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Experimental work pertaining to the molecular cytogenetics of malaria vector species of mosquitoes by the application of PCR technique has been carried out. The main objectives of the study included the sequence characterization of nuclear rDNA internal transcribed spacers 1 and 2 (ITS1, ITS2) and mitochondrial DNA COII gene as potential molecular markers for studying genetic relatedness and phylogenetic kinship among six important species of genus Anopheles. The present studies involved the extraction of genomic DNA from a single female mosquito followed by its amplification with specific primers. The total length of each DNA band with respect to the number of nucleotides was calculated along with GC:AT content, ratio of substitutions due to transitions and transversions (ts/tv), insertions/ deletions and identification of tandem and nontandem repeat sequences. Out of the three studied molecular markers, ITS1 and ITS2 were GC rich while COII gene was AT rich. As for the incidence of insertions/deletions (indels) of bases is concerned, it was found maximum in ITS1 and ITS2 and minimum in conserved COII gene sequence .From the present results it was evident that except for An. culicifacies the ITS2 sequence of the remaining five species is under rapid evolutionary changes due to less conserved nature of the sequence. To the contrary ITS1 sequence was found to have highly variable length ranging from 300-900bp. It was found that An. stephensi + An. culicifacies and An. annularis + An. splendidus share a close genetic homology while An. subpictus and An. maculatus have hypervariable non-homologous genomic qualities. The phylogenetic dendrograms revealed that An. stephensi and An. culicifacies grouped together only when analysed by MP in case of ITS1 gene only while they drifted apart in case of ITS2 and COII gene. This is attributed to their host and habitat feeding preferences which have given them the dubious status of urban and rural vectors respectively.
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Author(s): Monika Sharma S Chaudhry
Phylogeny, Anopheles, ITS1, ITS2, COII.