PLB143 - Lecture 05

Contemporary Methods in the Study of Crop Evolution

Plant Sciences

© Felix Fritschi and Paul Gepts 2009

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Readings

Required:

Hancock J.F. 1992. Chapter 1: Mutation and genetic variability: ../ucd-access/Hancock Chapter 1.pdf (Note: large file: 4.3 Mb)
Bowers et al. 1999. Historical Genetics: The Parentage of Chardonnay, Gamay, and Other Wine Grapes of Northeastern France. Science 285: 1562-1565 (accessible with campus login and password)

Additional readings:

Avise, J.C. 1994. Molecular markers, natural history and evolution. Chapman and Hall, New York, N.Y.
Zohary D, Hopf M. 1988. Domestication of plants in the Old World. Clarendon, Oxford, U.K.
Dvorak, J. et al. 1993. The evolution of polyploid wheats: identification of the A genome donor species. Genome 36: 21-31.
Doebley, J.F. and M.M. Goodman. 1984. Isoenzymatic variation in Zea (Gramineae). Syst. Bot. 9:203-218.
Freyre R, Ríos R, Guzmán L, Debouck DG, Gepts P. 1996. Ecogeographic Distribution of Phaseolus spp. (Fabaceae) in Bolivia. Econ. Bot. 50: 195-215

Lecture slides

Pdf format

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)	Close distance between maize (left) and its progenitor, teosinte (right) Distribution in Mexico and Guatemala of the major teosinte types :Genetic distances among teosinte and maize population Different dendrograms depending on the trait analyzed: isozymes (left) and tassel morphology (right)

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).	Proposed scenario for the origin of bread wheat Hybridizations with RFLP probes revealing the identitity of one of the parents of bread wheat

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	PCR RAPD Genetic distances among bean lines

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?	AFLP procedure Distribution of wild einkorn wheat Genetic relatedness among wild einkorn population

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	Example of microsatellite marker variation

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	Genetic distances among cassava populations Location of presumed progenitor of cassava

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