Arabidopsis Experiments Forward Genetic Screen Ethylene Insensitive Mutants

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

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

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

H+ (protons) ATP synthase Transporters - carriers, - channels ATP hydrolase (ATPase) Adapted from Biochemistry and Molecular Biology of Plants, pp. 1

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

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

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

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

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

Agrobacterium tumefaciens Ti Plasmid (Tumor inducing) Mother Nature wt plant chromosome hormone genes (i. e. auxins) Ti Plasmid (from agro) opaline virulence genes nopaline neoplastic transformation opaline, nopaline virulence genes hormone genes Agro food

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

Germination Done Ø Breaking Dormancy Ø H 2 O/Imbibition, Surface Sterilize Seeds Plant on Nutrient Media Germinate 1. EMS Treated Seeds on MS/ACC media. 2. aha 3 -1 on MS media. Ø O 2/Aeration, Ø Cold/Prechilling "scarification” Ø Inducing Germination Ø Light

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

Krysan et al. , 1999 Knockology Plants/Pools DNA/Pools

Set-Up DNA Pooling Maintain lines as pools of seed. Seeds (9) Germinate and grow seeds in liquid culture. Seedlings (225) Extract DNA, DNA (225) Super Pool DNA, 1 2 3 4 5 PCR Screen 6 … 30 Super Pools (2025)

5’--GCATTAT 5’--GCATTAGGCTACATCGACTAGCACTG--3’ 3’--GCTACGTAATCCGATGTAGCTGATCGTGAC--5’ 5’--GCATTAGGCTACATCGACTAGCACTG-3’ 5’--GCATTAGGCTACATCGACTAGCACTG--3’ 3’--GCTACGTAATCCGATGTAGCTGATCGTGAC--5’ 3’--CGTAATACGATGTAGCTGATCGTGAC--5’ 94 Synthesis ~1 minute/kb 72 o o Denature Step ~30 seconds PCR ~65 o Annealing Step ~30 seconds 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG-3’ CTGATCGTGAC--5’ 5’--GCATGCATTAT 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

PCR Strategy 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. Reaction: Product: 5’ Your gene amplified 3’

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

PCR Screens for Mutants

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

Find the Plant You are ~here

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

PCR Genotyping 5’ homozygote wt 5’ L t T L t 3’ 3’ 5’ 3’ heterozygote homozygote mutant 5’ 3’ T

Genetic Analysis F 2 Segregation T t T TT Tt tt 1 : 2 : 1 Not Lethal T t T TT Tt tt 1 wt : 2 het Lethal T t T TT Tt tt 1 wt : 1 het Gametophyte Lethal