Supplementary Table S1. The probes and primers used in this study.
Probes for northern blotsGhmiR156 / GTGCTCTCTATCTTCTGTCAA
GhmiR160 / TGGCATACAGGGAGCCAGGCA
GhmiR164 / TGCACGTGCCCTGCTTCTCCA
GhmiR165/166 / GGGGAATGAAGCCTGGTCCGA
GhmiR167 / TGAGATCATGCTGGCAGCTTCA
GhmiR171 / GATATTGGCACGGCTCAATCA
GhmiR172 / CTGCAGCATCATCAGGATTCT
GhmiR396 / AAGTTCAAGAAAGCTGTGGAA
GhmiR397 / CATCAACGCTGCACTCAATGA
GhmiR160-3p.2 / ATACATGCATGGCTCCTCATA
GhmiRn13 / TGAAGGAGACATCTAAAGTAA
GhmiRn22 / ATGACGTGGAATCACAGTTTCATA
GhmiRn33 / TCAAACGATTCCTGGCTGATT
U6 / TGTATCGTTCCAATTTTATCGGATGT [1]
Primers for 5′ RACE mapping of miRNA cleavage sites
DW504749 outer primer / GATGGGAAAGAGAAATGGGACGAA
DW504749 inner primer / TGCTGCTCAAGTTCCATGTGTTGCAC
ES822760 outer primer / CGATAGGATGGTATGTCGAA
ES822760 inner primer / ACAAACTGAACGAACGGCGAAACC
TC220992 outer primer / TGATGCTCAGATTTTCCGACACCT
TC220992 inner primer / CACTTAGTGCTGCCATATTCTCGTTC
TC154649 outer primer / ATCCTCCCGATCTCCTTCCACAGT
TC154649 inner primer / TTGCTGAACTCCAGGAAATGTGTCGG
DW224951 outer primer / TTTGTGATTATGATGGGATGGATG
DW224951 inner primer / TTTGAGCAGAACATTTGTGGTTTGGC
Primers for quantitative real-time PCR
DW504749 forward / GAGAATGCTTCACGGGAGT
DW504749 reverse / TTCGGTGTTCATCATGTCG
ES822760 forward / TTCTCACACGCGCAAGGGAT
ES822760 reverse / TAACAAACTGAACGAACGGC
TC220992 forward / ATTATCGGTCTCGTCTCA
TC220992 reverse / TTCTCTTCCTTTTGCTTG
TC154649 forward / TTATGAGTGGGGACGGGTTA
TC154649 reverse
TC82706 forward
TC82706 reverse
TC132940 forward
TC132940 reverse
DW226555 forward
DW226555 reverse / CACAGTTTCACCTTCTTCCAA
GTACCTAACATGCTTCCCGTCCAC [1]
TATGCCTGCAGGAGCATTATCAGA [1]
TAGTTAGGGATCTAAGGGCATTTGGG
CAAAGACAAGGCAACCATGGAAATGG
AACTTGATAAGGGGATTGGAGC
AGAAGGTAACGGGGGAAGGACA
Histone3 (AF024716.1) forward / TCAAGACTGATTTGCGTTTCCA [1]
Histone3 (AF024716.1) reverse / GCGCAAAGGTTGGTGTCTTC [1]
Reference
[1] Pang,M., Woodward,A.W., Agarwal,V., Guan,X., Ha,M., Ramachandran,V., Chen,X., Triplett,B.A., Stelly,D.M. and Chen,Z.J. (2009) Genome-wide analysis reveals rapid and dynamic changes in miRNA and siRNA sequence and expression during ovule and fiber development in allotetraploid cotton (Gossypium hirsutum L.). Genome Biol., 10, R122.
Supplementary Table S2. Overview of the deep sequencing data.
WT library / MU libraryAll reads / Distinct reads / All reads / Distinct reads
Raw reads / 7268821 / 6738944
High quality reads / 6137588 / 3855471
Clean reads / 5896335 / 3432703 / 3747020 / 2165655
Matching reads / 1500303 / 643656 / 990535 / 446657
Supplementary Table S3. Conserved cotton miRNA families.
