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  • Shows a small region on chromosome 2 with markers, polymorphisms, genes
  • Discuss the various advantages of each insertion point It’s a random event You have to collect many T2 plants
  • Advantages get intermediate alleles of your favorite gene--generating an allelic series is imperative Also can get LOF if no insertion allele for you Usually get 10 mutations in your gene Also used in other plant systems (don’t need to know the genome sequence, just primers) High throughput once the 96 well plates are made, you can just cycle through with each gene Can tell within 10bp where your mutation is inferred from the size of the fragment. Multiple insertion alleles in each round Disadvantage: expensive. All the problems associated with reverse genetics.
  • Point out from pe (2) that AP1, PI, AP3, and a SEP gene are required


  • 1. Plants: The Other Eukaryotic Organism Elizabeth Haswell Bio 5491 January 29, 2009
  • 2. OUTLINE
    • Introduction
    • Forward genetics
    • Reverse genetics
    • Genomic resources and strategies
  • 3. OUTLINE
    • Introduction
      • Why study plant genetics?
      • Model organisms
      • Arabidopsis thaliana
        • 1. Life cycle
        • 2. Genome
        • 3. Tools
  • 5.
    • 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 of plants:
      • Pest Resistance
      • Enhanced Nutrition
  • 6.
    • Practical Value of Plant Studies: Plants can be Green Machines
    • Plants are a source of biofuel.
    • Derived from recently living biomass:
      • Wood
      • Biodiesel (rapeseed)
      • Bioethanol (corn)
      • New plants with high biomass yield:
      • Switchgrass (prairie grass)
      • Miscanthus
      • Algae
  • 7. 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
  • 8. a. Plants share a common eukaryotic ancestor with animals. Examples: Chromatin Cytoskeleton Golgi, ER, usual organelles Gene expression components Ga = giga-annum = billion years From Meyerowitz, 2002
  • 9. Ga = giga-annum = billion years
    • b. Plants evolved multicellularity independently from animals.
      • Implications for:
      • pattern formation
      • cell-cell communication.
      • Example: flower development
    From Meyerowitz, 2002
  • 10.
    • c. Plants underwent two endosymbiotic events.
      • Horizontal transfer of bacterial genes that integrate into eukaryotic system
      • Example: chloroplast division proteins
    Ga = giga-annum = billion years From Meyerowitz, 2002
  • 12.
    • Plant Genetic Model Systems
    • Crop plants
      • Rice
      • Alfalfa
      • Tomato
      • Grapevine
      • Sugarcane
      • Tobacco
      • Maize
    • Considerations
    • Genome size
    • Polyploidy
    • Translation to crop plants
    • Synteny
    • Model systems
      • Mosses
      • Algae
      • Poplar
      • Arabidopsis
  • 13. 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
      • Genome
        • Diploid
        • 125 Mb, smallest known in plant kingdom
        • Little repetitive DNA
      • 3. Tools
        • Agrobacterium transformation
        • RFLP map between ecotypes
        • Tiling arrays
    Arabidopsis is a member of the mustard ( Brassicaceae ) family, which includes cultivated species such as cabbage and radish. Meyerowitz. Ann. Rev. Genet. 21 : 93-111(1987)
  • 14. 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) (D) Gametes produced by meiosis: pollen and ovule (E) Fusion of pollen and ovule to form new diploid generation (embryo).
    • Haploid generation is multicellular
    • No dedicated germline
  • 15. 1. 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.
  • 16. 2. The Arabidopsis genome (Nature, 408:796-815; 2000)
    • Sequenced by an international consortium:
    • European Union
    • Riken
    • US (CSH/WU/ABI did parts of chromosomes 4 and 5)
    • Strategies: BAC-end sequencing, physical map-based approaches
    • Error rate is < 1 error per 20 Kb
  • 17.
    • 125 Mb
    • Current status:The TAIR8 release (April 08) contains 27,235 protein coding genes(~same as humans)
      • 1 gene / 4.4 Kb.
