PLB143 - Lecture 17

Future directions in the study of crop evolution

© Paul Gepts 2011

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PLB143: Readings - Lecture 17

  • Required:
    • Erickson DL, Smith BD, Clarke AC, Sandweiss DH, Tuross N (2005) An Asian origin for a 10,000-year-old domesticated plant in the Americas. Proceedings of the National Academy of Sciences of the United States of America 102:18315-18320 Pdf version
    • Olsen KM, Caicedo AL, Polato N, McClung A, McCouch S, Purugganan MD (2006) Selection under domestication: Evidence for a sweep in the rice Waxy genomic region. Genetics 173:975-983 Pdf version
  • Additional:
    • Kami J, Becerra Velásquez VL, Debouck DG, Gepts P (1995) Identification of presumed ancestral DNA sequences of phaseolin in Phaseolus vulgaris . Proc. Nat. Acad. Sci. USA 92: 1101-1104
    • Kwak M, Kami JA, Gepts P (2009) The putative Mesoamerican domestication center of Phaseolus vulgaris is located in the Lerma-Santiago basin of Mexico. Crop Sci 49:554-563.
    • Matsuoka Y, Vigouroux Y, Goodman MM, Sanchez G. J, Buckler E, Doebley J (2002) A single domestication for maize shown by multilocus microsatellite genotyping. Proc Natl Acad Sci USA 99: 6080-6084.
    • Wang, RL; Stec, A; Hey, J; Lukens, L; Doebley, J.  The limits of selection during maize domestication. Nature, MAR 18, 1999, V398(N6724):236-239  (with correction published Nature 410, 718  (05 April 2001))
    • Cong B, Barrero LS, Tanksley SD (2008) Regulatory change in YABBY-like transcription factor led to evolution of extreme fruit size during tomato domestication. Nature Genetics 40:800-804Archetti M (2009) Evidence from the domestication of apple for the maintenance of autumn colours by coevolution. Proceedings of the Royal Society B-Biological Sciences 276:2575-2580
    • Jaenicke-Després V, Buckler ES, Smith BD, Gilbert MTP, Cooper A, Doebley J, and Paabo S, 2003. Early allelic selection in maize as revealed by ancient DNA. Science 302:1206-1208
  • Presentation slides

Some current topics

  • Search of the wild progenitor: specific populations or descendants thereof: the case of common bean (Phaseolus vulgaris):
    • additional field explorations
    • DNA sequence information: direct or indirect: Kami et al. 1995
    • Identification of the Mesoamerican center of domestication of common bean: Kwak et al. 2009
  • Center of domestication and pathways of origin:
    • Use of microsatellite markers
    • Calculations of age domestication: Matsuoka et al. 2002
  • Changes during domestication:
    • physiological and ecological studies
    • cloning of genes involved in the domestication syndrome: reduced branching in maize: Wang et al. 1999
    • extreme fruit size in tomato: Cong et al. 2008
    • discovery of domestication genes by identification of selective sweeps: Olsen et al. 2006
  • Interactions with pests or pathogens
    • domestication of the apple, aphids, and fall colors: Archetti 2009
  • "Back to the future":
    • further integration of plant science and archaeology:
      • analysis of old DNA: Jaenicke-Després et al. 2003
      • origin of the bottle gourd: Erickson et al. 2005

Searching for the progenitor of common bean

Kami et al. 1995

Phaseolin as an evolutionary marker

  • Phaseolin: major seed storage protein in beans; 35-50% of total seed N
  • Small multigene family: 6-8 genes; single, complex locus
  • Two major phaseolin types:
    • S: Mesoamerican
    • T: Andean
  • Two classes of genes within S and T phaseolin gene families:
    • alpha: have tandem direct repeats
    • beta: do not have tandem direct repeats

Interpretation of the distribution of repeats

  • Absence of repeats also in nearest relatives (P. coccineus )
  • Probability of generating tandem direct repeat is higher than losing one:
    • both suggest that absence of repeats is ancestral state
  • Intermediate geographic distribution:
    • suggests dispersal northwards and southwards of wild populations (how?) followed by domestications in Mesoamerica and southern Andes


  • Mexico map
    (from Smith 1995 © Scientific American Library, New York.)

Cloning of the first domestication gene

Doebley et al. 1997: The tb1 gene

Differences in growth habit ( = shape of the plant) between teosinte and maize, specifically with regard to lateral branches

Comparison teosinte and maize plants

Teosinte Maize
At most nodes At 2-3 nodes
Elongated Short
Tip of primary lateral branch: inflorescence = tassel 
(image from: Virtual Foliage , Univ. of Wisconsin, Madison)
Tip of primary lateral branch: inflorescence = ear 
(image from: Virtual Foliage , Univ. of Wisconsin, Madison)
Secondary lateral branch: carry ears No secondary lateral branch