miRNA family / Sequencea / Length / Count in WTb / Count in MUc / Target gene family156/157d / UUGACAGAAGAUAGAGAGCAC / 21 / 22808 / 32026 / Squamosa promoter-binding protein SPL9
159d / UUUGGAUUGAAGGGAGCUCUA / 21 / 592 / 790 / 3-Ketoacyl-CoA synthase 2, F-box family protein
160 / UGCCUGGCUCCCUGUAUGCCA / 21 / 566 / 386 / Auxin response factor
160-3p / GCGUAUGAGGAGCCAUGCAUG / 21 / 358 / 131 / Gibberellin induced protein
162d / UCGAUAAACCUCUGCAUCCAG / 21 / 221 / 286 / Ubiquitin-protein ligase
164d / UGGAGAAGCAGGGCACGUGCA / 21 / 792 / 302 / Transcriptional factor NAC35, UDP-glucuronic acid decarboxylase2, Ubiquitin-conjugating enzyme
165/166d / UCGGACCAGGCUUCAUUCCCC / 21 / 49838 / 22150 / Heat shock protein, SET domain-containing protein
167 / UGAAGCUGCCAGCAUGAUCUCA / 22 / 358826 / 539077 / LIM domain protein
168 / UCGCUUGGUGCAGGUCGGGAA / 21 / 1113 / 674 / Glucose-methanol-choline (gmc) oxidoreductase, Dead box ATP-dependent RNA helicase
169 / CAGCCAAGGAUGACUUGCCGA / 21 / 268 / 231 / Nuclear transcription factor Y subunit A-3, CCAAT-binding transcription factor family protein
170/171 / UGAUUGAGCCGUGCCAAUAUC / 21 / 753 / 570 / GRAS family transcription factor, SCL6
172d / AGAAUCCUGAUGAUGCUGCAG / 21 / 45629 / 50664 / Transcription factor APETALA2, Protein AINTEGUMENTA
390d / AAGCUCAGGAGGGAUAGCGCC / 21 / 1493 / 1179 / Leucine-rich repeat transmembrane protein kinase, BRASSINOSTEROID INSENSITIVE 1 precursor
393d / UCCAAAGGGAUCGCAUUGAUUU / 22 / 79 / 85 / Transport inhibitor response 1
394d / UUGGCAUUCUGUCCACCUCC / 20 / 49 / 47 / F-box family protein
395 / CUGAAGUGUUUGGGGGAACUC / 21 / 9 / 2 / Cellulose synthesis, Transferase
396 / UUCCACAGCUUUCUUGAACUU / 21 / 20 / 39 / ATP binding protein
396-3p / GCUCAAGAAAGCUGUGGGAGA / 21 / 127 / 83 / Flavonoid 3',5'-hydroxylase
397 / UCAUUGAGUGCAGCGUUGAUG / 21 / 70 / 20 / Laccase-like protein
399 / UGCCAAAGGAGAUUUGCCCGG / 21 / 57 / 25 / MYB transcription factor, Clathrin heavy chain, Ethylene-responsive element-binding protein
403 / UUAGAUUCACGCACAAACUCG / 21 / 11 / 25 / Argonaute protein group, Cytoplasmic superoxide dismutase
408 / AUGCACUGCCUCUUCCCUGGC / 21 / 4 / 6 / Basic blue copper protein
473/477 / ACUCUCCCUCAAGGGCUUCUG / 21 / 14 / 2 / Unknown
482d / UUGCCUACUCCACCCAUGCCAC / 22 / 113 / 127 / NBS-type resistance protein
530 / UGCAUUUGCACCUGCACCUUC / 21 / 12 / 13 / Transcription factor C2H2
535 / UGACAAUGAGAGAGAGCACGC / 21 / 1316 / 1495 / Unknown
827d / UUAGAUGACCAUCAACAAACA / 21 / 46 / 41 / ASPARTATE KINASE 3, Expp1 protein precursor
894 / CGUUUCACGUCGGGUUCACCA / 21 / 3137 / 3379 / Unknown
1310 / AGGCAUCGGGGGCGCAACGCC / 21 / 19 / 2 / Cyclin-dependent protein kinase
2111 / UAAUCUGCAUCCGGAGGUUUG / 21 / 67 / 68 / F-box family protein, Cryptochrome 2
2218 / UUGCCGAUUCCACCCAUGCCUA / 22 / 67 / 127 / NBS-type resistant protein, Cinnamate-4-hydroxylase
2911 / GGCCGGGGGACGGACUGGGA / 20 / 25 / 13 / Beta-tubulin 8
2916 / UGGGGGCUCGAAGACGAUCAG / 21 / 8 / 2 / Cytochrome b5
2947d / UAUACCGUGCCCAUGACU / 18 / 2959 / 2655 / Unknown
2948-5pd / UGUGGGAGAGUUGGGCAAGAAU / 22 / 221 / 36 / Cytochrome P450
2949d / ACUUUUGAACUGGAUUUGCCGA / 22 / 672 / 1089 / Metal-dependent phosphohydrolase HD domain-containing protein
2950d / UGGUGUGCAGGGGGUGGAAUA / 21 / 10 / 0 / Cyclopropane fatty acid synthase
2950-5pd / UUCCAUCUCUUGCACACUGGA / 21 / 8 / 17 / F-box family protein, SEP3-related MADS-box protein
3476d / UGAACUGGGUUUGUUGGCUGC / 21 / 669 / 678 / Putative phosphatidylinositol 4-phosphate 5-kinase
3711-3p / CAGGCCCUCCUUCUAGCGCCA / 21 / 48 / 36 / Glutathione s-transferase, putative
3954 / UUGGACAGAGUAAUCACGGUCA / 22 / 62 / 46 / Unknown
aSequences in this table represent the most abundant miRNA sequences in each miRNA family identified by Solexa sequencing.
bCount in WT indicates the original sequencing counts in the wild type.
cCount in MU shows the normalized results from the sequencing counts in the fl mutant, considering the difference of total sequencing reads between the wild type and the fl mutant.
dmiRNAs whose precursors have been identified and could adopt standard stem-loop structures.
