Methods

3.2.1. Gene isolation

Leaf material was collected from D. tenuifolia (Dt) Single Seed Descent 4 (SSD4) plants at different time points during a long day and at different developmental stages when it was expected that the genes of interest would be highly expressed. RNA was extracted from all the leaf material individually using the Z6 buffer method (Logemann et al., 1987) as outlined in section 2.2.2. DNase treatment was carried out on RNA from each sample using Ambion® Turbo DNA-freeTM DNase. 1 µg DNase-treated RNA was synthesized into cDNA using a BioRad iScript cDNA synthesis kit. Degenerate primers were designed and used to amplify regions of Dt specific sequence for all genes of interest. PCR products were purified and sequenced as outlined in sections 2.2.5 and 2.2.6. Once these sequences had been obtained, new primers specific to each Dt gene (Appendix A.1) were designed for GeneRacer™ Kit (RLM-RACE) (Invitrogen, see section 2.2.7). Further primers were designed for each gene using the sequence data from GeneRacer™ results which covered the start and stop codons, and other primers designed to 5’ and 3’ untranslated regions (UTR) (START/STOP and 5’/3’ primer pairs, see Appendix A.1) to enable the amplification by PCR of the entire coding sequence (CDS) for each gene. DNA was extracted from leaf material of Dt SSD4 plants and using the START/STOP and 5’/3’-UTR primer pairs, the full length genomic DNA (gDNA) sequence was PCRed, isolated and sequenced for each gene of interest using Kod Hot Start and Kod Xtreme™ PCR kits (Novagen®) as described in sections 2.2.4-

2.2.6. Additional sequence needed to be isolated for theFT-like genes; this was done using a PolyATtract® mRNA Isolation System (Promega). RNA was extracted using the Z6 buffer method outlined above from leaf material collected from Dt SSD6 plants at ZT16 close to the bolting date when theFTgene is likely to be most highly expressed. 1-5 mg of total RNA was used in the mRNA isolation and the procedure carried out according to the manufacturer’s instructions using Streptavidin MagneSphere® Paramagnetic Particles. The eluted mRNA was concentrated following the manufacturer’s instructions and used for cDNA synthesis. A Thermoscript cDNA synthesis kit (Invitrogen) was used for making the cDNA with Oligo dT as the primer and the reaction carried out according to the manufacturer’s instructions. The cDNA was used in PCRs with DtFT and DtTSF specific forward primers (Appendices A.1.5.3 and A.1.6.3) and Oligo dT as the reverse primer following the method set out in section 2.2.4. Product purification and sequencing was then undertaken as described in sections 2.2.5 and 2.2.6.

3.2.2. Functional complementation of Arabidopsis mutants

Functional complementation of Arabidopsis mutants was undertaken for each gene of interest in this study and was carried out according to the method set out in section 2.2.8.3 and 2.2.9. The final step of the GATEWAY reaction ligated the isolated CDS for each gene into the pB2GW7 vector (Figure 3.1) which was then transformed into E. Coli for verification before transforming into Agrobacterium for floral dipping. For the analysis of transgenic plants in T1, T2and T3generations, leaf material was collected and DNA extracted using the CTAB buffer method (Stewart and Via, 1993) adapted for use with low amounts of starting material. The DNA was diluted to 100 ng/µl and used in PCR with primers designed to amplify the transgene. The products were visualised on a 1% (w/v) agarose (AGTC BioProducts)/1xTAE gel stained with GelRed (Biotium) at 100V for 20 minutes and placing on a UV lightbox (Syngene G-box with GeneSnap software). For T2 generation of Arabidopsis complementation with DtFLC and T3 generation of Arabidopsis complementation with DtFT, RNA was extracted from leaf material using a half volume Z6 buffer method (Logemann et al., 1987). Real time PCR was then performed on these samples. AtActin, AtTIP41 and Atβ-tubulin were used as housekeeping genes for normalisation and the standard was made from PCR products amplified from cDNA using real time primer pairs for the housekeeping genes, and

DtFT and DtFLC (Appendices A.2.1.1 and A.2.1.2). These were combined and purified using a PCR purification kit (Qiagen) before diluting from 100to 10-10. The real time PCR was performed using S10-6– S10-9as laid out in section 2.2.10 with T2 FLC complementation samples run with iTaq™Universal SYBR® Green Supermix (BioRad) and T3 FT complementation samples run with Go-Taq® qPCR master mix (Promega) with the addition of 10 nm (final concentration) Fluorescein calibration dye (Bio-Rad).

Figure 3.1 – pB2GW7 plasmid used as binary vector in complementation experiments.

Using the GATEWAY reaction, the isolated CDS for each gene was ligated into the region between attR1 and attR2, replacing the ccdB gene. Sm/SpR – Spectinomycin resistance gene, LB – Left border, Bar – BASTA

resistance gene, p35S – 35S promotor, T35S – 35S terminator, RB – Right border (Karimiet al., 2002).

3.2.3. Diurnal time course experiment

Dt SSD6 seed was sown onto F2S soil in p24s and topped with vermiculite. These were placed into a controlled environment cabinet (Versatile plant growth MLR-352, Panasonic Co. Ltd) at 22°C with 16 hours light, 8 hours dark. After 2.5 weeks, seedlings were transplanted into 5 inch pots of M2 soil and placed into two controlled environment cabinets (Versatile plant growth MLR-352, Panasonic Co. Ltd) at 22°C with 16 hours light, 8 hours dark. The cabinets were off-set from each other by 12 hours to enable harvesting during a normal working day. At four weeks

from sowing, the diurnal time course began. Over 52 hours, samples were collected from two sets of eight plants at 3 hour intervals beginning 30 minutes after the lights come on (ZT0). Material was collected from the newest fully expanded leaf on each plant.

3.2.4. Developmental time course

DtSSD6 seed was sown into F2S soil and placed in a controlled environment cabinet (Versatile plant growth MLR-352, Panasonic Co. Ltd) at 22°C with a 16 hour photoperiod until initiation and bolting. Leaf material was collected at time point ZT15.5 once a week from sowing until two week post-initiation of bolting. Collecting material at the end of the long day enabled the levels of COandFT to be measured.

3.2.5. Gene expression analysis using real time PCR

RNA was extracted from leaf material collected from both diurnal and developmental experiments using the Z6 buffer method (Logemann et al., 1987) as laid out in section 2.2.2. RNA from each sample was DNase treated using an Ambion® Turbo DNA-freeTM DNase kit and finally resuspended in 12 µl DEPC- treated H2O. 2 µg RNA was synthesised into cDNA using a Thermoscript cDNA synthesis kit (Invitrogen). Standards for the real time PCR were made using PCR products amplified from Dt cDNA using gene-specific real time primers for all six key genes and three housekeeping genes (DtTIP41, DtCACS, Dtα-tubulin) (Appendix A.2.1.3). These were PCR purified using a PCR purification kit (Qiagen) and eluted in 20 µl H2O. Each was quantified and an equal concentration of each product was mixed together. This was diluted from 100 to 10-10 and the real time PCR performed using S10-6– S10-9. Analysis was done according to section 2.2.10.