Genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs in Arabidopsis
Supplementary Data
Hanna Lee1, Seong Jeong Yoo1, Jeong Hwan Lee1, Wanhui Kim1, Seung Kwan Yoo1, Heather Fitzgerald2,3, James C. Carrington2,3 and Ji Hoon
Ahn1,*
1
Creative Research Initiatives, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea; 2Department of Botany and
Plant Pathology, and 3Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR 97331, USA
*To whom correspondence should be addressed. Tel: +82-2-3290-3451; Fax: +82-2-927-9028; Email: [email protected]
The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint first authors.
1
Supplementary Results
Specificities of probes used in miRNA Northern hybridization analysis
As miRNAs are only 20–24 nucleotides long and often closely related, it is a prerequisite to test the degree of cross-hybridization of the probes
before performing miRNA Northern hybridization analysis. To test the probe specificity, we chose miR156 and miR157, two closely related
miRNAs, and performed Northern hybridization. miR156 and miR157 have eight (miR156a–h) and four (miR157a–d) loci, respectively. Six
paralogous loci of miR156 (a–f) give rise to an identical mature form (Supplementary Figure S2A); therefore, the mature sequences of
miR156a–f were indistinguishable. In contrast, miR156g and miR156h produce unique mature sequences. Three loci (a–c) of miR157 give the
same mature sequences, and miR157d produces a unique mature sequence. To test the cross-reactivity of the miR156/157 probes, an equal
amount (20 fmol) of synthesized RNA corresponding to miR156a, miR156g, miR156h, miR157a, and miR157d was loaded on 17%
polyacrylamide gel and hybridized with each DNA probe.
The Northern hybridization revealed that the miR156a probe strongly hybridized to itself and to miR156g with similar intensity, revealing
cross-hybridization (Supplementary Figure S2B). In contrast, the miR156a probe did not hybridize to miR156h, miR157a, and miR157d,
indicating that it retained selective hybridization properties. Our miR156g probe strongly hybridized to itself and to miR156a, but did not
hybridize to miR156h, miR157a, and miR157d, reminiscent of the miR156a probe (Supplementary Figure S2B). The miR156h probe strongly
hybridized to itself and weakly to miR157a, and very weak cross-hybridization to other loci was observed. Our miR157a probe strongly
hybridized to itself and to miR156h and miR157d with weaker intensity. The miR157d probe strongly hybridized with miR157a, and slightly
weakly to itself and miR156h.
To investigate the correlation between the selective hybridization properties and the sequence similarity, we predicted the hybridized
structures and their melting temperature (Tm) values (Supplementary Figure 2C and D). It was apparent that both the Tm values of the
hybridized sequences and the number of mismatches within the hybridized sequences were important in determining the probe specificity. DNA
probes did not hybridize to miRNAs with two mismatches (e.g., miR156a:miR156h and miR157d:miR156a). However, in the case of hybridized
sequences with one mismatch, it seemed that the hybridization strength depended upon their Tm values. For instance, although both
miR156h:miR157a and miR156h:miR156g had one mismatch, the hybridization signal of miR156h:miR156g (Tm = 61.2°C) was weaker than
that of miR156h:miR157a (Tm = 67.0°C). An exceptional observation was that the hybridization signal of miR157d:miR157a was stronger than
that of miR157d:miR157d. The stronger hybridization signal of miR157d:miR157a can be partly explained by its higher Tm value (70.8°C vs.
69.6°C). In addition, the Tm values between opposite configurations (e.g., miR156g:miR156a and miR56a:miR156g) were different (71.5°C vs.
