Intragenomic sequence variations in the second internal transcribed spacer (ITS2) ribosomal DNA of the malaria vectorAnopheles stephensi

Abstract

Second Internal Transcribed Spacer (ITS2) ribosomal DNA (rDNA) sequence is a widely used molecular marker for the species-identification or -delimitation due to observed concerted evolution which is believed to homogenize rDNA copies in an interbreeding population. However, intra-specific differences in ITS2 of Anopheles stephensi have been reported. This study reports the presence of intragenomic variation in the ITS2 region of An. stephensi and hypothesized that observed intra-specific differences in this species may have been resulted due to ambiguous DNA-chromatogram resulting from intragenomic heterogeneity. Anopheles stephensi collected from different parts of India were sequenced for complete ITS2 and the variable region of 28S rDNA (d1-d3 domains). Intragenomic variations at fixed loci were found in ITS2 of all An. stephensi sequenced, but no such variation was observed in d1 to d3 domains of 28S rDNA. Cloning and sequencing of ITS2 through the d3 domain of the 28S region of rDNA from representative samples from northern, central and southern India confirmed the presence of intragenomic variation in ITS2. The variations are due to three transitions and two bp indel in a di-nucleotide microsatellite locus at fixed loci. Multiple haplotypes were observed in ITS2 raised from such variations. Due to the absence of detectable intragenomic sequence variation in the d1 to d3 domain of 28S rDNA of An. stephensi, this region can serve as an ideal reference sequence for taxonomic and phylogenetic studies. The presence of intragenomic variation in rDNA should be carefully examined before using this as a molecular marker for species delimitation or phylogenetic analyses.