To better understand the evolutionary dynamics of chromosomal inversions, a physical map for an Asian malaria vector, Anopheles stephensi, was created and compared with the maps of the major African malaria vectors A. gambiae and A. funestus No interchromosomal transposition was observed between A. gambiae and A. stephensi. Several cases of euchromatin and heterochromatin transitions weridentified between A. gambiae and A. stephensi. The study of paracentric inversions between lineages in Anopheles mosquitoes demonstrated that X chromosome has the fastest rate of inversion fixations and highest density of repetitive elements. Among the autosomes, 2R evolved faster than other autosomes. The slowly evolved autosomes have more M/SARs than rapidly evolving arms. Breakpoint regions are enriched with repetitive elements. The study revealed that fixed inversions are distributed nonrandomly and breakpoint clustering is common in lineages of A. gambiae and A. stephensi. The parallel association between the density of inversion fixations and polymorphisms suggests that polymorphic inversions can be fixed during evolution.

To understand the direction of evolution in A. gambiae complex, the ancestral status of fixed inversions for this complex was identified. The presence of the 2La inversion in outgroups, A. stephensi and A. nili, confirmed the ancestral status of the 2La inversion. The presences of breakpoint structure of the 2Ro inversion in outgroup species, A. stephensi, indicated that the 2Ro is ancestral arrangement. The presence of SINE elements at the breakpoints of the 2R+p in A. gambiae PEST strain suggested that the 2R+p is a derived arrangement. Therefore, the carrier of 2Rop inversions, A. merus, was considered closest to the ancestral species.

We have developed a new protocol for laser microdissection and whole genome amplification of polytene chromosomal fragments to obtain DNA for sequencing and assembly. The chromosomal regions spanning both breakpoints of the 2La in A. arabiensis and A. merus were laser microdissected from the polytene chromosomes. Subsequently, DNA samples were amplified using Illustra GenomePhi V2 DNA and Whole-pool amplification methods for obtaining amplicons. Successful amplification of our target DNA was confirmed by PCR with specific primers followed by Sanger sequencing.