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A standard cytogenetic map of Culex quinquefasciatus polytene chromosomes in application for fine-scale physical mapping.

Unger MF, Sharakhova MV, Harshbarger AJ, Glass P, Collins FH - Parasit Vectors (2015)

Bottom Line: Six supercontigs were oriented and one was found putatively misassembled.Better assembled C. quinquefasciatus genome can serve as a reference for studying other vector species of C. pipiens complex and will help to resolve their taxonomic relationships.This, in turn, will contribute into future development of vector and disease control strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, USA. Munger1@alumni.nd.edu.

ABSTRACT

Background: Southern house mosquito Culex quinquefasciatus belongs to the C. pipiens cryptic species complex, with global distribution and unclear taxonomy. Mosquitoes of the complex can transmit human and animal pathogens, such as filarial worm, West Nile virus and avian malarial Plasmodium. Physical gene mapping is crucial to understanding genome organization, function, and systematic relationships of cryptic species, and is a basis for developing new vector control strategies. However, physical mapping was not established previously for Culex due to the lack of well-structured polytene chromosomes.

Methods: Inbreeding was used to diminish inversion polymorphism and asynapsis of chromosomal homologs. Identification of larvae of the same developmental stage using the shape of imaginal discs allowed achievement of uniformity in chromosomal banding pattern. This together with high-resolution phase-contrast photography enabled the development of a cytogenetic map. Fluorescent in situ hybridization was used for gene mapping.

Results: A detailed cytogenetic map of C. quinquefasciatus polytene chromosomes was produced. Landmarks for chromosome recognition and cytological boundaries for two inversions were identified. Locations of 23 genes belonging to 16 genomic supercontigs, and 2 cDNA were established. Six supercontigs were oriented and one was found putatively misassembled. The cytogenetic map was linked to the previously developed genetic linkage groups by corresponding positions of 2 genetic markers and 10 supercontigs carrying genetic markers. Polytene chromosomes were numbered according to the genetic linkage groups.

Conclusions: This study developed a new standard cytogenetic photomap of the polytene chromosomes for C. quinquefasciatus and was applied for the fine-scale physical mapping. It allowed us to infer chromosomal position of 1333 of annotated genes belonging to 16 genomic supercontigs and find orientation of 6 of these supercontigs; the new cytogenetic and previously developed genetic linkage maps were integrated based on 12 matches. The map will further assist in finding chromosomal position of the medically important and other genes, contributing into improvement of the genome assembly. Better assembled C. quinquefasciatus genome can serve as a reference for studying other vector species of C. pipiens complex and will help to resolve their taxonomic relationships. This, in turn, will contribute into future development of vector and disease control strategies.

No MeSH data available.


Related in: MedlinePlus

Typical chromosomal layouts from salivary glands of IV instar larvae C. quinquefasciatus, (a) Generation four of an iso-female JHB line, and (b) Generation seven of iso-female JHB line. Centromeres and chromosome arms are labeled. Thin arrows point to the ectopic contacts. Nucleolus is shown by thick arrow (a). Landmark of 3L is shown in subdivision 68C (a, b ). Telomeres are abbreviated as T, and shown where possible
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Fig1: Typical chromosomal layouts from salivary glands of IV instar larvae C. quinquefasciatus, (a) Generation four of an iso-female JHB line, and (b) Generation seven of iso-female JHB line. Centromeres and chromosome arms are labeled. Thin arrows point to the ectopic contacts. Nucleolus is shown by thick arrow (a). Landmark of 3L is shown in subdivision 68C (a, b ). Telomeres are abbreviated as T, and shown where possible

Mentions: In order to develop a cytogenetic map for the polytene chromosomes of C. quinquefasciatus we examined salivary glands, Malpighian tubules, and gastric cecum of 4th instar larvae, pupae and adults, for the presence of polytene chromosomes using lacto-aceto-orcein staining. All of these tissues develop many lower-polytenized nuclei but only a few are highly polytenized. We chose to work with salivary glands, because they appeared to have chromosomes which achieved the highest polyteny level and their banding patterns did not vary as much as in chromosomes of other tissues. We also utilized Imaginal Disks (IDs) as a pointer to the sub-staging within IV instar larval stage. Larvae possessing IDs with oval shape were found to be the best candidates for well-developed polytene chromosomes, with a more uniform banding pattern. In contrast, larvae with IDs round (earlier sub-stage), or with IDs leg-shaped (later sub-stage), had respectively under-developed or over-developed polytene chromosomes, respectively. For the map development we used iso-female inbred lines of JHB strain to decrease inversion polymorphism and asynapsis of homologs. The best chromosome photographs were obtained from iso-female lines of the JHB strain generation 4 (Fig. 1a) and generation 7 (Fig. 1b, Fig. 2). Approximately 25 % of all chromosome preparations contained more than one nucleus suitable for cytogenetic analysis. Images were taken from approximately 30 of the best slides. Chromosome arms were straightened and overlaid using AdobePhotoshop. Images were compared to identify a common banding pattern for the C. quinquefasciatus chromosomes and establish landmarks for chromosome arm recognition.Fig. 1


A standard cytogenetic map of Culex quinquefasciatus polytene chromosomes in application for fine-scale physical mapping.

