Enhancing Genome Investigations in the Mosquito Culex quinquefasciatus via BAC Library Construction and Characterization

Background Culex quinquefasciatus (Say) is a major species in the Culex pipiens complex and an important vector for several human pathogens including West Nile virus and parasitic filarial nematodes causing lymphatic filariasis. It is common throughout tropical and subtropical regions and is among the most geographically widespread mosquito species. Although the complete genome sequence is now available, additional genomic tools are needed to improve the sequence assembly. Findings We constructed a bacterial artificial chromosome (BAC) library using the pIndigoBAC536 vector and HindIII partially digested DNA isolated from Cx. quinquefasciatus pupae, Johannesburg strain (NDJ). Insert size was estimated by NotI digestion and pulsed-field gel electrophoresis of 82 randomly selected clones. To estimate genome coverage, each 384-well plate was pooled for screening with 29 simple sequence repeat (SSR) and five gene markers. The NDJ library consists of 55,296 clones arrayed in 144 384-well microplates. Fragment insert size ranged from 50 to 190 kb in length (mean = 106 kb). Based on a mean insert size of 106 kb and a genome size of 579 Mbp, the BAC library provides ~10.1-fold coverage of the Cx. quinquefasciatus genome. PCR screening of BAC DNA plate pools for SSR loci from the genetic linkage map and for four genes associated with reproductive diapause in Culex pipiens resulted in a mean of 9.0 positive plate pools per locus. Conclusion The NDJ library represents an excellent resource for genome assembly enhancement and characterization in Culex pipiens complex mosquitoes

Genome-Based Microsatellite Development in the Culex pipiens Complex and Comparative Microsatellite Frequency with Aedes aegypti and Anopheles gambiae

Mosquitoes in the Culex pipiens complex are among the most medically important vectors for human disease worldwide and include major vectors for lymphatic filariasis and West Nile virus transmission. However, detailed genetic studies in the complex are limited by the number of genetic markers available. Here, we describe methods for the rapid and efficient identification and development of single locus, highly polymorphic microsatellite markers for Cx. pipiens complex mosquitoes via in silico screening of the Cx. quinquefasciatus genome sequence.Six lab colonies representing four Cx. pipiens and two Cx. quinquefasciatus populations were utilized for preliminary assessment of 38 putative loci identified within 16 Cx. quinquefasciatus supercontig assemblies (CpipJ1) containing previously mapped genetic marker sequences. We identified and validated 12 new microsatellite markers distributed across all three linkage groups that amplify consistently among strains representing the complex. We also developed four groups of 3-5 microsatellite loci each for multiplex-ready PCR. Field collections from three cities in Indiana were used to assess the multiplex groups for their application to natural populations. All were highly polymorphic (Mean  = 13.0 alleles) per locus and reflected high polymorphism information content (PIC) (Mean  = 0.701). Pairwise F(ST) indicated population structuring between Terre Haute and Fort Wayne and between Terre Haute and Indianapolis, but not between Fort Wayne and Indianapolis. In addition, we performed whole genome comparisons of microsatellite motifs and abundance between Cx. quinquefasciatus and the primary vectors for dengue virus and malaria parasites, Aedes aegypti and Anopheles gambiae, respectively.We demonstrate a systematic approach for isolation and validation of microsatellites for the Cx. pipiens complex by direct screen of the Cx. quinquefasciatus genome supercontig assemblies. The genome density of microsatellites is greater in Cx. quinquefasciatus (0.26%) than in Ae. aegypti (0.14%), but considerably lower than in An. gambiae (0.77%)

Analysis of 14 BAC sequences from the Aedes aegypti genome: a benchmark for genome annotation and assembly

In order to provide a set of manually curated and annotated sequences from the Aedes aegypti genome, mapped BAC clones encompassing 1.57 Mb were sequenced, assembled and manually annotated using computational gene-finding, EST matches as well as comparative protein homology

Mitotic-chromosome-based physical mapping of the Culex quinquefasciatus genome

The genome assembly of southern house mosquito Cx. quinquefasciatus is represented by a high number of supercontigs with no order or orientation on the chromosomes. Although cytogenetic maps for the polytene chromosomes of this mosquito have been developed, their utilization for the genome mapping remains difficult because of the low number of high-quality spreads in chromosome preparations. Therefore, a simple and robust mitotic-chromosome-based approach for the genome mapping of Cx. quinquefasciatus still needs to be developed. In this study, we performed physical mapping of 37 genomic supercontigs using fluorescent in situ hybridization on mitotic chromosomes from imaginal discs of 4th instar larvae. The genetic linkage map nomenclature was adopted for the chromosome numbering based on the direct positioning of 58 markers that were previously genetically mapped. The smallest, largest, and intermediate chromosomes were numbered as 1, 2, and 3, respectively. For idiogram development, we analyzed and described in detail the morphology and proportions of the mitotic chromosomes. Chromosomes were subdivided into 19 divisions and 72 bands of four different intensities. These idiograms were used for mapping the genomic supercontigs/genetic markers. We also determined the presence of length polymorphism in the q arm of sex-determining chromosome 1 in Cx. quinquefasciatus related to the size of ribosomal locus. Our physical mapping and previous genetic linkage mapping resulted in the chromosomal assignment of 13% of the total genome assembly to the chromosome bands. We provided the first detailed description, nomenclature, and idiograms for the mitotic chromosomes of Cx. quinquefasciatus. Further application of the approach developed in this study will help to improve the quality of the southern house mosquito genome

An integrated linkage, chromosome, and genome map for the yellow fever mosquito Aedes aegypti.

