Breakpoint structure of the Anopheles gambiae 2Rb chromosomal inversion
© Lobo et al; licensee BioMed Central Ltd. 2010
Received: 27 July 2010
Accepted: 25 October 2010
Published: 25 October 2010
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.
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.
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.
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.
Anopheles gambiae sensu stricto, the most important vector of human malaria in Africa, is the nominal member of a group of at least seven morphologically indistinguishable and closely related mosquito species [1, 2]. Polytene chromosome analysis of this group, the An. gambiae complex, revealed an abundance of paracentric inversions [1, 3], characterized by the breakage and 180 degree rearrangement of chromosome segments excluding the centromere. More than 130 paracentric inversions have been detected across the group as a whole, with 10 inversions distinguishing six of these species. Given the absence of morphological differences, these fixed chromosomal landmarks provided the first routine basis for species identification . However, fixed inversion differences between species and populations are the exception in the An. gambiae complex. The majority of inversions remain polymorphic in natural populations. Although most species in the complex carry at least one polymorphic inversion, only two species--An. gambiae s. s. (hereafter, An. gambiae) and Anopheles arabiensis--possess most of the known inversion polymorphisms, potentially helping to explain their vast geographic and ecological distributions across much of tropical Africa [1, 3].
The abundance of inversions in the An. gambiae complex, and their nonrandom taxonomic, genomic and ecological distributions suggested that they may be playing more than a passive role in the diversification of these mosquitoes. Indeed, chromosomal inversions have been viewed as a mechanism for ecotypic differentiation in An. gambiae [3, 5, 6]. A recent model that could explain the spread and distribution of inversions proposes that through suppressed recombination in inversion heterozygotes, allelic combinations beneficial in particular environments are protected from recombination with other genetic backgrounds adapted to alternative ecological settings [7, 8]. The maintenance of polymorphic inversions would thereby confer greater ecological flexibility to the species. Evidence consistent with an adaptive role for inversion polymorphisms in An. gambiae are the stable geographic clines in inversion frequency associated with climatic factors, such as aridity, replicated across Africa [1, 9–11]; the microhabitat differences in inversion frequencies of indoor- or outdoor-resting populations, also associated with aridity ; the temporal cycling of inversion frequencies in concert with rainy and dry seasons [1, 13]; and the heterotic maintenance of inversions in laboratory colonies . Of particular relevance to malaria transmission and control is the connection between inversions and indoor/outdoor resting, as this mosquito behavioral response to aridity impacts the probability of vector-human contact and vector contact with insecticide-treated walls or bed nets . Moreover, the process of ecotypic differentiation, potentially leading to speciation , implies restricted gene flow between populations and may be accompanied by other physiological and behavioral differences that could alter the efficiency of vector control efforts in unanticipated ways.
To date, only three inversions have been molecularly characterized in An. gambiae s.l., one on 2L (2La ) and two on 2R (2Rj ; 2Rd' ). Based on detailed molecular analysis of the breakpoint junctions, no clear consensus has emerged on the likely mechanism of breakage, nor have precise mechanisms responsible for inversion maintenance yet been identified. However, characterization of these breakpoint regions led to the development of molecular diagnostics for 2La  and 2Rj , which allow much higher throughput karyotyping of natural populations than allowed by laborious cytological methods, and have facilitated ongoing functional analysis [21–23]. Toward the long-term goal of understanding mechanisms responsible for the origin and maintenance of inversions in An. gambiae, the breakpoint structure of inversion 2Rb is presented along with a rapid molecular karyotyping method.
Assembly of 2Rb breakpoint proximal sequences
Using PCR primers designed from the chromosomally standard An. gambiae PEST reference genome ; , a fosmid library prepared from the An. gambiae BKO strain (2Rj+ b cu/j+ b cu; ) was screened for clones that possessed unique (non-repetitive) sequence corresponding to the centromere-proximal breakpoint region of the 2R+ b arrangement. (This strategy was part of a larger, ongoing effort to molecularly characterize other inversion breakpoints on chromosome 2R). Based on localization with in situ hybridization and sequence comparison, Fosmid clone 332D was identified as going to the breakpoints, and end-sequenced using the Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) and the ABI PRISM 3700 DNA Analyzer (Applied Biosystems). Sequences verified using Lasergene Seqman software (DNASTAR) were submitted to GenBank (accession numbers HN153245-HN153246).
