Supplementary Table 1List of primers used for validation and relative quantification of identified P. somniferum miRNAs

Primer Name / Primer Sequence
pso-MIR397a Forward: / 5'-GCGGCGGCATTGAGCGCAGC-3'
Universal Reverse: / 5'-GTGCAGGGTCCGAGGT-3'
pso-MIR414 Forward: / 5'-GCGGCGGCGATCTTCATCATC-3'
pso-MIR171b Forward: / 5'-GCGGCGGATATTGACAGCCGTGC-3'
pso-MIR408 Forward: / 5'-GCGGCGGATGCACTGCCTCTTCC-3'
pso-MIR167 Forward: / 5'-TCGCGTGAAGCTGCCAGCAT-3'
pso-MIR169a Forward: / 5'-GCGGCGGGGCAAATCATCC-3'

Supplementary Table 2 qRT-PCR primers designed for some of the opium poppy miRNA target gene expression level measurement

Primer Name / Primer Sequence / Product Size of Primer Pair
Pso- MIR169a Targ F / 5’-tctgtcatggcaaatcatcc -3’
Pso- MIR169a Targ R / 5’-aaaacccacccatacccatc-3’ / 96 bp
pso-MIR397a Targ F / 5’-aaaatggcatggctggtg-3’
pso-MIR397a Tar R / 5’-agcaattcctcgtgtcgtct-3 / 99 bp
pso-MIR171bTargF / 5’-gcactcacacaccgctctc-3’
pso-MIR171bTarg R / 5’-ctccggctgtataagaaacctc-3’ / 94 bp
pso-MIR414 Targ F / 5’-caatgagccaacaatggtaaaa-3’
pso-MIR414 Targ R / 5’-atcaggcaaaggtgttgtgg-3’ / 100 bp
miRNA / pre-miRNA sequence / A% / C% / G% / U%
pso-MIR169a / CCCAUUUGGCAAAUCAUCCUUGGCUGUGUUACUAAUUAUCAUUAUAUGAAAUCAUGUGUUUCUUUCAUUAUGUUUGCUUUUUAUUUUGAUGAACAGACAAUGAUGAUGUUGACGAACGAA / 27,5 / 15 / 16,7 / 40,8
pso-MIR397a / GGGUGUGAUUGGCAAUGCUGAAGAAAAGCUCGUCAUUGAGCGCAGCGUUGAUUAAUCGAAGA / 29 / 14,5 / 32,3 / 24,2
pso-MIR771 / AAUGUUUUUGCUUUUGGAAAACCUUGACGCGUUUGUUUCCCAACUAAACAUGAUUCACAGAUCCUUGAUUCAUCGUUUAUCUGUGGUAGCCCUCGAACCA / 24 / 23 / 17 / 36
pso-MIR781 / AUCAGAAUGGUUAACUCUGAAGGUGUGGCUAUUUUUAUGACUUGUAUCAGAAUAUGGAAC
UGUCUUGCCGAAUGUAAGCUAUCAGUUCUUGGUGUUUUUCUGGAUACUUAUGUG / 23,7 / 13,2 / 23,7 / 39,5
pso-MIR835 / AACUUGCAUAUGUUCUUUAGCUACAAACAUCCCACAUACAUUCUAAUAAACACACAUAAGCAAUUACUUGCGUGUGACAUUCAACCUGAUUGCAAAACUCCACUAAGAUAGUCAUCGUACUUGUUGCAACGUUUAUCACAACACUCGCAUACUGAAAUCACAUGGAUAGAUAAGAAAAUGGAGAUGGAAAAGUGAAGUGUGCAAAUGUUA / 37,1 / 20 / 15,7 / 27,1
pso-MIR844 / UUCUUGUGUUCUGUGUGGAAUUGGUGUAGAAUCU
CAGGAUCAUUUAUUCUUACAUUGUAAGAUUGCUUAUAAGAUUUG / 23,1 / 10,3 / 21,8 / 44,9