      • ~30% unknown.
      • 11,000 families.
    • Recent large-scale genome duplication events.
      • Recent tetraploid ancestor, now reducing.
    2. The Arabidopsis genome
  • 18. 2. The Arabidopsis genome Duplications: red (recent) and blue (old) sister regions Blanc, et al. Genome Res . 13 : 137-44 (2003).
  • 19.
    • Arabidopsis Molecular Genetic Tools A. Agrobacterium-mediated plant transformation
    Agrobacterium tumefaciens & crown gall tumor Agrobacterium is a genus of soil bacteria that infects wounded plants and leads to gall formation Galls = benign tumor that feeds the extracellular agrobacteria
  • 20. Agrobacteria induce tumors by genetically engineering plant cells at the wounded site
  • 21. Ti plasmid ~200 Kb LB RB Virulence genes LB RB T-DNA introduced into plant chromosomal DNA LB RB T-DNA plasmid for transformation Selectable marker T-DNA acts as an insertional mutagen
  • 22. Agrobacterium-mediated plant transformation “Floral Dip” Selection T1 Solution of Agrobacterium harboring a T-DNA plasmid
    • Complementation experiments
    • Method of mutagenesis
    • GFP fusions, reporter genes, etc.
  • 23. T-DNA insertion at random locations in the genome T-DNA plasmid Gene A Gene B Gene C Examples of possible insertions: LB Selection RB YFG *
  • 24.
    • A complete database of polymorphisms between Columbia and Landsberg ecotypes (and others!)
      • CAPS and SSLPs
      • Point mutations
      • Insertions or deletions
      • All occur randomly in one ecotype and not in the other .
    Jander, 2002; Lukowitz, 2000
    • Arabidopsis Molecular Genetic Tools B. Polymorphism database
  • 25. Positional Cloning in Arabidopsis: Why it feels good to have a genome initiative working for you
  • 26. Positional Cloning in Arabidopsis: Why it feels good to have a genome initiative working for you
  • 27. OUTLINE
    • Introduction
    • Forward genetics
    • Reverse genetics
    • Genomic resources and strategies
  • 28. II. Forward genetics B. Generate a mutant population C. Identify interesting mutants A. Select a biological process pi-1 D. Map and clone mutation Infer mechanism & Generate hypotheses Mutant phenotype Responsible gene
  • 29. A. Floral Organ Development in Arabidopsis 4 sepals 4 petals 6 stamen Male gametes pollen 1 carpel Female gametes ovules
  • 30. A. Arabidopsis Floral Organs are Arranged in Whorls
    • Four concentric whorls of organs
    • Stereotyped pattern of number and position.
    ca st pe se
  • 31. II. Forward genetics B. Generate a mutant population C. Identify interesting mutants A. Select a biological process (Flower development) pi-1 D. Map and clone mutation
  • 32. Mutagens commonly used in Arabidopsis
    • EMS
    • Insertional mutagenesis
      • T-DNA
      • Transposon
    • Irradiation
    • Fast neutron
    • Natural variation
    B. Generate a mutant population
  • 33. B1. Chemical Mutagenesis with EMS
    • EMS = ethylmethanesulfonate
    • Generates G/C to A/T mutations
    • Advantages:
    • • wide range of mutants possible
    • • mutagenesis easy to perform
    • • high mutation rate
    • • can combine many lesions in the same line
    • Disadvantages:
        • background mutations
        • cloning the gene can be time-consuming
        • not all mutations are transmitted to the second generation
  • 34. B2. T-DNA insertional Mutagenesis ATG STOP A random gene
    • Advantages:
    • • tagged, therefore easier to isolate gene involved
    • Disadvantages:
        • low mutation rate
        • unlinked mutations
        • preference for promoter regions
    • • will limit type of alleles; often severe loss of function alleles
  • 35. II. Forward genetics B. Generate a mutant population C. Identify interesting mutants A. Select a biological process Flower development pi-1 D. Map and clone mutation EMS
  • 36. C. Identify Interesting Mutants
    • Selection vs. Screen
    • False positives
    • False negatives
    • Etc.
