Tandem Inserts, Phenotypic Segregation, Hypocotyl Length, and More… James Reilly Department of Molecular and Cellular Biology University of California Davis, CA 95616

Recent Activities Isolation and verification of homozygous phyD and phyE mutants Isolation of YHB/WT, YHB/hy5, YHB/hyh, and YHB/hy5 hyh mutants Phenotypic segregation analysis of PHYC-OE / phyAB, WT PhyC-OE / Ler (WT), and YHC-OE / PhyAB lines on M.S. + kanamycin media. Digital hypocotyl measurements of different 4-day old phytochrome mutants under different growth conditions

Isolation and verification of homozygous PhyD and PhyE mutants phyD-2 (Salk_027336) T-DNA insertion In PHYD (463bp) PHYD Allele (485 bp)

Isolation and verification of homozygous PhyD and PhyE mutants PHYE-2 – Salk_040132 Genotyping Primer Scheme Left Primer + Right Primer: WT allele - 652 BP Right Primer + Salk LB Primer: PhyE tDNA - 401 BP Salk Insert: 5-6kb (large)

Isolation and verification of homozygous PhyD and PhyE mutants PHYE-2 – Salk_040132 Unexpected Double Band Right Flanking Primer CAGGTGATCCGGCTTTGACCCTTGCAGGCGCAGTTCAGTCTCAGAAGCTAGCCGTTAGGGCCATTTCTAGGCTGCAGTCACTTCCCGGAGGAGATATTGGTGCCTTGTGTGATACTGTTGTGGAAGATGTTCAGAGACTTACCGGTTATGACCGTGTTATGGTCTATCAGTTTCATGAAGATGATCATGGTGAAGTTGTTTCTGAGATTAGAAGGTCTGATTTGGAGCCTTATTTGGGTTTACATTATCCGGCAACAGATATTCCTCAGGCTGCTCGGTTCTTGTTCAAACAGAACCGTGTCCGAATGATTTGTGACTGCAATGCAACTCCGGTTAAGGTTGTTCAGAGTGAGGAACTCAAGAGACCACTTTGTTTAGTTAATTCTACTCTAAGAGCTCCTCATGGCTGCCATACGCAGTATATGGCG||AATATGGGCTCTGTAGCTTCTCTTGCACTCGCAATTGTAGTAAAAGGCAAAGATTCGAGCAAGCTTTGGGGATTAGTTGTTGGTCATCATTGTTCTCCTAGATACGTTCCATTCCCGTTGCGGTATGCTTGTGAGTTTCTGATGCAAGCATTTGGGCTTCAGCTTCAAATGGAACTTCAGTTAGCATCACAGTTAGCCGAGAAGAAGGCTATGCGGACGCAGACCTTGTTGTGCGATATGCTTCTCCGTGATACTGTTTCCGCTATTGTTACACAA Anticipated Insertion Site Right Flanking Primer

Isolation and verification of homozygous PhyD and PhyE mutants PHYE–2 – Salk_040132 Initial Run WT PHYE Allele (652bp) Salk 040132 T-DNA insertion In PHYE (401bp) PHYB allele with WT template (assures PCR conditions) PHYE allele with WT template (assures primer condition) Salk-PhyE-2 allele with #4 DNA PHYB allele + WT PHYE allele (with WT DNA) PHYB allele + Salk-PhyE-2 allele with #4 DNA Controls

Isolation and verification of homozygous PhyD and PhyE mutants PhyE-2 – Salk_040132 Wei’s PCR Verification Using Both Flanking and Salk LB Primers Expected Fragment Amplified from Right Flanking Primer and Salk LB Primer (401 BP) Amplified Fragment from WT Phy E (652 BP) (Left and Right Flanking Primers)

Isolation and verification of homozygous PhyD and PhyE mutants PHYE – Salk_040132 Unexpected Double Band - Not a WT Allele Flanking Primers, WT DNA as template Salk LB Primer + Left Flanking Primer, mutant DNA as template Band is slightly smaller than the WT fragment!

