PLB143 - Lecture 05

Contemporary Methods in the Study of Crop Evolution

Plant Sciences

© Felix Fritschi and Paul Gepts 2009


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Readings

Lecture slides


Sources of evidence for the origin and spread of cultivated plants

(see Lecture 02)
  • de Candolle :
    • Botanical
    • Archaeological
    • Historical
    • Linguistic
  • Harlan :
    • Plants
      • Living
      • Dead
    • Humans
      • Living
      • Dead

Evidence from living plants


  • Major contribution: Identification of the wild ancestor of crop plants
    • Distribution area of the wild ancestor
    • Organization of genetic diversity
    • Introgression between wild and cultivated forms
  • Comparison between wild and cultivated materials
    • Morphological
    • Physiological
    • Genetic
  • Identification of changes relevant to adaptation
    • New adaptive syndromes evolved under domestications
    • Selective forces
    • Genetic systems involved

Plant Science methods (Overview)


  • External phenotype-based analysis: Classical Taxonomy
    • Recognition by close morphological resemblance
    • Oldest method
    • Comparative morphology and anatomy
    • Excess of variation (repeated mutations) and convergent evolution (similar selection in different areas) makes establishing relationships difficult
  • Molecular and Biochemical analysis
    • Chromosome level: Cytogenetic analysis
    • Protein level: Biochemical markers
    • DNA level: Molecular markers

Plant Science methods


Hybridization


  • Crosses between wild and cultivated taxa followed by examination of hybrids
    • Cross-incompatibility
    • Inviability
    • Sterility
    • Other types of reproductive isolation barriers...
      • Definition of "primary", "secondary" and "tertiary" gene pools of Harlan and de Wet (1975)
 
      • Modified version to take into account genomics and genetic engineering

  • Chromosome pairing at Metaphase I of meiosis: may indicate the degree of chromosomal homology between the two parents
    • Fully fertile hybrids showing normal chromosome pairing in meiosis point to close relationship between the tested parents and implicate the wild plant in the ancestry of the crop.

  • Changes in chromosome numbers: Polyploidy, Haploidy and Aneuploidy
    • Evolution by polyploidy is common in the plant kingdom
      • Autopolyploids: One chromosome set is doubled: tuber plants, potato, alfalfa, blueberry.......
      • Allopolyploids: Two or more differentiated chromosome sets are doubled: Cotton, bread wheat , tobacco...
  • Limitations of cytogenetic analysis
    • Species with large chromosomes
    • Wild progenitor diverged not too long ago and is not extinct
    • Time-consuming and cumbersome; many crosses involved
    • In some cases, simple genetic control of pairing:
      • Example: Ph locus in wheat


Biochemical markers, i.e. proteins


Basis: Separation of proteins with different net charge and/or different molecular weight and/or conformation migrate at different rates through matrix of starch or acrylamide gels.
  • Seed storage proteins, e.g., phaseolin of Phaseolus vulgaris (common bean)
Basis: SDS (ionic detergent) will bind to proteins in a constant ratio and confers a negative charge so migration is proportional to polypeptide size.
  • Method:
    • Homogeneize seed
    • Add buffer, centrifuge and collect supernatant
    • Electrophorese
    • Stain and score

  • Example: Phaseolin marker confirmed morphological and viability studies pointing to two different areas of domestication: Mesoamerica and the Andes (Gepts et al. 1986; Koenig and Gepts 1989)
    • Where was common bean domesticated?
      • double (or triple) domestications in Mexico and South America

  • Various types of phaseolin (the principal seed protein of beans) revealed by electrophoresis



  • Distribution of wild beans in Latin America



  • Seed protein patterns revealing two major domestication centers for beans in Latin America


  • Isozymes: Different molecular forms of an enzyme that catalyze the same reaction,e.g., isozymes of Zea mays (maize or corn).
    • Basis: Non-denatured proteins with different net charge migrate at different rates
    • Method:  from Hancock 1992 , © Prentice-Hall)
      • Extract proteins under "protective" conditions: i.e. low temperature
      • Soak extract onto a paper wick
      • Place wicks side by side along a slit cut in the gel (starch gel)
      • Electrophoresis
      • Gel removed and sliced horizontally
      • Slices incubated with histochemical stains specific for the enzymes
    • Result: Zymograms helped identify the closest wild progenitor of maize (Doebley et al. 1984)
      • Is teosinte the wild ancestor of maize?
      • Distribution of teosinte: (from B. Smith, The Emergence of Agriculture, © Scientific American Library 1995)
      • Principal component analysis of isozyme frequencies: (from Doebley et al. 1984)
      • A comparison of dendrograms establised from isozyme and morphological data:  (from Doebley et al. 1984)
 
 



