500bp
Figure S1 – RAPD-PCR amplification profiles of genomic DNA of: (O) cv. Onward and
(OR) rogue paramutant line JI2723, with primers OPAL01 to OPAL10. (M) - 100 bp
ladder marker. Notice the identical molecular patterns exhibited by the two epigenotypes.
Figure S2 – SSR amplification profiles (markers:
AD146, AB146, AC58) of: (O) cv. Onward; (OR)
rogue paramutant line JI2723; (F) cv. Frilene; (S)
cv. Solara; (P) cv. Progreta and (PM) Progreta
mutant. (M) - 100 bp ladder marker. Notice the
identical molecular patterns exhibited by the two
epigenotypes
Table S1. MS-AFLP adapters
5’-CTCGTAGACTGCGTACC-3’
3’-CATCTGACGCATGGTTAA-5’
5’-GATCATGAGTCCTGCT
3’-AGTACTCAGGACGAGC-5’
EcoRI- Adapter
HpaII/MspI-Adapter
Table S2. MS-AFLP primers
Name
EcoRI + 0
E- AAC
E-AAG
E-ACA
E-ACG
E-ACT
E-AGC
E-AGG
E-TA
Sequence 5`-3`
Name
Pre-amplification primers
GACTGCGTACCAATTC
HpaII/MspI + 0
Selective primers
GACTGCGTACCAATTCAAC
HM-AA
GACTGCGTACCA ATTCAAG
HM-AC
GACTGCGTACCAATTCACA
HM-AG
GACTGCGTACCAATTCACG
HM-AT
GACTGCGTACCAATTCACT
HM-CA
GACTGCGTACCAATTCAGC
HM-CC
GACTGCGTACCAATTCAGG
HM-CG
GACTGCGTACCAATTCATA
HM-CT
Sequence 5`-3`
ATCATGAGTCCTGCTCGG
ATCATGAGTCCTGCTCGGAA
ATCATGAGTCCTGCTCGGAC
ATCATGAGTCCTGCTCGGAG
ATCATGAGTCCTGCTCGGAT
ATCATGAGTCCTGCTCGGCA
ATCATGAGTCCTGCTCGGCC
ATCATGAGTCCTGCTCGGCG
ATCATGAGTCCTGCTCGGCT
Table S3. Tested MS-AFLP primer combinations.
HM-AA
HM-AC
HM-AG
HM-AT
HM-CA
HM-CC
HM-CG
HM-CT
E-AAC
√
√
√
√
√
√
√
√
E-AAG
√
√
√
√
√
√
√
√
E-ACA
√
√
√
√
√
√
√
√
E-ACG
√
√
√
√
√
√
√
√
E-ACT
√
√
√
√
√
√
√
√
E-AGC
√
√
√
√
√
√
√
√
E-AGG
√
√
√
√
√
√
√
√
E-TA
√
√
√
√
√
√
√
√
TABLE S4. Genomic sequences differentially methylated in cv. Onward and paramutant line
JI2723.
