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 decompressor are need ed to see this picture. • 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 decompressor are need ed to see this picture. 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 QuickTime™ and a decompressor are neede d to see this picture. 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 decompressor (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 QuickTime™ an d a decompressor are need ed to see this p icture . QuickTime™ and a decompressor are neede d to see this picture. 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 QuickTime™ and a decompressor are neede d to see this picture. B A C ag QuickTime™ an d a decompressor are need ed to see this p icture . 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 decompressor 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 decompressor 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.