Supporting Information
Notes S1 Microarray design and production
EST sequence production and analysis
Five cDNA libraries were constructed from primary or secondary stem tissues, or from leaf
tissues of young trees (80–120 cm tall) grown under greenhouse conditions (see Supporting
Information Table S4). Four of the libraries (GQ009–GQ012) used healthy plants of Populus
trichocarpa (Torr. & Gray) x P. deltoides hybrid (clone H11-11) subjected to conditions that
altered plant N status, and one library (GQ014) was made from leaves of naturally occurring
Populus deltoides x P. balsamifera (jackii) inoculated with the rust-causing fungal pathogen
Melampsora medusae Thüm. (1878). A complete description of the tissues and methods used for
these libraries, together with other Populus libraries developed by the Arborea project
(Université Laval), has been made available at www.arborea.ulaval.ca. For each library, cDNA
was prepared from 500 ng poly A+ selected RNA, directionally ligated into the pBluescript II SK
(+) vector (Stratagene, La Jolla, CA, USA), and transformed by electroporation into Escherichia
coli DH10B cells (Invitrogen) for propagation. The high-throughput sequencing procedures,
which included colony selection, plasmid isolation, sequencing reactions and capillary analysis,
were conducted as previously described by Pavy et al. (2005). Sequencing of 7,680 random
cDNA inserts was performed from the 5’ end by using purified plasmid as templates.
The sequence data from the 7680 cDNAs were trimmed to remove vector and low quality
portions of the reads. In total, 5851 quality reads with at least 100 contiguous nucleotides that
had Phred20 quality scores were obtained. Cluster analysis was carried out using as described by
Pavy et al. (2005) and produced 4628 unique sequences, comprising 956 contigs (alignment of
sequences from two or more cDNAs) and 3052 singleton ESTs.
Functional annotations of the resulting unique consensus sequences (from contigs and
singletons) were performed by sequence similarity searches using BLAST and HMM against
several databases (nr, pfam, PopGI) as well as gene ontology assignment and classification into
multigene families. The similarity searches were updated in March 2009.
Selection of cDNA clones, amplicon production and microarray printing
We selected 3444 unique cDNA clones to use in microarray production, based on the following
criteria and procedures. We counter-selected against redundant sequences by forming clusters of
sequences sharing greater than 95% identity over 100 bp and selecting a single cDNA per cluster
to create a target, as described in Pavy et al. (2008). We also selected cDNAs to restrict the insert
length to between 400 bp and 2000 bp for efficient PCR amplification and to ensure relative
uniformity of amplicon concentrations (Pavy et al., 2008). Finally, to avoid including putative
Melampsora cDNAs from library GQ014, we removed the sequences unique to that specific
library and which did not match sequences from the first (unassembled) release of reads from the
poplar genome sequence.
The cDNA clones selected for inclusion on the microarray were robotically rearrayed
from daughter glycerol stock and PCR amplified as described in Pavy et al. (2008). All of the
rearrayed clones were sequenced from the 5’ end to enable validation of the identity of each
cDNA spotted on the microarray. A sequence was obtained for 3120 (90%) of the original
clones. Among the quality sequences (Phred 20 over 100 nt), 95% gave an exact match with the
original sequence of the cDNA clone.
Plasmid inserts were PCR-amplified in a GeneAmp PCR System 9700 (Applied
Biosystems) by using 2.5 µl of lysed culture, 0.2 µM M13 reverse, 0.2 µM -21M13 forward, 15
mM MgCl2, 100 mM Tris-HCl, 500 mM KCl, 250 µM dNTPs, 9U AmpliTaq, and nuclease-free
H2O to bring the final volume to 100 µl. PCR conditions were as follows: 5 min at 94°C
denaturation; 31 cycles of 30 s at 94°C, 30 s at 50°C, and 2 min at 72°C; and 7 min at 72°C.
Three microliters of each PCR product were run on a 1.2% agarose gel (E-Gel® Pre-cast
Agarose Electrophoresis System, Invitrogen) to assess quality and yield was determined at 260
nm with a SpectraMax plus (Molecular Devices, Sunnyvale, CA, USA). Of the 3444 clones,
more than 94% of the amplicons consisted of a single product, which had sufficient intensity and
the expected size.
PCR products were robotically purified on Multiscreen PCR filter plates (Millipore,
Etobicoke, ON), consolidated into 384-well plates, lyophilized by speed-Vac, and resuspended in
20 µl 50% DMSO. The cDNAs were printed on Corning UltraGaps slides with the Chipwriter
Pro microarray printer (Bio-Rad Laboratories, Hercules, CA, USA). SMP3 Microspot quill pins
(TeleChem International, Sunnyvale, CA, USA) in a 48-pin tool were used to deposit ~7 nl (~0.2
ng cDNA) per spot onto the slide. The PCR products were randomized prior to spotting to avoid
a biased distribution of cDNA from libraries or tissues. The resulting microarrays have a 4 × 6
subgrid layout with 160 spots per subgrid, with each spot having an approximate diameter and
pitch of 100 µM and 200 µM, respectively.