Sequence availability

Three pea VHPO gene sequences (VHPO1, VHPO2 and VHPO3) were obtained from the ‘Caméor’ Unigene set consisting of annotated pea orthologs available

online through The Pea RNA-Seq gene atlas

(http://bios.dijon.inra.fr/FATAL/cgi/pscam.cgi) (Alves-Carvalho et al. 2015). Details

of sequences shown in Table 5.2 were used in this study for phylogenetic analysis.

Table 5.2 Details of vHPO related sequences of various plants used for phylogenetic tree construction

Species Gene name Sequence

Pisum sativum PsVHPO1 PsCam021226

PsVHPO2 PsCam043648 PsVHPO3 PsCam021353

Medicago truncatula MtVHPO1 XM_003601766

MtVHPO2 XM_003596891 MtVHPO3 TC202187

Mt_1 XM_003596891

Mt_2 BT134107

Mt_out XM_003595828

Lotus japonicus LjVHPO1 BT135522

LjVHPO2 CM0105.840 LjVHPO3 CM0105.840

Lj_1 BT140406

Lj_out CM0996.190

Cicer arietinum Ca_1 XM_004502262

Ca_2 XM_004502261

Ca_3 XM_004487375

Ca_4 XM_004490438

Ca_5 XM_004497416

Trifolium pratense Tp_1 GAOU01044928

104 5.2.2 Plant materials

Wild-type (VHPO1) pea plants used for this study was the dwarf line Caméor. Mutant (vhpo1) pea plants originated from TILLING program in France were also in the genetic background of the dwarf line Caméor. All plants were grown as described previously (Jager et al., 2007). Seeds from these plants were harvested for the quantification of IAA and 4-Cl-IAA levels.

5.2.3 Primers

Degenerate primers for gene isolation were designed using CODEHOP strategy (Rose et al., 1998) on conserved domains identified from protein sequence

alignments using BLOCK MAKER application

(http://blocks.fhcrc.org/codehop.html). Selected primers were manually optimised by

reference to the input legume sequences. Other primers were designed using Primer3

(http://bioinfo.ut.ee/primer3-0.4.0/) based on legume sequences to target conserved

regions in pea. The details of primer sequences are recorded in Appendix 1.

5.2.4 Gene isolation 5.2.4.1 DNA extraction

Genomic DNA was isolated from individual plants using a modified CTAB protocol. Harvested tissue was frozen in liquid nitrogen and ground into fine powder in a mill mixer by adding tungsten carbide bead to each tube. 500μL of extraction buffer (100mM Tris-HCl pH8, 1.4M NaCl, 20mM EDTA, 2% w/v CTAB, 20mM 2-β- mercaptoethanol) was added into each tube prior to incubation at 60°C for 15 min. 500µL of Chloroform:Isoamyl alcohol mix (24:1) was then added to the tubes, and the contents were mixed by gentle inversion. The samples were centrifuged at 14000 rpm for 1 min and the upper aqueous phase was transferred to new tubes and again extracted. 1mL of precipitation buffer (50mM Tris-HCL pH8, 10mM EDTA, 1%w/v CTAB) was added after the second extraction. The contents of the tube were mixed gently and incubated at room temperature for 10-15 min to allow the formation of thread-like precipitate. The precipitate was collected by centrifugation at 14000 rpm for 10min and dissolved in 300µL of 1.5M NaCl containing 1µL RNase A (25mg/mL). The solution was incubated at 50°C for 15 min or until the pellet was fully dissolved. DNA was precipitated by adding 600µL of 100% ethanol and

105 collected by centrifugation at 14000 rpm for 10 min. After centrifugation, the DNA pellet was washed in 200µL of 70% ethanol, air-dried and resuspended in 50µL of sterilised water.

5.2.4.2 DNA quantification

DNA concentrations were determined using a NanoDrop 8000 Spectrophotometer (Thermo Scientific, https://www.thermofisher.com/order/catalog/product/ND-8000-

GL)

5.2.4.3 Polymerase Chain Reaction (PCR)

Most PCR reactions were performed in 50µL, which included 5µL of DNA template and 45µL of master mix. The master mix was prepared according to Table 5.3 and scaled up to the required number of reactions.

Table 5.3 Reagents required for a 50µL PCR reaction.

