Ctcgaacttgtttttggttcatctctcaaaaccaaaatcactaaagaggagaagattgctaaagtttgataaaacattccaaaatca ATGGCTGATAGGATCAAAGGTCCATGGAGTCCTGAAGAAGACGAGCAGCTTCGTAGGCTTGTTGTTAAATACGGTCCAAGAAACTGG ACAGTGATTAGCAAATCTATTCCCGGTAGATCGGGGAAATCGTGTCGTTTACGGTGGTGCAACCAGCTTTCGCCGCAAGTTGAGCAT CGGCCGTTTTCGGCTGAGGAAGACGAGACGATCGCACGTGCTCACGCTCAGTTCGGTAATAAATGGGCGACGATTGCTCGTCTTCTC AACGGTCGTACGGACAACGCCGTGAAGAATCACTGGAACTCGACGCTCAAGAGGAAATGCGGCGGTTACGACCATCGGGGTTACGAT GGTTCGGAGGATATCGGCCGGTTAAGAGATCGGTGAGTGCGGGATCTCCACCTGTTGTTACTGGGCTTTACATGAGCCCAGGAAGCC CAACTGGATCTGATGTCAGTGATTCAAGTACTATCCCGATATTACCTTCCGTTGAGCTTTTCAAGCCTGTGCCTAGACCTGGTGCTG TTGTGCTACCGCTTCCTATCGAAACGTCGTCTTCTTCCGATGATCCACCGACTTCGTTAAGCTTGTCACTTCCTGGTGCCGACGTAA GCGAGGAGTCAAACCGTAGCCACGAGTCAACGAATATCAACAACACCACTTCGAGCCGCCACAACCACAACAATACGGTGTCGTTTA TGCCGTTTAGTGGTGGGTTTAGAGGTGCGATTGAGGAAATGGGGAAGTCTTTTCCCGGTAACGGAGGCGAGTTTATGGCGGTGGTGC AAGAGATGATTAAGGCGGAAGTGAGGAGTTACATGACGGAGATGCAACGGAACAATGGTGGCGGATTCGTCGGAGGATTCATTGATA ATGGCATGATTCCGATGAGTCAAATTGGAGTTGGGAGAATCGAGTAGacaaagtgagattattaggaaactgtttaaattggagaag Aagaaaaatgctctgtttttttctcctttggattaggcttaagaattttgggttttaaggaaatgtatagaggaaatcgagtgaaca aagctcgagagctggggacgtagtgacgaagacgaagatcaaatttctcttaagctattcaggaaaataaaataaatttttattt “AT5G67300” -AtMYB44, AtMYBr1 Tomohiro Mochizuki Junior at Kyoto Univ. HC70AL S04 Final “Farewell, Thank You, Arigatou, Sayounara” Presentation

AT5G67300 (K8K14.2, ATMYB44, AtMYBr1) Putative myb-related protein cDNA 1217bp UTRUTR UTRUTR WHAT is AT5G67300? HOW important is it in embryo development? HOW is it expressed in Arabidopsis Thaliana? WHAT happens if you knockout AT5G67300? WHERE can you find knockout AT5G67300? HOW can you find the knockout line?

Madison insertion search Round1 within 30Superpools Control 1Kb PCR

Madison insertion search Round2 within 9 DNA Pools 5 1Kb Madison insertion; SUPERPOOL#4 DNAPOOL#34 (7th of SP4) Between +582 and +583 (right in the middle of the Exon!)

4FW FW FW FW RV RV RV &24FW 2914 AT5G FW FW 7514 AT5G67290 AT5G RV AT5G67310 FWRV RV ’ 3’ AT5G67300 situation AT5G67290 FAD dependent oxidoreductase family protein, contains Pfam FAD dependent oxidoreductse. AT5G67310 Cytochrome P450 family protein. At5G67320 WD-40 repeat family protein. 5’ 3’ ?

SALK Genotyping Sample #1-9Sample #10-19Sample #20-29Sample #30-36 G.S. FW/RV primers G.S. FW/LB (LBb1) primers Tubulin control

SALK result W WM M W W W WW W W W WW W W W W W W WW W WW WW W W WW WW WW W W W W W Mutation homozygous Wild/Mutation heterozygous Wildtype homozygous NO tDNA insertion within AT5G67300 in line# Brandon, call SALK!!! Checked 36samples of SALK # but……

How is AT5G67300 expressed in SRB? Leaf Stem FlowerUnf.-ovule 14-DAP- embryo 30-DAP axis Leaf Flower AT5G67300 Control Expressed highly in leaves. Higher in mature seeds. mRNA exists in all stages

Arabidopsis Thaliana vs. others % % dbEST_ID Embryo region: Suspensor Functional category : MYB Protein identification: (AF122051) tuber-specific and sucrose- responsive element binding factor [Solanum tuberosum]; myb DNA-binding protein (AtMYB73); protein id: At4g37260 UTRUTR UTRUTR Solanum tuberosum (Potato) 364a.a. 47% Identity, 56% Positive similarity tuber-specific and sucrose- responsive element binding factor Nicotiana Tabacum (tobacco) 329a.a. 62% Identity, 73%Positive similarity Myb-like protein

HOW can you find the knockout line? WHERE can you find knockout AT5G67300? WHAT happens if you knockout AT5G67300? HOW is it expressed in Arabidopsis Thaliana? HOW important is it in embryo development? WHAT is AT5G67300? By PCR and Southern blotting Pools. At Madison Facility. Superpool#4, DNAPool#34 Not quite sure. Mainly in leaves. Post maturation in seeds. Not very crucial. SALK Facility. Not reliable. “SALK sometimes gives weird seeds.” Erin Marsh et.al 2004