Candidate Gene Primer Design

Supplementary material S2 Candidate gene primer design For EuCAD2, the cDNA and mRNA sequences of E. gunnii (X75480), E. botryoides (D16624), E. globulus (AF038561) and E. saligna (AF294793) were aligned and the primers designed in the conserved regions. 11 overlapping primers were designed with the OLIGO EXPLORER v1.1.0 software (Teemu Kuulasmaa, 2000-2002). For half of them, the amplicons were sequenced on both sides (Macrogen). To check for the uniqueness of the EuCAD2 sequence, 3 individuals were cloned on the whole intron-exon length of 2410 bp. Seven to 12 clones was sequenced for each individual. For EuC4H1 and EuC4H2, no Eucalyptus sequences were found in the NCBI database in 2009. The cDNA and mRNA sequences of C4Hs from different species, Arabidopsis thaliana (ATU71080), Ginkgo biloba (AY748324), Brassica napus (DQ485132, DQ485130), Populus tomentosa (EU760387) were aligned with CodonCode software (CodonCode Corp.). In 2010, the Eucalyptus database: http://eucalyptusdb.bi.up.ac.za/ (DOE Joint Genome Institute and Eucalyptus genome Network, EUCAGEN) was released, and fragments of conserved sequences were blasted. Two sequences from the 4.5X genome scaffold sequence were recovered and aligned with the previous species. Consensus sequences were generated and primers were designed for the conserved regions. For EuCesAs primer design, because no cellulose synthase sequences were available for E. urophylla, sequences were based on E. grandis (Creux et al. 2008; Ranik and Myburg 2006) or E. globulus sequences. Cellulose synthase sequences such as EuCesA1 (EU737100), EuCesA2 (DQ014506, EU737101) and EuCesA3 (AB368175, EU737102, DQ014507) were downloaded from the GenBank database. For the EuCesA3 sequence, exon boundaries were estimated by aligning the complete coding sequence for E. globulus and the cDNA sequence for E. grandis using Spidey (Wheelan et al., 2001). For EuCesA1 and EuCesA2, because there were no complete sequences available in 2008, exon boundaries were estimated by aligning the E. grandis EgCesA cDNA sequence against the Populus trichocarpa genomic sequence (Tuskan et al. 2006) using Spidey (Wheelan et al. 2001), after homologous sequences of CesAs in Populus genomes were identified by BLAST (Altschul et al. 1990) searches. Primers were designed by the Primer3 (Rozen and Skaletsky 2000) program in Primer3Plus (Untergasser et al., 2007) taking special care to adjust PCR product size suitable for sequencing and to avoid exon-intron boundaries that were expected from the above alignments. Because CesA genes comprise a multigene family, a mismatch library that contains all of the CesA cDNAs except a target sequence was constructed for each CesA before designing primers. We designed X, Y and Z primer pairs for EuCesA1, EuCesA2 and EuCesA3, respectively.

PCR amplification, cloning and DNA sequencing

PCRs were performed in 10 L reaction mixtures according to the following conditions: 10 mM incubation buffer 1X CORE Kit Q-Biogene (Tampon CORE Kit Q-Biogene, MP Biomedical) containing 1.5 mM MgCl2, 0.20 mM dNTPs CORE Kit Q-Biogene, 0.30 µM forward and reverse primers, 1.2 units of Taq DNA polymerase (Invitrogen) and 15 ng of template DNA. The following PCR program was used for the amplification: 94 °C for 4 min, then 35 cycles (40 cycles for EuCesAs) of 94 °C for 30 sec, an annealing step at the primer’s optimized annealing temperature for 1 min and 72 °C for 1 min (2 min for C4Hs), followed by a final extension at 72 °C for 5 min. Then PCR products were loaded on agarose gel for electrophoresis and the amount was estimated by image analysis with the freeware Image J v1.45b (Wayne Rasband, NIH). PCR products (100 ng of each) were sent to HtSeq (High Throughput Sequencing, Seattle, WA, USA) for sequencing. For cloning, each fragment was generated by PCR amplification and the bands were cut out and extracted with the Qiaquick PCR purification kit (Qiagen). The DNA concentration was checked on agarose gel before ligation in the pGEMT or T-easy plasmid (Promega). A ratio insert/vector of 5/1 was used to calculate the amount of DNA to ligate to 50 ng of pGEM vector. The ligation reaction was performed according to the pGEM®-T or pGEM®-T Easy vector system manual and the solution was incubated at 4°C overnight. The transformation was performed as followed: 25 µL of XL1-Blue competent cells (Stratagene) were mixed with 1.7 µL of -mercaptoethanol, incubated on ice for 10 min, and then transformed with 1 µL of recombinant plasmid. The tube was incubated for 30 min on ice, then 45 sec at 42°C followed by 2 min on ice. 900 µL of SOC media prewarmed at 37°C was added before the final incubation at 37°C for 1 h. During the incubation, a 10 cm agar Petri plate was prepared by adding 100 µL of 100 mM IPTG stock solution and 50 µL of 20 mg/mL X-Gal stock solution. The two reagents were spread on the agar plate with glass beads, and the plate was incubated for 30 min at 37°C. 50 µL to 200 µL of transformed bacteria was plated on the 10 cm Petri plate and then incubated at 37°C overnight. The clones were sent to GATC Biotech (Mulhouse, France) for sequencing.

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