Isolation and verification of homozygous PhyD and PhyE mutants PHYE – Salk_040132 Unexpected Double Band – What Is It? Flanking Primers with WT DNA Salk LB Primer + Left Flanking Primer with transformant DNA Salk LB Primer + Both Flanking Primers. 4. Salk LB Primer + Left Flanking Primer with WT DNA A. Salk LB Primer + Left Flanking Salk LB Primer + Right Flanking Band is not from WT allele, instead it is amplified from opposite flanking primer and Salk LB Primer

Isolation and verification of homozygous PhyD and PhyE mutants PHYE – Salk_040132 Unexpected Double Band – Sequencing Results GAATCCTGTTTTGGTCCATTCTAGGACGACCCAGAAGCCTTTTTATGCTATTCTTCACAGGATTGATGCAGGGATTGTCATGGATTTGGAGCCTGCTAAATCAGGTGATCCGGCTTTGACCCTTGCAGGCGCAGTTCAGTCTCAGAAGCTAGCCGTTAGGGCCATTTCTAGGCT|GCAGTCACTTCCCGGAGGAGATATTGGTGCCTTGTGTGATACTGTTGTGGAAGATGTTCAGAGACTTACCGGTTATGACCGTGTTATGGTCTATCAGTTTCATGAAGATGATCATGGTGAAGTTGTTTCTGAGATTAGAAGGTCTGATTTGGAGCCTTATTTGGGTTTACATTATCCGGCAACAGATATTCCTCAGGCTGCTCGGTTCTTGTTCAAACAGAACCGTGTCCGAATGATTTGTGACTGCAATGCAACTCCGGTTAAGGTTGTTCAGAGTGAGGAACTCAAGAGACCACTTTGTTTAGTTAATTCTACTCTAAGAGCTCCTCA|Left_Border_Salk|TGGCTGCCATACGCAGTATATGGCG|Salk_Left_Border|AATATGGGCTCTGTAGCTTCTCTTGCACTCGCAATTGTAGTAAAAGGCAAAGATTCGAGCAAGCTTTGGGGATTAGTTGTTGGTCATCATTGTTCTCCTAGATACGTTCCATTCCCGTTGCGGTATGCTTGTGAGTTTCTGATGCAAGCATTTGGGCTTCAGCT|TCAAATGGAACTTCAGTTAGCATCACAGTTAGCCGAGAAGAAGGCTATGCGGACGCAGACCTTGTTGTGCGATATGCTTCTCCGTGATACTGTTTCCGCTATTGTTACACAATCTCCGGGTATTATGGACCTTGTGAAATGTGATGGAGCTGCGTTATATTACAAGGGGAAATGTTGGTTGGTTGGTGTTACT Left Flanking Primer and Salk Left Border Primer Right Flanking Primer and Salk Left Border Primer

Isolation and verification of homozygous PhyD and PhyE mutants PHYE – Salk_040132 Unexpected Double Band – Conclusion: Tandem Inserts phyE-2 (Salk_040132) Flanking sequences (2 T-DNA insertions) TCTAAGAGCTCCTCA|LBRB|TGGCTGCCATACGCAGTATATGGCG|RBLB|AATATGGGCTCTGTA

Hypocotyl Measurement of 4-day old Arabidopsis Seedlings Under Different Light Conditions

Hypocotyl Measurement of 4-day old Arabidopsis Seedlings Under Different Light Conditions

Hypocotyl Measurement of 4-day old Arabidopsis Seedlings Under Different Light Conditions

Phenotypic Segregation Analysis Determining Genotype of Transgenic Parents Through Kanamycin-Resistance Ratio of Offspring

Phenotypic Segregation Analysis Results for YHC-OE and PHYC-OE Transgenic Parents in WT and PhyAB Mutant Backgrounds Target Genotype Homozygous Offspring Sub-line YHC-OE / phy AB # 1 4, 5, 6 YHC-OE / phy AB # 2 1, 2, 6 PHYC-OE / phy AB # 5 7, 8 PHYC-OE / phy AB # 3 PHYC-OE / phy AB # 8 PHYC-OE / phy AB # 6 PHYC-OE / phy AB # 1 3 PHYC-OE / phy AB # 7 3, 6, 12 PHYC-OE / Ler #9 4, 7 PHYC-OE / Ler #3 6, 11 PHYC-OE / Ler #7 7, 9

Extra: PCR Dependence on dNTP Concentration WT PHYB Allele (1099 BP) PCR Conditions: Annealing Temp: 56° x 30” Extension Time: 72° x 1’30” Results: 80uM – 250uM is an adequate concentration of dNTP in a PCR reaction to yield acceptable results for an ~1100BP fragment.

Current Project: Isolation of YHB/WT, YHB/hy5, YHB/hyH, and YHB/hy5 hyH mutants Progress: F3 seeds have been harvested and are ready for planting. Their phenotypic segregation will be analyzed next week.

Ongoing Project: Isolation of YHBg/hy1hy2phyABCDE from the cross between YHBg/phyABCDE and YHBg/hy1hy2phyAB Progress: 2 different F2 plants are currently growing, despite numerous hardships.