Supplementary Table S4. Conserved cotton miRNA families homologous to known miRNAs from other species.
miRNA / Coniferophyta & Embryophyta / MagnoliophytaEudicotyledons / Monocotyledons
pab / pta / ppt / ath / ptc / vvi / mtr / peu / csi / osa / sbi / zma
156/157 / + / + / ++ / ++ / ++ / ++ / + / + / + / +
159 / + / ++ / ++ / ++ / ++ / ++ / + / + / +
160 / ++ / ++ / ++ / ++ / ++ / ++ / + / ++ / ++ / ++
160-3p / +
162 / ++ / ++ / ++ / ++ / ++ / ++ / ++ / +
164 / ++ / ++ / ++ / ++ / ++ / ++ / ++ / ++
165/166 / ++ / ++ / ++ / ++ / ++ / ++ / ++ / ++ / + / ++
167 / + / + / + / ++ / + / + / + / + / +
168 / ++ / ++ / ++ / + / + / + / +
169 / ++ / ++ / ++ / ++ / + / ++ / ++ / ++
170/171 / + / ++ / ++ / ++ / + / ++ / ++ / ++
172 / + / + / + / ++ / + / + / + / +
390 / + / ++ / ++ / ++ / ++ / ++ / ++ / ++ / ++ / ++
393 / + / + / + / + / + / + / + / +
394 / ++ / ++ / ++ / ++ / ++ / ++ / ++
395 / + / ++ / ++ / ++ / + / ++ / + / + / +
396 / + / ++ / ++ / ++ / + / ++ / + / ++ / ++ / ++
396-3p / +
397 / + / ++ / ++ / ++ / ++ / ++ / ++ / +
399 / ++ / ++ / ++ / + / + / + / + / +
403 / ++ / ++ / ++ / ++
408 / ++ / + / ++ / ++ / ++ / ++ / + / + / +
473/477 / + / + / + / +
472/482 / + / + / + / + / + / +
530 / + / + / +
535 / + / + / + / + / +
827 / + / + / ++ / + / +
894 / +
1310 / +
2111 / + / + / +
2218 / +
2911 / +
2916 / +
2947
2948-5p
2949
2950 / ++
2950-5p / ++
3476
3711 / ++
3954 / +
Sequence used for conservation analysis represents the most abundant sequence in each miRNA family identified by deep sequencing. The plus symbols indicate: ++, miRNA sequences of cotton were precisely identical to those in other species; +, miRNA sequences of cotton were conserved in other species but have variations in some nucleotide positions. pab, Picea abies; pta, Pinus taeda; ppt, Physcomitrella patens; ath, Arabidopsis thaliana; ptc, Populus trichocarpa; vvi, Vitis vinifera; mtr, Medicago truncatula; peu, Populus euphratica; csi, Citrus sinensis; osa, Oryza sativa; sbi, Sorghum bicolor; zma, Zea mays.
Supplementary Table S5. Target prediction for novel miRNAs.
