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					Plants: The Other Eukaryotic Kingdom.


         Advanced Genetics
          January 22, 2008
            OUTLINE

I.     Introduction
II.    Forward genetics
III.   Reverse genetics
IV.    Genomic resources and
       strategies
                OUTLINE
I.    Introduction
     A. Why study plant genetics?
     B. Model organisms
     C. Arabidopsis thaliana
       1. Life cycle
       2. Genome
       3. Tools
A. WHY STUDY PLANT GENETICS?

1. Application
2. Basic Biology
1.     Practical Value of Plant Studies:
     Plants are the Foundation of Our Diet


                 According to the Food and Agriculture
                 Organization of the United Nations, more
                 than 25,000 people died of starvation every
                 day in 2003 and about 800 million people
                 were chronically undernourished.




              Genetic engineering:
                 •Pest Resistance
                 •Enhanced Nutrition
         1.    Practical Value of Plant Studies:
                           Biofuels

Fuel derived from recently living biomass
  – Wood
  – Biodiesel (rapeseed)
  – Bioethanol (corn)

  –   New plants with high biomass yield:
  –   Switchgrass (prairie grass)
  –   Miscanthus
  –   Algae



                                            switchgrass
     2. Value of Plant Genetic
     Studies for Basic Biology


1.   For comparison
2.   As additional examples
3.   Because they are part of the natural world
4.   Aesthetics
a. Plants share a
   common eukaryotic
   ancestor with
   animals.
Examples:
   Chromatin
   Cytoskeleton
   Golgi, ER, usual organelles
   RNAi




     Ga = giga-annum = billion years
b. Plants evolved
   multicellularity
   independently from
   animals.

  Implications for:
  pattern formation
  cell-cell communication.

  Example: flower development



  Ga = giga-annum = billion years
c. Plants underwent
   two endosymbiotic
   uptake events.
  Horizontal transfer of bacterial
      genes that integrate into
      eukaryotic system

  Example: ethylene receptors




  Ga = giga-annum = billion years
         B.     Plant Genetic Model Systems

• Crop plants
   –   Rice
   –   Alfalfa
   –   Tomato
   –   Grapevine
   –   Sugarcane
   –   Tobacco
   –   Maize
• Model systems
                       Considerations
   –   Mosses
                       •Genome size
   –   Algae
                       •Polyploidy
   –   Poplar          •Translation to crop
   –   Arabidopsis     plants
                       •Synteny
     C. Arabidopsis thaliana: A model system for flowering plants

                                            Advantages:

                                                 1. Life cycle
                                                     • 6 weeks
                                                     • Small plant, easy to grow
                                                     • High fecundity (10,000
                                                         seed/individual)
                                                     • Self and cross-fertilization

                                                 2.   Genome
                                                      • Diploid
                                                      • 125 Mb, Smallest known in
                                                         plant kingdom
                                                      • Little repetitive DNA

                                                 3. Tools
  Arabidopsis is a member of the                     • RFLP map between ecotypes
  mustard (Brassicaceae) family,
                                                     • Agrobacterium transformation
  which includes cultivated species
  such as cabbage and radish.
Meyerowitz. Ann. Rev. Genet. 21 : 93-111(1987)
                            1. Arabidopsis Life Cycle




Life cycle of higher plants.
(A) The dominant diploid generation
(B) flowers
(C) male and the female reproductive structures (anthers and siliques)    • Haploid generation
(D) Gametes produced by meiosis: pollen and ovule
(E) Fusion of pollen and ovule to form new diploid generation (embryo).   • No dedicated germline
Two Features of Plant Development Relevant to
              Genetic Analyses

• Gametes in plants are formed by a separate
  multicellular haploid generation called the
  gametophyte.
  – Multiple rounds of division in the haploid phase.
  – Implication for essential genes.



