Supplementary Material and Methods
cDNA-amplified fragment length polymorphism (AFLP) analysis and DNA
cloning
The cDNA-AFLP method was performed according to Bachem et al. (1996, 1998).
The cDNA was synthesized starting with 1 µg of total RNA using the Super SMART
cDNA synthesis kit (Clontech). 300 ng double-stranded cDNA was restricted with 5 U
PstI and 2.5 U MseI (New England Biolabs) at 37ºC for 3 hours. To the restricted
cDNA fragments were ligated 2 pmol PstI adapter and 20 pmol MseI adapter
complementary to the restriction sites (supplemantary table 2). The ligation was
performed overnight at 4ºC in 12.5 µl mix containing 1x NEB2 buffer, 1 mM ATP,
BSA (0.1 mg/ml) and 1 U T4-Ligase (New England Biolab). Ligated fragments were
diluted 1:4 with 0.1x TE. 5 µl was used for PCR-preamplification with 6 pmol of each
primers MseI 00 and PstI 03 (suppl. table 3), 0.2 mM dNTP, 1.5 mM MgCl2, 1x
reaction buffer and 1U Taq polymerase (Qiagen). PCR conditions were: 3 minutes
95ºC; 20 cycles: 30 seconds 95ºC, 30 seconds annealing 60ºC and extension 2
minutes 72ºC. The preamplified mixture was diluted 40 times, and 3 µl was used for
final selective PCR-amplification according to Vos et al. (1995). MseI and PstI
primers containing three selective nucleotides at the 3’-prime end were used. PCR
mix contained 6 pmol of each primers MseI and PstI (supplementary table 3), 0.2
mM dNTP, 1.5 mM MgCl2, 1x reaction buffer and 1U Taq polymerase (Qiagen). PCR
conditions included following steps:1) 3 minutes 95ºC, 2) 9 cycles: 30 seconds 95ºC,
30 seconds annealing (touch-down profile 65ºC-1ºC/cycle) and extension 2 minutes
72ºC, 3) 23 cycles: 30 seconds 95ºC, 30 seconds annealing 56ºC and extension 2
minutes 72ºC. PCR products were separated in 5% acrylamide gel containing 8 M
urea, 1x TBE using sequencing gel electrophoresis apparatus (Gibco BRL). Silver
staining, performed according to Sanguinetti et al. (1994) with slight modifications,
was used to visualize cDNA-AFLP fragments. First, the gel was fixed in 10% ethanol,
5% acetic acid solution for 5 minutes. Subsequently, the gel was stained in 0.3 %
AgNO3 for 15 minutes and developed in developing solution (1.5% NaOH, 0.4%
formaldehyde, 100 µg/l NaBH3) during 15 - 20 minutes. After each step, gels were
rinsed in distilled water. The cDNA-AFLP fragment patterns were compared between
plants with and without B chromosomes. Fragments of interest were excised from the
acrylamide gel and resuspended in 50-100 µl elution buffer (50 mM KCl, 10mM Tris
HCl (pH 9.0) and 0.1% Triton-X-100) for 20 minutes at room temperature (Sanguinetti
et al., 1994). 5 µl of eluted DNA was used for reamplification using the same PCR
conditions and primer combinations as been used for selective amplification. PCR
products were checked by 1.2% TAE agarose gel electrophoresis, cloned into pGEMT Easy vector (Promega), and sequenced.
References
Bachem, C.W.B., Oomen, R.J.F.J., and Visser, R.G.F. (1998) Transcript imaging with
cDNA-AFLP: A step-by-step protocol. Plant Molecular Biology Reporter
16:157-173.
Bachem, C.W.B., vanderHoeven, R.S., deBruijn, S.M., Vreugdenhil, D., Zabeau, M.,
and Visser, R.G.F. (1996) Visualization of differential gene expression using a
novel method of RNA fingerprinting based on AFLP: Analysis of gene
expression during potato tuber development. Plant Journal 9:745-753.
Sanguinetti, C.J., Neto, E.D., and Simpson, A.J.G. (1994) Rapid silver staining and
recovery of PCR products separated on polyacrylamide gels. Biotechniques
17:914.
Vos, P., Hogers, R., Bleeker, M., Reijans, M., Vandelee, T., Hornes, M., Frijters, A.,
Pot, J., Peleman, J., Kuiper, M., and Zabeau, M. (1995) AFLP - a new
technique for DNA fingerprinting. Nucleic Acids Research 23:4407-4414.