Sexing: Sampling, DNA Extraction, PCR, and Sexing Markers

Hermit Thrushes

  • winter in E US and NE Mexico

  • Do females and males winter in separate areas?


General Considerations

Live animals: invasive and non-invasive sampling

  • Invasive

    • Blood

      • Mammalian red blood cells have no nuclei or mitochondria

      • Avian, reptilitian, amphibian, and fish do have nuclei and mitochondria (except salamanders)

    • Tissue clips, Darts, plucked feathers

  • Non-invasive

    • Shed feathers, scat, hair, Environmental DNA (ex. leeches)


Invasive sampling pros

  • 10x more DNA

  • Near the limit of DNA available in a feather

    • Allelic dropout in low DNA, replication necessary

    • No spare DNA for development of primers

  • Animals identity is known


Non-invasive sampling pros

  • no harm to the animal

  • sometimes don’t have to catch the animal (hair traps, scat samples)


Storing field samples for effective DNA extraction

  • freezing

  • lysis buffer

  • DMSO buffer

  • Ethanol (shipping issues)

  • dried blood on filter paper

  • dried slowly at a low temp (less than 30 C)


cross-contamination

  • mixing DNA from 2 or more samples

  • change needles/scalpel blades between samples

  • decontaminate scissors/drill bits/knives with 10% bleach then rinse with distilled water

  • is any sample on gloves, change them

  • note that dna is not destroyed by ethanol


Collecting samples: animals that died a long time ago

  • museum samples or subfossils

  • dried flesh (ex. toepads from birds)

  • feathers

  • bones

  • teeth

  • insect legs

  • careful of the preservation technique


DNA extraction

  • break down cell membranes (soap: SDS = SLS)

    • need to break down to get rid of hydrophobic bilayer

  • Digest cell proteins: histones attached to DNA and nucleases (enzyme proteinase K)

  • Isolate DNA from cellular debris and inhibitors


DNA Amplification

  • need to amplify DNA before most analyses

  • amplification: polymerase chain reaction (PCR)

    • advantages: almosy any tissue can be used, tiny amounts of tissue required

    • disadvantages: primer of known sequence usually required

  • Template DNA, primers, dNTPs, polymerase (Taq), buffer


Primer development

  • primers are on either side of a gene or locus and are crucial to amplification

  • Gene: a sequence of DNA, usually coding for a specific protein

  • Locus: (L = location) a site on a chromosome

  • primers must be developed for a new study species

  • you must have high quality and a lot of DNA to develop primers

  • primers need to be optimized: all quantities can be adjusted in a PCR reaction and affect amplification

    • process consumes DNA

  • human forensic analyses: primers and optimization well established


Common types of DNA markers

  • Marker: a gene or DNA sequence that allows us to detect genetic variation

    • SNPs

    • Microsatellites

    • DNA sequences

    • RFLPs, AFLPs, and many others


Markers on sex chromosomes

  • can be used to determine sex

  • Birds: female ZW, male ZZ

  • 2 copies of CHD (chromohelicase DNA) gene in the avian genome

  • one copy on the Z chromosome, the other on the W chromosome

  • CHD genes on Z and W vary in size and base sequence

  • females, with CHD-Z and CHD-W forms of the gene, can be distinguished from males, which only have CHD-Z

  • CHD dimorphism doesn’t exist for ratites


Load PCR product on a gel: gel electrophoresis


Hermit Thrush results

  • sex-biased latitudinal distribution, with the female mean 1.2 degrees south of male mean


Sexing other species

  • mammals: typically XX/XY with some exceptions, females are homogametic

    • in birds and mammals, heterogamety (ZW in birds and XY in mammals) is completely different genetically and evolutionarily stable

      • not the same for reptiles, fish, and amphibians

        • heterogametic sexes are not always evolutionarily stable: only a tiny genetic difference between them

      • The frog Rana rugosa has both male and female heterogametic systems: females are XX in some pops and ZW in others

  • Reptiles:

    • genetic sex determination

      • some spp of turtles and lizards are like mammals where females are homogametic and males are heterogametic

      • snakes are like birds, males homogametic

    • temp dependent sex determination

      • turtles (cool = male, warm = female)

      • crocodilians (low/high = females, middle = male)

    • Both (e.g. central bearded dragon)

  • Fish:

    • XX/XY, ZZ/ZW to polygenic, sometimes determined by environmental factors, dominance

    • who knows for some spp (like sturgeon)

  • Amphibians: XX/XY, ZZ/ZW, temperature

  • Invertabrates:

    • large variation: XX/XY, XX/X0, ZZ/ZW, hermaphrodites, haplodiploidy, sex reversal, parthenogenesis