End-sequence of fosmid 332D was used to walk toward the 2Rb proximal breakpoint using trace sequence reads generated from whole genome sequencing of the An. gambiae S form (Pimperena strain; Lawniczak et al, submitted). The Pimperena strain carries the opposite (inverted) orientation of the 2Rb arrangement (i.e., 2Rb/b). Pimperena trace sequences matching sequence from fosmid 332D were used to initiate an iterative BLAST procedure in which the Pimperena trace archive was queried and contigs from this 2Rb background were built using mate pair information and sequence similarity. These 2Rb contigs were compared to the PEST (2R+ b ) assembly to find evidence of the rearrangement breakpoint. Highly repetitive regions that could not be assembled manually were excluded. Manually assembled 2Rb sequences flanking the putative breakpoint were used to identify scaffolds assembled independently during the An. gambiae M (Mali-NIH, 2Rbc/bc) and S genome sequencing project [26, 25] and compared to the PEST genome.
Mosquito sampling and cytological determination of karyotype
Collections of indoor resting An. gambiae were made by spray catch from Mali and Cameroon. Samples from Mali were collected from seven villages in the southern part of the country in Aug-Sep 2004, as previously described . Samples from Cameroon were collected from five villages in a forest/savanna mosaic zone in the eastern part of the country in Sep-Oct 2009: Gado-Badzere and Zembe Borango (4°58.435'N, 13°2.403'E), Nkoumadjap (4°21.575'N, 13°38.391'E), Mayos I (4°20.505'N, 13°33.490'E), and Daiguene (4°46.608'N, 13°50.668'E). Mosquitoes were sorted morphologically to An. gambiae s.l. and by gonotrophic stage. Ovaries of semi-gravid females were dissected and placed into a micro-tube containing Carnoy's solution (1 part glacial acetic acid, 3 parts ethanol); the carcass was placed in a correspondingly numbered micro-tube and stored over desiccant for DNA-based analysis. Preparation and scoring of polytene chromosomes followed .
PCR determination of karyotype
Genomic DNA was isolated from individual mosquitoes using the DNeasy Extraction Kit (Qiagen, Valencia, CA). Anopheles gambiae sensu stricto and its molecular forms were identified using an rDNA-based PCR diagnostic assay .
Sequence comparisons between the PEST (2R+ b ), Pimperena (2Rb), and Mali-NIH (2Rbc) genome assemblies were used to design primers to differentially amplify alternative arrangements of the b inversion. Diagnostic primers include: b-For (5'-CGGGAGCAAAGATAAGTAGCA), b+-Rev (5'-CCGGATAATCGACGCTCTAC), and b-Rev (5' AACCCTACCATATACCAGTACCAACG 3'). The 25 μl PCR reaction contained 20 mM Tris-Cl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 μM of each dNTP; 5 pmol b-For primer, 2.5 pmol b-Rev primer, 5 pmol b+-Rev primer, 1 U Taq polymerase, 5% DMSO and 1% BSA. PCR was performed on 1 μl genomic DNA template using an initial incubation at 94°C for 2 min, followed by 35 cycles of 94°C for 30s, 58°C for 30s, and 72°C for 45s, concluding with a 10 min incubation at 72°C and a 4°C hold. Mosquitoes from established colonies with known karyotypes were used as PCR controls. These included Mali-NIH M form (2Rbc; 2La) and Pimperena S form (2Rb; 2La/+ a ) available from MR4, and colonies selected at the University of Notre Dame to be homokaryotypic for chromosomal inversions on 2R: CAM M form (2R+; 2L+, derived from parental colony Yaounde from Cameroon), KIST S form (2R+; 2L+, derived from parental colony KISUMU from Kenya), and SUCAM M form (2R+; 2La/+ a , derived from a cross between CAM and Sua2La ).
Results and Discussion
Sequence assembly across the 2Rb breakpoints
Molecular organization of the 2Rb inversion breakpoints
Assemblies of the 2Rb inversion breakpoints from Mali-NIH (2Rbc) and Pimperena (2Rb) traces were nearly identical, with only minor SNP and insertion-deletion differences. A schematic diagram of their molecular structure, in comparison to the corresponding regions of the uninverted chromosome, is provided in Figure 3. Together, these data reveal that the 2Rb inversion encompasses 7.55 Mb and extends from subdivision 11C to 12E on the cytogenetic map of PEST (position 19,023,925 to 26,758,676 on 2R; red arrows in Figure 3).
