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In JoVE (2)
- High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization
- Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes
Other Publications (17)
- Science (New York, N.Y.)
- Science (New York, N.Y.)
- Proceedings of the National Academy of Sciences of the United States of America
- Journal of Medical Entomology
- Genome Biology
- Journal of Computational Biology : a Journal of Computational Molecular Cell Biology
- Gene
- PloS One
- BMC Genomics
- Malaria Journal
- The American Journal of Tropical Medicine and Hygiene
- PloS One
- BMC Evolutionary Biology
- The Journal of Heredity
- PLoS Neglected Tropical Diseases
- Parasites & Vectors
- Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases
Articles by Maria V. Sharakhova in JoVE
JoVE General
High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization
Department of Entomology, Virginia Tech
Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.
JoVE Immunology and Infection
Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes
Department of Entomology, Virginia Tech
Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.
Other articles by Maria V. Sharakhova on PubMed
The Genome Sequence of the Malaria Mosquito Anopheles Gambiae
Anopheles gambiae is the principal vector of malaria, a disease that afflicts more than 500 million people and causes more than 1 million deaths each year. Tenfold shotgun sequence coverage was obtained from the PEST strain of A. gambiae and assembled into scaffolds that span 278 million base pairs. A total of 91% of the genome was organized in 303 scaffolds; the largest scaffold was 23.1 million base pairs. There was substantial genetic variation within this strain, and the apparent existence of two haplotypes of approximately equal frequency ("dual haplotypes") in a substantial fraction of the genome likely reflects the outbred nature of the PEST strain. The sequence produced a conservative inference of more than 400,000 single-nucleotide polymorphisms that showed a markedly bimodal density distribution. Analysis of the genome sequence revealed strong evidence for about 14,000 protein-encoding transcripts. Prominent expansions in specific families of proteins likely involved in cell adhesion and immunity were noted. An expressed sequence tag analysis of genes regulated by blood feeding provided insights into the physiological adaptations of a hematophagous insect.
Evolution of Supergene Families Associated with Insecticide Resistance
The emergence of insecticide resistance in the mosquito poses a serious threat to the efficacy of many malaria control programs. We have searched the Anopheles gambiae genome for members of the three major enzyme families- the carboxylesterases, glutathione transferases, and cytochrome P450s-that are primarily responsible for metabolic resistance to insecticides. A comparative genomic analysis with Drosophila melanogaster reveals that a considerable expansion of these supergene families has occurred in the mosquito. Low gene orthology and little chromosomal synteny paradoxically contrast the easily identified orthologous groups of genes presumably seeded by common ancestors. In A. gambiae, the independent expansion of paralogous genes is mainly a consequence of the formation of clusters among locally duplicated genes. These expansions may reflect the functional diversification of supergene families consistent with major differences in the life history and ecology of these organisms. These data provide a basis for identifying the resistance-associated enzymes within these families. This will enable the resistance status of mosquitoes, flies, and possibly other holometabolous insects to be monitored. The analyses also provide the means for identifying previously unknown molecules involved in fundamental biological processes such as development.
Breakpoint Structure Reveals the Unique Origin of an Interspecific Chromosomal Inversion (2La) in the Anopheles Gambiae Complex
Paracentric chromosomal inversions are major architects of organismal evolution and have been associated with adaptations relevant to malaria transmission in anopheline mosquitoes. The processes responsible for their origin and maintenance, still poorly understood, can be illuminated by analysis of inversion breakpoint sequences. Here, we report the breakpoint structure of chromosomal inversion 2La from the principal malaria vector Anopheles gambiae and its relatives in the A. gambiae complex. The distal and proximal breakpoints of the standard (2L+a) arrangement contain gene duplications: full-length genes and their truncated copies at opposite ends. Intact genes without pseudogene copies in the alternative arrangement (2La) imply that 2L+a is derived and was viable despite damage to genes, because duplication preserved gene function. A unique origin for the interspecific 2La inversion was challenged previously by indirect genetic evidence, but breakpoint sequences determined from members of the A. gambiae complex strongly suggest their descent from a single event. The derived position of 2L+a, long considered ancestral in this medically important group, has significant implications for the phylogenetic history and the evolution of vectorial capacity in the A. gambiae complex.