AAC/AA_174_R
GAATTCAACCGTGACTGGTTAAGTCTCACATTTGCTAGAAATGAAAGAAATGTTGGATATATAAGGGA
AAGAACTCATACACATTACGCACGTTGCCTTAAGGTTTTGGGTCAGACAGATATGTAGTGTCAAAGTC
TCTTGGTTCTTAAATGTTTAGTCTATTAAAATTTCCGG
AAC/AA_300_O
GAATTCAACGGTAGGAAAATTGCATGGAGAGTGAAGTATAGCGAGTGGTAGTAACACTGAGGCAAAAT
TGTTGGCGGAGATCGGGGCGAGTGTAGAAGACAGATCCAAGTTGGCAATTTTATAGTCAATTTTGGAT
ATTTTGGAGATATTTTTGGTCTATCTATCTCTCTCACGCTTTTCTCGTATCATCGTCGATTTTGATTA
GGTTAAGATTTCAGAGAGAGGGATGGGAAGAGAAGACAACGGAAAGTGGAAACAGGGATGGGGTTTAC
TACCCGACAAGACACTTTACTGTTCCGG
AAG/AA_197_R
GAATTCAAGTGAGTGGTTAGTCCCACATCGACTAAAATGAAGAAAATGTTGGATATACAATAGAGGTA
ACCCATATATCTAATGCTTTAAGGTTTTTGGTGAAAATATGGTGTAAAGGTCTCTTATAAAAAAAGTA
TTCCACCTTCCCTTTTGCCCCATGCTATTATTCTTAGTTATAGTTATGATGTCTTTTCCGG
AAG/AA_325_R
GAATTCAAGGTGGGAGCTCAACCAGGAAAGGAAAGGAAGCGAGAATTTGTAGACAACCCAAATGGTCT
ATTCTCCGCACAAGCATCACCAAAACCTCCACCAGCCATGTACACAATAGTTGAAGGAATGAGAGTCG
GAGGGAAGGTGATTGAAATACCCCCTCTTGATTTTGTTCTAGTGTGTATTATCTGATGTTTTTATTAG
CTATGATTCACGTGTCACTTCTTTTCTTATTCTTATTGTTCTCTTAAATGATGGTTGCAGCGAACTGT
GATTGTTCCTCCTGAAAAAGGGTATGCACAAAAGGGAATGAATGAGATTCCGG
AAG/AC_613_R
GAATTCAAGAGACTCGTCTATTGTCAAATTATTGCCACCTATGACGAGTGATTCAAGGTCTCTTAAAT
TTCCAAATGAGTTGGGTATAACTCCGCTAAATTCATTCGAACTCAAGTCAATTATGACGAGATTTGAG
GCATTGGATATGCTATTTGGAATATTTCCAACAAACCTATTTCCATACATGTGTAGTTGTTGTAGGTT
TGGAAGGCCTAATCCCATATTTGATGGAAGCATACCCGAGAGAGAATTTAGTTCAAGATGAAGATTTT
TCAATCTTGAAATGTTGAAGACATTTGAAGGAACATATCCACTCAAACTGTTATTTCCCAATTGAAGA
AACTGAAGTTGATTGAGATTGCCAATCTCCATAGGTATCAAACCTAAAATCACATATCATGAGATGAA
GCATGAGAGTAAAATGATGCATGAAATGCTTAACCAAAAGATGAAGATAGAAATTTGAGGATTTACCT
GAAAAAAAGTTATCCTGTAATGTTAATATTTGCAACAATGTGCAATTGCCAATCGATCGTGGAATGGT
TCCTTCCAAACGATTACCGAATAGAGTAAATTTTTCAAGTTGAGGAAGTTGAAGACACATCTCGTCCG
G
AAG/AG_366_O
GAATTCAAGGCATGAGTCCAAAGGTAATCAAATATCCCTTTTAAACTCGTAGCATCATCAATCCAAGC
ATTTCCTATCTGAATTTTTTTTAAATACAAGATAAGAAAGTTAGACATATATGATTGTGAGTCATATT
TCATCATCAGTGTTTGAAGTTTGAACAACATACGGAAATTCCTTTGAGGTTAATAATGATTTGGCTAT
AGAGTTTATTGTTGCGAAGAATAGTGGATGCAAGTTGAGGAACATAATGACCAGCATAACTCTCTCCA
GTCATGTAAAAATCTCTAGTTTTGTACTGTGGAAACCTCTCCAGCCAGTTGATGAGGAAGGCATAAGC
ATCCCTCGCAGTTGTCTTATCTCCGG
ACA/AA_749_R
GAATTCACACTAAGCATTCTTAGGACAGAGATTCGCACAACAAAATTCACAAAATTGTTTCACATTAT