  • Attributes and limitations of protein analyses
    • Many loci and individuals can be measured simultaneously
    • Seed proteins
      • High level of polymorphism
      • Low number of loci
      • Environmental stability
    • Isozymes
      • Although more than 100 isozyme systems have been described only 40 or 50 are available for a given taxon
      • Low level of polymorphism
      • Simplicity and low cost
      • Environmentally influenced
    • Variability assessed only at the level of gene product


Molecular markers


  • Restriction fragment length polymorphisms (RFLPs)
    • Basis: Differences in restriction patterns between two related sequences indicative of a modification in the DNA primary structure
    • Method:  (from Hancock 1992 , © Prentice-Hall)
      • Cutting (restricting) DNA with one or more endonucleases
      • Separation of restriction fragments according to molecular weight
      • Denature the DNA
      • Transfer by capillarity to a membrane
      • Hybridize to a given probe
      • Types of DNA and implications (mt, cp,  nDNA )
    • Result: The donors of 2 out of 3 of the the basic genomes of breadwheat have been unequivocally identified (Dvorak et al. 1993).


  • Attributes and limitations of RFLPs:  
    • Slow and expensive
    • Higher level of polymorphism than isozymes
    • Environmental stability
    • Larger number of loci
    • Selective neutrality



  • Random Amplified Polymorphic DNA (RAPDs)
    • Basis: Detection of differences in patterns of DNA amplification from short primers of arbitrary sequence
    • Method: Compare PCR and  RAPD
      • Denaturation of DNA and annealing of primers
      • Primer extension
      • Repeat cycling for 20 x
      • Electrophorese PCR products
      • Stain and score
    • Result: RAPDs have confirmed and complemented inferences from protein and RFLP studies in Phaseolus vulgaris (Freyre R, Ríos R, Guzmán L, Debouck DG, Gepts P. 1996. Ecogeographic Distribution of Phaseolus spp. (Fabaceae) in Bolivia. Econ. Bot. 50: 195-215



    • Advantages and disadvantages of RAPDs:
      • More polymorphic than RFLPs
      • Simple and quick
      • Selective neutrality
      • Reproducibility among labs may be a problem



  • AFLP: Amplified Fragment Length Polymorphism
    • Basis: A combination of RFLP and PCR
    • Procedure: DNA digestion
      • Ligation of adaptors to ends of restriction fragments
      • PCR with primers complementary to adaptors but with 3' overhangs
      • Visualization after separation on sequencing gel: 
    • Advantages / disadvantages:
      • highly sensitive
      • highly reproducible (repeatable)
      • selective neutrality
      • technically demading
      • expensive
    • Example: Site of Einkorn Wheat Domestication Identifed by DNA Fingerprinting (Heun et al., 1997)
      • Fingerprinting of 338 lines based on the presence vs. absence of 288 AFLP bands
      • Lines were assigned into groups based on geographical origin.
      • Cultivated einkorns are closely related among themselves and show a close phylogenetic similarity to group D
      • => Are the D lines the closest relatives of the wild progenitors that gave rise to cultivated einkorn?



  • Microsatellites or Simple Sequence Repeats (SSRs)
    • Basis:
      • variable number of short tandemly repeated sequences
    • Method:
      • sequence SSR and adjacent DNA
      • design PCR primers and conduct a PCR amplification of DNA
      • separate amplified DNA by electrophoresis: 
    • Result:
      • genealogy of wine grape cultivars
    • Advantages:
      • high level of polymorphism
      • easy and fast to run
      • robust and reproducible
    • Disadvantage:
      • time-consuming and expensive to develop
    • Example: The origin of Chardonnay, Gamay Noir, etc. Bowers et al. 1999
      • Pinot noir x Gouais blanc
      • Identified some 16 varieties as progeny of this cross
      • Probability: P x G is 1012 to 1015 times more likely than other combinations
      • Morphological ressemblances were not a good predictor of relatedness
      • Parents:
        • Pinot noir: already known by Romans
        • Gouais blanc: poor varietal
        • Both: widespread during Middle Ages




  • DNA Sequencing
    • Basis: Differences in nucleotide sequences of specific regions (locus/loci)
    • Method:
      • DNA extraction
      • PCR
      • DNA sequencing
    • Advantages and disadvantages
      • highly reproducible
      • very informative
      • expensive
      • knowledge of sequence for probe required
    • Example: Evidence on the origin of cassava: Phylogeography of Manihot esculenta. Olsen and Schaal. 1999. PNAS 96:5586-5591
      • Analysis of G3pdh sequences of 212 accessions
      • 64 polymorphic sites found in the sequenced region (max. 962 bp)
      • 28 haplotypes were detected; 5 of them occurred in cultivated and wild cassava: 
      • Geographically the shared haplotypes occur along the southern border of the Amazon basin



Summary of the lecture
  • Plant sciences can help identify the wild ancestor of crops by analyzing variability present at different levels
  • Molecular techniques have improved crop evolution studies by allowing:
    • Broader sampling of the genome
    • Quicker screening of a larger number of plants
    • Sampling of variability that is "selectively neutral" in general



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