• Plants have no dedicated germline. Instead,
  cells giving rise to the germline develop de
  novo from the somatic tissues.
  – Implications for environmental inputs into the production of
    the germline.
                    2. The Arabidopsis genome

•Sequenced by an international
consortium:
•European Union
•Riken
•US (CSH/WU/ABI did parts of
chromosomes 4 and 5)
•convinced Jim Watson to
support it--NSF funding

•Strategies: BAC-end
sequencing, physical map-
based approaches

•Error rate is < 1 error per 20
Kb




(Nature, 408:796-815; 2000)
              2. The Arabidopsis genome
•   125 Mb

•   Current status:The TAIR7 release contains 27,029 protein coding
    genes(~same as humans)
     – 1 gene / 4.5 Kb.
     – ~30% unknown.
     – 11,000 families.




                                   QuickTime™ and a
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•   Recent large-scale genome duplication events.
     – Recent tetraploid ancestor, now reducing.
         2. The Arabidopsis genome




Duplications: red (recent) and blue (old) sister regions
         Blanc, et al. Genome Res. 13 : 137-44 (2003).
     3. Arabidopsis Molecular Genetic Tools
         Agrobacterium-mediated plant transformation
    Uses:
    Ability to transform plants with foreign or
    recombinant DNA
    Complementation experiments
                                                  Crown gall tumor

Agrobacterium tumifaciens



                QuickTime™ an d a
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3. Arabidopsis Molecular Genetic Tools
    Agrobacterium-mediated plant transformation

 Virulence genes            LB                           RB

                                 2. T-DNA
                                 introduced into plant
                                 chromosomal DNA

    ~200 Kb
                        LB Selection          YFG         RB




   1. Ti plasmid
                      3. T-DNA plasmid for transformation
   3. Arabidopsis Molecular Genetic Tools
Agrobacterium-mediated plant transformation


                              “Floral Dip”
                It is the ovules that are transformed




 Solution of                                      Selection
 Agrobacterium                                    T1
 harboring a T-DNA
 plasmid
 3. Arabidopsis Molecular Genetic Tools
      T-DNA insertion at random locations in the genome


                        LB Selection           YFG   RB


                                   T-DNA plasmid


Examples of possible insertions:




          Gene A    Gene B            Gene C
3. Arabidopsis Molecular Genetic Tools
           Polymorphism database
3. Arabidopsis Molecular Genetic Tools
            Polymorphism database
            OUTLINE

I.     Introduction
II.    Forward genetics
III.   Reverse genetics
IV.    Genomic resources and
       strategies
              II. Forward genetics
                   B.             C.          D.
A. Select a     Generate a     Identify    Map and
 biological      mutant      interesting    clone
  process       population     mutants     mutation




                               pi-1
     A. Floral Organ Development in Arabidopsis
6 stamen
Male gametes
pollen



                                        1 carpel
    4 petals                            Female
                                        gametes
                                        ovules

    4 sepals
    A. Arabidopsis Floral Organs are
          Arranged in Whorls




                                        ca
                                             st pe
                                                  se




• Four concentric whorls of organs
• Stereotyped pattern of number and position.
                       II. Forward genetics
                            B.             C.          D.
 A. Select a             Generate a     Identify    Map and
  biological              mutant      interesting    clone
   process               population     mutants     mutation




(Flower development)



                                        pi-1
     B. Generate a mutant population

Mutagens commonly used in Arabidopsis
  • EMS
  • Insertional mutagenesis
     – T-DNA
     – Transposon


  • Irradiation
  • Fast neutron
        B1. Generate a mutant population
 EMS = ethylmethanesulfonate                    Guanine alkylation



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Pitfalls
                    background mutations
                    cloning the gene can be time-consuming
                    not all mutations are transmitted to the
                    second generation
 B2. Generate a mutant population           T-DNA insertion

                        LB     Selectable Marker          RB


                                   T-DNA plasmid


Examples of possible insertions:


      ATG                                          STOP

                       A random gene
Easy to clone
Pitfalls:
        unlinked mutations
        preference for promoter regions
                       II. Forward genetics
                            B.                            C.          D.
 A. Select a             Generate a                    Identify    Map and
  biological              mutant                     interesting    clone
   process               population                    mutants     mutation




                                      QuickTime™ and a
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(Flower development)           are need ed to see this picture.