72.6°C); the discrepancy may be due to the different DNA probe in the pairs (miR156g[DNA]:miR156a[RNA] and
miR156a[DNA]:miR156g[RNA]). Together, this analysis suggested that the other probes used in the miRNA Northern hybridization analysis
2
weakly cross-hybridize to other sequences with one mismatch. However, as it was also likely that the probes did not cross-hybridize to miRNA
loci with a sequence difference of more than one nucleotide, the probes that we used in the miRNA Northern hybridization could efficiently
discriminate between closely related miRNA members.
3
Supplementary Table S1. The porbes used in the imRNA microarray and northern hybridization analyses.
Probe used for
miRNA
microarray
Paralogous
loci
ath-miR156a
b,c,d,e,f
ath-miR156g
ath-miR156h
ath-miR157a
b,c
Sequence
Probe sequence (probe)
Probe used
for miRNA
northern
Paralogous
loci
b,c,d,e,f
5'-UGACAGAAGAGAGUGAGCAC-3'
5'-GTGCTCACTCTCTTCTGTCA-3'
1
miR156a
5'-CGACAGAAGAGAGUGAGCACA-3'
5'-TGTGCTCACTCTCTTCTGTCG-3'
2
miR156g
5'-UUGACAGAAGAAAGAGAGCAC-3'
5'-GTGCTCTCTTTCTTCTGTCAA-3'
3
miR156h
5'-UUGACAGAAGAUAGAGAGCAC-3'
5'-GTGCTCTCTATCTTCTGTCAA-3'
4
miR157a
b,c
Sequence
Probe sequence (probe)
5'-TGACAGAAGAGAGTGAGCAC-3'
5'-GTGCTCACTCTCTTCTGTCA-3'
5'-CGACAGAAGAGAGTGAGCACA-3'
5'-TGTGCTCACTCTCTTCTGTCG-3'
5'-TTGACAGAAGAAAGAGAGCAC-3'
5'-GTGCTCTCTTTCTTCTGTCAA-3'
5'-TTGACAGAAGATAGAGAGCAC-3'
5'-GTGCTCTCTATCTTCTGTCAA-3'
ath-miR157d
5'-UGACAGAAGAUAGAGAGCAC-3'
5'-GTGCTCTCTATCTTCTGTCA-3'
5
miR157d
5'-TGACAGAAGATAGAGAGCAC-3'
5'-GTGCTCTCTATCTTCTGTCA-3'
ath-miR158a
5'-UCCCAAAUGUAGACAAAGCA-3'
5'-TGCTTTGTCTACATTTGGGA-3'
6
miR158a
5'-TCCCAAATGTAGACAAAGCA-3'
5'-TGCTTTGTCTACATTTGGGA-3'
ath-miR158b
5'-CCCCAAAUGUAGACAAAGCA-3'
5'-TGCTTTGTCTACATTTGGGG-3'
7
miR158b
5'-CCCCAAATGTAGACAAAGCA-3'
5'-TGCTTTGTCTACATTTGGGG-3'
ath-miR159a
5'-UUUGGAUUGAAGGGAGCUCUA-3'
5'-TAGAGCTCCCTTCAATCCAAA-3'
8
miR159a
5'-TTTGGATTGAAGGGAGCTCTA-3'
5'-TAGAGCTCCCTTCAATCCAAA-3'
ath-miR159b
5'-UUUGGAUUGAAGGGAGCUCUU-3'
5'-AAGAGCTCCCTTCAATCCAAA-3'
9
miR159b
5'-TTTGGATTGAAGGGAGCTCTT-3'
5'-AAGAGCTCCCTTCAATCCAAA-3'
ath-miR159c
5'-UUUGGAUUGAAGGGAGCUCCU-3'
5'-AGGAGCTCCCTTCAATCCAAA-3'
10
miR159c
5'-TTTGGATTGAAGGGAGCTCCT-3'
5'-AGGAGCTCCCTTCAATCCAAA-3'
5'-UGCCUGGCUCCCUGUAUGCCA-3'
5'-TGGCATACAGGGAGCCAGGCA-3'
11
miR160a
5'-TGCCTGGCTCCCTGTATGCCA-3'
5'-TGGCATACAGGGAGCCAGGCA-3'
5'-UUGAAAGUGACUACAUCGGGG-3'
5'-CCCCGATGTAGTCACTTTCAA-3'
12
miR161a.1
5'-TTGAAAGTGACTACATCGGGG-3'
5'-CCCCGATGTAGTCACTTTCAA-3'
13
miR161a.2
5'-TCAATGCATTGAAAGTGACTA-3'
5'-TAGTCACTTTCAATGCATTGA-3'
ath-miR160a
b,c
ath-miR161a
b,c
ath-miR162a
5'-UCGAUAAACCUCUGCAUCCAG-3'
5'-CTGGATGCAGAGGTTTATCGA-3'
14
miR162a
5'-TCGATAAACCTCTGCATCCAG-3'
5'-CTGGATGCAGAGGTTTATCGA-3'
ath-miR163a
5'-UUGAAGAGGACUUGGAACUUCGAU-3'
5'-ATCGAAGTTCCAAGTCCTCTTCAA-3'
15
miR163a
5'-TTGAAGAGGACTTGGAACTTCGAT-3'
5'-ATCGAAGTTCCAAGTCCTCTTCAA-3'
ath-miR164a
5'-UGGAGAAGCAGGGCACGUGCA-3'
5'-TGCACGTGCCCTGCTTCTCCA-3'
16
miR164a
5'-TGGAGAAGCAGGGCACGTGCA-3'
5'-TGCACGTGCCCTGCTTCTCCA-3'
ath-miR164c
5'-UGGAGAAGCAGGGCACGUGCG-3'
5'-CGCACGTGCCCTGCTTCTCCA-3'
17
miR164c
5'-TGGAGAAGCAGGGCACGTGCG-3'
5'-CGCACGTGCCCTGCTTCTCCA-3'
ath-miR165a
b
5'-UCGGACCAGGCUUCAUCCCCC-3'
5'-GGGGGATGAAGCCTGGTCCGA-3'
18
miR165a
b
5'-TCGGACCAGGCTTCATCCCCC-3'
5'-GGGGGATGAAGCCTGGTCCGA-3'
ath-miR166a
c,d,e,f,g
5'-UCGGACCAGGCUUCAUUCCCC-3'
5'-GGGGAATGAAGCCTGGTCCGA-3'
19
miR166a
c,d,e,f,g
5'-TCGGACCAGGCTTCATTCCCC-3'
5'-GGGGAATGAAGCCTGGTCCGA-3'
ath-miR167a
b
5'-UGAAGCUGCCAGCAUGAUCUA-3'
5'-TAGATCATGCTGGCAGCTTCA-3'
20
miR167a
b
5'-TGAAGCTGCCAGCATGATCTA-3'
5'-TAGATCATGCTGGCAGCTTCA-3'
5'-UUAAGCUGCCAGCAUGAUCUU-3'
5'-AAGATCATGCTGGCAGCTTAA-3'
21
miR167c
5'-TTAAGCTGCCAGCATGATCTT-3'
5'-AAGATCATGCTGGCAGCTTAA-3'
5'-UGAAGCUGCCAGCAUGAUCUGG-3'
5'-CCAGATCATGCTGGCAGCTTCA-3'
22
miR167d
5'-TGAAGCTGCCAGCATGATCTGG-3'
5'-CCAGATCATGCTGGCAGCTTCA-3'