A Standard Cytogenetic Photomap for the Mosquito Anopheles Stephensi (Diptera: Culicidae): Application for Physical Mapping
To facilitate physical genome mapping, we have developed a new cytogenetic photomap for Anopheles stephensi (Liston) (Diptera: Culicidae), an important malaria vector in Asia. The high-resolution images of the ovarian polytene chromosomes have been straightened and divided by numbered divisions and lettered subdivisions. The exact chromosomal locations of eight DNA probes have been determined by fluorescent in situ hybridization. Using the DNA sequences, we have established correspondence between chromosomal arms among An. stephensi, Anopheles gambiae (Patton), and Anopheles funestus (Giles). The results support previous cytogenetic observations of arm translocations taking place during diversification of the species. To make the cytogenetic map useful for population genetics studies, we have indicated the chromosomal positions for the breakpoints of 19 polymorphic inversions.
Update of the Anopheles Gambiae PEST Genome Assembly
The genome of Anopheles gambiae, the major vector of malaria, was sequenced and assembled in 2002. This initial genome assembly and analysis made available to the scientific community was complicated by the presence of assembly issues, such as scaffolds with no chromosomal location, no sequence data for the Y chromosome, haplotype polymorphisms resulting in two different genome assemblies in limited regions and contaminating bacterial DNA.
Reconstructing Ancestral Autosomal Arrangements in the Anopheles Gambiae Complex
Members of the Anopheles gambiae complex have remarkably distinct ecological adaptations, behaviors, and degrees of vectorial capacity. Inferring phylogenetic relationships in the complex is crucial for identifying the genomic changes associated with the origin and loss of epidemiologically important traits. However, the high level of sequence similarity, genetic introgression, and shared molecular ancestral polymorphisms makes reconstruction of the A. gambiae complex phylogeny difficult. Phylogenetic relationships among the members of species complexes can be inferred from the distribution of fixed chromosomal inversions if outgroup arrangements are known. The aim of this work is to test a possibility of determining ancestral autosomal arrangements in the A. gambiae complex using outgroup chromosomes and a combination of bioinformatic and cytogenetic approaches. The minimum number of inversions between members of the A. gambiae complex and the outgroup species A. funestus and A. stephensi was calculated using the Multiple Genome Rearrangements (MGR) and Sorting Permutation by Reversals and block-INterchanGes (SPRING) programs. The physical mapping of A. merus chromosomes identified molecular coordinates of the proximal 2Ro+ inversion breakpoint in A. gambiae. DNA probes from 2La+ and 2Ro+ inversion breakpoints of the A. gambiae were mapped to the A. stephensi chromosomes. Assuming monophyletic origin of the inversions, this study concludes that physical mapping of ingroup and outgroup species can be used for identifying inversion breakpoints and ancestral autosomal arrangements within species complexes. Molecular characterization of the breakpoints in both ingroup and outgroup species will provide a solid basis for reconstructing the inversion history in the A. gambiae complex.
Molecular Organization of Heterochromatin in Malaria Mosquitoes of the Anopheles Maculipennis Subgroup
Although heterochromatin makes up a significant portion of the malaria mosquito genome, its organization, function, and evolution are poorly understood. Sibling species of the Anopheles maculipennis subgroup, the European malaria mosquitoes, are characterized by striking differences in the morphology of pericentric heterochromatin; however, the molecular basis for the rapid evolutionary transformation of heterochromatin is not known. This study reports an initial survey of the molecular organization of the pericentric heterochromatin in nonmodel species from the A. maculipennis subgroup. Molecular identity and chromosomal localization were established for short DNA fragments obtained by microdissection from the pericentric diffuse beta-heterochromatin of A. atroparvus. Among 102 sequenced clones of the Atr2R library, twenty had sequence similarity to transposable elements (TEs) from the Anopheles gambiae and Aedes aegypti genomes. At least six protein-coding single-copy genes from A. gambiae and four single-copy genes from Drosophila melanogaster were homologous to eight clones from the library. Most of these conserved genes were heterochromatic in A. gambiae but euchromatic in D. melanogaster. The remaining 74 clones were characterized as noncoding repetitive DNA. Comparative chromosome mapping of twelve clones in the sibling species A. atroparvus and A. messeae demonstrated that the noncoding repetitive sequences and the TEs have undergone independent chromosome-specific and species-specific gains and losses in the morphologically different pericentric heterochromatic regions, in accordance with the "library model."