                                                         pi-1
       C. Identify Interesting Mutants

• Selection.
• Screen.
• Design.

• Our example: visual screen for floral development
  mutants of Arabidopsis.
                       Arabidopsis Homeotic Mutants
Homeotic mutations cause conversion from one organ to
another.            ap2          ap3 or pi         ag


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      ca                    ca                               ca
           st                    st                            ca                                pe
                pe                 st                                                                 pe
                                                                se
                  se                    ca                           se                                 se
      The ABC Model of Floral
           Development




Coen and Meyerowitz, Nature 1991
ap3 or pi


                    ca
                      ca
                       se
                            se




                B

            A       C
ap2

                                   ca
                                        st
                                          st
                                               ca
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                     B

      A                             C
ag
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                                                  pe
                                                       pe
                                                         se




                                          B

                A                             C
                       II. Forward genetics
                            B.                             C.                                                              D.
 A. Select a             Generate a                     Identify                                                        Map and
  biological              mutant                      interesting                                                        clone
   process               population                     mutants                                                         mutation


                                                           QuickTime™ an d a
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                                                    are need ed to see this p icture .




                                                                                         QuickTime™ an d a
                                                                                            decompressor
                                                                                   are need ed to see this p icture .




                                      QuickTime™ and a
                                        decompressor
(Flower development)           are need ed to see this picture.




                                                                  pi-1
     C1. Map and Clone Mutation--EMS
   Map-based cloning:
      – Mutant plants are crossed to another ecotype
      – The mutant gene is identified by virtue of its
        association with nearby genetic markers that differ
        between ecotypes (polymorphisms).


Ecotype A
             mutant
             mutant                          mutant
Ecotype B                  X                 mutant
            Wild-type
            Wild-type
   C1. Map and Clone Mutation--EMS

First, map to a chromosome arm:




Then, narrow down further and further.
C2. Map and Clone Mutation--T-DNA



                    Your favorite mutant
    ATG                                                STOP




   LB      Selectable Marker          RB


        •Degenerate PCR
        •Sequence PCR product
        •Test for altered mRNA or protein production
        •Complement with transgene
The floral homeotic genes encode MADS box
transcription factors that activate organ-specific gene
expression in a combinatorial manner.
      Homeotic mutants in Drosophila
• Homeotic mutants
  – Antennapedia
• Genes direct                             QuickTime™ an d a
                                             decompressor
  anterior-posterior                are need ed to see this p icture .


  positioning of the
  embryo.
• Hox genes.                Wild type                           Antennapedia



Thus homeotic genes arose once in animals and once in
plants, accomplishing the same function using different types of
transcription factors.
             II. Forward Genetics
                phenotype      genotype


Uses:
   – Unbiased search for genes involved in a biological
     process.

Pitfalls :
   – You get what you screen for
   – Will miss redundant or essential genes
            III. Reverse genetics
                  genotype         phenotype
Uses:
    – can get additional alleles to corroborate data on previous EMS
      alleles
    – can study genes that are interesting because of evolutionary,
      biochemical, or expression data.



Pitfalls:
    – Requires guessing
    – Common outcome: no phenotype
              III. Reverse genetics:
             the Arabidopsis toolbox

1. Systematically generated T-DNA insertion/transposon lines
      SALK, GABI-Kat, CHSL

2. TILLING

3. RNAi, artificial miRNAs

4. Overexpression
   III. Reverse genetics: the Arabidopsis toolbox
SIGnAL= Salk Institute Genomic Analysis Laboratory
http://signal.salk.edu/

                                     AP3




Available SALK insertion lines for AP3
   III. Reverse genetics: the Arabidopsis toolbox
SIGnAL= Salk Institute Genomic Analysis Laboratory
http://signal.salk.edu/
                            PI




