SUPPLEMENTAL MATERIALS AND METHODS
Microarray Hybridizations and Microarray Analysis
The construction of the spruce 16.7K array and further details of the microarray
experiment will be described elsewhere (Ralph et al., in prep.). Briefly, hybridizations
were performed using the Genisphere Array350 kit (Genisphere, Hatfield, USA)
following manufacturer’s instructions. Forty µg total RNA was reverse transcribed using
Superscript II reverse transcriptase (Invitrogen) and oligo d(T18) primers with a 5’ unique
sequence overhang specific to either the Cy3 or Cy5 labeling reactions. The RNA strand
of the resulting cDNA:RNA hybrid was hydrolyzed in 0.075 M NaOH / 0.0075 M EDTA
at 65°C for 15 min followed by neutralization in 0.175 M Tris-HCl (pH 8.0). Following
pooling of the appropriate cDNAs, samples were precipitated with linear acrylamide and
resuspended in a 45 µL hybridization solution consisting of 0.25 M NaPO4, 1 SSC,
0.5% SDS, 2 Denhardt’s solution, 1 mM EDTA, 4.0 µL LDNA d(T) blocker, 0.5 µg/µL
sheared salmon testes DNA (Invitrogen) and 0.3 µL of Cy5-labeled GFP cDNA (Cy5dUTP and Ready-To-Go labeling beads, Amersham Pharmacia Biotech). Immediately
prior to use, arrays were pre-washed 3 in 0.1% SDS at room temperature for 5 min each,
followed by two washes in MilliQ-H2O for 2 min each, 3 min at 95°C in MilliQ-H2O,
and dried by centrifugation (5 minutes at 2000 rpm in an IEC Centra CL2 centrifuge with
rotor IEC 2367-00 in 50 mL conical tube). The cDNA probe was heat denatured at 80°C
for 10 min, then maintained at 65°C prior to adding to a microarray slide heated to 55°C,
1
covered with a 22  60  1.5 mm glass coverslip (Fisher Scientific), and incubated for 16
h at 60°C. Arrays were washed in 2 SSC, 0.2% SDS at room temperature for 5 min to
remove the coverslip, followed by 15 min at 65°C in the same solution, then three washes
of 5 min in 2 SSC at room temperature, and three washes of 5 min in 0.2 SSC at room
temperature, and dried by centrifugation. The Cy3 and Cy5 3DNA capture reagent
(Genisphere) were then hybridized to the bound cDNA on the microarray in a 45 µL
volume consisting of 0.25 M NaPO4, 1 SSC, 0.5% SDS, 2 Denhardt’s solution, 1 mM
EDTA, 2.5 µL Cy3 capture reagent and 2.5 µL Cy5 capture reagent. The 3DNA capture
reagent is bound to its complementary cDNA capture sequence on the Cy3 or Cy5 oligo
d(T) primers. The second hybridization was performed for 3 h at 60°C, then washed and
dried as before.
Fluorescent images of hybridized arrays were acquired by using
ScanArray Express (Perkin Elmer, Foster City, USA). The Cy3 and Cy5 cyanine fluors
were excited at 543 nm and 633 nm, respectively. All scans were performed at the same
laser power (90%), but with the photomultiplier tube settings for the two channels
adjusted such that the ratio of the mean signal intensities was ~1, and the percentage of
saturated array elements was < 0.5% but > 0%, while minimizing background
fluorescence.
Fluorescent intensity data were extracted by using the ImaGene 5.5
software (Biodiscovery, El Segundo, USA).
Before normalization, the lowest 10% of median foreground intensities was
subtracted from the median foreground intensities to correct for background intensity.
After quantification of the signal intensities, data were normalized to compensate for
nonlinearity of intensity distributions using the vsn method (Huber et al., 2002). A linear
model was applied to obtain a single estimate and standard error of the ΔH difference
2
statistic of expression for each parameter examined (i.e. mechanical wounding versus
untreated control at 2 h) (Smyth, 2004). The ratio of the estimate to the standard error
was used to calculate a t statistic, from which a P value was obtained.
3
Supplemental Table V. Gymnosperm and angiosperm DIR and DIR-like genes
represented in phylogenetic analyses.
Species
Gymnosperms
Picea glauca (white spruce)
Picea sitchensis (Sitka spruce)
Picea glauca x engelmannii (interior spruce)
Tsuga heterophylla (western hemlock)
Thuja plicata (western red cedar)
Angiosperms
Arabidopsis thaliana (thale cress)
Oryza sativa (Japonica) (rice)
DIR
Nomenclature
FLcDNA
Accession
Number
Genomic ORF
Accession/AGI
Number
PDIR1
PDIR7
PDIR10
PDIR12
PDIR18
PDIR11
PDIR15
PDIR16
PDIR17
PDIR19
PDIR2
PDIR3
PDIR4
PDIR5
PDIR6
PDIR8
PDIR9
PDIR13
PDIR14
ThDIR1
ThDIR2
TpDIR1
TpDIR2
TpDIR3
TpDIR4
TpDIR5
TpDIR6
TpDIR7
TpDIR8
TpDIR9
DQ395241
DQ395247
DQ395250
DQ395252
DQ395258
DQ395251
DQ395255
DQ395256
DQ395257
DQ395259
DQ395242
DQ395243
DQ395244
DQ395245
DQ395246
DQ395248
DQ395249
DQ395253
DQ395254
AF210071
AF210072
AF210063
AF210064
AF210065
AF210066
AF210067
AF210068
AF210069
AF210070
AF487405
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
AtDIR1
AtDIR2
AtDIR3
AtDIR4
AtDIR5
AtDIR6
AtDIR7
AtDIR8
AtDIR9
AtDIR10
AtDIR11
AtDIR12
AtDIR13
AtDIR14
AtDIR15
AtDIR16
AtDIR17
AtDIR18
AtDIR19
AtDIR20
AtDIR21
AtDIR22
AtDIR23
AtDIR24
AtDIR25
OsDIR1
OsDIR2
OsDIR3
OsDIR4
OsDIR5
OsDIR6
OsDIR7
OsDIR8
OsDIR9
OsDIR10
OsDIR11
OsDIR12
OsDIR13
n.a.