Unger MF, Sharakhova MV, Harshbarger AJ, Glass P, Collins FH - Parasit Vectors (2015)

Typical chromosomal layouts from salivary glands of IV instar larvae C. quinquefasciatus, (a) Generation four of an iso-female JHB line, and (b) Generation seven of iso-female JHB line. Centromeres and chromosome arms are labeled. Thin arrows point to the ectopic contacts. Nucleolus is shown by thick arrow (a). Landmark of 3L is shown in subdivision 68C (a, b ). Telomeres are abbreviated as T, and shown where possible
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4465148&req=5

Fig1: Typical chromosomal layouts from salivary glands of IV instar larvae C. quinquefasciatus, (a) Generation four of an iso-female JHB line, and (b) Generation seven of iso-female JHB line. Centromeres and chromosome arms are labeled. Thin arrows point to the ectopic contacts. Nucleolus is shown by thick arrow (a). Landmark of 3L is shown in subdivision 68C (a, b ). Telomeres are abbreviated as T, and shown where possible
Mentions: In order to develop a cytogenetic map for the polytene chromosomes of C. quinquefasciatus we examined salivary glands, Malpighian tubules, and gastric cecum of 4th instar larvae, pupae and adults, for the presence of polytene chromosomes using lacto-aceto-orcein staining. All of these tissues develop many lower-polytenized nuclei but only a few are highly polytenized. We chose to work with salivary glands, because they appeared to have chromosomes which achieved the highest polyteny level and their banding patterns did not vary as much as in chromosomes of other tissues. We also utilized Imaginal Disks (IDs) as a pointer to the sub-staging within IV instar larval stage. Larvae possessing IDs with oval shape were found to be the best candidates for well-developed polytene chromosomes, with a more uniform banding pattern. In contrast, larvae with IDs round (earlier sub-stage), or with IDs leg-shaped (later sub-stage), had respectively under-developed or over-developed polytene chromosomes, respectively. For the map development we used iso-female inbred lines of JHB strain to decrease inversion polymorphism and asynapsis of homologs. The best chromosome photographs were obtained from iso-female lines of the JHB strain generation 4 (Fig. 1a) and generation 7 (Fig. 1b, Fig. 2). Approximately 25 % of all chromosome preparations contained more than one nucleus suitable for cytogenetic analysis. Images were taken from approximately 30 of the best slides. Chromosome arms were straightened and overlaid using AdobePhotoshop. Images were compared to identify a common banding pattern for the C. quinquefasciatus chromosomes and establish landmarks for chromosome arm recognition.Fig. 1

Bottom Line: Six supercontigs were oriented and one was found putatively misassembled.Better assembled C. quinquefasciatus genome can serve as a reference for studying other vector species of C. pipiens complex and will help to resolve their taxonomic relationships.This, in turn, will contribute into future development of vector and disease control strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, USA. Munger1@alumni.nd.edu.

ABSTRACT

Background: Southern house mosquito Culex quinquefasciatus belongs to the C. pipiens cryptic species complex, with global distribution and unclear taxonomy. Mosquitoes of the complex can transmit human and animal pathogens, such as filarial worm, West Nile virus and avian malarial Plasmodium. Physical gene mapping is crucial to understanding genome organization, function, and systematic relationships of cryptic species, and is a basis for developing new vector control strategies. However, physical mapping was not established previously for Culex due to the lack of well-structured polytene chromosomes.

Methods: Inbreeding was used to diminish inversion polymorphism and asynapsis of chromosomal homologs. Identification of larvae of the same developmental stage using the shape of imaginal discs allowed achievement of uniformity in chromosomal banding pattern. This together with high-resolution phase-contrast photography enabled the development of a cytogenetic map. Fluorescent in situ hybridization was used for gene mapping.

Results: A detailed cytogenetic map of C. quinquefasciatus polytene chromosomes was produced. Landmarks for chromosome recognition and cytological boundaries for two inversions were identified. Locations of 23 genes belonging to 16 genomic supercontigs, and 2 cDNA were established. Six supercontigs were oriented and one was found putatively misassembled. The cytogenetic map was linked to the previously developed genetic linkage groups by corresponding positions of 2 genetic markers and 10 supercontigs carrying genetic markers. Polytene chromosomes were numbered according to the genetic linkage groups.

Conclusions: This study developed a new standard cytogenetic photomap of the polytene chromosomes for C. quinquefasciatus and was applied for the fine-scale physical mapping. It allowed us to infer chromosomal position of 1333 of annotated genes belonging to 16 genomic supercontigs and find orientation of 6 of these supercontigs; the new cytogenetic and previously developed genetic linkage maps were integrated based on 12 matches. The map will further assist in finding chromosomal position of the medically important and other genes, contributing into improvement of the genome assembly. Better assembled C. quinquefasciatus genome can serve as a reference for studying other vector species of C. pipiens complex and will help to resolve their taxonomic relationships. This, in turn, will contribute into future development of vector and disease control strategies.

No MeSH data available.


Related in: MedlinePlus