal inversion
Anopheles gambiae
Inversion genotyping
Mosquito Vectors
Biology
Molecular karyotyping
010603 evolutionary biology
01 natural sciences
Polymerase Chain Reaction
lcsh:Infectious and parasitic diseases
03 medical and health sciences
Cytogenetics
PCR-RFLP
Genotype
Anopheles
Animals
Humans
Tag SNP
lcsh:RC109-216
anopheles gambiae complex
chromosomal inversion
inversion genotyping
malaria vector
molecular karyotyping
pcr-rflp
tag snp
Genotyping
Genetics
Anopheles gambiae complex
Polymorphism
Genetic
Research
biology.organism_classification
3. Good health
Malaria
Restriction enzyme
030104 developmental biology
Infectious Diseases
Malaria vector
Karyotyping
Africa
Chromosome Inversion
Parasitology
Restriction digest
Restriction fragment length polymorphism
Polymorphism
Restriction Fragment Length
Zdroj: Parasites & Vectors, Vol 13, Iss 1, Pp 1-9 (2020)
Parasites & Vectors
ISSN: 1756-3305
Popis: Background Chromosomal inversion polymorphisms play a role in adaptation to heterogeneous environments. Inversion polymorphisms are implicated in the very high ecological flexibility of the three main malaria vector species of the Afrotropical Anopheles gambiae complex, facilitating the exploitation of anthropogenic environmental modifications and promoting a strong association with humans. In addition to extending the species’ spatial and temporal distribution, inversions are associated with epidemiologically relevant mosquito behavior and physiology, underscoring their medical importance. We here present novel PCR-RFLP based assays strongly predictive of genotype for the cosmopolitan 2Rb inversion in An. coluzzii and An. gambiae, a development which overcomes the numerous constraints inherent to traditional cytological karyotyping. Methods We designed PCR-RFLP genotyping assays based on tag SNPs previously computationally identified as strongly predictive (> 95%) of 2Rb genotype. We targeted those tags whose alternative allelic states destroyed or created the recognition site of a commercially available restriction enzyme, and designed assays with distinctive cleavage profiles for each inversion genotype. The assays were validated on 251 An. coluzzii and 451 An. gambiae cytologically karyotyped specimens from nine countries across Africa and one An. coluzzii laboratory colony. Results For three tag SNPs, PCR-RFLP assays (denoted DraIII, MspAI, and TatI) reliably produced robust amplicons and clearly distinguishable electrophoretic profiles for all three inversion genotypes. Results obtained with the DraIII assay are ≥ 95% concordant with cytogenetic assignments in both species, while MspAI and TatI assays produce patterns highly concordant with cytogenetic assignments only in An. coluzzii or An. gambiae, respectively. Joint application of species-appropriate pairs of assays increased the concordance levels to > 99% in An. coluzzii and 98% in An. gambiae. Potential sources of discordance (e.g. imperfect association between tag and inversion, allelic dropout, additional polymorphisms in the restriction target site, incomplete or failed restriction digestion) are discussed. Conclusions The availability of highly specific, cost effective and accessible molecular assays for genotyping 2Rb in An. gambiae and An. coluzzii allows karyotyping of both sexes and all developmental stages. These novel tools will accelerate deeper investigations into the role of this ecologically and epidemiologically important chromosomal inversion in vector biology.
Databáze: OpenAIRE
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