Cytogenetics in Wildlife Conservation

Karyotyping by M-FISH

Introduction to Cytogenetics

• Cytogenetics is the study of the structure and function of cells, particularly chromosomes.

• It is one of the oldest forms of genetic study, dating back to 1882 when Salamander chromosomes were described by Walther Fleming.

• Walther Fleming used dyes to identify chromatin in the cell nucleus and discover mitosis.

Learning Outcomes

• Use cytogenetic approaches to identify species.

• Assess numerical and structural changes in chromosomes.

• Determine gender relevant to wildlife conservation.

Mitosis

*Process:

• Parental cell.

• Each chromosome duplicates itself to form two chromatids joined at the centromere.

• At this stage, chromosomes are visible under a light microscope.

• Each duplicated chromosome lines up at the center of the cell.

The Cell Cycle

Phases:

• Interphase: Cell growth and preparation for division; includes G1, S, and G2 phases.

• Prophase: Chromatin condenses into visible chromosomes.

• Prometaphase: Nuclear envelope breaks down; spindle microtubules attach to kinetochores.

• Metaphase: Chromosomes align at the cell's center.

• Anaphase: Sister chromatids separate and move to opposite poles.

• Telophase: Chromosomes arrive at the poles; nuclear envelope reforms.

• Cytokinesis: Cell divides into two daughter cells.

Key Components & Processes:

• Spindle Assembly Checkpoint: Ensures proper chromosome alignment and segregation.

• Microtubule-kinetochore attachment: Attachment of microtubules to kinetochores on chromosomes.

• APC/C regulation: Regulation of the Anaphase-Promoting Complex/Cyclosome.

• Spindle elongation: Elongation of the spindle during anaphase.

• Cleavage furrow formation: Formation of the cleavage furrow during cytokinesis.

• Chromosome segregation: Separation of chromosomes during anaphase.

• Mitotic entry and M: Entry into mitosis.

• G1/S transition: Decision point to proceed with cell division.

• G2/M transition: Checkpoint before entering mitosis.

• Centrosome, Centriole, Microtubule: Structures involved in spindle formation and organization.

Metaphase Spreads

Process:

• Chromosome Reproduction (Doubling of the DNA Content).

• Illustrations show the progression from interphase through prophase, prometaphase, metaphase, anaphase, and telophase, eventually returning to interphase.

Metaphase Spread Preparation

• Sampling: Taking a blood sample.

• Culturing: Growing cells in a medium.

• Harvesting:

  • Adding Colcemid.

  • Using a Hypotonic Solution.

  • Fixation (x3).

• Slide Preparation: Applying 5% acetic acid solution, Chromosome staining.

Karyotype Examples

• Human karyotype: shows the arrangement of human chromosomes.

• Rat karyotype.

• Dog karyotype: Diploid number 2n = 78. There are 78 chromosomes arranged and numbered accordingly.

• Chicken karyotype: Diploid number 2n = 18.

• Chicken karyotype showing microchromosomes: Actual diploid number 2n = 78.

• Platypus karyotype: Diploid number 2n = 52 (21 autosomes + 10 sex chromosomes).

Ideograms

• Diagrammatic representation of chromosomes, showing banding patterns.

• Example: Ideogram for Physical Mapping in the Saltwater Crocodile (Crocodylus porosus).

The Centromere

• The region of the chromosome where microtubules of the spindle attach during cell division.

• Chromosomes can be classified based on the position of the centromere.

Cytogenetic Techniques

• Banding

• Fluorescent in situ hybridization (FISH)

• Gene mapping with FISH

• Fiber FISH

• Chromosome painting

• Comparative genomic hybridization

• Next-generation sequencing techniques

Case Studies using Cytogenetic Techniques

The African Grey Parrot

• Gene mapping with FISH.

Zoo-FISH

• Using chicken chromosomes 1-9 and Z.

• Multiple fluorescent colors can show different things in the one image.

• Mapping chicken chromosomes to the African grey parrot chromosomes in a metaphase spread.

The Tammar Wallaby

• Comparative genomic hybridizations.

• Helps to physically locate where homologous regions or loci map on the chromosomes between species (human and Tamar wallaby).

Rock Wallabies

• Distributed throughout Australia.

• Twenty-one (21) taxa of rock wallabies had been described as species or subspecies up to 1976.

• Chromosomal differences (structure and/or number) within rock wallabies indicate different species and subspecies.

• Differences in chromosome structure between species:

  • Petrogale persephone (2n=22)

  • Petrogale celeris (2n=22)

  • Petrogale inorta (2n=22)

  • Petrogale burbidgei (2n=16)

  • Petrogale brachyotis (2n=18)

  • Petrogale godmani godmani (2n=20)

• Differences in chromosome number between species (morphologically distinct).

• Differences in chromosome number and structure within a species:

  • Petrogale lateralis lateralis (2n=22)

  • Petrogale lateralis purpureicollis (2n=22)

  • Petrogale lateralis hacketti (2n=20) (morphologically similar).

Extant Peccary Species

• Chacoan peccary (Catagonus wagneri) - 2n = 20

• White-lipped peccary (Tayassu pecari) - 2n = 26

• Collared peccary (Pecari tajacu) - 2n = 30

Collared Peccaries

• Demonstration of how chromosomal abnormalities arise in meiosis.

• Illustrates the consequences of unbalanced gametes in the offspring (partial trisomy/monosomy).

Chromosomal Aberrations

Numerical Changes

• Polyploidy (auto or allo)

• Aneuploidy

Structural Rearrangements

• Deletions

• Duplications

• Inversions

• Translocations

• Somatic mosaicism

Summary

• All genetic material is part of the genome.

• The number and ploidy of chromosomes can vary between species.

• Chromosomal changes can impact selection/evolution – not just base pair changes.

Applications of Karyotypes and Cytogenetic Techniques in Conservation

• Species determination

• Identifying hybridization

• Understanding genome evolution and species relationships