Outside of and immediately flanking these breakpoints on both 2R+ b and 2Rb arrangements is a repetitive structure comprising tandemly arrayed copies of unit length ~30 bp, (ACTTTTGCGATTGTCGCAAAAACTTCTGCGA)N. At the telomeric end of the 2R+ b arrangement of PEST, this tandem repeat structure extends for at least 3.2kb (i.e., >100 tandem repetitions) and is flanked by a ~10 kb assembly gap. At the centromeric end of 2R+ b , the repetitive structure is 72 bp in length. The alternative 2Rb arrangement in both Mali-NIH and Pimperena also was flanked by the same tandem repeat sequence. At the telomeric breakpoint of 2Rb, the tandem repeat sequence spans 519 bp and is embedded in a palindrome, flanked by a ~2.5 kb assembly gap. At its centromeric end, the 2Rb breakpoint also abuts at least 1 kb of the same tandem repeat, but the highly repetitive nature of this sequence caused gaps in both WGS and manual assemblies.
The position of these tandem repeats, presumably at the precise breakpoints of both arrangements (2Rb and 2R+ b ), implicates the tandem repeat in the generation of this chromosomal inversion, although the ancestral-descendant relationship between the alternative gene orders based on these data is ambiguous. Other molecularly characterized inversion breakpoints on 2R in An. gambiae also possess flanking repetitive sequences, but only on the derived arrangement: 2Rj is flanked by nearly identical 14.6 kb complex inverted repeat structures reminiscent of segmental duplications, while 2L+ a is bordered by homologous repetitive elements [17, 19]. In neither case were these repetitive sequences found adjacent to either breakpoint of the ancestral arrangement, in contrast to the situation for 2Rb. Either the 2Rb or 2R+ b arrangements could have arisen via non-allelic homologous recombination between flanking tandem repeats on the ancestral chromosome, leading to inversion of the intervening sequence. Notably, two unrelated palindromes with short internal spacers were present at the breakpoints of the 2Rb inversion in both Pimperena and Mali-NIH 2Rb arrangements, one at each end. As the arms of both palindromes involve sequences apparently present only once in the PEST genome (near each breakpoint), it is tempting to suggest that the 2R+ b arrangement in PEST may be ancestral.
Gene annotations adjacent to the 2Rb breakpoints
Gene predictions were compared between the 2Rb and 2R+ b arrangements, within the ~10 kb region immediately internal to the breakpoints. Most of the sequence consisted of transposons and low complexity sequence. There were no genes annotated in the 10 kb region proximal to the centromeric breakpoint of 2R+ b in PEST. At the telomeric end of this arrangement, one gene has been predicted of unknown function (AGAP002299, annotated as a conserved hypothetical protein). This gene has putative orthologs in other mosquitoes (Culex quinquefasciatus, CPIJ008031; Aedes aegypti, AAEL007792) and Drosophila melanogaster (CG18635). The corresponding gene at the centromeric end of 2Rb contained several SNP differences and a deletion in the predicted 5' UTR. Although limited EST evidence suggests that this gene may have alternative transcripts, potential functional consequences of sequence differences in AGAP002299 between alternative arrangements (if any) remain to be investigated.
Molecular karyotyping by PCR
As a first step in the validation of this assay, its performance was tested on at least 25 mosquitoes (50 chromosomes) sampled from each of five different An. gambiae laboratory colonies of known and monomorphic 2Rb karyotype (determined from polytene chromosome banding pattern), originating from geographic locations as diverse as Mali, Cameroon, Liberia, and Kenya: Mali-NIH M form (2Rbc/bc), CAM M form (2R+ b /+ b ), SUCAM M form (2R+ b /+ b ), KIST S form (2R+ b /+ b ), and Pimperena S form (2Rb/b). Without exception, PCR amplicons of the expected size were generated. Moreover, when DNA was mixed in 1:1 proportion from mosquitoes carrying 2Rb or 2R+ b karyotypes prior to PCR, both bands were amplified (suggesting that the assay is capable of detecting 2Rb/+ b heterozygotes). Additionally, at least 15 mosquitoes from an An. arabiensis colony (Dongola) selected to be homokaryotypic for 2Rb (i.e., 2Rb/b) were tested, and each generated the expected 429 bp PCR fragment (and only this fragment), consistent with other evidence that the 2Rb inversion in An. arabiensis and An. gambiae shares a common origin (e.g., see ).