Genome Landscape and Evolutionary Plasticity of Chromosomes in Malaria Mosquitoes
Nonrandom distribution of rearrangements is a common feature of eukaryotic chromosomes that is not well understood in terms of genome organization and evolution. In the major African malaria vector Anopheles gambiae, polymorphic inversions are highly nonuniformly distributed among five chromosomal arms and are associated with epidemiologically important adaptations. However, it is not clear whether the genomic content of the chromosomal arms is associated with inversion polymorphism and fixation rates.
Genome Mapping and Characterization of the Anopheles Gambiae Heterochromatin
Heterochromatin plays an important role in chromosome function and gene regulation. Despite the availability of polytene chromosomes and genome sequence, the heterochromatin of the major malaria vector Anopheles gambiae has not been mapped and characterized.
Breakpoint Structure of the Anopheles Gambiae 2Rb Chromosomal Inversion
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.
A Physical Map for an Asian Malaria Mosquito, Anopheles Stephensi
Physical mapping is a useful approach for studying genome organization and evolution as well as for genome sequence assembly. The availability of polytene chromosomes in malaria mosquitoes provides a unique opportunity to develop high-resolution physical maps. We report a 0.6-Mb-resolution physical map consisting of 422 DNA markers hybridized to 379 chromosomal sites of the Anopheles stephensi polytene chromosomes. This makes An. stephensi second only to Anopheles gambiae in density of a physical map among malaria mosquitoes. Three hundred sixty-three (363) probes hybridized to single chromosomal sites, whereas 59 clones yielded multiple signals. This physical map provided a suitable basis for comparative genomics, which was used for determining inversion breakpoints, duplications, and origin of novel genes across species.
Evolutionary Dynamics of the Ty3/gypsy LTR Retrotransposons in the Genome of Anopheles Gambiae
Ty3/gypsy elements represent one of the most abundant and diverse LTR-retrotransposon (LTRr) groups in the Anopheles gambiae genome, but their evolutionary dynamics have not been explored in detail. Here, we conduct an in silico analysis of the distribution and abundance of the full complement of 1045 copies in the updated AgamP3 assembly. Chromosomal distribution of Ty3/gypsy elements is inversely related to arm length, with densities being greatest on the X, and greater on the short versus long arms of both autosomes. Taking into account the different heterochromatic and euchromatic compartments of the genome, our data suggest that the relative abundance of Ty3/gypsy LTRrs along each chromosome arm is determined mainly by the different proportions of heterochromatin, particularly pericentric heterochromatin, relative to total arm length. Additionally, the breakpoint regions of chromosomal inversion 2La appears to be a haven for LTRrs. These elements are underrepresented more than 7-fold in euchromatin, where 33% of the Ty3/gypsy copies are associated with genes. The euchromatin on chromosome 3R shows a faster turnover rate of Ty3/gypsy elements, characterized by a deficit of proviral sequences and the lowest average sequence divergence of any autosomal region analyzed in this study. This probably reflects a principal role of purifying selection against insertion for the preservation of longer conserved syntenyc blocks with adaptive importance located in 3R. Although some Ty3/gypsy LTRrs show evidence of recent activity, an important fraction are inactive remnants of relatively ancient insertions apparently subject to genetic drift. Consistent with these computational predictions, an analysis of the occupancy rate of putatively older insertions in natural populations suggested that the degenerate copies have been fixed across the species range in this mosquito, and also are shared with the sibling species Anopheles arabiensis.
Arm-specific Dynamics of Chromosome Evolution in Malaria Mosquitoes
The malaria mosquito species of subgenus Cellia have rich inversion polymorphisms that correlate with environmental variables. Polymorphic inversions tend to cluster on the chromosomal arms 2R and 2L but not on X, 3R and 3L in Anopheles gambiae and homologous arms in other species. However, it is unknown whether polymorphic inversions on homologous chromosomal arms of distantly related species from subgenus Cellia nonrandomly share similar sets of genes. It is also unclear if the evolutionary breakage of inversion-poor chromosomal arms is under constraints.
