methods

• Phylogenetic Approach: Methods and Analyses

  1. Collecting plant specimens

  2. Choice of material

  3. Plant pressing

  4. Collecting data

  5. Mounting of specimens

  6. DNA extraction

  7. PCR amplification

  8. Sequencing

  9. Data Analysing

  10. Writing

  
• Workflow


  • Field Work
  • Lab Work
  • Data analyzing
  • Writing everything up
• Collection of Raw Materials

  1. Field Trips

  2. Conducted different times of the year depending on flowering times and season

  3. Eg: Daisies = April – June, Roses = May – July, Succulents = July – September

  4. Whole plant, leaves, seeds, flowers – depending on what is available
• Collecting plant specimens


  • Herbarium specimens serve as permanent records of species and populations of plants
  • Good quality specimens must be collected
  • Material must be representative of the population and complete (flowers and/or fruit, underground parts)
  • Complete collecting data must accompany the specimen
  • Specimens must be properly dried and processed
  • Some of the specimen must be placed in silica gel for DNA extraction

  
• Collection of herbarium voucher in the field

  1. Use a press

  2. Make sure the samples dry up correctly (even changing papers if needed)

  3. Label the specimen with a voucher number

  4. Collect all morphological characters. E.g. flowers fruits

  5. Collect material for DNA extraction in silica

  6. For succulent plants cut off the green material

  7. For plants with small leaves place in a material bag before the silica

  8. Collect data: Collecting number, Date of collection, Country/province, GPS, Altitude, Identification, Plant family, Field observations, Picture numbers, Collectors

  
• Herbarium specimens


  • Whole plants, leaves, flowers, seed/capsules are dried and pressed
  • Called specimen sheets
  • Some are collected and replanted in greenhouses and botanical gardens
  • Pressed samples can take up to 1 – 3 months to dry

  
• Herbaria


  • 3000 herbaria in over 165 countries
  • The National Herbarium in Pretoria – Largest collection of plant specimens in Southern Africa (over one million)
  • Collection of preserved plant specimens
  • Botanical gardens to replant and cultivate the plants
  • Function: document specimen for formal research

  
• Lab work - Extraction

  1. DNA is extracted and isolated from plant material (roots, leaves)

  2. Plant material is crushed

  3. CTAB method

  4. Extraction kits

  5. (1Hr30min – 3Hrs)

  
• Lab work – Extraction steps

  1. Cell Lysis (Break Open Cells)

  2. DNA Purification (Clean Up)

  3. Resuspension (Get the DNA)

  4. Quantification (Check Quality)

  
• Gel electrophoresis


  • Electric Field: Apply an electric field to a gel containing molecules (like DNA or proteins)
  • Gel Matrix Separation: The gel acts like a sieve, slowing down larger molecules more than smaller ones
  • Visualize and Analyze: After running, visualize the separated molecules using UV lights
• PCR (Polymerase Chain Reaction)


  • A technique used in molecular biology that amplifies a specific segment of DNA
  • Allows scientists to make millions to billions of copies of a particular DNA sequence