    • Our example: visual screen for floral development mutants of Arabidopsis.
      • Deviations in floral organ number
      • Deviations in floral organ location
  • 37. Arabidopsis Homeotic Mutants Homeotic mutations cause conversion from one organ to another. ca st pe se apetala1 (ap1) apetala2 (ap2) ca se ca se pistillata (pi) apetala 3 (ap3) agamous (ag) ca st st ca pe se pe
  • 38. The ABC Model of Floral Development Coen and Meyerowitz, Nature 1991
    • Three classes of homeotic genes
      • A function (AP1, AP2)-->sepals, petals
      • B function (AP3, PI)-->petals, stamen
      • C function (AG)--stamen, carpels
  • 39. ap3 or pi ca se ca se B-function genes are required for the production of petals and stamens A C B
  • 40. ap2 A function genes are required for the production of sepals and petals A C B ca st st ca
  • 41. ag C-function genes are required for the production of stamen and carpels A C B pe se pe
  • 42. ABC Model of Floral Organ Development
    • Provides framework for future work.
    • Comparison to other species.
  • 43. II. Forward genetics B. Generate a mutant population C. Identify interesting mutants A. Select a biological process pi-1 D. Map and clone mutation Flower development EMS ag-1 pi
  • 44.
    • 1. Uses linkage analysis: test for genetic linkage between previously identified genetic markers and your gene.
    • 2. Based on the principle that the frequency of recombination between genes decreases along with the distance between them.
    How to identify mutant alleles? Genetic Mapping
  • 45. 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 B Ecotype A mutant Wild-type Wild-type mutant X mutant mutant
  • 46.
    • Look for co-segregation of the mutant phenotype and the ecotype A marker
    C1. Map and Clone Mutation--EMS Michelmore, 1991 A B het First, map to a chromosome arm: Then, narrow down further and further.
  • 47. C2. Map and Clone Mutation--T-DNA ATG STOP Your favorite mutant
    • Degenerate PCR
    • Sequence PCR product
    • Test for altered mRNA or protein production
    • Complement with transgene
    LB Selectable Marker RB
  • 48. The floral homeotic genes encode MADS box transcription factors that activate organ-specific gene expression in a combinatorial manner.
  • 49. Homeotic mutants in Drosophila
    • Homeotic mutants
      • Antennapedia
    • Genes direct anterior-posterior 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.
  • 50. II. Forward Genetics
    • Uses:
      • Unbiased search for genes involved in a biological process.
    • Pitfalls :
      • You get what you screen for
      • Will miss redundant or essential genes
    phenotype genotype
  • 51. III. Reverse genetics
    • 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.
      • Can do a step-by-step analysis of the redundant functions of members of a gene family
    • Pitfalls:
      • Requires guessing.
      • Common outcome: no phenotype because gene is redundant or conditionally required.
    genotype phenotype
  • 52. Reverse genetics B. Generate mutant plants C. Evaluate mutant phenotype A. Identify gene or genes of interest pi-1 D. Identify the function of the genes Infer mechanism & Generate hypotheses Mutant phenotype Responsible gene
  • 53. III. Reverse genetics: the Arabidopsis toolbox
    • Sequence-indexed T-DNA insertion/transposon lines
      • SALK, GABI-Kat, CHSL, RIKEN, SAIL, Wisconsin, etc.
    • TILLING.
    • Engineered Post-Transcriptional Gene Silencing.
    • Overexpression of wild-type or dominant-negative alleles.
    • Gene replacement.
  • 54. Available SALK insertion lines for AP3 AP3 SIGnAL= S alk I nstitute G e n omic A nalysis L aboratory http://signal.salk.edu/ III. Reverse genetics: the Arabidopsis toolbox
  • 55. Available SALK insertion lines for PI PI III. Reverse genetics: the Arabidopsis toolbox SIGnAL= S alk I nstitute G e n omic A nalysis L aboratory http://signal.salk.edu/
  • 56. 2. TILLING T argeted I nduced L ocal L esions IN G enomes
    • A 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.
