1. Arabidopsis Experiments Forward Genetic Screen (Ethylene Insensitive Mutants) Reverse Genetic Screen / PCR Genotyping (H + - ATPase Mutants)

2. Forward vs. Reverse Genetics Treat thousands of organisms with a mutagen, Treat thousands of organisms with a mutagen, - random mutagenesis, Identify an individual with a phenotype of interest, Identify an individual with a phenotype of interest, Identify the gene. Identify the gene. Treat thousands of organisms with a mutagen (usually),Treat thousands of organisms with a mutagen (usually), –random mutagenesis, Identify an individual with a genotype of interest,Identify an individual with a genotype of interest, Identify the phenotype.Identify the phenotype. Forward Reverse

3. Proton Pumps in planta Stems transport; sucrose hormones Leaves stomata (gas exchange) sucrose transport Anthers cell elongation Pollen tip growth Embryo/Seeds loading Roots root hair growth mineral uptake Arabidopsis

4. Adapted from Biochemistry and Molecular Biology of Plants, pp. 115 H + (protons) ATP synthase ATP hydrolase (ATPase) Transporters - carriers, - channels.

5. Arabidopsis Genome ~125 Mb (Megabases, million base pairs), Rice: 420 Mb, Human: 3 Gb, Rice: 420 Mb, Human: 3 Gb, 25,498 genes from 11,000 gene families, Rice: 32,000 - 50,000, Human: 20,000 - 25,000. Rice: 32,000 - 50,000, Human: 20,000 - 25,000.

6. Proton Pumps in planta Stems transport; sucrose hormones Leaves stomata (gas exchange) sucrose transport Anthers cell elongation Pollen tip growth Embryo/Seeds loading Roots root hair growth mineral uptake Arabidopsis

7. Arabidopsis H + -ATPase Gene Family Phylogenetic Family Tree (ClustalW --> Phylip: protdist, fitch) Baxter et al., Plant Physiol, 123, (2003)

8. Reverse Genetics Functional Genomics Gene DNA Sequence Gene Disruption Phenotype Analysis Function Mutate DNA Sequence Development Physiology Cell Biology Genetically Link

9. Agrobacterium Plant Cells Nature Ti-Plasmid T-DNA Hormones Opines Lab Selectable Markers Reporter Genes Genes Out : Ti genes, opine genes, In : DNA of choice. T-DNA

10. wtplantchromosome Ti Plasmid (from agro ) hormone genes (i.e. auxins) opaline nopaline virulencegenes virulencegenes hormone genes opaline, nopaline neoplastic transformation Agrobacterium tumefaciens Ti Plasmid (Tumor inducing) Mother Nature Agrofood

11. Construct T-DNA selection genes virulencegenes infect plant, select for plants with T-DNA T-DNA (Transfer DNA) Laboratory transform, select for agro with T-DNA Agrobacterium …if the T-DNA lands in a gene, the gene is disrupted. …can put other genes.

12. DoneGermination  Breaking Dormancy  Breaking Dormancy  H 2 O/Imbibition,  H 2 O/Imbibition,  O 2 /Aeration,  Cold/Prechilling "scarification”  Cold/Prechilling "scarification”  Inducing Germination  Inducing Germination  Light Surface Sterilize Seeds Plant on Nutrient Media Germinate 1. EMS Treated Seeds on MS/ACC media. 2. aha3-1 on MS media.

13. Probability of Finding an Insert in a Specific Gene thousands of inserts p = 1-(1-f) n p = probability of insertion event f = 1-(Genome/Size of Gene) n = number of T-DNA inserts

14. Knockology Plants/PoolsDNA/Pools Krysan et al., 1999

15. Set-Up DNA Pooling Set-Up DNA Pooling Seeds (9) Seedlings (225) DNA (225) 123456…30 Super Pools (2025) Germinate and grow seeds in liquid culture. Extract DNA, Super Pool DNA, Maintain lines as pools of seed. PCR Screen

16. 94 o 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG-- 3’ 5’--GCATGCATTAT CTGATCGTGAC--5’ Denature Step ~30 seconds ~65 o 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG-- 3’ 5’--GCATGCATTAT CTGATCGTGAC--5’ Annealing Step ~30 seconds 72 o 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG-- 3’ 5’--GCATGCATTAT CTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’ Synthesis ~1 minute/kb PCR

17. PCR Strategy Polymerase Chain Reaction (PCR), Polymerase Chain Reaction (PCR), with oligonucleotide primers with homology to the 5’ and 3’ ends of your gene, amplify the DNA sequence between the primers. with oligonucleotide primers with homology to the 5’ and 3’ ends of your gene, amplify the DNA sequence between the primers. 5’ 3’ Your gene Reaction: Product: Your gene amplified

18. Reverse Genetic PCR Strategy T-DNA Reaction: Product: Reaction: Product: none.

19. PCR Screens for Mutants

20. PCR Strategy T-DNA Reaction: Product: T-DNA Reaction: Product:

21. Find the Plant You are ~here

22. T-DNA Mutants Genetic Analysis tagged seed line tagged seed line isolate homozygous mutant isolate homozygous mutant backcross to wildtype backcross to wildtype 2x phenotype analysis phenotype analysis tt x TT (wt) Tt T-DNA Segregation TTTt tt T t T t F2

23. PCR Genotyping LtT 5’5’ 3’3’ 5’5’ 3’3’ heterozygote LtT 5’5’ 3’3’ 5’5’ 3’3’ homozygote wt LtT 5’5’ 3’3’ 5’5’ 3’3’ homozygote mutant

24. Genetic Analysis F2 Segregation Genetic Analysis F2 Segregation 1 : 2 : 1 TTTt tt T t T t Not Lethal 1 wt : 2 het TTTt tt T t T t Lethal 1 wt : 1 het TTTt tt T t T t Gametophyte Lethal