pso-MIR859 / UAAACAACUGGCGACCACAGUGGGAGAUUAAUCUCUCAGUUGUGAAGUCAAUUUCUUCAAUACCCAAUCCCAUUUGGUUGAUUUCAAAAUGGUUGGUCUUAUGACUCCCCUAACAACUC / 28,6 / 23,5 / 16,8 / 31,1
pso-MIR172a / AAGUAUGUUUGAAGAAUUGAUGAAUGAAAAUCCAUGGAUGAUCCAGAAGUUGCUGCUCUU
GAUGAUGCUGCAGCUGCUCC / 28,8 / 16,2 / 25 / 30
pso-MIR169b / CGUUCGUCAACAUCAUCAUUGUCUGUUCAUCAAAAUAAAAAGCAAACAUAAUGAAAGAAACACAUGAUUUCAUAUAAUGAUAAUUAGUAACACAGCCAAGGAUGAUUUGCCAAAUGGGGGA / 41,3 / 16,5 / 16,5 / 26,6
pso-MIR169d / AGAAUUGGAAGGAGCCAAGGAUGACUUACUGAGGAAACCGCUAUUGAUCAGAUAAGGACUAGCGGCUUCAAAGGGUAAGAAAAUCCAAAGUACCUUUACA / 38 / 17 / 25 / 20
pso-MIR171b / GAGAAGUUAUGGCCUACGCCCCCCAGGCGCGCCAGCGAAAUUUUCCAGUCUGUCACAUGAAUGAUAUUGACAGCCGUGCCAAUAUCGACUAACG / 26,6 / 28,7 / 23,4 / 21,3
pso-MIR172d / UUAAGCUUCAACUGAGAAACAUGAAAAACAGGAUG
GAUCUUGAUGAUGCUGGUAACUGAAGCUUGU / 34,8 / 13,6 / 24,2 / 27,3
pso-MIR397b / UGGUUAUUGAGUGCAGCAUUGAUGAUUCUAAGCAAAUAGGUCUUGCCUUUUUCGACUUGGAUUUUGUGCGUCUGGUUUGCAGAACAUGGGAAUUGAUCACCAAGGCGGCCAUUGAUCGUA / 22,5 / 16,7 / 27,6 / 34,2
pso-MIR399k / UAUGCAUUUCUUGAUGGAGAUUUGGAGGAAGAAGUGUAUAUGAAGAUACCUCCAGGUUUUGCCAAAGGAAAUUUUCAAUAUGGUUUGUAGAAUGAAA / 33 / 9 / 24,7 / 33
pso-MIR406 / AAUCGUUUGAAUGCUAUUGUAAUUAUGUAUGGGUAUGAGCUAAAUAUUUCAUCCUAGGAAACAAUGUUUUACAUUAGUUUAAAGGAA / 34,5 / 9 / 18,4 / 37,9
pso-MIR159c / AGAGUGUUGAUUGGAUUGAAGGGAAAUUUCCUAAUUAUCUUAGUCUUGCUUUGAUGUCUUU / 23 / 9 / 23 / 44,3
pso-MIR169n / ACACAAGAACCAAGCCAAGAAUGAAUUGCCUGAGAAACACCAGUUACCCUGGGGGGAAUCCACUUCUGGUUGCUAGGGUUCAUAUUCCAGCAUCACUGUUGAU / 30,1 / 24,3 / 22,3 / 23,3
pso-MIR166g / AUGUGAUGGGCAUGUUCAUAUGUAUUUUCAUCAUGGAUAGAUCUCGGACCAGGCUUCAUUCCUUACU / 22,4 / 19,4 / 22,9 / 37,3
pso-MIR159 / AUGACCCAUCAACCAGUGAAUAUAAGAUAGAACCCGUAUAUGUUCCCUAGAUCAAGAAAAAAGAAAGAAAAAAAUUGUUUGGCCUUUGAAGGGAGCUCUGCAUUGUAUAAAUAAGUUGAGGGGAAAUUUAUCAAUAC / 35,5 / 28,9 / 11,9 / 20,7
pso-MIR446 / CUUUAGCUUCUUAGCUAGGUUGGAUCAUAUGAAUAUGGGAAGUGGUCAUCCAAGGCUAAGCUUAGGGGUUCGACUCCAUUCUUCUUCCUUUCAUUACCGUUAUGUUACCUAGCCUUAGUC / 20,8 / 21,7 / 20,8 / 36,7
pso-MIR476a / GUUUUACUGACUAGGGUUUUCCCAAUAUUCCUAACAGAGAAGAGCGAUGGUGCUUUGAUAUCAUACCAAUCCUUCUUUGCAAAUGGAU / 27,3 / 19,3 / 19,3 / 34,1
pso-MIR414 / CAUUUGCAGCAGGGUCAUCAUUAUCUUCAUCAUCCUCGUCACUCUCGGAGUUUUCAUGAG
AAGGAAUAUUUGAAGGAGAGGAAGAACGGAUUUUUCUCUUC / 25,7 / 19,8 / 21,8 / 32,7