5'-UCGCUUGGUGCAGGUCGGGAA-3'
5'-TTCCCGACCTGCACCAAGCGA-3'
23
miR168a
5'-TCGCTTGGTGCAGGTCGGGAA-3'
5'-TTCCCGACCTGCACCAAGCGA-3'
5'-CAGCCAAGGAUGACUUGCCGA-3'
5'-TCGGCAAGTCATCCTTGGCTG-3'
24
miR169a
5'-CCGGCAAGTCATCCTTGGCTG-3'
25
miR169b
5'-CGGCAAGTCATCCTTGGCTCA-3'
26
ath-miR167c
ath-miR167d
ath-miR168a
b
ath-miR169a
ath-miR169b
c
5'-CAGCCAAGGAUGACUUGCCGG-3'
ath-miR169d
e,f,g
5'-CAGCCAAGGAUGACUUGCCGG-3'
ath|miR169g*
ath-miR169h
b
5'-CAGCCAAGGATGACTTGCCGA-3'
5'-TCGGCAAGTCATCCTTGGCTG-3'
c
5'-CAGCCAAGGATGACTTGCCGG-3'
5'-CCGGCAAGTCATCCTTGGCTG-3'
miR169d
e,f,g
5'-TGAGCCAAGGATGACTTGCCG-3'
5'-CGGCAAGTCATCCTTGGCTCA-3'
i,j,k,l,m,n
5'-AGCCAAGGTCAACTTGCCGGA-3'
5'-UAGCCAAGGAUGACUUGCCUG-3'
5'-CAGGCAAGTCATCCTTGGCTA-3'
27
miR169h
5'-TAGCCAAGGATGACTTGCCTG-3'
5'-CAGGCAAGTCATCCTTGGCTA-3'
ath-miR170a
5'-UGAUUGAGCCGUGUCAAUAUC-3'
5'-GATATTGACACGGCTCAATCA-3'
28
miR170a
5'-TGATTGAGCCGTGTCAATATC-3'
5'-GATATTGACACGGCTCAATCA-3'
ath-miR171a
5'-UGAUUGAGCCGCGCCAAUAUC-3'
5'-GATATTGGCGCGGCTCAATCA-3'
29
miR171a
5'-TGATTGAGCCGCGCCAATATC-3'
5'-GATATTGGCGCGGCTCAATCA-3'
5'-UUGAGCCGUGCCAAUAUCACG-3'
5'-CGTGATATTGGCACGGCTCAA-3'
30
miR171b
5'-TTGAGCCGTGCCAATATCACG-3'
5'-CGTGATATTGGCACGGCTCAA-3'
ath-miR172a
5'-AGAAUCUUGAUGAUGCUGCAU-3'
5'-ATGCAGCATCATCAAGATTCT-3'
31
miR172a
5'-AGAATCTTGATGATGCTGCAT-3'
5'-ATGCAGCATCATCAAGATTCT-3'
ath-miR172b*
5'-GCAGCACCAUUAAGAUUCAC-3'
5'-GTGAATCTTAATGGTGCTGC-3'
32
miR172b
5'-GCAGCACCATTAAGATTCAC-3'
5'-GTGAATCTTAATGGTGCTGC-3'
ath-miR171b
i,j,k,l,m,n
c
4
c
ath-miR172c
d
ath-miR172e
ath-miR173a
ath-miR319a
b
ath-miR319c
ath-miR390a
b
ath-miR391a
ath-miR393a
b
5'-AGAAUCUUGAUGAUGCUGCAG-3'
5'-CTGCAGCATCATCAAGATTCT-3'
33
miR172c
5'-GGAAUCUUGAUGAUGCUGCAU-3'
5'-ATGCAGCATCATCAAGATTCC-3'
34
miR172e
5'-UUCGCUUGCAGAGAGAAAUCAC-3'
5'-GTGATTTCTCTCTGCAAGCGAA-3'
35
miR173a
5'-UUGGACUGAAGGGAGCUCCC-3'
5'-GGGAGCTCCCTTCAGTCCAA-3'
36
miR319a
5'-UUGGACUGAAGGGAGCUCCU-3'
5'-AGGAGCTCCCTTCAGTCCAA-3'
37
miR319c
5'-AAGCUCAGGAGGGAUAGCGCC-3'
5'-GGCGCTATCCCTCCTGAGCTT-3'
38
miR390a
5'-UUCGCAGGAGAGAUAGCGCCA-3'
5'-TGGCGCTATCTCTCCTGCGAA-3'
39
miR391a
5'-UCCAAAGGGAUCGCAUUGAUC-3'
5'-GATCAATGCGATCCCTTTGGA-3'
40
miR393a
d
b
b
b
5'-AGAATCTTGATGATGCTGCAG-3'
5'-CTGCAGCATCATCAAGATTCT-3'
5'-GGAATCTTGATGATGCTGCAT-3'
5'-ATGCAGCATCATCAAGATTCC-3'
5'-TTCGCTTGCAGAGAGAAATCAC-3'
5'-GTGATTTCTCTCTGCAAGCGAA-3'
5'-TTGGACTGAAGGGAGCTCCC-3'
5'-GGGAGCTCCCTTCAGTCCAA-3'
5'-TTGGACTGAAGGGAGCTCCT-3'
5'-AGGAGCTCCCTTCAGTCCAA-3'
5'-AAGCTCAGGAGGGATAGCGCC-3'
5'-GGCGCTATCCCTCCTGAGCTT-3'
5'-TTCGCAGGAGAGATAGCGCCA-3'
5'-TGGCGCTATCTCTCCTGCGAA-3'
5'-TCCAAAGGGATCGCATTGATC-3'
5'-GATCAATGCGATCCCTTTGGA-3'
ath-miR394a
b
5'-UUGGCAUUCUGUCCACCUCC-3'
5'-GGAGGTGGACAGAATGCCAA-3'
41
miR394a
b
5'-TTGGCATTCTGTCCACCTCC-3'
5'-GGAGGTGGACAGAATGCCAA-3'
ath-miR395a
d,e
5'-CUGAAGUGUUUGGGGGAACUC-3'
5'-GAGTTCCCCCAAACACTTCAG-3'
42
miR395a
d,e
5'-CTGAAGTGTTTGGGGGAACTC-3'
5'-GAGTTCCCCCAAACACTTCAG-3'
ath-miR395b
c,f
5'-CUGAAGUGUUUGGGGGGACUC-3'
5'-GAGTCCCCCCAAACACTTCAG-3'
43
miR395b
c,f
5'-CTGAAGTGTTTGGGGGGACTC-3'
5'-GAGTCCCCCCAAACACTTCAG-3'
5'-UUCCACAGCUUUCUUGAACUG-3'
5'-CAGTTCAAGAAAGCTGTGGAA-3'
44
miR396a
5'-TTCCACAGCTTTCTTGAACTG-3'
5'-CAGTTCAAGAAAGCTGTGGAA-3'
ath-miR396b
5'-UUCCACAGCUUUCUUGAACUU-3'
5'-AAGTTCAAGAAAGCTGTGGAA-3'
45
miR396b
5'-TTCCACAGCTTTCTTGAACTT-3'
5'-AAGTTCAAGAAAGCTGTGGAA-3'
ath-miR397a
5'-UCAUUGAGUGCAGCGUUGAUG-3'
5'-CATCAACGCTGCACTCAATGA-3'
46
miR397a
5'-TCATTGAGTGCAGCGTTGATG-3'
5'-CATCAACGCTGCACTCAATGA-3'
ath-miR397b
5'-UCAUUGAGUGCAUCGUUGAUG-3'
5'-CATCAACGATGCACTCAATGA-3'
47
miR397b
5'-TCATTGAGTGCATCGTTGATG-3'
5'-CATCAACGATGCACTCAATGA-3'
5'-UGUGUUCUCAGGUCACCCCUU-3'
5'-AAGGGGTGACCTGAGAACACA-3'
48
miR398a
5'-UGUGUUCUCAGGUCACCCCUG-3'
5'-CAGGGGTGACCTGAGAACACA-3'
49
miR398b
5'-UGCCAAAGGAGAUUUGCCCUG-3'
5'-CAGGGCAAATCTCCTTTGGCA-3'
50
miR399a
5'-UGCCAAAGGAGAGUUGCCCUG-3'
5'-CAGGGCAACTCTCCTTTGGCA-3'
51
miR399b