AY093095
n.a.
n.a.
AK175255
BT002439
AK118030
n.a.
BT010722
BT002889
AK176442
BT004016
BT009718
n.a.
CNS0A3JZ
BT008336
n.a.
AY081267
AK117899
AY128336
n.a.
BT015420
BT005788
CNS0A5KQ
n.a.
AK109288
n.a.
AK065027
AK108186
AK108922
n.a.
n.a.
n.a.
AK108101
n.a.
AK106022
n.a.
n.a.
At5g42510
At5g42500
At5g49040
At2g21110
At1g64160
At4g23690
At3g13650
At3g13662
At2g39430
At2g28670
At1g22900
At4g11180
At4g11190
At4g11210
At4g38700
At3g24020
CAB67637
At4g13580
At1g58170
At1g55210
At1g65870
At3g13660
At2g21100
At3g55230
At1g07730
NM_186138
NM_186143
AP006186
XM_482075
NM_196355
NM_189817
NM_189810
NM_184035
NM_194246
NM_184603
XM_479255
XM_479260
XM_479267
4
Hordeum vulgare (barley)
Triticum aestivum (wheat)
Saccharum officinarum (sugarcane)
Pisum sativum (pea)
Forsythia intermedia (shrub)
Podophyllum peltatum (mayapple)
Sorghum bicolor (sorghum)
Gossypium barbadense (cotton)
Zea mays (corn)
OsDIR14
OsDIR15
OsDIR16
OsDIR17
OsDIR18
OsDIR19
OsDIR20
OsDIR21
OsDIR22
OsDIR23
OsDIR24
OsDIR25
OsDIR26
OsDIR27
OsDIR28
OsDIR29
OsDIR30
HvDIR1
HvDIR2
HvDIR3
TaDIR1
TaDIR2
TaDIR3
SoDIR1
SoDIR2
SoDIR3
PsDIR1
PsDIR2
FiDIR1
FiDIR2
PpDIR1
SbDIR1
GbDIR1
ZmDIR1
n.a.
AK108983
n.a.
n.a.
n.a.
AK121408
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
U43497
U43496
AF021258
U32427
AF483596
AB012103
AY421731
AJ626722
AY781903
AF115574
U11716
AF210061
AF210062
AF352736
AF527807
AY560544
AF232008
5
NM_188847
XM_479273
XM_479258
NM_195796
XM_450817
AC135595
AC119796
NM_184427
AP003856
NM_196360
AP003369
NM_195790
NM_195802
XM_481885
XM_481843
XM_481834
XM_481890
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
Supplemental Table VI. Oligonucleotides used in real-time PCR.
Isoform
PDir2
Primer name
Dir2F
Dir2R
PDir5
Dir5F
Dir5R
PDir6
Dir6F
Dir6R
PDir8
Dir8F
Dir8R
PDir13
Dir14F
Dir14R
PDir19
Dir15F
Dir15R
PDir1
Dir1F
Dir1R
Dir10F
PDir10
Dir10R
Dir16F
PDir12
Dir16R
ActinF
ß-Actin
ActinR
1
Primer position from stop codon.
Sequence
5’-TGTTTCATTCTCATGCAGATTTG-3’
5’-TTATTTCACTCTACGCTAGCTG-3’
5’-TCCTTCTGCTCTCCATTTCTATG-3’
5’-CATTATTTCACTGCACGCTACAG-3’
5’-CTTTTGCAAATCTGCATTGGTAG-3’
5’-TTCAATCAATCATGTGAGGAGC-3’
5’-GCGTGCAGTGAAATAATGTCTAC-3’
5’-GAGAACGATTCTGACAAAATCTC-3’
5’-ACTGCTTATCACATGAGTTGCG-3’
5’-GGACCTTCATGGAGATCCCTC-3’
5’-TCTGCCACCGTAAGCAAGGACC-3’
5’-GATGAGCATAGCTCCACACAAATC-3’
5’-GCTGTGGGTAGGTATTCTGATC-3’
5’-TATGCACAGAAGACACGGCCATG-3’
5’-TGCCCGGCAGTGGTATATACATTG-3’
5’-GCAAGCAGCATTGGATACTGTCAC-3’
5’-TGTAGAGGTGAAATCAAGGCTCAGTC-3’
5’-TGGTCATGATTAGACTACCCTTTG-3’
5’-GGTATCCATGAGACTACATAC-3’
5’-CAGGAAACATGGTAGAACCAC-3’
6
Description1
+93 to +116
+142 to +164
+30 to +52
+104 to +127
+101 to +124
+190 to +213
+88 to +111
+182 to +205
+12 to +34
+104 to +125
+1 to +23
+74 to +95
+70 to +91
+136 to +158
+46 to + 69
+119 to +142
+79 to +103
+161 to +184