Breakpoint structure of the Anopheles gambiae 2Rb chromosomal inversion

Abstract

Background: Alternative 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.

Methods: Sequence 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.

Results: The 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.

Conclusions: The 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.

Figure 1

Schematic diagram (after [13]) of common inversions 2Rj, 2Rb, 2RC and 2Ru on chromosome 2R in An. gambiae.

Figure 2

Schematic diagram of scaffolds analysed in M and S relative to the An. gambiae PEST reference. Horizontal rectangle (labeled at left) represents genomic sequence from the PEST reference genome, with green shaded sections indicating flanking sequence at the telomeric and centromeric ends, red sections indicating repetitive sequence at both breakpoints, and the blue section indicating the 2R+^b arrangement. Horizontal arrows indicate the relative position (not to scale) and orientation of assembly or manual scaffolds used in the analysis. The scaffold number (if applicable), approximate length, and source (An. gambiae M or S genome sequence) is indicated at left. Vertical gray rectangles highlight the ~10 kb breakpoint regions whose sequence was compared between the PEST, M and S genomes.

Figure 3

Structure of the 2Rb/+^b breakpoint junctions. At top and bottom is a schematic overview of the 2Rb/+^b and 2Rc/+^c arrangements as represented by the PEST and M reference sequences, respectively. Horizontal black bar represents a segment of chromosome 2R. The relative position and orientation of chromosomal arrangements is indicated by labeled brackets, and centromeric/telomeric ends of 2R are indicated as Cen and Tel, respectively. Shaded arrows indicate the orientation of the arrangements, and labels inside the arrows (e.g., 11C) provide the cytogenetic subdivision in which the breakpoint junction occurs. Blue and gray boxes labeled with the corresponding cytogenetic subdivision represent flanking sequence outside of chromosomal rearrangements; red boxes represent repetitive DNA. The central part of the diagram provides a more detailed structural analysis of the color-coded breakpoint regions. Throughout, color is used to indicate homologous sequences between alternative arrangements, except for rectangles filled by patterns, which represent exclusive insertion events. Horizontal blunt arrows shaded in olive green and orange are sequences present once in the 2R+^b arrangement that have been duplicated into a palindrome in the alternative 2Rb arrangement. The red vertical arrows represent the putative breakpoints, positions where unique sequence ends and repetitive sequence framing both ends of the arrangement (red blunt arrows) begins. Black arrows at the ends of each diagram represent continuing chromosomal sequence; dotted lines represent gaps in the assembly. Blue curved lines represent sequence linked by mate-pair information. Asterisk framed by a white box indicates the chromosomal region targeted by the PCR diagnostic assay; see text and Figure 4 for details. Not to-scale.

Figure 4

Schematic diagram of the three-primer PCR assay for molecular karyotyping of 2Rb. The white box with an asterisk in Figure 3 is represented here. Areas common to both arrangements are connected by dotted lines. The grey box represents an insertion exclusive to the 2Rb arrangement, to which primer bRev anneals. Primer bFor is a universal primer that anneals to a region common to both arrangements. Although +^bRev can anneal to both arrangements at different distances from bFor, size limitations on successful PCR amplification restrict the product to a 2R+^b-specific fragment of 630 bp. In combination with bFor,primer bRev amplifies a 429 bp sequence diagnostic of the 2Rb arrangement.