ath-miR399d
5'-UGCCAAAGGAGAUUUGCCCCG-3'
5'-CGGGGCAAATCTCCTTTGGCA-3'
52
miR399d
ath-miR399e
5'-UGCCAAAGGAGAUUUGCCUCG-3'
5'-CGAGGCAAATCTCCTTTGGCA-3'
53
ath-miR399f
5'-UGCCAAAGGAGAUUUGCCCGG-3'
5'-CCGGGCAAATCTCCTTTGGCA-3'
54
ath-miR400a
5'-UAUGAGAGUAUUAUAAGUCAC-3'
5'-GTGACTTATAATACTCTCATA-3'
ath-miR401a
5'-CGAAACUGGUGUCGACCGACA-3'
5'-TGTCGGTCGACACCAGTTTCG-3'
ath-miR402a
5'-UUCGAGGCCUAUUAAACCUCUG-3'
ath-miR403a
5'-UUAGAUUCACGCACAAACUCG-3'
ath-miR404a
5'-AUUAACGCUGGCGGUUGCGGCAGC-3'
5'-GCTGCCGCAACCGCCAGCGTTAAT-3'
ath-miR405a
5'-AUGAGUUGGGUCUAACCCAUAACU-3'
5'-AGTTATGGGTTAGACCCAACTCAT-3'
ath-miR406a
5'-UAGAAUGCUAUUGUAAUCCAG-3'
5'-CTGGATTACAATAGCATTCTA-3'
ath-miR407a
5'-UUUAAAUCAUAUACUUUUGGU-3'
5'-ACCAAAAGTATATGATTTAAA-3'
ath-miR408a
5'-AUGCACUGCCUCUUCCCUGGC-3'
5'-GCCAGGGAAGAGGCAGTGCAT-3'
ath-miR413a
5'-AUAGUUUCUCUUGUUCUGCAC-3'
5'-GTGCAGAACAAGAGAAACTAT-3'
ath-miR414a
5'-UCAUCUUCAUCAUCAUCGUCA-3'
5'-TGACGATGATGATGAAGATGA-3'
ath-miR415a
5'-AACAGAGCAGAAACAGAACAU-3'
5'-ATGTTCTGTTTCTGCTCTGTT-3'
ath-miR416a
5'-GGUUCGUACGUACACUGUUCA-3'
5'-TGAACAGTGTACGTACGAACC-3'
ath-miR417a
5'-GAAGGUAGUGAAUUUGUUCGA-3'
5'-TCGAACAAATTCACTACCTTC-3'
ath-miR396a
ath-miR398a
ath-miR398b
c
ath-miR399a
ath-miR399b
c
c
5'-AAGGGGTGACCTGAGAACACA-3'
5'-CAGGGGTGACCTGAGAACACA-3'
5'-TGCCAAAGGAGATTTGCCCTG-3'
5'-CAGGGCAAATCTCCTTTGGCA-3'
5'-TGCCAAAGGAGAGTTGCCCTG-3'
5'-CAGGGCAACTCTCCTTTGGCA-3'
5'-TGCCAAAGGAGATTTGCCCCG-3'
5'-CGGGGCAAATCTCCTTTGGCA-3'
miR399e
5'-TGCCAAAGGAGATTTGCCTCG-3'
5'-CGAGGCAAATCTCCTTTGGCA-3'
miR399f
5'-TGCCAAAGGAGATTTGCCCGG-3'
5'-CCGGGCAAATCTCCTTTGGCA-3'
55
miR400a
5'-TATGAGAGTATTATAAGTCAC-3'
5'-GTGACTTATAATACTCTCATA-3'
5'-CAGAGGTTTAATAGGCCTCGAA-3'
56
miR402a
5'-TTCGAGGCCTATTAAACCTCTG-3'
5'-CAGAGGTTTAATAGGCCTCGAA-3'
5'-CGAGTTTGTGCGTGAATCTAA-3'
57
miR403a
5'-TTAGATTCACGCACAAACTCG-3'
5'-CGAGTTTGTGCGTGAATCTAA-3'
58
miR408a
5'-ATGCACTGCCTCTTCCCTGGC-3'
5'-GCCAGGGAAGAGGCAGTGCAT-3'
59
miR416a
5'-GGTTCGTACGTACACTGTTCA-3'
5'-TGAACAGTGTACGTACGAACC-3'
ath-miR418a
5'-UAAUGUGAUGAUGAACUGACC-3'
5'-GGTCAGTTCATCATCACATTA-3'
ath-miR419a
5'-UUAUGAAUGCUGAGGAUGUUG-3'
5'-CAACATCCTCAGCATTCATAA-3'
ath-miR420a
5'-UAAACUAAUCACGGAAAUGCA-3'
5'-TGCATTTCCGTGATTAGTTTA-3'
5
c
5'-TGTGTTCTCAGGTCACCCCTT-3'
5'-TGTGTTCTCAGGTCACCCCTG-3'
ath-miR426a
5'-UUUUGGAAAUUUGUCCUUACG-3'
5'-CGTAAGGACAAATTTCCAAAA-3'
5'-UUGGGGACGAGAUGUUUUGUUG-3'
5'-CAACAAAACATCTCGTCCCCAA-3'
60
miR447a
5'-TTGGGGACGAGATGTTTTGTTG-3'
5'-CAACAAAACATCTCGTCCCCAA-3'
ath-miR447c
5'-UUGGGGACGACAUCUUUUGUUG-3'
5'-CAACAAAAGATGTCGTCCCCAA-3'
61
miR447c
5'-TTGGGGACGACATCTTTTGTTG-3'
5'-CAACAAAAGATGTCGTCCCCAA-3'
62
miR472a
5'-TTTTTCCTACTCCGCCCATACC-3'
5'-GGTATGGGCGGAGTAGGAAAAA-3'
ath-miR771a
5'-UGAGCCUCUGUGGUAGCCCUC-3'
5'-GAGGGCTACCACAGAGGCTCA-3'
63
miR771a
5'-TGAGCCTCTGTGGTAGCCCTC-3'
5'-GAGGGCTACCACAGAGGCTCA-3'
ath-miR772a
5'-UUUUUCCUACUCCGCCCAUAC-3'
5'-GTATGGGCGGAGTAGGAAAAA-3'
ath-miR773a
5'-UUUGCUUCCAGCUUUUGUCUC-3'
5'-GAGACAAAAGCTGGAAGCAAA-3'
64
miR773a
5'-TTTGCTTCCAGCTTTTGTCTC-3'
5'-GAGACAAAAGCTGGAAGCAAA-3'
ath-miR774a
5'-UUGGUUACCCAUAUGGCCAUC-3'
5'-GATGGCCATATGGGTAACCAA-3'
65
miR774a
5'-TTGGTTACCCATATGGCCATC-3'
5'-GATGGCCATATGGGTAACCAA-3'
ath-miR775a
5'-UUCGAUGUCUAGCAGUGCCAA-3'
5'-TTGGCACTGCTAGACATCGAA-3'
66
miR775a
5'-TTCGATGTCTAGCAGTGCCAA-3'
5'-TTGGCACTGCTAGACATCGAA-3'
ath-miR776a
5'-UCUAAGUCUUCUAUUGAUGUU-3'
5'-AACATCAATAGAAGACTTAGA-3'
67
miR776a
5'-TCTAAGTCTTCTATTGATGTT-3'
5'-AACATCAATAGAAGACTTAGA-3'
ath-miR777a
5'-UACGCAUUGAGUUUCGUUGCU-3'
5'-AGCAACGAAACTCAATGCGTA-3'
68
miR777a
5'-TACGCATTGAGTTTCGTTGCT-3'
5'-AGCAACGAAACTCAATGCGTA-3'
ath-miR778a
5'-UGGCUUGGUUUAUGUACACCG-3'
5'-CGGTGTACATAAACCAAGCCA-3'
69
miR778a
5'-TGGCTTGGTTTATGTACACCG-3'
5'-CGGTGTACATAAACCAAGCCA-3'
ath-miR779a
5'-UUCUGCUAUGUUGCUGCUCAU-3'
5'-ATGAGCAGCAACATAGCAGAA-3'
70
miR779a
5'-TTCTGCTATGTTGCTGCTCAT-3'
5'-ATGAGCAGCAACATAGCAGAA-3'
ath-miR780a