Aedes aegypti, the yellow fever mosquito, is an efficient vector of arboviruses and a convenient model system for laboratory research. Extensive linkage mapping of morphological and molecular markers localized a number of quantitative trait loci (QTLs) related to the mosquito's ability to transmit various pathogens. However, linking the QTLs to Ae. aegypti chromosomes and genomic sequences has been challenging because of the poor quality of polytene chromosomes and the highly fragmented genome assembly for this species.Based on the approach developed in our previous study, we constructed idiograms for mitotic chromosomes of Ae. aegypti based on their banding patterns at early metaphase. These idiograms represent the first cytogenetic map developed for mitotic chromosomes of Ae. aegypti. One hundred bacterial artificial chromosome clones carrying major genetic markers were hybridized to the chromosomes using fluorescent in situ hybridization. As a result, QTLs related to the transmission of the filarioid nematode Brugia malayi, the avian malaria parasite Plasmodium gallinaceum, and the dengue virus, as well as sex determination locus and 183 Mbp of genomic sequences were anchored to the exact positions on Ae. aegypti chromosomes. A linear regression analysis demonstrated a good correlation between positions of the markers on the physical and linkage maps. As a result of the recombination rate variation along the chromosomes, 12 QTLs on the linkage map were combined into five major clusters of QTLs on the chromosome map.This study developed an integrated linkage, chromosome, and genome map-iMap-for the yellow fever mosquito. Our discovery of the localization of multiple QTLs in a few major chromosome clusters suggests a possibility that the transmission of various pathogens is controlled by the same genomic loci. Thus, the iMap will facilitate the identification of genomic determinants of traits responsible for susceptibility or refractoriness of the mosquito to diverse pathogens

Genome-based polymorphic microsatellite development and validation in the mosquito <it>Aedes aegypti </it>and application to population genetics in Haiti

<p>Abstract</p> <p>Background</p> <p>Microsatellite markers have proven useful in genetic studies in many organisms, yet microsatellite-based studies of the dengue and yellow fever vector mosquito <it>Aedes aegypti </it>have been limited by the number of assayable and polymorphic loci available, despite multiple independent efforts to identify them. Here we present strategies for efficient identification and development of useful microsatellites with broad coverage across the <it>Aedes aegypti </it>genome, development of multiplex-ready PCR groups of microsatellite loci, and validation of their utility for population analysis with field collections from Haiti.</p> <p>Results</p> <p>From 79 putative microsatellite loci representing 31 motifs identified in 42 whole genome sequence supercontig assemblies in the <it>Aedes aegypti </it>genome, 33 microsatellites providing genome-wide coverage amplified as single copy sequences in four lab strains, with a range of 2-6 alleles per locus. The tri-nucleotide motifs represented the majority (51%) of the polymorphic single copy loci, and none of these was located within a putative open reading frame. Seven groups of 4-5 microsatellite loci each were developed for multiplex-ready PCR. Four multiplex-ready groups were used to investigate population genetics of <it>Aedes aegypti </it>populations sampled in Haiti. Of the 23 loci represented in these groups, 20 were polymorphic with a range of 3-24 alleles per locus (mean = 8.75). Allelic polymorphic information content varied from 0.171 to 0.867 (mean = 0.545). Most loci met Hardy-Weinberg expectations across populations and pairwise F_STcomparisons identified significant genetic differentiation between some populations. No evidence for genetic isolation by distance was observed.</p> <p>Conclusion</p> <p>Despite limited success in previous reports, we demonstrate that the <it>Aedes aegypti </it>genome is well-populated with single copy, polymorphic microsatellite loci that can be uncovered using the strategy developed here for rapid and efficient screening of genome supercontig assemblies. These loci are suitable for genetic and population studies using multiplex-PCR.</p

Multicolor FISH on mitotic and polytene chromosomes of <i>Aedes aegypti</i>.

<p>DNA probes labeled with fluorescein, Cy3, Cy5, and combinations of these dyes were hybridized to the prophase (A, B, C) and polytene chromosomes (D) stained by DAPI. The maximum resolutions of ∼0.5 Mb between markers LF179 and LF314 (A) and 300 kb between markers LF407 and <i>Amyl</i> (C) are obtained on prophase and polytene chromosomes, respectively.</p

Chromosome locations of genetic markers and genomic supercontigs.

<p>Major signals are indicated by asterisks, NA – not applicable, LG – Linkage Group.</p

Localization of QTLs to various pathogens on linkage and chromosome maps of <i>Aedes aegypti</i>.

<p>The linkage positions of QTLs in cM on linkage groups I, II, and III are indicated on the left side. QTLs to dengue virus 2 (DEN2), midgut infection barrier (MIB), DEN2 midgut escape barrier (MEB), malaria parasite, and filarial worm are shown on the right sides of chromosomes 1, 2, and 3.</p