ATTGTTCAATACTAATTTATTAAAGGTGTTCAGCATCAAAATATCAAGTAATATTGACACCTAAAATA
GTTCCTGACCTCCTGCTCAAGTTTAAACAGATGCCGATTAAAATGTTGTTGAAGCCTTTCATTGGCAT
AATTTATACAAAACTGTTCAAAGTTGTTCTTCTGAAAAAGAGAAGCAAGAAACTGTTAGAACATATAC
CATATTCTTTGAAGCATGAATATATCAATACAAATCGCCTACCTGGAATGACTGAAACCCATAAAAAT
CAAGGATACTAATGGATTTCTCTGTATGTTTTTCATTCACTTCAAGTGACTTGTCAAGTTGTTGTACA
AGCCATTCAAACAAGCTTGAATAAATAAATTTGGCAATTGCATCTCTTGTTCCAATGGCCTGGCATAG
AATGTGAAAAAGATAGATAAATAAATAAACAGCACACATATACACATATTGAATAAAGAGCATTTATA
ACCACGGAAAATACAAGAATGAAATGTTCGAATAATGAGCAGACCTGCAATAATGTCAAACTTTTGGA
AACAGTATCTTTGTCAGATTGGACTTTATGGGTAGATAATGCTGCCATTAAGTCCTGGGAACTGCAAC
CCATCAGCAGGCAGCACTGGTTACAGCTGCGAAAAAAAAAAATCACGTATTCTTCAAAATCATTTCCG
G
ACA/AG_560_O
GAATTCACAATCGAACCTTCAATCAGAAGTAATTCTTGGAGATGTCGTTGCAATCCACAACATATAAA
ACTGAACCGTGAAATTCGTTGGAACCAATCACCGAACCAATCATCCCGAATGAACCCCTCTTCTGTGG
ACTTCTCGCACCTCCTCTCGACCGCCTTGACTCCATAACAAAACTACTTTCTACCAATGACACAAAGC
CAACAACTATCAAACCATACACACGCCAGCAACAATATAGCAGGAACACCGCGATTGCATGAAAGATG
CACCAAGCTGCAAAACACAAAACGAAGCGCATCAACGGCGTATCAAATAAGATAAGCGCGAGCTCAAA
ACGAAAGCATGTACGAGCTAACCGAAATGCAGCTACCAGAAGGCATAAAAGGTAGTGAAACCGCACAA
GCTGCCCCATCTTTTCCTGCAGCAAACGCAACCATTGCACTAACTGGGCTCAACTCCAACCGGCAGAA
TCGCTTGCTCATTTAACTTCCTCCCTCGCATCTAACTGAACCTGCAAAAGAAGCTTAATATAATTCAA
TAATATGAATCTCCGG
ACA/AG_735_R
GAATTCACACATGCATCTATGTAGTCCGTCCACGTTCTCACACACGCATTGTTTTCCTCTCTCTAAAA
CTTGGTAATCCATGAAAAGCACAGAATTGACATTTGAGCTACAAAAAGTGAAATGATGAATTGAATTA
ATTGAAGGCGGTGGGATCCACGTTTGTGACATTCCACACATGGATAGAAGTTGTATCACAGTTAGATT
AAGCTGGAAATGTTTGGTAGCTAGTAACAACTCTCATCATCATACACAACCTTGCGTTACTTAGAATC
TAAATCTAAACTCTCTCTCCCTATTTAACCTCTCTCCTCCAACCAAACTCTTAACTCTCTCTTCACCT
CAATCTCTTCCGTTCTGAGATTGAGATATACATTGTATTGTATTATATAGAGGTATAAACAAATCAAC
ACAAGAATATGGGCACAAATATAGTTTTGGTGGTTGCTTTGATCTGCACTGTGTTTGCATTTGTAGGA
GCACAACAACCTCCAAGTCCAAACACCTCTCCAGCACCACCTACTCCTCCTGCTAATACTCCACCAAC
CACTCCTCAAGCTTCTCCTCCACCTGTTCAGTCTTCTCCTCCACCTGTCCAGTCTTCTTCTCCTCCAC
CTGCTCAATCTTCTCCACCTCCAGTTCAATCTTCTCCTCCACCTGCTCAATCTCCTCCACCAGTTCAA
TCTTCTCCTCCACCTGTATCCACTCCACCACCTGTTCCGGCTTCCCCTCCTCCGG
ACA/CT_546_O
GAATTCACATTTATTGGACCATATCCTAGTTAGTTAACGAAATCTTCACGTTTTCCCTCAATAACACA