5'-UUUCUUCGUGAAUAUCUGGCA-3'
5'-TGCCAGATATTCACGAAGAAA-3'
71
miR780a
5'-TTTCTTCGTGAATATCTGGCA-3'
5'-TGCCAGATATTCACGAAGAAA-3'
ath-miR781a
5'-UUAGAGUUUUCUGGAUACUUA-3'
5'-TAAGTATCCAGAAAACTCTAA-3'
72
miR781a
5'-TTAGAGTTTTCTGGATACTTA-3'
5'-TAAGTATCCAGAAAACTCTAA-3'
ath-miR782a
5'-ACAAACACCUUGGAUGUUCUU-3'
5'-AAGAACATCCAAGGTGTTTGT-3'
ath-miR783a
5'-AAGCUUUGCUCGUUCAUGUUC-3'
5'-GAACATGAACGAGCAAAGCTT-3'
73
miR822
5'-TGCGGGAAGCATTTGCACATG-3'
5'-CATGTGCAAATGCTTCCCGCA-3'
74
miR823a
5'-TGGGTGGTGATCATATAAGAT-3'
5'-ATCTTATATGATCACCACCCA-3'
75
miR824a
5'-TAGACCATTTGTGAGAAGGGA-3'
5'-TCCCTTCTCACAAATGGTCTA-3'
76
miR825a
5'-TTCTCAAGAAGGTGCATGAAC-3'
5'-GTTCATGCACCTTCTTGAGAA-3'
77
miR826a
5'-TAGTCCGGTTTTGGATACGTG-3'
5'-CACGTATCCAAAACCGGACTA-3'
78
miR827a
5'-TTAGATGACCATCAACAAACT-3'
5'-AGTTTGTTGATGGTCATCTAA-3'
79
miR828a
5'-TCTTGCTTAAATGAGTATTCCA-3'
5'-TGGAATACTCATTTAAGCAAGA-3'
80
miR829a
5'-CAAATTAAAGCTTCAAGGTAG-3'
5'-CTACCTTGAAGCTTTAATTTG-3'
81
miR830a
5'-TCTTCTCCAAATAGTTTAGGTT-3'
5'-AACCTAAACTATTTGGAGAAGA-3'
82
miR831a
5'-TGATCTCTTCGTACTCTTCTTG-3'
5'-CAAGAAGAGTACGAAGAGATCA-3'
83
miR832a
5'-TGCTGGGATCGGGAATCGAAA-3'
5'-TTTCGATTCCCGATCCCAGCA-3'
84
miR833a
5'-TAGACCGATGTCAACAAACAAG-3'
5'-CTTGTTTGTTGACATCGGTCTA-3'
85
miR834a
5'-TGGTAGCAGTAGCGGTGGTAA-3'
5'-TTACCACCGCTACTGCTACCA-3'
86
miR835a
5'-TTCTTGCATATGTTCTTTATC-3'
5'-GATAAAGAACATATGCAAGAA-3'
87
miR836a
5'-TCCTGTGTTTCCTTTGATGCGTGG-3'
5'-CCACGCATCAAAGGAAACACAGGA-3'
88
miR837a
5'-ATCAGTTTCTTGTTCGTTTCA-3'
5'-TGAAACGAACAAGAAACTGAT-3'
89
miR838a
5'-TTTTCTTCTACTTCTTGCACA-3'
5'-TGTGCAAGAAGTAGAAGAAAA-3'
90
miR839a
5'-TACCAACCTTTCATCGTTCCC-3'
5'-GGGAACGATGAAAGGTTGGTA-3'
91
miR840a
5'-ACACTGAAGGACCTAAACTAAC-3'
5'-GTTAGTTTAGGTCCTTCAGTGT-3'
92
miR841a
5'-TACGAGCCACTTGAAACTGAA-3'
5'-TTCAGTTTCAAGTGGCTCGTA-3'
93
miR842a
5'-TCATGGTCAGATCCGTCATCC-3'
5'-GGATGACGGATCTGACCATGA-3'
94
miR843a
5'-TTTAGGTCGAGCTTCATTGGA-3'
5'-TCCAATGAAGCTCGACCTAAA-3'
ath-miR447a
b
6
b
95
miR844a
5'-AATGGTAAGATTGCTTATAAG-3'
5'-CTTATAAGCAATCTTACCATT-3'
96
miR845a
5'-CGGCTCTGATACCAATTGATG-3'
5'-CATCAATTGGTATCAGAGCCG-3'
97
miR845b
5'-TCGCTCTGATACCAAATTGATG-3'
5'-TCGCTCTGATACCAAATTGATG-3'
98
miR846
5'-TTGAATTGAAGTGCTTGAATT-3'
5'-AATTCAAGCACTTCAATTCAA-3'
99
miR847
5'-TCACTCCTCTTCTTCTTGATG-3'
5'-CATCAAGAAGAAGAGGAGTGA-3'
100
miR848
5'-TGACATGGGACTGCCTAAGCTA-3'
5'-TAGCTTAGGCAGTCCCATGTCA-3'
101
miR849
5'-TAACTAAACATTGGTGTAGTA-3'
5'-TACTACACCAATGTTTAGTTA-3'
102
miR850
5'-TAAGATCCGGACTACAACAAAG-3'
5'-CTTTGTTGTAGTCCGGATCTTA-3'
103
miR851
5'-TCTCGGTTCGCGATCCACAAG-3'
5'-CTTGTGGATCGCGAACCGAGA-3'
104
miR852
5'-AAGATAAGCGCCTTAGTTCTGA-3'
5'-TCAGAACTAAGGCGCTTATCTT-3'
105
miR853
5'-TCCCCTCTTTAGCTTGGAGAAG-3'
5'-CTTCTCCAAGCTAAAGAGGGGA-3'
106
miR856
5'-TAATCCTACCAATAACTTCAGC-3'
5'-GCTGAAGTTATTGGTAGGATTA-3'
107
miR857
5'-TTTTGTATGTTGAAGGTGTAT-3'
5'-ATACACCTTCAACATACAAAA-3'
108
miR858
5'-TTTCGTTGTCTGTTCGACCTT-3'
5'-AAGGTCGAACAGACAACGAAA-3'
109
miR859
5'-TCTCTCTGTTGTGAAGTCAAA-3'
5'-TTTGACTTCACAACAGAGAGA-3'
110
miR860
5'-TCAATAGATTGGACTATGTAT-3'
5'-ATACATAGTCCAATCTATTGA-3'
111
miR861
5'-GATGGATATGTCTTCAAGGAC-3'
5'-GTCCTTGAAGACATATCCATC-3'
112
miR862
5'-TCCAATAGGTCGAGCATGTGC-3'
5'-GCACATGCTCGACCTATTGGA-3'
113
miR863
5'-TTGAGAGCAACAAGACATAAT-3'
5'-ATTATGTCTTGTTGCTCTCAA-3'
114
miR864
5'-TCAGGTATGATTGACTTCAAA-3'
5'-TTTGAAGTCAATCATACCTGA-3'
115
miR865
5'-ATGAATTTGGATCTAATTGAG-3'
5'-CTCAATTAGATCCAAATTCAT-3'
116
miR866
5'-ACAAAATCCGTCTTTGAAGA-3'
5'-TCTTCAAAGACGGATTTTGT-3'
117
miR867
5'-TTGAACATGGTTTATTAGGAA-3'
5'-TTCCTAATAAACCATGTTCAA-3'
118
miR868
5'-CTTCTTAAGTGCTGATAATGC-3'
5'-GCATTATCAGCACTTAAGAAG-3'
119
miR869
5'-TCTGGTGTTGAGATAGTTGAC-3'
5'-GTCAACTATCTCAACACCAGA-3'
120
miR870
5'-TAATTTGGTGTTTCTTCGATC-3'
5'-GATCGAAGAAACACCAAATTA-3'
*Asterisk indicates the microRNA from opposite arm of the precursor reported by northern blot evidence (Wang et al., 2004).