Performance of the 2Rb molecular karyotyping PCR assay in field collections of An. gambia e
Molecular karyotypes congruent with banding pattern
2R+ b /+ b
2R+ b /b
Molecular karyotyping discrepancies in relation to banding patterns on 2R in Mali
Source of anomalies based on 2Rb karyotype
Complete 2R karyotype
Exp. molecular karyotype
Obs. molecular karyotype
2R + b / + b (N = 17)
2R j+ b cu / j+ b cu (N = 13)
2R + b
2R+ j + b cu/+ j + b cu (N = 3)
2R + j + b cu / + j + b + c + u (N = 1)
2R + b
2R+ b /b (N = 6)
2Rj+ b cu/jbcu (N = 4)
2R+ b /b
2R + j b+ c + u / + j + b cu (N = 2)
2R + b / b
Application of this PCR assay in Mali in conjunction with cytogenetic analysis, raised the intriguing possibility of 2Rb homoplasy through breakpoint reuse. However, it appears that the PCR assay as presented here has limited application by itself, for molecular karyotyping of 2Rb in natural populations of An. gambiae, even in Cameroon where the degree of 2R chromosomal polymorphism is low, due to a relatively high rate of 2Rb "miscalls". The rate of miscalls is based on the assumption that the cytogenetic analysis was error-free, which is unlikely. Thus, the miscall rate may be over-estimated. Nevertheless, it is possible that high repeat content near the 2Rb breakpoints is associated with relatively high genetic instability and sequence rearrangement, which can result in elimination or alteration of primer binding sites as well as unexpected changes in amplicon length due to insertions and deletions. On the other hand, the PCR assay yields results that are perfectly congruent with cytology in all laboratory colonies tested thus far, suggesting that the diagnostic assay will be useful in experimental manipulations and crosses where rapid karyotype analysis of living mosquitoes, both males and females across all developmental stages, is desired. Moreover, the 2Rb PCR assay may prove useful in Mali in the future, in combination with yet-to-be-developed molecular diagnostic assays for other arrangements on 2R.
Elucidation of the molecular breakpoint structure of the 7.5 Mb 2Rb inversion points to the involvement of repetitive DNA-- specifically, extensive tandem arrays of short unit length flanking both breakpoints-- in the rearrangement process. Although the ancestral-descendant relationship between standard and inverted arrangements is uncertain, molecular karyotyping based on a newly developed PCR diagnostic assay suggests two things. First, the 2Rb inversion shared between the sibling species An. gambiae and An. arabiensis has a common origin. Second, the polytene chromosome banding pattern indicative of the 2R+ b standard arrangement may have arisen twice through breakpoint reuse. Sequence instability and high repeat content near the breakpoints complicate the application of the PCR diagnostic assay for molecular karyotyping of natural populations, although the assay represents a novel and powerful tool for functional genomic studies of 2Rb in laboratory colonies, and may hold promise for future field application in combination with other molecular tools. The current impediments to analysis of inversion breakpoints posed by repetitive DNA may be overcome by powerful new technologies , such as single DNA molecule platforms capable of mapping and even sequencing repetitive DNA, enabling further insights into the origin and stability of 2R rearrangements in natural populations of An. gambiae.
M. Kern and M. Belton provided laboratory assistance, O. Grushko and J. Agbor assisted with karyotyping, M. Coulibaly and S. Traore' managed the 2004 field collections in Mali. Thanks to the entomological teams of MRTC (Mali) and OCEAC (Cameroon) for fieldwork. The Mali-NIH and Pimperena genome sequence data used to assemble breakpoint junctions were produced by The Genome Center at Washington University School of Medicine, St. Louis and J. Craig Venter Institute, Rockville with funding from NHGRI (08UHG003079). Research was supported by NIH R01 AI076584 to NJB. DMS was supported by NIH Clinton Funds.
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