Supplementary Table 4. List of computer basedpredicted targets ofP. somniferummiRNAs using miRU plant target finder

miRNA / Sequence / miRU / mismatch
pso-MIR169a / UUGGCAAAUCAUCCUUGGCUG / At4g09780.1 68417.m01607 meprin and TRAF homology domain-containing protein / MATH domain-containing protein low similarity to ubiquitin-specific protease 12 [Arabidopsis thaliana] GI:11993471; contains Pfam profile PF00917: MATH domain / 4
pso-MIR397a / UCAUUGAGCGCAGCGUUGAUU / At2g29130.1 68415.m03541 laccase, putative / diphenol oxidase, putative similar to laccase / 2
pso-MIR771 / GUUUAUCUGUGGUAGCCCUCG / At3g62600.1 68416.m07032 DNAJ heat shock family protein similar to DnaJ homolog subfamily B member 11 precursor / 4
pso-MIR781 / UUGGUGUUUUUCUGGAUACUU / At5g48570.1 68418.m06007 peptidyl-prolyl cis-trans isomerase, putative / FK506-binding protein, putative similar to rof1 [Arabidopsis thaliana] GI:1373396 / 3
pso-MIR835 / AACUUGCAUAUGUUCUUUAGC / At4g14560.1 68417.m02242 auxin-responsive protein / indoleacetic acid-induced protein 1 (IAA1) identical to SP|P49677 Auxin-responsive protein IAA1 (Indoleacetic acid-induced protein 1) {Arabidopsis thaliana} / 4
pso-MIR844 / UUGUAAGAUUGCUUAUAAGAU / At2g17820.1 68415.m02064 histidine kinase 1 99% identical to GP:4586626 / 3
pso-MIR859 / UCUCUCAGUUGUGAAGUCAAU / At3g04250.1 68416.m00449 F-box family protein-related contains TIGRFAM TIGR01640: F-box protein interaction domain / 3
pso-MIR172a / CUGCUCUUGAUGAUGCUGCAG / At3g45630.1 68416.m04928 RNA recognition motif (RRM)-containing protein similar to SP|P34909 General negative regulator of transcription subunit 4 {Saccharomyces cerevisiae}; contains InterPro entry IPR000504: RNA-binding region RNP-1 (RNA recognition motif) (RRM) / 5
pso-MIR169b / CAGCCAAGGAUGAUUUGCCAA / At5g37020.1 68418.m04440 auxin-responsive factor (ARF8) identical to auxin response factor 8 GI:4104931 from [Arabidopsis thaliana] / 4
pso-MIR169d / GGAGCCAAGGAUGACUUACUG / At1g72830.1 68414.m08421 CCAAT-binding transcription factor (CBF-B/NF-YA) family protein contains Pfam profile: PF02045 CCAAT-binding transcription factor (CBF-B/NF-YA) subunit B / 4
pso-MIR171b / UUGACAGCCGUGCCAAUAUC / No hit found
pso-MIR172d / GGAUCUUGAUGAUGCUGGUA / No hit found
pso-MIR397b / UUAUUGAGUGCAGCAUUGAUG / >At2g29130.1 68415.m03541 laccase, putative / diphenol oxidase, putative similar to laccase [Liriodendron tulipifera][GI:1621467] / 2
pso-MIR406 / UUGAAUGCUAUUGUAAUUAUG / At1g30350.1 68414.m03711 pectate lyase family protein similar to pectate lyase GP:14289169 from [Salix gilgiana];contains Pfam profile: PF00544: Pectate lyase / 4
pso-MIR169n / AAGCCAAGAAUGAAUUGCCUG / At3g20910.1 68416.m02643 CCAAT-binding transcription factor (CBF-B/NF-YA) family protein contains Pfam profile: PF02045 CCAAT-binding transcription factor (CBF-B/NF-YA) subunit B / 2
pso-MIR166g / UCGGACCAGGCUUCAUUCCUU / At1g52150.1 68414.m05884 homeobox-leucine zipper family protein / lipid-binding START domain-containing protein similar to to HD-zip transcription factor (athb-8) (GI:7270235) [Arabidopsis thaliana]; contains Pfam profiles PF01852: START domain, PF00046: Homeobox domain / 3
pso-MIR159 / UUUGGCCUUUGAAGGGAGCUCUG / At3g30843.1 68416.m03970 hypothetical protein / 6
pso-MIR446 / GAUCAUAUGAAUAUGGGAAGUGG / At2g19130.1 68415.m02233 S-locus lectin protein kinase family protein contains Pfam domains PF00954: S-locus glycoprotein family, PF00069: Protein kinase domain and PF01453: Lectin (probable mannose binding) / 6
pso-MIR476a / UACCAAUCCUUCUUUGCAAAU / At3g07550.1 68416.m00901 F-box family protein (FBL12) contains similarity to F-box protein FBL6 GI:6456737 / 3
pso-MIR414 / UAUCUUCAUCAUCCUCGUCA / No hit found