“Prediction and identification of Arabidopsis thaliana microRNAs and their mRNA targets”
Wang XJ, Reyes JL, Chua NH, Gaasterland T. Genome Biol. 5:R65 (2004).
7
Supplementary Table S2. The oligonucleotides used for RT-PCR and qRT-PCR.
Gene
Annealing
Temperature
(°C)
AGO1
62
AP11
AP2
At1g17590
At1g54160
At1g66690
At1g66700
At1g66720
At3g15640
At3g20910
At5g06510
CSD2
DCL1
60
60
60
60
57
57
57
60
60
60
60
58
Primer
Sequence
JH5521
5'-GGCCTGGTAAAGGACAGAGT-3'
JH5522
5'-AAGAACCTGCAGAGCTTCCT-3'
JH5128
5'-CATGGGTGGTCTGTATCAAGAAGAT-3'
JH5129
5'-CATGCGGCGAAGCAGCCAAGGTT-3'
JH4587
5'-ACAACAAGATTCTCTCCACTCTAATGA-3'
JH4588
5'-GCAGCCAATTTTGATGAGGAGTA-3'
JH4575
5'-GAATCCCCAAATGACTCGAGTT-3'
JH4576
5'-TCATTCAAATGATATCTGGACAAAGC-3'
JH4577
5'-GCCATTGCCTCATCACATTC-3'
JH4578
5'-TAGTTTGACAATGATTTGAAAGTTCGTC-3'
JH4068
5'-TGGATATGGCCCTGAAAGTTAC-3'
JH4069
5'-TAGCGGAAACTCATTCTTAGAGTGA-3'
JH4509
5'-ATAAACCAAAGTATCAAACAATAAAAGATT-3'
JH4071
5'-GTTGAAATCATTGAGGCTGAAATCA-3'
JH4072
5'-TAAAATAAACGCGGTGATTTCAA-3'
JH4073
5'-AGACTCCACCTTGTCTTTCTCAA-3'
JH4533
5'-GAATAAAACATTTGGGGAGTTTCTT-3'
JH4535
5'-ATAGTAGGACTTCACAATAGCAGGAGC-3'
JH4579
5'-CCACTGCCGCCTGAGATG-3'
JH4580
5'-TACTCTTTCATGGCTGCTACCG-3'
JH4581
5'-AGGCTGAAAAACTGAGTAGATGCCGTAA-3'
JH4582
5'-TCAGAGATCTTATGAAGATGCGTAGAA-3'
JH3333
5'-TGACACACGGAGCTCCAGAA-3'
JH3334
5'-CCTGCGTTTCCAGTGGTCAG-3'
JH5517
5'-CTTTCCTTGAGACCGGTGC-3'
JH5518
5'-AACTTGGTCTCGAGGTTACG-3'
8
qPCR4
SPL2
60
SPL3
60
SPL4
60
SPL5
59
SPL6
60
SPL9
58
SPL10
58
SPL11
60
SPL13
58
SPL15
60
SVP
62
SVP-qPCR
60
TOE1*
58
TOE2*
58
TOE3*
58
JH5003
5'-CCTCGATTGAGCATGTTCCTATG-3'
JH4039
5'-CCATCCAAGAAGTGAGGAAAAGTT-3'
JH4040
5'-CAGACCGGTGAGCTACGGG-3'
JH4041
5'-AGATACTTTTGAAGAAGAAGAGGCT-3'
JH4042
5'-CATGTCGTAGGTTTAGCAGATAGC-3'
JH4043
5'-TAGGTAGGGATAGAGTTAGAGGGTC-3'
JH4044
5'-GCTTAGCGTTTGCATATAGCTGAT-3'
JH4045
5'-TAGCACTGACCGTGTTCCATC-3'
JH4046
5'-CGTGTAGGATTTAATACCATGACC-3'
JH4047
5'-ATAAGCTTCTTCGCACCTCTCA-3'
JH4048
5'-ATAAGCTTCTTCGCACCTCTCA-3'
JH4049
5'-TCTCTCTGTGTTAGCTTCTCGTTACG-3'
JH4050
5'-AGAAGATTCTCTGATGCAAAGACA-3'
JH4051
5'-TCTCTTTCTCTGCGTTTCAAACA-3'
JH4052
5'-AGTTGTCATACCAACAGAATCCAG-3'
JH4053
5'-GTATGTTCTCTACATCTCAAACCTCAGG-3'
JH4054
5'-TCCACCAACTGAGTAACAGGTTTAC-3'
JH4055
5'-GAATGACGAGTTGCATCCATGAC-3'
JH4056
5'-CTTTTACGCCATAATATGTGAACA-3'
JH4057
5'-ATTACTCTCGAATCGCTCCATCT-3'
JH4058
5'-GATGTGTTGAGATGGGCGG-3'
JH2107
5'-GAAGGAAGTCCTAGAGAGGCATAA-3'
JH2108
5'-AATTGTTCCATCTCTAACCACCAT-3'
JH3776
5'-GAAGAGAACGAGCGACTTGG-3'
JH3777
5'-GAGCTCTCGGAGTCAACAGG-3'
JH4583
5'-TTTACTGGAACGGAGCATGC-3'
JH4584
5'-GTGTGGATAAAAGTAACCACGTGTT-3'
JH4589
5'-GGCATGTGATACGCCTTTCA-3'
JH4590
5'-ATAGAGACCGGGCTGATTCAGAT-3'
JH4591
5'-TATGATAAAGCGGCAATAAAGTGT-3'
JH4592
5'-AGGAATGCGGTAAGGGGAAG-3'
10
FLC
FLC-qPCR
FT
FT-qPCR2
HAP2A
HAP2B
HAP2C
HYL13
PHO2
SE3
SMZ
SMZ-qPCR
SNZ
SNZ-qPCR
62
60
62
60
60
60
60
60
60
62
60
60
60
60
SOC1
62
SOC1-
60
MB73
5'-GTAGCCGACAAGTCACCTTCTCCA-3'
MB74
5'-GAGATTTGTCCAGCAGGTGACATCT-3'
JH3778
5'-GCCAAGAAGACCGAACTCAT-3'
JH3779
5'-TTTGTCCAGCAGGTGACATC-3'
JH1002
5'-ACTATAGGCATCATCACCGTTCGTTAC-3'
JH1003
5'-ACAACTGGAACAACCTTTGGCAATG-3'
JH6163
5'-TCCCTGCTACAACTGGAACAACCTTTG-3'
JH6164
5'-CGCAGCCACTCTCCCTCTGACAATTGT-3'
JH4506
5'-CGAAGAAAAGTGAGGTAGAAGCG-3'
JH4443
5'-TAAACCCACACAATATGAGACTCTGA-3'
JH4444
5'-CAGATTCAGTCTCAGCCTAAGCC-3'
JH4507
5'-CACACTACTAGTTGGAACAAGTGGG-3'
JH4446
5'-CAAAAAACTTCTTCAAGAATCCGA-3'
JH4508
5'-TGGTCGTTCTTGTGATGTCTAACA-3'
JH5515
5'-ATGACCTCCACTGATGTTTCCTC-3'
JH5516
5'-CAGTTCTCCCAGCGCTAATC-3'
JH4537
5'-CTGTTTCCCATTTATACTTCAGATTCTA-3'
JH4539
5'-CATAATCAGAGAGTGAAATAGAACGCA-3'
JH5513
5'-CTGATTCCGTCGATAACCGTCTCC-3'
JH5514
5'-CAGGCCTCCCACCCATTTCAC-3'
JH5070
5'-CGAAGATCAAGATCGGAAAGTACC-3'
JH5071
5'-CCTGTTTTGGAAGAGATGAATCTGA-3'
JH6225
5'-CATCATCATCGGAAAGTATAAAGTTGAC-3'
JH6226
5'-GTCTTCAGAGGTTTCATGGTTGCCATG-3'
JH4585
5'-CTTAGACGAGCGAGTGCAAGC-3'
JH4586
5'-CATGGATCAAAACAAGATAAGGACA-3'
JH6173
5'-CAGCAGCAGCAAAATGCAATGAG-3'
JH6174
5'-CACCGATCGATTCAAACCCATGT-3'
JH1145
5'-GGATCGAGTCAGCACCAAACC-3'
JH1146
5'-CCCAATGAACAATTGCGTCTC-3'
JH5002
5'-CTTCTAAACGTAAACTCTTGGGAGAAG-3'
9
TSF-qPCR
TUB-qPCR
UBQ10
62
60
62
JH5829
5'-GTGGATCCAGATGTGCCGAGTC-3'
JH5830
5'-TCCCTCTGGCAGTTGAAGTAAGAGG-3'
JH3792
5'-TGTTCAGGCGAGTGAGTGAG-3'
JH3793
5'-ATGTTGCTCTCCGCTTCTGT-3'
JH1011
5'-GATCTTTGCCGGAAAACAATTGGAGGATGGT-3'
JH1012
5'-CGACTTGTCATTAGAAAGAAAGAGATAACAGG-3'
*These primers are used for both RT-PCR and qRT-PCR.
1
(Liu et al. 2008)
(Mathieu et al. 2009)
3
(Yang et al. 2006)