CCACTCTTGTGTTGTTTTATAGCCAGTGGCAAATGCCAAATCATAACGCCTTTGGAAGGATAAGCTTT
GTTAGTATGACAGACATTTTGTTTTTTTTCCCCAATATAAGAAAGGGATTGAACCTACATAACATCAA
AGAGTAAACAACTTCTCTTTATCCTTAAAGAGTATTTTGATTTTTTTATATCAGTATTTTACTCGATA
GTTTAACCCTGAAATGTTTGTATGTTCTATTATTCATTTTGATCAATAAAATTACCAACAAAATACAC
AATTGAAGTTCTTTTAGTAATTTTAATATATTAAAAAAAATGTACAATTGTCTCGCCCATTTAAGATC
AAATCAAAATGTTATTTTAAGATAAGTTTTAAAATTGTGTATAGAACAATTGCGATACAATTATTACT
ACCATAACAATTTTTTCTTCCCAAATTTTAAAACCAGTGGTTAGAAAAATATAAGTTTTGTTGAAGCC
GG
ACG/CC_81_O
GAATTCACGGAACAAAAATAGAACTCGTCCATCGGATAAGGTGTTTGACTGGTTTGATACCTCATGTG
CGAAGAAGGCCGG
ACT/AC_451_R
GAATTCACTGTGGTTAAAGTAAAATCTCACAAGGTAGAAATGAAAATGAGAAATGGTCAGTAGTGAAC
AGCCTCGTTAAGTAAACCCTTTAAAAAGCTTTTGAAATTTGGAGTCATGCTATCAGGATATTTAACCG
GATCCTGGATTATTCAATTAGTGAATAGTCAATCCTTCAGTAAAATAACATAAGTGATTGTATAACAA
ATCTTGTCCGATTGAAAATGTACGTTAACAATGCGCCTGACGAGAGTATAAACATAATTTGTGTAAAA
TGGTGGTTGGCCTACAAATAACTCGTACCTGTTATAGGCATAAAAAAGCAGGCTTCAATTCAATGTGA
ATAACAAAGAACATTGAGAGTGTGAAAGCCATGTTTAAACAACACTTGCTACATAACTAATAGTTAGA
TAACGTCATCAATCAGAACACTTGCAAATTGCAACAGGTCCGG
ACT/AG_449_O
GAATTCACTAAACAGAAACAGAAAGAGAAGTAGATGAGAGAATTTCTAGGATAAATGAGGGAGTAAAT
GAAACTCACAAATTTTTGGGAGGAGAGAACTTCACTCTTCATCATTCATCATCGTGAATCCTTCAACA
AACAATTCGTGAAGAAAACGGATTCATCGCTTCAATCACGAATCTCATCACACTTCCAACACTTCACT
CTTCATCATCAACGGAAAATCAAAGAATCAACAGCAACGACGCAAACGCAACCCAATTCAGAATCGGA
CAACAACAACACATAGCACGCGCACAAACCTGAAGGCTTCATCTTCGTCTCAACGGAACGCACGGATC
CAAACGAAGTTGCATCTTCATCCTAATCAACCACGAGTGAAAGAGTACATCGAACGTTGAGGTGAGTT
TTCATTGAGTCGTGGATGCCATCGAGTGAATTTTTCTCCGG
ACT/CA_584_R
GAATTCACTGCAGTGATCAAAATAGATTTGAAATATTCACCCGAGAAATTCATGGTCTCGGAACCATT
GTCGAAACTTTTAGGGATTGAGGTCGAGAGTCGCCCGAGAATAATAGCTGCTCTTTGGCATTATGTGA
AGTCCAGGAGGCTACAATGTCCGGACGAGCCTTCCTTCTTCATCTGTGATCCTTCCCTCCAAAGGATT
TTCGGGGCAGAGAAGATGGGATTCCCCCTGGCTGCTCAGAAGTTGTTAGACCATTTGTCGCAACCAAA
GCCTATATATTTGGAGCATAACATCAAGCTTTCTGGAGACTGTCCTGCTGGAACTGCATGTTACGACG
TACAGGTTGACGTGCCTATACCACTGCAGAAAGAAATGTCTGGTTTTTTGGCTAGCAACAGCGTTGAT
AGTCACAAAGAGATTGAAGCTTCTGATGAGATTATTTCCTGTAACCTAAAGAAAATCCAAGAGCATCG
TAGAAGACGTGCTTTCTTTCTTAGCTTTAGTCAGTCTCCATCAGAGTTTATCAACACTATGATTGCTT
CTCGGAGCAAGGATCCAAAGCCTGCTGCTGGAGATGCCGG
AGC/AA_202_R
GAATTCAGCCTCTCAACAGCGATCGATGTCGGGTTCTTCTGCCGTGTTCTCGGGCTTTGTACACTCAA