Previous results from genetic analyses

  • Analysis of domestication syndrome in maize (see Lecture 16 )
    • LBIL: Lateral branch internode length; genewith largest effect for this trait, located on chromosome 1, in same region as tb1 (teosinte branched)
  • Tb1, when introduced from maize into teosinte by hybridization, converts teosinte to maize plant type
  • Effects of tb1:
    • Loss of apical dominance --> marked growth of axillary buds
    • Bottom of the plant: tillers; top of the plant: long branches ending in tassles (>< normal maize has short branches ending in ears)
    • F2 generation: single recessive allele

  • Linkage map of maize with location of domestication genes

Cloning of tb1 gene

  • Transposon tagging:
    • tb1-ref/tb1-ref x Tb1/Tb1;Mu/Mu

      F1: Tb1/tb1-ref;Mu/- + tb1-mum/tb1-ref; -/-

      3 new mutant/26,000 F1 plants

  • Are these new mutants due to transposon insertion in Tb1 locus?
tb1-mum/tb1-ref x Tb1/Tb1
tb1-mum/Tb1 + tb1-ref/Tb1
    • distinguish using markers closely linked to Tb1
    • use Mu probe to identify Mu elements co-segregating with tb1-mum mutation

  • Cloning of DNA fragments containing Mu --> further analysis showed that all 3 mutations have actually a Mu insertion.

Analysis of gene expression

  • Tb1 teosinte, maize: 1.5 kb message; tb1 maize: 2.5 kb message
  • tb1 expressed in same tissues as Tb1, but at lower levels
  • isolated a message from a cDNA library with sequence similar to that of genomic clones;
  • sequence of Tb1/tb1: short sequences (62 aa.) similar to sequences in cycloidea gene of snapdragon (Antirrhinum majus )

Model for the evolution of tb1 in maize

  • Teosinte: tb1 is functional
    • normally expressed in secondary axillary meristems, where it controls conversion to ear- shoots
    • not expressed in in primary axillary meristems --> elongated primary branches
  • Domestication of maize
    • selection of tb1 allele that is expressed in primary axillary meristems --> forms ear-shoots
  • Therefore, evolution would not have proceeded by loss/gain or change in tb1 function but by change in gene regulation. This hypothesis was tested by Wang et al. (1999)

Tb1 sequencing (Wang et al. 1999)

  • Sample of plants:
    • Maize (13 entries): mostly Mex (6), also Ecd, Gua, Ven; AZ, ND, WI
    • Teosinte parviglumis (9): Gue (5), Mex, Jal (2), Mich
    • Teosinte mexicana (8): Mex (4), Jal, Dur, Mich, Chi
    •  Teosinte diploperennis (1)
  •  Sample of sequence:
    • 2.9 kb: transcriptional unit (TU) and 1.1. kb of 5' non transcribed region

Nucleotide polymorphism

  • Tb1: TU
    •  Maize: pi = 1.74; parviglumis: pi = 4.62
  • Tb1: NTR
    •  Maize: pi = 0.47; parviglumis: pi = 28.68
  •  Adh1:
    •  Maize: pi = 15.72; parviglumis: pi = 17.38

from Wang et al. 1999: Fig. 1; © Macmillan Publishers Ltd 1999 [with correction published Nature 410, 718  (05 April 2001)]


  • Tb1 TU sequences falling in multiple clades
  • Tb1 NTR sequences in single clade, associated with parviglumis sequences

from Wang et al. 1999: Fig. 2; ©  Macmillan Publishers Ltd 1999

Further analyses

  • HKA test: subject to selective sweep?
    • Compare ratio (polymorphism w/in species/divergence to outgroup) for tb1 to same ratio for neutral gene
    • Test not significant for TU but well for NTR
    • Test also significant when TU used as neutral control; very short region of hitchhiking
  • Regulatory sequence is key
    • But: no fixed differences in NTR between maize and teosinte --> further upstream?
  • Selection coefficient: s = 0.04 to 0.08
  • Time to fixation: 315 to 1,000 years

Microsatellite marker analysis of maize origin (Matsuoka et al. 2002)

  • Plant material: Broad cross-section: 246 plants
  • Markers: 99 microsatellites 
  • Phylogenetic analyses:
    • trees:  
    • principal component analysis:  
  • Age of domestication:
    • 33 microsatellite markers: stepwise variation
    • estimate mutation rate in a known population: F11
      • 4.28 × 10^-4
    • result:
      • 9,188 B.P. (95% confidence limits of 5,689-13,093 B.P.)