Reagent For 1 reaction (50µL)

DNA template (added separately) 5µL

PCR buffer (5x) 10µL dNTPs (10mM) 1µL Forward primer (10mM) 1µL Reverse primer (10mM) 1µL DNA polymerase 0.2µL Sterilised water 31.8µL

PCR was carried out in a thermal cycler with heated lid as follows: an initial template denaturation step at 95°C for 1 min, followed by 35 cycles of 95°C for 15seconds (denaturation of DNA into single strands), Tm (annealing temperature for 15 seconds (annealing of primer), and 72°C for 1 min and 30 seconds (extension of newly synthesised DNA strands). Reaction were concluded by heating at 72°C for 10 min and then held at 12°C. The Tm varied according to the length and composition of the primers used and the extension temperature varied according to the specific DNA polymerase used.

106 5.2.4.4 Visualisation of DNA

To visualise PCR and enzyme digest products, DNA was separated on an agarose gel at appropriate agarose content percentage in TAE buffer (40mM Tris Acetate and 1mM EDTA), containing GoldView Nucleic Acid Stain (Acridine orange; SBS Genetech Co., Ltd, Beijing, China) with appropriate DNA ladder and visualised under UV light.

5.2.4.5 PCR product purification

Prior to sequencing, PCR products were purified using Promega Wizard SV Gel and PCR Clean-Up System (Promega, https://www.promega.com.au/) with the microcentrifuge protocol.

5.2.4.6 Sequencing

Purified PCR fragments were sequenced by Macrogen (South Korea,

https://dna.macrogen.com/eng/) or Australian Genome Research Facility (AGRF,

http://www.agrf.org.au/). Sequencing results were aligned and analysed using

Sequencher 4.0 (Genecodes, http://www.genecodes.com/).

5.2.5 Gene expression 5.2.5.1 RNA extraction

Harvested tissue was frozen in liquid nitrogen and homogenised by using tungsten carbide beads and mill mixer. Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen, https://www.qiagen.com/us/shop/sample-technologies/rna/total-rna/rneasy-

plant-mini-kit/#productdetails).

5.2.5.2 Quantification

RNA concentration was determined using NanoDrop 8000 Spectrophotometer (Thermo Scientific, https://www.thermofisher.com/order/catalog/product/ND-8000-

107 5.2.5.3 Reverse transcription

First strand cDNA was synthesised from equal amount of (5µg) of total RNA using the SuperScript III Reverse Transcriptase (Thermo Fisher,

https://www.thermofisher.com/), an engineered version of M-MLV RT with reduced

RNase H activity and increased thermal stability. RNA was first incubated with 1µL of oligo(dT)20 (50µM), 1µL 10mM dNTP mix and sterile water to a total of 13µL at 65°C for 5 min and then on ice for at least 1 min. A master mix was prepared (Table 5.4) and a 7µL aliquot was added to each tube. A negative control without reverse transcriptase (RT-) was included for each sample to check for the presence of genomic DNA contamination in the RNA sample. Reverse transcription was performed at 50°C for 30 - 60 min with a final incubation at 70°C for 15 min for enzyme inactivation.

Table 5.4 Reagents required for a 20µL reverse transcription reaction.

Reagent RT+ RT-

RNA + oligo(dT)20 (50µM) + 10mM dNTP mix + sterile water

13µL 13µL

First-Strand Buffer (5x) 4µL 4µL

DDT (0.1M) 1µL 1µL

RNaseOUT Recombinant RNase Inhibitor 1µL 1µL

Superscript III RT 1µL (1µL of water)

5.2.5.4 Quantitative reverse transcription PCR (qRT-PCR)

For analysis of relative gene expression, qRT-PCR was conducted using a Rotor- Gene 3000 Real-time Thermal Cycler with Rotor-Gene 6 Version 6.1 (Corbett Research, Australia). A Corbett Robotics CAS-1200TM pipetting robot (Corbett Research, Australia) with CAS Robotics 4 Version 4.9.8 (1.6.61) software was used to prepare reactions. Each 10µL reaction comprised 2µL cDNA template, 5µL 2x Quantace SensiMixPlus SYBR reagent (Alexandria, NSW, Australia), 0.3µL each of forward and reverse primer (10µM) and 2.4µL autoclaved Milli-Q water. A no- template control (containing water instead of cDNA) was included for each run to check for contamination, and each sample was run in duplicate for increased accuracy. For each cDNA sample, actin was run on the reverse transcriptase negative

108 control (RT-) to check for contamination. Reactions were run for 50 cycles. A standard curve for the target gene was included in every run. Standard curves were generated from a 10-fold dilution series from 1 x 10-2 to 10-6 ng/µL. Standard curve regression was considered acceptable if the R2 value was equal to or higher than 0.99. Actin was chosen as the reference gene for evaluating transcript levels of flowering genes as previously described (Foo et al., 2005; Hecht et al., 2011; see Appendix 1 for primer details). Calculations of gene expression relative to actin were based on non-equal amplification efficiencies and deviation in threshold cycle using the means for two technical replicates.