GCTTTCACTGACACTACTATTACTCCTATTGTCATATGGAGAATTTGAAGCCTGATCACTTGTAATAT
CTATTGATTCTTTGTATCTGTGCATTGCTTCAAGAATCTGTTTAAGGGCTCTAAGATCCTTTCCGG
AGC/AT_134_O
GAATTCAGCATTAAAAGATCAGTCTTCTCACTAGCTTTAGCTTGAAAAACAAATTTATGCAATTGCAA
TGCCAAGTACAAATCGCCAACACTTCCACAAGCCGATGTAACGCTAACCATGGTAATCCCATCCGG
AGC/AT_466_O
GAATTCAGCAACTATGAGTCGAATGACCTCCAGAATGTTATGAAAGTTATGCAGTACACATTTATGGG
ATTCTTTATCTGATGCCCATGATTATGCACGTGTGTAAGCCAATACTACAACTGCCGAAGAGTGAACC
AAGAACTTGAACATGGCATTAGACACGCTGTTGTATGTGGTGACTTCGGCCAAAATCCTAATTCAATG
TTATATTAATACTGGTCCAAAACTTAACTCAAACTACACCGCTGCCTGTTGTAACTGCGATAACAATG
CCTTTACGGAAAAAGTTGCCTAGGTTTTTGGTTTTGTTAAGAAAGTAAAAACATCTGATAGAGTGAAA
GAAGAAGTTGAAATTTTCATCGCAGCCTATCCATATTTCAATGTCAATGAAATGCTTGTTGAGAAGTT
GTCTTGAATACTGAAAACAGATTATTTTTAATGGAGAACTCGACCGAATCAGATCCGG
AGG/AC_384_R
GAATTCAGGGTCAACAAGCAGGTGGTGAGTGACTTAAGATGAAGTTATGGGTTGCCATTTATAATCGT
CATTTTCGGGTCATGAATGGAGAAGTTTCATTTGGACTGAGTTTAAGTGTTTATTTTTCGCATTGTGT
GACTATAAGAAATAGACAGGTCTTTGTCTGCGGATTTCATGCATAACGTCATCTGGTGTGGGTATTAG
CTTTGATTTATGATTTTTGAACCACAAGTTTCAATTTACAAACAAAGTTGTTTCAAATACATTAATAA
AACCATACCACAAGACCGTTCATGACAACCCTGTGACTTAAGATACATTACAGTCAAGCCAAATTAAA
GAAAGAGATTTCCAAAAATCTAACAGGCAACATGCCATGTCCGG
AGG/AG_705_R
GAATTCAGGGAAAGGTTTTCAATTTAGTTTTTTAATGATAAACTGATCTATGAGACTCTTGCTTGATT
TTTTTTTACCCCGTGTCAAACTGGCTCTTGTTGAGATTGAGAATGAGATGACTCGGTGGCCTACTCGT
GTATCTCTGTTTCTTACTTTCACTGCAGTTTGTACTGTATGCCGGCTTCTCTAGCTGTTTCTTGCTCC
AGCATGCAATGTAAAAGCAGCAATCTTATGCTTGGCAATGTTACCGTGCAACAGACTAGTGGTGGTTG
TAATGTGACTTCTTGCAGTTGTGATGGTCTTGTTAATGGCACCATAGCGACAATGTATGTTTCTCCTC
TGCATTCACGTCTTTCTCTTTGGGACATTTCAATTATGTATATTTTACTGCACCGTTTCTGAGTTTAT
GCGCGTTGAATGTTTCTTTCCTCCCTGCAGGTTGACACCGTCTCTTCAGCCTCGATGCCCAGGTCAGT
GTGCTGCAGCCATTATAATTTATGTATTTTCTGAGACGCCTTTGAAGTGCTAAATATATTTTAAATGA
TGCTGCAGGGACACAGGAATTTCCTCCTCTTATCGCCCCACCTACAACTGTTGCGAGAGAATCAGTAT
TTGCACCGGCACCGTCTCCATCATCGCTGTTAGATGGAACTGATGGGAGATCACCCAAATCCTCTGTG
GTGCCTTCAACAGGATTTACTCCGG
AGG/AT_139_O
GAATTCAGGTTGAGTTGAAGGTGAGGTTTGGAACTGATTCACTGATCAAATGTAGTTGATGATTGTGA
TGGATGAAGGTGAGGGAATACGTGGGGGTGGAGCAATGACTGATGAAGGTTGAAGATTGATTGATATC
CGG
AGG/AT_302_O
GAATTCAGGTGAGCGGTTAGTCCCACATCGACTAAAATGAAGAAAATGTTGGATATACAATAGAGGTA
