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.
References S2
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search
tool. J Mol Biol 215:403-10
Creux NM, Ranik M, Berger DK, Myburg AA (2008a) Comparative analysis of orthologous
cellulose synthase promoters from Arabidopsis, Populus and Eucalyptus: evidence of
conserved regulatory elements in angiosperms. The New phytologist 179:722-37
Ranik M, Myburg AA (2006) Six new cellulose synthase genes from Eucalyptus are
associated with primary and secondary cell wall biosynthesis. Tree Physiol 26:545-56
Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist
programmers, in: K S and M S (Eds), Bioinformatics Methods and Protocols: Methods in
Molecular Biology, Humana Press, Totowa, NJ. pp. 365-386
Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, et al. (2006) The
genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313:1596-604
Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JA (2007) Primer3Plus,
an enhanced web interface to Primer3. Nucleic Acids Res 35:W71-4
Wheelan SJ, Church DM, Ostell JM (2001) Spidey: a tool for mRNA-to-genomic alignments.
Genome Res 11:1952-7