research article peculiar evolutionary history of mir390...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 924153 14 pageshttpdxdoiorg1011552013924153

Research ArticlePeculiar Evolutionary History of miR390-GuidedTAS3-Like Genes in Land Plants

Maria S Krasnikova1 Denis V Goryunov1 Alexey V Troitsky1 Andrey G Solovyev1

Lydmila V Ozerova2 and Sergey Y Morozov1

1 Belozersky Institute of Physico-Chemical Biology Lomonosov Moscow State University Moscow 119991 Russia2Main Botanic Garden Russian Academy of Sciences Botanicheskaya 4 Moscow 127276 Russia

Correspondence should be addressed to Lydmila V Ozerova lyozerovayandexru

Received 6 July 2013 Accepted 27 August 2013

Academic Editors B D Gregory and S Mlotshwa

Copyright copy 2013 Maria S Krasnikova et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

PCR-based approach was used as a phylogenetic profiling tool to probe genomic DNA samples from representatives of evolutionarydistant moss taxa namely classes Bryopsida Tetraphidopsida Polytrichopsida Andreaeopsida and Sphagnopsida We foundrelatives of all Physcomitrella patens miR390 and TAS3-like loci in these plant taxa excluding Sphagnopsida Importantly cloningand sequencing ofMarchantia polymorpha genomic DNA showedmiR390 and TAS3-like sequences which were also found amonggenomic reads of M polymorpha at NCBI database Our data suggest that the ancient plant miR390-dependent TAS molecularmachinery firstly evolved to target AP2-like mRNAs in Marchantiophyta and only then both ARF- and AP2-specific mRNAs inmossesThe presented analysis shows that moss TAS3 families may undergone losses of tasiAP2 sites during evolution toward fernsand seed plants These data confirm that miR390-guided genes coding for ARF- and AP2-specific ta-siRNAs have been graduallychanged during land plant evolution

1 Introduction

MicroRNAs (miRNAs) are a class of small interfering RNAs(siRNAs) that are 21ndash24 nt in length and predominantlyfunction to repress gene expression at posttranscriptionallevel Many miRNAs found to be ubiquitously present in awide range of animals plants (including algae) and someanimal viruses [1ndash7] Various miRNA families were foundto be highly conserved across the plant kingdom [3 7ndash10]Plant miRNAs are transcribed by RNA polymerase II to gen-erate primary capped and polyadenylated miRNA (pri-miR)transcripts These pri-miRs range from few hundred bases toseveral thousand bases and contain a partly complementarydouble-stranded hairpin structure [1 3 4 7 8 11 12] Anonperfect duplex of 21 nt RNAs (miRNAmiRNAlowast duplex)with characteristic two-nucleotide 31015840 overhangs is excisedfrom this hairpin precursor by a DCL (Dicer-like) proteinand accessory factors (Figure 1(a)) Each strand of duplex ismethylated on its 31015840 nucleotide by HEN1 Then one strand of

the excised duplex is incorporated into a complex containingan AGO slicer protein thereby becoming a mature miRNA[1 3 8 9 11 12]

Highly conserved plantmiRNAmiR390 forms a special-ized complex with slicer AGO7 [13] The activity of the latterprotein is required to stably associate this complex with the 51015840site and cleave the 31015840 site of miR390 dual target sites in TAS3precursor transcripts to start pathway resulting in trans-acting siRNAs (ta-siRNAs) [1 14 15] Following miRNA-directed cleavage tasiRNAprecursor transcripts enter into anRDR6- and SGS3-dependent pathway and are processed byDICER-like4 (DCL4) protein into phased 21 nucleotide ta-siRNAs [16] In general ta-siRNAs are subclass of so-calledphasiRNAs [1 14 15] In dicots the TAS3 ta-siARF RNAsregulate leaf patterning developmental timing and rate oflateral root growth by repressing the Auxin Response Factors(ARF-2 -3 and -4) [17ndash19] It is currently known that manyimportant processes of plant development and physiology(including those in mosses see [20 21]) are regulated by

2 The Scientific World Journal

5998400 3998400

5998400-a

rm

3998400-a

rm

(a)

P patens ppt-MIR390a (minimum free energy of minus6440 kcalmol)CAUGAUACAAUUACGAAGCUCAGGAGGGAUAGCGCCAUUCUCUGUUCUUCUCUAUCGACACUUGGUAAACCAGCUUGUGCUAGCUAUGGGCAGAGAUAUGGCGUUAUCCAUUCUGAGCUUUGCAAGUGUCUCGUG

(((((((((((((((((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))))))))))))))))

ppt-MIR390b (MFE of minus6400 kcalmol)UGCUGGGAGAUACAAUUACGAAGCUCAGGAGGGAUAGCGCCCAGCCCUGCUUUUACUGUUCAAUCAGUUAGAGUGCUUCUGAUAGUAAGAUGCAGAUGCAUGACGCUAUCCAUUCUGAGCUUUGCAACUGUGUCCCCCUAAUUUGGGCA

((((((((((((((((((((((((((((((((((((((((((((((((((((((((()))))))))))))))))))))))))))))))))))))))))))))))))))))))))

ppt-MIR390c (MFE of minus6230 kcalmol)GGUAUAAUUACAGAGCUCAGGAGGGAUAGCGCCCAUCUCAGUCUUUUUUAGCAUCCAAUUGAAAUGGUUAACAUUCCUUUGGAUAGCUAGACGGCGAAGCAUGGCGCUGUCCAUUCUGAGCAUUGCAUUUGUACC

(((((((((((((((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))))))))))))))