4
(Mockler et al. 2004)
2
“Direct interaction of AGL24and SOC1integrates flowering signals in Arabidopsis”
Liu, C., Chen, H., Er, H.L., Soo, H.M., Kumar, P.P., Han, J.H., Liou, Y.C. and Yu, H. Development 135, 1481-1491. (2008)
“Repression of Flowering by the miR172 Target SMZ”
Mathieu, J., Yant, L.J., Murdter, F., Kuttner, F. and Schmid, M. PLoS Biol, 7, e1000148. (2009)
“SERRATE is a novel nuclear regulator in primary microRNA processing in Arabidopsis”
Yang L, Liu Z, Lu F, Dong A, Huang H. Plant J. 47:841-850 (2006)
“Regulation of flowering time in Arabidopsis by K homology domain proteins.”
Mockler, T.C., Yu, X., Shalitin, D., Parikh, D., Michael, T.P., Liou, J., Huang, J., Smith, Z., Alonso, J.M., Ecker, J.R. Proc Natl Acad Sci U S A,
101, 12759-12764. (2004)
11
Supplementary Table S3. Fold change values of the miRNA loci in the microarray analysis.
Name
Sequence of probe
Length
Fold(23/16)
PM_016_1
PM_016_2
PM_023_1
PM_023_2
ath|miR169a
TCGGCAAGTCATCCTTGGCTG
21
-1.56
22358.50
34275.50
18649.13
16972.93
ath|miR156g
TGTGCTCACTCTCTTCTGTCG
21
-1.47
28612.25
49487.43
25143.88
26052.96
ath|miR169b
CCGGCAAGTCATCCTTGGCTG
21
-1.46
16437.25
24556.89
14567.25
13048.57
ath|miR319c
AGGAGCTCCCTTCAGTCCAA
20
-1.41
1636.00
1889.98
1593.25
972.55
ath|miR169h
CAGGCAAGTCATCCTTGGCTA
21
-1.37
13863.50
19087.02
12865.63
10904.48
ath|miR156a
GTGCTCACTCTCTTCTGTCA
20
-1.36
20129.00
32267.99
20348.13
17199.27
ath|miR159c
AGGAGCTCCCTTCAATCCAAA
21
-1.35
9917.50
11454.76
9899.00
6289.10
ath|miR169d
CGGCAAGTCATCCTTGGCTCA
21
-1.34
15131.00
22487.37
15302.63
12362.75
ath|miR319a
GGGAGCTCCCTTCAGTCCAA
20
-1.32
1030.88
1161.10
963.50
711.69
ath|miR156h
GTGCTCTCTTTCTTCTGTCAA
21
-1.21
8678.50
10330.23
9077.38
6780.76
ath|miR173
GTGATTTCTCTCTGCAAGCGAA
22
-1.18
13363.63
18441.62
13584.00
13070.35
ath|miR158b
TGCTTTGTCTACATTTGGGG
20
-1.12
1024.13
977.19
986.38
802.64
ath|miR399b
CAGGGCAACTCTCCTTTGGCA
21
-1.12
1273.75
2219.38
1545.25
1459.38
ath|miR169g*
AGCCAAGGTCAACTTGCCGGA
21
-1.11
112.38
144.43
120.75
108.90
ath|miR400
GTGACTTATAATACTCTCATA
21
-1.11
150.25
175.70
171.50
125.48
ath|miR157a
GTGCTCTCTATCTTCTGTCAA
21
-1.10
24159.88
38917.11
28069.88
27658.13
ath|miR158a
TGCTTTGTCTACATTTGGGA
20
-1.08
1272.25
1136.45
1303.50
950.90
ath|miR157d
GTGCTCTCTATCTTCTGTCA
20
-1.06
16918.75
25467.30
21648.88
17576.09
ath|miR775
TTGGCACTGCTAGACATCGAA
21
-1.05
745.50
1112.12
935.67
799.76
ath|miR447a
CAACAAAACATCTCGTCCCCAA
22
-1.04
172.67
290.26
215.83
216.48
ath|miR395b
GAGTCCCCCCAAACACTTCAG
21
-1.03
778.25
1243.12
944.75
963.27
ath|miR159a
TAGAGCTCCCTTCAATCCAAA
21
-1.01
31295.25
44259.24
39615.13
34136.56
ath|miR159b
AAGAGCTCCCTTCAATCCAAA
21
1.02
30486.88
41892.92
39142.88
33761.85
ath|miR162a
CTGGATGCAGAGGTTTATCGA
21
1.02
604.75
831.31
782.00
665.65
ath|miR168a
TTCCCGACCTGCACCAAGCGA
21
1.02
4664.75
8222.16
6347.25
6338.85
ath|miR170
GATATTGACACGGCTCAATCA
21
1.02
205.00
290.04
279.25
223.62
ath|miR393a
GATCAATGCGATCCCTTTGGA
21
1.09
1712.88
2308.95
2625.25
1796.85
ath|miR165a
GGGGGATGAAGCCTGGTCCGA
21
1.10
2849.00
4528.19
4516.63
3450.52
ath|miR390a
GGCGCTATCCCTCCTGAGCTT
21
1.11
4405.25
7950.93
6450.63
6711.46
ath|miR394a
GGAGGTGGACAGAATGCCAA
20
1.12
254.50
288.98
340.50
272.37
ath|miR160a
TGGCATACAGGGAGCCAGGCA
21
1.14
6711.25
10075.70
9854.25
8991.43
12
ath|miR166a
GGGGAATGAAGCCTGGTCCGA
21
1.16
5170.00
8199.76
8477.38
6725.32
ath|miR172e
ATGCAGCATCATCAAGATTCC
21
1.18
676.13
855.02
1013.38
788.16
ath|miR161
CCCCGATGTAGTCACTTTCAA
21
1.19
10995.00
16771.72
16341.75
15968.82
ath|miR396b
AAGTTCAAGAAAGCTGTGGAA
21
1.24
187.50
235.06
315.75
213.84
ath|miR171a
GATATTGGCGCGGCTCAATCA
21
1.24
2193.88
3440.36
3737.00
3127.66
ath|miR396a
CAGTTCAAGAAAGCTGTGGAA
21
1.25
241.00
332.71
410.75
305.29
ath|miR164a
TGCACGTGCCCTGCTTCTCCA
21
1.26
1641.50
2971.58
2884.50
2673.62
ath|miR403
CGAGTTTGTGCGTGAATCTAA
21
1.26
849.38
1336.66
1444.38
1245.17
ath|miR164c
CGCACGTGCCCTGCTTCTCCA
21
1.26
2111.88
3552.46
3519.25
3401.15
ath|miR167c
AAGATCATGCTGGCAGCTTAA
21
1.27
12149.50
14235.54
18035.00
15358.74
ath|miR399f
CCGGGCAAATCTCCTTTGGCA
21
1.28
376.63
550.14
633.13
534.48
ath|miR399a
CAGGGCAAATCTCCTTTGGCA
21
1.30
415.50
648.06
740.75
615.90
ath|miR399d
CGGGGCAAATCTCCTTTGGCA
21
1.31
289.88
467.33
530.75
434.86
ath|miR399e
CGAGGCAAATCTCCTTTGGCA
21
1.31
142.75
187.89
236.13
196.39
ath|miR172a
ATGCAGCATCATCAAGATTCT
21
1.32
639.13
740.54
1003.00
816.63
ath|miR167a
TAGATCATGCTGGCAGCTTCA
21