miRNA / Predicted target / Mismatch / E-value / Target gene annotationGhmiRn1 / DT458988 / 1 / 7.00E-34 / Root phototropism protein, putative [Ricinus communis]
7.00E-33 / unnamed protein product [Vitis vinifera]
4.00E-32 / predicted protein [Populus trichocarpa]
DT051920 / 1.5 / 6.00E-19 / unnamed protein product [Vitis vinifera]
4.00E-17 / hypothetical protein [Vitis vinifera]
1.00E-15 / PREDICTED: hypothetical protein [Vitis vinifera]
CO110739 / 2 / 2.00E-05 / unknown [Medicago truncatula]
2.00E-04 / Inhibitor of growth protein, putative [Ricinus communis]
0.006 / unknown [Glycine max]
CO123727 / 2 / 4.00E-14 / unnamed protein product [Vitis vinifera]
4.00E-14 / PREDICTED: hypothetical protein isoform 1 [Vitis vinifera]
4.00E-14 / PREDICTED: hypothetical protein isoform 2 [Vitis vinifera]
TC178897 / 2 / 4.00E-45 / dehydroascorbate reductase, putative [Ricinus communis]
4.00E-45 / predicted protein [Populus trichocarpa]
5.00E-45 / DHAR class glutathione transferase DHAR1
TC155150 / 2.5 / 2.00E-23 / protein binding protein, putative [Ricinus communis]
2.00E-23 / unnamed protein product [Vitis vinifera]
2.00E-23 / predicted protein [Populus trichocarpa]
TC133459 / 2.5 / 6.00E-11 / predicted protein [Populus trichocarpa]
4.00E-09 / predicted protein [Populus trichocarpa]
5.00E-08 / myosin vIII, putative [Ricinus communis]
CO077276 / 2.5 / 3.00E-24 / predicted protein [Populus trichocarpa]
3.00E-24 / unknown [Populus trichocarpa x Populus deltoides]
4.00E-22 / unnamed protein product [Vitis vinifera]
TC179336 / 3 / 2.00E-101 / ABC transporter [Gossypium hirsutum]
3.00E-99 / hypothetical protein [Vitis vinifera]
3.00E-99 / PREDICTED: hypothetical protein [Vitis vinifera]
ES831616 / 3 / 2.00E-41 / unnamed protein product [Vitis vinifera]
8.00E-41 / PREDICTED: hypothetical protein [Vitis vinifera]
4.00E-40 / zinc finger protein, putative [Ricinus communis]
ES799029 / 3 / 4.00E-09 / GTP binding protein, putative [Ricinus communis]
3.00E-08 / predicted protein [Populus trichocarpa]
2.00E-07 / PREDICTED: hypothetical protein [Vitis vinifera]
DW488974 / 3 / 2.00E-08 / hypothetical protein [Zea mays]
6.00E-08 / unknown [Glycine max]
6.00E-08 / predicted protein [Populus trichocarpa]
DT464501 / 3 / 3.00E-45 / predicted protein [Arabidopsis lyrata subsp. lyrata]
3.00E-45 / CPK16; ATP binding / calcium ion binding / calmodulin-dependent protein kinase/ protein kinase/ protein serine/threonine kinase [Arabidopsis thaliana]
6.00E-45 / PREDICTED: hypothetical protein [Vitis vinifera]
CO103802 / 3 / 7.00E-61 / unnamed protein product [Vitis vinifera]
7.00E-61 / PREDICTED: hypothetical protein [Vitis vinifera]
4.00E-60 / protein phosphatase 2c, putative [Ricinus communis]
CO073602 / 3 / 2.00E-76 / predicted protein [Populus trichocarpa]
6.00E-75 / PREDICTED: hypothetical protein [Vitis vinifera]
6.00E-72 / Ethanol tolerance protein GEKO1, putative [Ricinus communis]
GhmiRn2 / TC146877 / 2.5 / 1.00E-56 / defender against cell death 1 [Gossypium hirsutum]
6.00E-53 / hypothetical protein LBL11 [Panax quinquefolius]
6.00E-53 / DAD1 [Petunia x hybrida]
TC140391 / 2.5 / 2.00E-42 / defender against cell death 1 [Gossypium hirsutum]
2.00E-42 / homolog to defender against apoptotic death 1 [Citrus unshiu]
9.00E-42 / defender against cell death, putative [Ricinus communis]
FL576578 / 3 / 3.00E-11 / gag/pol polyprotein [Pisum sativum]
4.00E-11 / gag/pol polyprotein [Pisum sativum]
2.00E-09 / RNA-directed DNA polymerase (Reverse transcriptase); Ribonuclease H [Medicago truncatula]
TC161005 / 3 / 6.00E-53 / PREDICTED: hypothetical protein [Vitis vinifera]
6.00E-53 / hypothetical protein [Vitis vinifera]
3.00E-52 / PREDICTED: hypothetical protein [Vitis vinifera]
GhmiRn3 / - / - / - / -
GhmiRn4 / - / - / - / -
GhmiRn5 / CO073333 / 1 / 1.00E-75 / ABC transporter [Gossypium hirsutum]
6.00E-73 / PREDICTED: hypothetical protein [Vitis vinifera]
6.00E-73 / hypothetical protein [Vitis vinifera]
DR456441 / 1 / 3.00E-05 / ABC transporter [Gossypium hirsutum]
6.00E-05 / PREDICTED: hypothetical protein [Vitis vinifera]
6.00E-05 / hypothetical protein [Vitis vinifera]
TC179336 / 1 / 2.00E-101 / ABC transporter [Gossypium hirsutum]
3.00E-99 / hypothetical protein [Vitis vinifera]
3.00E-99 / PREDICTED: hypothetical protein [Vitis vinifera]
TC140355 / 1.5 / 2.00E-92 / calcium ion binding protein, putative [Ricinus communis]
1.00E-89 / predicted protein [Populus trichocarpa]
3.00E-89 / predicted protein [Populus trichocarpa]
TC171089 / 1.5 / 2.00E-25 / unnamed protein product [Arabidopsis thaliana]