ACCCATATATCTAATGCTTTAAGGTTTTTGGTGAAAATATGGTGTAAAGGTCTCTTATAAAAAAAGTA
TTCCACCTTCCCTTTTGCCCCATGCTATTATTCTTAGTTATAGTTATGATGTCTTTTCCGGCCCAAAA
ACTTGAGAATCTTTATACACATCTTTGTTTTGGTTATGCAATCTTATGATAGAAATTTCATTAAGTAA
AACACTTTGGTAAACTTTTGTAGGATCCGG
TA/CA_260_R
GAATTCTATGGTTGTGGGGTTGATTAGGAATGGTGATTTGGAGGAAGCGAGAAAGGTTTTTGATGACA
CCCCTTTTAAAAATGTGGTGTCTTGGAATGCTATGATTGAGAGTTATGTTGAAAATGGTAGAATGGAG
GAAGCAAGAGTTTTGTTTGATCAAATGGGGTTTAGGAATGTGATCACTTGGACTAGCATGATATCTGG
GTATTGTCGTGTGGGTGATGTGGATGAGGCTTGTCGTTTGTTCTGGATTATGCCGG
Table S5 - Primers for confirmation of the differential methylation of the
identified sequences.
Sequences
AAC/AA_174_R
AAC/AA_300_O
AAG/AA_197_R
AAG/AA_325_R
AAG/AC_613_R
AAG/AG_366_O
ACA/AA_749_R
ACA/AG_735_R
ACA/CT_546_O
ACA/GA_560_O
ACG/CC_81_O
ACT/AC_451_R
ACT/AG_449_O
ACT/CA_584_R
AGC/AA_202_R
AGC/AT_134_O
AGC/AT_466_O
AGG/AC_384_R
AGG/AG_705_R
AGG/AT_139_O
AGG/AT_302_O
TA/CA_260_R
Primers (5'- 3')
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
GCGTACCAATTCAACCG
GAGTCCTGCTCGGAAATT
TACCAATTCAACGGTAGGAA
CTCGGAACAGTAAAGTGTC
GCGTACCAATTCAAGTGAG
AGTCCTGCTCGGAAAAG
GCGTACCAATTCAAGGTG
TCCTGCTCGGAATCTCA
TGCGTACCAATTCAAGAGAC
CTGCTCGGACGAGATG
CGTACCAATTCAAGGCATG
GTCCTGCTCGGAGATAAG
GCGTACCAATTCACACTAAG
GTCCTGCTCGGAAATGA
GCGTACCAATTCACACATGC
AGTCCTGCTCGGAGGAG
GACTGCGTACCAATTCACATT
CCTGCTCGGCTTCAAC
CTGCGTACCAATTCACAATC
GTCCTGCTCGGAGATTC
TGCGTACCAATTCACGGAAC
GTCCTGCTCGGCCTTC
TGCGTACCAATTCACTGTGG
TCCTGCTCGGACCTGT
GCGTACCAATTCACTAAACAG
CCTGCTCGGAGAAAAATTC
CTGCGTACCAATTCACTGCA
GTCCTGCTCGGCATCTC
CCAATTCAGCCTCTCAAC
TGCTCGGAAAGGATCTTAG
ACTGCGTACCAATTCAGCATT
TCCTGCTCGGATGGGA
CGTACCAATTCAGCAACT
CCTGCTCGGATCTGATTC
GCGTACCAATTCAGGGTCA
GTCCTGCTCGGACATGG
CGTACCAATTCAGGGAAAG
GTCCTGCTCGGAGTAAA
CGTACCAATTCAGGTTGAG
GTCCTGCTCGGATATCAA
CGTACCAATTCAGGTGAGC
CCTGCTCGGATCCTACA
TGCGTACCAATTCTATGGTT
TCCTGCTCGGCATAATC
Annealing
temperature (ºC)
Expected
Size (bp)
65
189
63
304
61
211
60
337
63
624
65
378
65
762
65
749
67
561
65
575
67
95
67
464
66
460
69
599
64
206
61
149
63
476
68
397
59
717
63
151
65
312
65
273
TABLE S6. Methylation state of the 22 genomic sequences in leaves and pollen of cv. Onward and “rogue” line JI2723.