1.32
12223.75
15967.84
21594.50
15659.33
ath|miR395a
GAGTTCCCCCAAACACTTCAG
21
1.33
270.00
437.91
456.13
456.14
ath|miR408
GCCAGGGAAGAGGCAGTGCAT
21
1.35
1023.25
1403.71
1662.63
1575.46
ath|miR167d
CCAGATCATGCTGGCAGCTTCA
22
1.36
6605.50
8807.67
11804.13
9101.69
ath|miR171b
CGTGATATTGGCACGGCTCAA
21
1.41
2024.00
3804.34
3931.75
3872.39
ath|miR172c
CTGCAGCATCATCAAGATTCT
21
1.53
377.38
423.47
697.75
536.70
ath|miR163
ATCGAAGTTCCAAGTCCTCTTCAA
24
1.61
981.38
1998.10
2130.50
2371.67
ath|miR398b
CAGGGGTGACCTGAGAACACA
21
1.88
1088.50
1493.54
2766.75
2069.72
ath|miR398a
AAGGGGTGACCTGAGAACACA
21
1.97
777.63
1046.22
2076.13
1519.16
The miRNA loci that did not satisfy our selection criterion and thus converted to the background value (see Materials and Methods) were
excluded.
13
Supplementary Figure S1. Comparison of sensitivity between enhanced miRNA Northern hybridization analysis using EDC and a procedure
using UV cross-linking. miR156a was used as a probe. Total RNAs isolated at the indicated time points were used for this analysis. The
autoradiograms were exposed for an equal amount of time (1 h) in a BAS phosphorimager (Fuji BAS, Japan). The numbers below each panel
indicate the quantification value of each band. The quantification value of a band from 6-day-old wild-type plants was set to 1.0 to calculate the
relative values for the other bands. This analysis revealed that the method using EDC was approximately 100 times more sensitive than the UV
cross-linking method.
14
Supplementary Figure S2. Degree of cross-hybridization of the miR156 and miR157 probes used in the miRNA Northern hybridization
analysis. (A) Sequence comparison of the mature forms of miR156 and miR157 loci. Different nucleotides within the miR156/157 family are
indicated in bold type. (B) Test of cross-hybridization using different miR156 and miR157 probes in the miRNA Northern hybridization analysis.
Note that the DNA probes were hybridized to synthesize the miRNA RNA sequences. (C) Predicted Tm value (°C) of the hybridized sequences
between a DNA probe and a miRNA. The Tm values were calculated by using DINAMelt (Zuker, 2003). The highest value in different possible
combinations from a single probe is shown in bold type. (D) Prediction of the hybridized sequences between different miR156/157 probes by
using the Two-state Hybridization program at the DINAMelt Server (http://dinamelt.bioinfo.rpi.edu/twostate.php) (Zuker, 2003).
“Mfold web server for nucleic acid folding and hybridization prediction.” Zuker, M. Nucleic Acids Res., 31:3406-3415 (2003).
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Supplementary Figure S3. The miRNAs that were undetectable in our miRNA Northern hybridization analysis. The numbers above the blots
indicate the times of harvest (ZT). Ethidium bromide-stained rRNAs are shown below each blot to demonstrate an equal amount of loading.
Note that a probe hybridizes to its paralogous loci as well as itself, because of their identical sequences.
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Supplementary Figure S4. The miRNAs that did not show significant changes in their expression or that showed inconsistent results between
two biological replicates or ZT time points in our miRNA Northern hybridization analysis. The numbers above the blots indicate the times of
harvest (ZT). Ethidium bromide-stained rRNAs are shown below each blot to demonstrate an equal amount of loading. Note that a probe
hybridizes to its paralogous loci as well as itself, because of their identical sequences.
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Supplementary Figure S5. qRT-PCR analysis of miR172 target genes in 8-day-old wild type plants grown under LD conditions at 23°C and
16°C.
Supplementary Figure S6. Flowering time of the miRNA biogenesis mutants at 23°C and 16°C under LD conditions. The numbers listed below
the genotypes denote the ratios of the flowering time at 16°C and 23°C (16°C/23°C). The error bars denote the standard deviation.
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Supplementary Figure S7. Expression of the miRNA biogenesis genes in 10-day-old wild-type plants grown under LD conditions at 23°C and
16°C. The numbers to the right of every panel indicate the number of PCR cycles used in the RT-PCR analysis. UBQ10 was used as an internal
control.
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