Abstract
BackgroundAlternative arrangements of chromosome 2 inversions in Anopheles gambiae are important sources of population structure, and are associated with adaptation to environmental heterogeneity. The forces responsible for their origin and maintenance are incompletely understood. Molecular characterization of inversion breakpoints provides insight into how they arose, and provides the basis for development of molecular karyotyping methods useful in future studies.MethodsSequence comparison of regions near the cytological breakpoints of 2Rb allowed the molecular delineation of breakpoint boundaries. Comparisons were made between the standard 2R+b arrangement in the An. gambiae PEST reference genome and the inverted 2Rb arrangements in the An. gambiae M and S genome assemblies. Sequence differences between alternative 2Rb arrangements were exploited in the design of a PCR diagnostic assay, which was evaluated against the known chromosomal banding pattern of laboratory colonies and field-collected samples from Mali and Cameroon.ResultsThe breakpoints of the 7.55 Mb 2Rb inversion are flanked by extensive runs of the same short (72 bp) tandemly organized sequence, which was likely responsible for chromosomal breakage and rearrangement. Application of the molecular diagnostic assay suggested that 2Rb has a single common origin in An. gambiae and its sibling species, Anopheles arabiensis, and also that the standard arrangement (2R+b) may have arisen twice through breakpoint reuse. The molecular diagnostic was reliable when applied to laboratory colonies, but its accuracy was lower in natural populations.ConclusionsThe complex repetitive sequence flanking the 2Rb breakpoint region may be prone to structural and sequence-level instability. The 2Rb molecular diagnostic has immediate application in studies based on laboratory colonies, but its usefulness in natural populations awaits development of complementary molecular tools.
Highlights
Alternative arrangements of chromosome 2 inversions in Anopheles gambiae are important sources of population structure, and are associated with adaptation to environmental heterogeneity
Of 2Rb breakpoint proximal sequences Using PCR primers designed from the chromosomally standard An. gambiae PEST reference genome [24]; [25], a fosmid library prepared from the An. gambiae BKO strain (2Rj+bcu/j+bcu; [17]) was screened for clones that possessed unique sequence corresponding to the centromere-proximal breakpoint region of the 2R: CAM M form (2R+)b arrangement. (This strategy was part of a larger, ongoing effort to molecularly characterize other inversion breakpoints on chromosome 2R)
The An. gambiae PEST reference is homokaryotypic standard for all chromosomal arrangements including 2R+b [24,29], while the S and M form assemblies were derived from colonies (Pimperena and Mali-NIH, respectively) carrying the opposite (2Rb inverted) arrangement [26]
Summary
Alternative arrangements of chromosome 2 inversions in Anopheles gambiae are important sources of population structure, and are associated with adaptation to environmental heterogeneity. More than 130 paracentric inversions have been detected across the group as a whole, Cytological studies have demonstrated the highly non-random occurrence of inversions along the five arms of the polytene complement in An. gambiae [1,3,4]. 2R represents less than 30% of the complement, this arm is the source of 6/7 (86%) common, and 67/82 (82%) rare, polymorphic chromosomal inversions in An. gambiae and 18/31 (58%) common polymorphic inversions in the species complex as a whole–a highly significant bias [3,4]. Inversion breakpoints cluster in particular regions, and appear coincident at the cytological level at a much higher rate than expected by chance [4], suggesting that a nonrandom process could be responsible for their origin and/or maintenance. Coluzzi et al [3] identified the central part of chromosome arm 2R, the area corresponding to polymorphic inversions 2Rb, bc and u in An. gambiae, as being involved in independent interspecific inversions in the sibling species Anopheles merus, Anopheles melas and An. arabiensis, leading him to speculate that these parallel chromosomal changes may underlie adaptations to ecologically distinct larval breeding sites characteristic of these different species
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