Suppl. Materials and methods

PCR-based genotyping

Presence of a T-DNA was detected by PCR with primers specific to one position in the T-DNA and another one in the flanking chromosomal DNA. Absence of a T-DNA was tested by PCR with primers specific to positions in the flanking chromosomal DNA at both sides of the T-DNA insertion site (Table A and B). Thus, for plants homozygous for a particular T-DNA insertion, the “presence” test was positive and the “absence” test negative, while for plants not containing the T-DNA of interest, the “presence” test was negative and the “absence” test positive. For hemizygous plants, both tests were positive.

Table A Primers specific for transgenes containing lacO tandem repeat arrays

transgene / test for / primer sequences / product / reference
CCP4.193 / presence - LB / 5’TCTGTCTCTGATCTTATTGTTGGTG / 0.39 kb / Rosin et al. 2008
5’GCTTGCTGCAACTCTCTCTCAGG
presence - RB / 5’GTTGCGGTTCTGTCAGTTCC / 0.49 kb
5’TCGACGAGAAGACTTGACTTGG
absence / 5’TCTGTCTCTGATCTTATTGTTGGTG / 0.58 kb
5’TCGACGAGAAGACTTGACTTGG
CCT431 / presence - LB / 5’TCCGTTGTCTGCTTCCATCA / 0.62 kb
5’CCGTCCCGCAAGTTAAATATG
presence - RB / 5’TACGATAACGGTCGGTACGG / 0.46 kb
5’AAGATCGGGAAGAGGCGAAG
absence / 5’TCCGTTGTCTGCTTCCATCA / 0.89 kb
5’AAGATCGGGAAGAGGCGAAG
26 / presence / 5`TTCTGTCAGTTCCAAACGTAAAAC / 0.9 kb / Matzke et al. 2005
5`ATTGATTACAACGAAACGAACA
absence / 5`TGATGGTATTTTGTATGCATGCC / 1.1 kb
5`ATTGATTACAACGAAACGAACA
112 / presence / 5`TTCTGTCAGTTCCAAACGTAAAAC / 0.8 kb
5`CCTTGGAGACTTCGGCATAA
absence / 5`CCTATTCTTCCTCTCTCTGCC / 1.0 kb
5`CCTTGGAGACTTCGGCATAA
EL702C / presence / 5’GGGATCCTGTACTCCACCAAAGAAGAAGAG / 1.4 kb / Watanabe et al. 2005
5’AGATTAGGAAAGCGTGAGAGTAAAACCCTA
absence / 5’GCCAAACGAGATCACATACAATATCTTGAT / 2.1 kb
5’AGATTAGGAAAGCGTGAGAGTAAAACCCTA

Table B Primers specific for T-DNA-insertion mutant alleles

allele / test for / primer sequences / product / reference
suvh5-1 / presence / 5´CGTGTGCCAGGTGCCCAACGGAATAGT / 0.55 kb / Ebbs et al. 2006
5´GCTTATTCAGACAGACAGAACTGAAC
absence / 5´GATGGTCTTTGCAATGTTG / 0.84 kb
5´GCTTATTCAGACAGACAGAACTGAAC
suvh6-1 / presence / 5´TAGCATCTGAATTTCATAACCAATCTCGATACAC / 0.79 kb
5´CCTGTGCGAAGAACATCACGTG
absence / 5´CTGAAAGAGCCTGAAGACC / 1.02 kb
5´CCTGTGCGAAGAACATCACGTG
cmt3
GABI-
Kat 106B03 / presence
- 5´ / 5´ATAGCGAGAGAAAGAGATGGGCC / 0.4 kb / Rosso et al. 2003
5´TGGGTATCTGGGAATGGCGAAATG
presence
- 3´ / 5´GCTGTACCTGAAACAAAACAAACA / 0.67 kb
5´TGGGTATCTGGGAATGGCGAAATG
absence / 5´ATAGCGAGAGAAAGAGATGGGCC / 0.60 kb
5´GCTGTACCTGAAACAAAACAAACA
suvh2
SALK
079574 / presence / 5’ATAGTCGACATGAGTACATTGTTACCA / Naumann et al. 2005
5’TCTCGCTAGGTCTCTCGGTC / 0.4 kb
absence / 5’TGGTTCACGTAGTGGGCCATCG
5’TCTCGCTAGGTCTCTCGGTC / 0.8 kb
suvh4
SALK
105816 / presence / 5’AGAAATTGAGTGTTTCTCTGAATGG
5’GCATTGTCTAGATCATCCTCGTACT / 0.95 kb
absence / 5’AGAAATTGAGTGTTTCTCTGAATGG
5’TGGTTCACGTAGTGGGCCATCG / 1.2 kb

Line cmt3 GABI-Kat 106B03 was found to contain two T-DNA copies in a “head-to-head” arrangement with left-border (LB)-sides pointing outwards in the CMT3 open reading frame. Thus, PCR with primers specific to positions in the flanking chromosomal DNA at both sides of the T-DNA insertion site will give PCR products in combination to a primer specific to a position at the LB end of the T-DNA construct.

In the case of a single nucleotide polymorphism in an EMS-generated mutant allele, a cleaved amplified polymorphic sequence (CAPS) test was employed. This involved amplification of PCR products, which differed between mutant and wild-type allele in fissility by a restriction enzyme (RE) (Table C).

Table C Primers for CAPS analysis specific for EMS mutant alleles

allele / primer sequences / RE / product(s) / reference
suvh4 R302* / 5´TCAGGATTTACAGTGTATAAGTACCGACTGAAT / none / 144 b / Ebbs et al. 2006
5´TGGAACTTTGGTATATGGAACG / ClaI / 144 b
SUVH4 / 5´TCAGGATTTACAGTGTATAAGTACCGACTGAAT / none / 144 b
5´TGGAACTTTGGTATATGGAACG / ClaI / 111 b + 33 b

Chip-quantitative PCR

Table D Primers for ChIP-quantitative PCR

target / primer sequences / product / reference
lacO / 5´AATTGTGAGCGTATAACA / 0.35 kb / Kato and Lam 2001
5´GTTATCCGCTCACAATTC
Ta3 / 5’TAGGGTTCTTAGTTGATCTTGTATTGAGCTC / 0.2 kb / Johnson, et al. 2002
5’TTTGCTCTCAAACTCTCAATTGAAGTTT
180 bp / 5’GTTATCTGTTCCTAAAAGATAATAGTGTTC / 0.65 kb / Mathieu et al. 2005
5’ACTCCCACTCATGTATTTCCTATCATAGCG
PFK / 5’ccatgtgcatctctctcaagc / 0.55 kb / Banai Moghaddam et al. 2011
5’gtcttagccgaaggaaacgtc

The PCR were set up in the following way:

10µl DNA preparation

1.25µl Primer 1 (5pmol)

1.25 µl Primer 2 (5pmol)

12.5µl super-mix

PCR was performed withan iCycler IQ thermal cycler system (BioRad) using the program:

1. 10 min 95°C

2. 15 sec 95°C

3. 30 sec 62°C

4. 30 sec 72°C

5. goto 2. 40 times

6. 10 sec 65°C (Start)

7. +0.5°C

8. goto 6 60 times

9. 4°C endless

All data were imported in MS Excel and calculations were performed according to the comparative Ct method (Pfaffl 2001;Bustin et al. 2009).

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