1. Searching for Genes Important in Seed Development At1g19000 At1g74840 BY: Mike Douglas

2. MYB-Related Transcription Factor At1g19000 (GENE MODEL)

3. Genotyping Expected Wild Type Band: 1104 bp T-DNA Mutant Band: 1076 bp T-DNA Mutant Band 325 bp

4. Plant Layout Chart & Genotyping Results 15 : 13 : 3 Homozygous Wild-Type : Heterozygous : Homozygous Mutant

5. Where is AT1g19000 Expressed?

6. Cloning of Promoter Region in E. Coli Plasmid Vector ECO-RI Digestion 1 kb Ladder (+) Colony Colony I Colony II Colony III Colony IV (+) (-) 1617 1287 3500 Sequence using T7 and SP6 Primers Blast: T7 Sequencing vs. Actual Promoter Sequence Identities = 637/637 (100%) T7 Sequencing Blast: SP6 Sequencing vs. Actual Promoter Sequence Identities = 708/709 (99%) 1 MUTATION SP6 Sequencing

7. Mutant Phenotypes No Obvious Physical Phenotype Nomarski Imaging WILD TYPE EMBRYOS HOMOZYGOUS MUTANT EMBRYOS

8. At1g74840 (GENE MODEL) MYB-Related Transcription Factor

9. Genotyping 1 kb ladder 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 WILD TYPE (+) (-) 1 kb Ladder (-)

10. Plant Layout Chart & Genotyping Results ALL 18 Plants are WILD TYPE

11. Where is AT1g74840 Expressed? Expected RT-PCR product: 603 bp Ladder Leaf +RT Silique +RT gDNA TA Leaf +RT TA Seed +RT H20 Ladder NOTE: No Tubulin Band

12. Cloning of Promoter Region in E. Coli Plasmid Vector ECORI Digestion T7 and SP6 Sequencing Blast: T7 Sequencing vs. Actual Promoter Sequence Identities = 719/722 (99%) Plus/Plus Blast: SP6 Sequencing vs. Actual Promoter Sequence Identities = 705/706 (99%) Plus/Minus 4 MUTATIONS Colony I Colony II Colony III 1 kb Ladder 1888 3500

13. Phylogenic Relationship of At1g19000 and At1g74840 Cross to make DOUBLE MUTANT- may eliminate possible redundancy for mutant phenotype