An Integrated Chromosome Map of Microsatellite Markers and Inversion Breakpoints for an Asian Malaria Mosquito, Anopheles Stephensi
Anopheles stephensi is one of the major vectors of malaria in the Middle East and Indo-Pakistan subcontinent. Understanding the population genetic structure of malaria mosquitoes is important for developing adequate and successful vector control strategies. Commonly used markers for inferring anopheline taxonomic and population status include microsatellites and chromosomal inversions. Knowledge about chromosomal locations of microsatellite markers with respect to polymorphic inversions could be useful for better understanding a genetic structure of natural populations. However, fragments with microsatellites used in population genetic studies are usually too short for successful labeling and hybridization with chromosomes. We designed new primers for amplification of microsatellite loci identified in the A. stephensi genome sequenced with next-generation technologies. Twelve microsatellites were mapped to polytene chromosomes from ovarian nurse cells of A. stephensi using fluorescent in situ hybridization. All microsatellites hybridized to unique locations on autosomes, and 7 of them localized to the largest arm 2R. Ten microsatellites were mapped inside the previously described polymorphic chromosomal inversions, including 4 loci located inside the widespread inversion 2Rb. We analyzed microsatellite-based population genetic data available for A. stephensi in light of our mapping results. This study demonstrates that the chromosomal position of microsatellites may affect estimates of population genetic parameters and highlights the importance of developing physical maps for nonmodel organisms.
Imaginal Discs--a New Source of Chromosomes for Genome Mapping of the Yellow Fever Mosquito Aedes Aegypti
The mosquito Aedes aegypti is the primary global vector for dengue and yellow fever viruses. Sequencing of the Ae. aegypti genome has stimulated research in vector biology and insect genomics. However, the current genome assembly is highly fragmented with only ~31% of the genome being assigned to chromosomes. A lack of a reliable source of chromosomes for physical mapping has been a major impediment to improving the genome assembly of Ae. aegypti.
Improving the Population Genetics Toolbox for the Study of the African Malaria Vector Anopheles Nili: Microsatellite Mapping to Chromosomes
Anopheles nili is a major vector of malaria in the humid savannas and forested areas of sub-Saharan Africa. Understanding the population genetic structure and evolutionary dynamics of this species is important for the development of an adequate and targeted malaria control strategy in Africa. Chromosomal inversions and microsatellite markers are commonly used for studying the population structure of malaria mosquitoes. Physical mapping of these markers onto the chromosomes further improves the toolbox, and allows inference on the demographic and evolutionary history of the target species.
Cytogenetic Map for Anopheles Nili: Application for Population Genetics and Comparative Physical Mapping
Anopheles nili is one of the major malaria vectors in Africa with a wide geographic distribution. However, the taxonomic and population genetic studies on this species are scarce. New research tools are urgently needed to genetically characterize this important malaria vector. In this study, a high-resolution cytogenetic map was developed for An. nili polytene chromosomes. Chromosomes were straightened and subdivided into 46 numbered divisions according to the banding pattern. Population analysis of An. nili females collected in Burkina Faso revealed the presence of two highly polymorphic inversions on the 2R chromosomal arm. A statistically significant departure from Hardy-Weinberg equilibrium due to a deficit in heterozygotes was detected for inversion 2Rb. To determine chromosome homologies and gene order conservation between An. nili and other major malaria vectors, PCR probes based on the An. gambiae coding sequences were mapped to An. nili chromosomes. Comparative mapping demonstrated that An. nili chromosomes have an An. stephensi-like arm association and that whole-arm translocations and paracentric inversions were the major types of rearrangement in evolution of these mosquitoes. The minimum number of fixed inversions among An. nili, An. gambiae, and An. stephensi was calculated using the Multiple Genome Rearrangements (MGR), Genome Rearrangements In Man and Mouse (GRIMM), and Sorting Permutation by Reversals and block-INterchanGes (SPRING) programs. The data suggest that the An. nili is, at least, as diverged from An. gambiae as An. stephensi. We provide evidence that 2La/a arrangement of An. gambiae is present in outgroup species An. nili and An. stephensi confirming the ancestral status of the 2La inversion in the An. gambiae complex. Availability of the new polytene chromosome map, polymorphic inversions, and physically mapped DNA markers for An. nili will further stimulate population genetic, taxonomic, and genomic studies of this neglected malaria vector.