ONWARD
LEAVES
Sequences /
Number
CpG
CHG
CHG
CpG
CpG
CHG
CHG
CpG
CpG
CHG
CHG
CpG
CpG
Full
Hemi
Yes
No
Full
Hemi
Yes
No
Full
Hemi
Yes
No
Full
Hemi
Yes
No
5
6
8
3
5
2
6
9
L/P*
5
5
3
9
2
4
7
9
L/P
*
X
N
X
Y
X
N
X
X
X
AAG/AA_325_R
X
AAG/AC_613_R
X
X
X
X
X
AAG/AG_366_O
X
X
ACA/AA_749_R
X
X
ACA/AG_560_O
X
X
X
X
X
X
X
AGG/AC_384_R
X
Y
X
X
Y
Y
X
N
X
X
X
Y
X
X
X
Y
Y
X
Y
X
X
X
X
N
N
Y
X
X
Y
Y
X
X
Y
N
X
X
X
N
X
Y
Y
X
X
Y
Y
X
X
Y
Y
X
X
X
Y
X
X
AGC/AT_466_O
Y
N
X
X
AGC/AT_134_O
X
X
X
AGC/AA_202_R
X
Y
X
N
X
X
X
ACT/AG_449_O
X
N
Y
X
X
X
N
Y
X
ACG/CC_81_O
X
Y
X
ACA/CT_546_O
AGG/AG_705_R
POLLEN
CpG
AAG/AA_197_R
ACT/AC_584_R
LEAVES
CHG
AAC/AA_300_O
ACT/AC_451_R
POLLEN
CHG
AAC/AA_174_R**
ACA/AG_735_R
ROGUE
X
Y
X
X
X
Y
Y
AGG/AT_139_O
X
X
N
X
X
Y
AGG/AT_302_O
X
X
N
X
X
Y
X
X
Y
X
X
TA/CA_260_R
Y
* L/P - Maintenance in pollen of the leaf methylation state. Y - Yes; N - No. ** Underlined sequences show altered but identical methylation patterns in the pollen DNA of both
epigenomes.
Table S7. Expression analysis of the 22 differentially methylated sequences.