  • Plant material
     Origin of maize plants
  • Phylogenetic tree
  • Principal component analysis

Analysis of "old DNA" in maize

Jaenicke-Després et al. (2003)
  • What is old DNA?
    • technical challenges: degraded DNA: small size --> small fragments for PCR that distinguish maize and teosinte
    • contamination: controls, location of analysis: here, two locations: Leipzig and Cambridge!
  • Application to crop evolution studies:
    • Cloning of domestication genes: in maize:
      • tb1
      • prolamin box binding factor (pbf: seed storage protein expression)
      • sugary1 (su1: starch composition --> texture of tortillas) Availability of archaeological samples
Archaeological maize cobs from Ocampo Caves (Valenzuela cave), dated to 3890 ± 60 years before the present
ancient maize cob

Choice of DNA sequences

  • tb1: 56 bp fragment: distinguishes maize and teosinte; 1 allele: 100% in maize, 36% in teosinte (total of 6 alleles in teosinte)
  • pbf: 25 bp fragment: two alleles: 97% and 3% in maize; 17% and 83% in teosinte
  • su1: 60 bp fragment: two alleles: 30% and 62% in maize; both 7% in teosinte)
Variability at tb1, pbf, and su1


  • Archaeological:
    • Material:
      • 5 cobs from Ocampo Caves, N.E. Mexico
      • 6 cobs from Tularosa Caves, New Mexico
    • Dating by AMS
      • Ocampo: 4,400 BP to 2,300 BP
      • Tularosa: 1,900 BP to 650 BP
  • DNA:
    • 150-200 mg of tissue
    • PCR amplification & sequencing

Geographic and chronological differentiation

  • Alleles of modern maize were already present 4,500 years ago 
  • Possible exception: su1, where 2,000 yr old cobs still carried alleles known now only from teosinte 
  • Southwest U.S.: possible origin of Northern Flint, one of the two parents of Corn Belt maize.

  • Conclusion: In conclusion, by 4400 years ago, early farmers had already had a substantial homogenizing effect on allelic diversity at three genes associated with maize morphology and biochemical properties of the corn cob. Thus, selection by farmers had profound genomic effects relatively early in the history of this crop.
geographic and temporal differentiation in archaeological maize cobs

Origin of the Bottle Gourd

Erickson DL, Smith BD, Clarke AC, Sandweiss DH, Tuross N (2005) An Asian origin for a 10,000-year-old domesticated plant in the Americas. Proceedings of the National Academy of Sciences of the United States of America 102:18315-18320

  • Bottle gourd: Lagenaria siceraria
  • Belongs to the family Cucurbitaceae: Cucumis (C. melo, C. sativus), Citrullus
  • Viny plant; used primarily as a "utilitarian" crop for the containers made from its fruits



  • L. siceraria:
    • African plant: wild population in Zimbabwe
    • Ancient dispersal to Asia: archaeological remains in China and Japan (8000-9000 BP)
  • Grown mostly as (durable & lightweight) containers, musical instruments, and fishing floats



Early dispersal

  • Early diffusion by ocean currents:
    Buoyant fruits yield still viable seeds after floating in sea water > 7 months
  • Found in close association with earliest New World crops
  • Alternative explanations: Asia vs. Africa, wild vs. domesticated, ocean current vs. human transport?


(1) Temporal context of arrival of bottle gourd in the Americas

  • AMS radiocarbon dates of bottle gourd rind fragments
  • In Mexico, Guila Naquitz site: same age as earliest documented food plant (C. pepo): 10,000-9,000 BP)
  • Widespread distribution of earliest occurrences: Mexico, Florida (Windover), Peru (Quebrada Jaguay) = widespread early dispersal

from Erickson et al. 2005

(2) Morphological evidence: increase in thickness of the rind

  • Selection pressures:
    Loss of natural seed dispersal
    Human selection for thick and durable exocarp
  • Measurement:
    Wild: < 2mm; Archaeological remnants: 3-7 mm


(3) What is the source of the introduction of bottle gourd in the New World?

  • Use DNA (paternity test!) :
    • Compare DNA of archaeological remains of New World with DNA of existing varieties in Africa and Asia
  • Which DNA?
    • Checked nuclear DNA (ribosomal DNA) and mtDNA: no polymorphism or not amplifiable by PCR
    • pDNA: after screening:
      • trnC-trnD intergenic region
      • trnS-trnG intergenic region
  • Ancient DNA extraction:
    • Avoid contaminations: different labs, 1 sample + control (no plant tissue) at a time
  • PCR amplification:
    • Avoid contaminations: 2 controls: template blank (no plant tissue), water
    • Reamplification of weak samples
  • Cloning & sequencing of PCR products


From Erickson et al. 2005


  • All archaeological rind fragments predating European arrival:
    • DNA identical to modern Asian DNA
  • Post-European arrival: A.D. 1660:
    • DNA corresponds to modern African DNA; i.e., African landraces have spread rapidly and almost completely replaced Asian landraces
  • Distinctive shape of early bottle gourd seeds in pre-European contexts: similar to Asian materials


Introduction into Americas from Asia:

  • Wild? Unlikely because thin rind; limited floating capability & usefulness to humans

  • Domesticated? More likely
    • Pacific Ocean currents: independent data from buoyant cargo spills in N. Pacific
    • Humans: not seafarers, but on foot or near-shore watercrafts along southern Beringia, together with dog
    • But: no archaeological remains western N. Am.
  • Suggest domestication in Asia first, some 12,000-13,000 yrs ago, in same time frame as dog, and way before other plant and animal species!
  • Also, potential later domestication in Africa


  • Importance of molecular analyses:
    • identification of wild progenitor
    • evolution of genetic diversity
    • genetics of the domestication syndrome
  • Active field with progress made continuously

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