  • 57. 2. TILLING T argeted I nduced L ocal L esions IN G enomes EMS-mutagenized plant population * * CEL1 * * * * * * x x x Heat, anneal wild type and mutant versions Digest with CEL1 endonuclease digests heteroduplexes ONLY * * * * * * * * Pooled DNA from individual plants in 96 well plates PCR amplify your gene with fluorescently tagged primers from DNA in each pool Pool #104
  • 58. Nature Biotechnology 18 : 455-457 (2000). Run on gel Screen individual samples, sequence TILLING T argeted I nduced L ocal L esions IN G enomes pools
    • Arabidopsis
    • Other plants
    • Animals
  • 59. 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?
  • 60. The SEPELLATAS : A fourth class of floral organ identity genes From Current Opinion in Genetics and Development 2001 11 : 449
    • SEP1 identified as an AP3-interacting protein in the Y2H.
    • SEP1,2,3,4 are all highly similar MADS-box proteins.
  • 61. The SEPALLATA genes are required for floral organ formation Wild type sep1sep2sep3sep4 Ditta, et al. Current Biology 14 :1935 (2004).
  • 62. 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)
  • 63. The ABCE Model of Floral Development
  • 64. OUTLINE
    • Introduction
    • Forward genetics
    • Reverse genetics
    • Genomic resources and strategies
  • 65. IV. Genomic Resources and Strategies 1. genome gene genotype phenotype 2. Whole genome vs. one-by-one strategies
    • Genomics
    • Transcriptomics
    • Proteomics
    • Phenomics = the total phenotypic outcome of disrupting the genome
    • Epigenomics = the total epigenetic modifications made in a genome
    • Metabolomics = totality of metabolites in an organism
    Changes in response to environmental or genetic conditions
  • 67. Occurrence of Related Genes in Clusters of Co-expressed Genes during Floral Development
    • Five clusters of genes with similar expression profiles during flower development.
    • Black bars indicate the percentage of closely related sequences in each cluster.
    • White and gray bars are randomly chosen genes.
    • Suggests genetic buffering by functional redundancy.
    Wellmer et al. PLOS Genetics 2(7) 1012-1024 (2006).
  • 68. Metabolomics
    • Study of the complement of metabolites:
      • ions
      • osmolytes
      • sugars
      • biosynthetic intermediates
      • etc.
  • 69.  
  • 70. Ionomics
    • High-throughput, quantitative mass spectroscopy
    • The altered ionome of a mutant plant can reflect defects in transport or metabolism
    Nature Biotechnology 21, 1215 - 1221 (2003) frd-3 WT msl9-1; msl10-1 msl9-1; msl10-1 msl4-1; msl5-2; msl6-1 msl4-1; msl5-2; msl6-1 Fe transporter Other ions assayed: Na, Mg, K, Ca, Mn, Co, Ni, Cu, Zn, Mo, B, P
  • 71. Epigenomics
    • Characterizing the “methylome”:
      • Wild type and methylase/demethylase mutants
      • Natural variation in methylation sites.
      • Genome-wide bisulfite sequencing and subsequent mapping to Arabidopsis genome (Lister et al., 2008).
      • Chromatin IP with antibodies against methyl-C, then hybridize to a microarray covering entire genome (Zilberman et al., 2006).
      • Digestion with methylation-sensitive enzymes , then hybridize to microarray covering the entire genome Zhang et al., 2008).
    • Plants are interesting and important.
      • practical value
      • opportunity to gain insight into basic life processes
    • Arabidopsis is an excellent model system for molecular genetics
      • but--inefficient homologous recombination
      • --redundancy
    • Forward and reverse genetic approaches help reveal the genes involved in a particular biological process
    • State of the art genomics-level resources are available to understand the interaction between plant genomes and their environment.