Blast
Unigenes
Putative
expression
Expression
RT-PCR
Medicago truncatula clone mth2-20l8,
complete sequence
-
No
No
-
Predicted Cicer arietinum
uncharacterized LOC101505144
-
Sequence
AAC/AA_174_R
AAC/AA_300_O
AAG/AA_197_R
AAG/AA_325_R
AAG/AC_613_R
AAG/AG_366_O
ACA/AA_749_R
ACA/AG_560_O
ACA/AG_735_R
ACA/CT_546_O
ACG/CC_81_O
ACT/AC_451_R
ACT/AG_449_O
ACT/CA_584_R
AGC/AA_202_R
Blast NCBI
Yes
Medicago truncatula chromosome 2
clone mth2-4g10,
P. sativum v1
contig976
Yes
Predicted peptidyl-prolyl cis-trans
isomerase FKBP18, [Cicer arietinum]
P.sativum wa1
_contig26417
Yes
Predicted LRR serine/threonine-protein
kinase FLS2-like [Cicer arietinum]
P. sativum wa1
contig28359
Yes
Serine carboxypeptidase II-3 [Medicago
truncatula]
-
Yes
Predicted unconventional myosin-Va-like
[Cicer arietinum]
P. sativum v2_
contig4504
Yes
-
No
Medicago sativa proline-rich protein
mRNA, complete cds
-
Predicted Cicer arietinum serine
/threonine-protein kinase TIO-like
P. sativum v2
contig634
Yes
-
No
-
-
No
Yes
P. sativum wa1
contig29580
Yes
Chromatin remodeling complex subunit
[Medicago truncatula]
-
Yes
Predicted protein LONGIFOLIA 1-like
[Cicer arietinum]
-
-
Yes
Yes
No
Yes
Yes
Yes
Yes
No
Yes
No
No
No
Yes
Yes
Yes
No significant
differences
Primers (5´-3´)
F
TTCAACCGTGACTGGT
R
AACCAAGAGACTTTGACAC
F
GAGTGTAGAAGACAGATCC
R
CGTGAGAGAGATAGATAGAC
141
87
F
CAAGTGAGTGGTTAGTCC
R
CCGGAAAAGACATCATAACT
-
192
No significant
differences
F
GAATTCAAGGTGGGAGC
R
TGTGTACATGGCTGGTG
No significant
differences
F
CAAGAGACTCGTCTATTGTC
R
CGGAGTTATACCCAACTC
114
89
No significant
differences
F
TAGTGGATGCAAGTTGAGG
R
TCATCAACTGGCTGGAG
No significant
differences
F
GCTCAAGTTTAAACAGATGC
R
AGAAGAACAACTTTGAACAG
F
GCAACCATTGCACTAAC
R
GGTTCAGTTAGATGCGA
102
No significant
differences
F
GATCTGCACTGTGTTTGC
R
AGTGGTTGGTGGAGTATTAG
F
TGGACCATATCCTAGTTAGT
R
TTATCCTTCCAAAGGCG
-
87
83
100
117
F
GAATTCACGGAACAAAAATAG
R
CCTTCTTCGCACATGAG
F
TGGAGTCATGCTATCAGG
R
GTACGAGTTATTTGTAGGCC
-
77
No significant
differences
Expected
size (bp)
107
F
TTCGTCTCAACGGAACG
R
ACTCACCTCAACGTTCG
No significant
differences
F
TTCACCCGAGAAATTCAT
R
CATAATGCCAAAGAGCAG
No significant
differences
F
GAATTCAGCCTCTCAACA
R
TTACAAGTGATCAGGCTTC
92
98
133
AGC/AT_134_O
AGC/AT_466_O
AGG/AC_384_R
AGG/AG_705_R
AGG/AT_139_O
AGG/AT_302_O
TA/CA_260_R
Predicted pentatricopeptide repeatcontaining protein [Cicer arietinum]
Medicago truncatula hypothetical protein
(MTR_5g027220) mRNA, complete cds
-
-
Yes
P. sativum wa1
contig22324
Yes
-
No
LysM domain-containing GPI-anchored
protein [Medicago truncatula]
P. sativum wa1
contig21148
Yes
-
P. sativum wa1
contig22939
Yes
Medicago truncatula chromosome 5
clone mth2-57m18,
P. sativum v1
Contig976
Yes
Pentatricopeptide repeat-containing
protein [Medicago truncatula]
-
Yes
Yes
Yes
No
Yes
Yes
No
Yes
No significant
differences
F
GAATTCAGCATTAAAAGATCAGTC
R
CATGGTTAGCGTTACATCG
No significant
differences
F
TTCATCGCAGCCTATCC
R
GGATCTGATTCGGTCGAG
F
TCAGGGTCAACAAGCAG
R
GTTGTCATGAACGGTCTTG
120
109
No significant
differences
No significant
differences
296
F
GTGCAACAGACTAGTGG
R
CTGATTCTCTCGCAACAG
F
GGTTTGGAACTGATTCAC
R
TCAATCTTCAACCTTCATCA
166
F
CGGTTAGTCCCACATCG
R
GATCCTACAAAAGTTTACCAAAGTG
F
GGTTGATTAGGAATGGTGA
R
ACTCTCAATCATAGCATTCC
No significant
differences
104
286
95