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

2. 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

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

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

5. 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

6. Isolation and verification of homozygous PhyD and PhyE mutants
PHYE–2 – Salk_040132
Initial Run
WT PHYE
Allele
(652bp)
Salk
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

7. 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)

8. 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!

9. 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

10. 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

11. 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

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

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

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

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

16. 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

17. 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.

18. 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.

19. 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.