Available SALK insertion lines for PI
 III. Reverse genetics: the Arabidopsis toolbox
2. TILLING      Targeted Induced Local Lesions IN Genomes



 A powerful new high-throughput strategy for generating
 and isolating point mutations in your favorite gene
 Exploits a nuclease that recognizes and digests
 heteroduplexes.
 Use:
    •   Mutations in genes that are not found in the
        insertional database.
    •   Partial loss of function.
    •   Conditional alleles.
 TILLING Targeted Induced Local Lesions IN Genomes
EMS-mutagenized     Pooled DNA from                PCR amplify your gene with
plant population    individual plants in 96        fluorescently tagged primers
                    well plates                    from DNA in each pool
                                                    *
                                                                           *
                                                    *                             Pool
                                                                           *      #104
                                                    *
                                                                           *
                                                    *          x
                                                               x
                                                                           *
                                              Heat, anneal wild type
                                              and mutant versions

                                                         x



           Digest with CEL1 endonuclease                CEL1
           digests heteroduplexes ONLY


                                              *                    *   *                 *
                                              *                    *   *                 *
TILLING Targeted Induced Local Lesions IN Genomes
Run on gel
Screen individual samples, sequence
  pools

                                      Nature Biotechnology 18: 455-457
                                      (2000).
                                      Seattle Tilling Project
                                      Arcadia Biosciences, Davis, CA




                                         • Arabidopsis
                                         • Other plants
                                         • Animals
Reverse genetics gave additional
 insight into floral development
 • We know MADS-box genes are required for
   floral development:




 • But they are not sufficient.
 • What are the missing factors?
         Phylogenetic Tree of MADS
                Box Genes

    •AP3 interacts with
    another MADS box
    gene in the Y2H.                                             SEP4




    •sep1 mutant appears
    wild-type.

    •SEP1,2,3,4 are all
    highly similar.




From Current Opinion in Genetics and Development 2001 11 : 449
   The SEPALLATA genes are
required for floral organ formation




                      Wild type             sep1sep2sep3sep4



Ditta, et al. Current Biology 14 :1935 (2004).
The ABCE Model of Floral
     Development
  Turning Leaves into Petals


Wild type




                      Expression of AP1, AP3,
                      PI, and SEP is sufficient
                      to convert seedling
                      leaves into petals.

               Pelaz, et al. Current Biology 11 : 182-184 (2001)
            OUTLINE

I.     Introduction
II.    Forward genetics
III.   Reverse genetics
IV.    Genomic resources and
       strategies
 IV. Genomic Resources and Strategies


1. genome     gene      genotype       phenotype



     2. Whole genome vs. one-by-one strategies
    Cool Arabidopsis Genomics
            Resources
• Co-expression analysis with
  transcriptome datasets.

• Whole-genome tiling arrays.

• The metholome: A genome-wide map
  of DNA methylation.
    Occurrence of Related Genes in Clusters
           of Co-expressed Genes
• Five clusters of genes with
  similar expression profiles
  during development.

• Black bars indicate the                                  QuickTime™ an d a
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  percentage of closely related                      are need ed to see this picture .


  sequences in each cluster.

• White and gray bars are
  randomly chosen genes.




                                  Wellmer et al. PLOS Genetics 2(7) 1012-1024 (2006).
         Whole genome vs. One-by-One Strategies




Alonso et al. Nature Reviews Genetics advance online publication;
published online 06 June 2006 | doi:10.1038/nrg1893
      TAKE-HOME MESSAGES
1. Plants are interesting and important.
      practical value
      opportunity to gain insight into basic life processes

2. Arabidopsis is an excellent model system for molecular
   genetics
      but--no homologous recombination
         --redundancy


3. Forward and reverse genetic approaches help reveal
   the genes involved in a particular biological process

4. Genomics-level resources available to understand the
   interaction between plant genomes and their
   environment.