Pohlia nutans (MFE of minus5720 kcalmol)GGAUGAUACCAUUACGAAGCUCAGGAGGGAUAGCGCCUGCCUCUGUUUCUUCCUUGCAGCAGUCGUCGCAUGGCGCUAUCCAUUCUGAGCUUUGCAACUGUGUCGUCC

(((((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))))

Ceratodon purpureus (MFE of minus6400 kcalmol) GGACGAUACAAUUACCAAGCUCAGGAGGGAUAGCGCCUAGCUCUGUUCUUCUGUCACACUGUGAUAACAGAGUGCCAGACGUAUGGCGCUAUCCAUUCUGAGCUUUGCAAGUGUGUCGUCC

(((((((((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))))))))

Brachitecium rivulare (MFE of minus4980 kcalmol) GGACGACACAGUUGCAAAGCUCAGAAUGGAUAGCGCCGUGCUACUGUCCCAAACUUGAUUUCAACAGAGGUAGGCGCUAUCCCUCCUGAGCUUCGUAACGGUAUCAUCC

(((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))

Marchantia polymorpha (MFE of minus6960 kcalmol) AGGUUGGUCAGAGCGACAAAGCUCAGGAGGGAUAGCGUCUGCUUUUCCGUCAGGCUCGUGUGGGGCACGGGUACUUGUUCCUGGCUGUGGAGUUUACGCUAUCCAUUGUGAGCUCUGUCGCUCUCCCUUCCU

((((((((((((((((((((((((((((((((((((((((((((((((()))))))))))))))))))))))))))))))))))))))))))))))))

Timmia austriaca Tau-miR390a (MFE of minus5010 kcalmol)AGCUCAGGAGGGAUAGCGCCCAGCUCUGUUCUUCUGUAUCAUUGCUUGGUAAACAAGUAUUUGACAGUGAAGCGCAGAUGUAUGGCGCUCUCCAUUCUGAGCA

((((((((((((((((((((((((((((((((((((()))))))))))))))))))))))))))))))))))))

Timmia austriaca Tau-miR390b (MFE of minus5610 kcalmol)AGCUCAGGAGGGAUAGCGCCUAUGUUUUCUGUUACUCUGUUACAUUGCUUAUUGUUUAAGCUUGUGACAGUUAAGUGCAGAAGUAAGGCGUUAUCCAUUCUGAGCU

((((((((((((((((((((((((((((((((((((((((((()))))))))))))))))))))))))))))))))))))))))))

Tetraphis pellucida (MFE of minus4810 kcalmol)AGCUCAGGAGGGAUAGCGCCUAGCUCUUGGUUUCUGUGGCAUUGCUGUCAUUUGAAGCUCUGUAGCAGGAAAGGCUUUGAAGCAAUGCGCUAUCCAUUCUGAGCU

((((((((((((((((((((((((((((((((((((((()))))))))))))))))))))))))))))))))))))))

Polytrichum commune (MFE of minus6380 kcalmol)AGCUCAGGAGGGAUAGCGCCUAGCUCUGGAGCCACUCUGCUGUUUCACCAGUUGUGGAAAACUGGUUGUUCACCAGUCAGUCCCAGACGUACAGCGCUAUCCCUCCUGAGCU

(((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))

Oedipodium griffithianum (MFE of minus4950 kcalmol)AGCUCAGGAGGGAUAGCGCCUAGCCCUGGUCUCUUUGUGGCAUUGCUUUUCAUCGGAAGCUGUGUACCAAUUAGGCUCAGAAGGAAAGCGCUAUCCAUUCUGAGCU

(((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))

Andreaea rupestris (MFE of minus4910 kcalmol)AGCUCAGGAGGGAUAGCGCCCAACUCUGUACUAGAACUUCUGUAACUAUAUCUCAUUGUUUGGUAAACAGCCUGCAUUUGACAGUUCUUUGCAGAAGUGUGGCGUUAUCCAUUCUGAGCU

(((((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))))

Harpanthus flotovianus (MFE of minus4710 kcalmol) AGCUCAGGAGGGAUAGCGCCAAAUUCUGGCCUUUUUGUGGGAUCGCUUUCGAUGAAAGCUUGCACAGUUUGGUUCAGAUGCAAAUCGCUAUCCAUUCUGAGCU

((((((((((((((((((((((((((((((((((((()))))))))))))))))))))))))))))))))))))

(b)

Figure 1 (a) Schematic representation of a typicalmiRNAhairpin showing the position of the lowerDCL processing site (filled and open darkgray arrowheads) upper DCL processing site (filled and open gray arrowheads) and the positions of potential mature miRNA or miRNAlowast(filled and open arrowheads resp) (b) Primary and predicted secondary structures of stem-loop elements in Bryophyta andMarchantiophytamiR390 precursors Sequences of the highly conserved AGCU tetranucleotides at the lower DCL processing sites of miRNA or miRNAlowast areunderlined Names of plant species with sequenced portions limited by conserved AGCU tetranucleotides are underlined

The Scientific World Journal 3

numerous classes of phasiRNAs [1 14 15] The discovery thatorganisms share similar ldquodevelopmental genesrdquo as well as fun-damental aspects of their ontogeny opens the door towards amolecular understanding of development using comparisonof gene structuresThus orthologous genesmay be studied inspecies occupying key phylogenetic positions to deduce cor-relations between the molecular changes that were responsi-ble for evolutionary events in distinct species of interest [22]

Previously we described a new method for identifi-cation of plant ta-siARF precursor genes based on PCRwith oligodeoxyribonucleotide primers mimicking miR390The method was found to be efficient for genomic DNAand cDNA of dicotyledonous plants cycads and conifers[23] The PCR-based approach was used as a comparativeprofiling tool to probe genomic DNA samples from speciesbelonging to classes Bryopsida and Sphagnopsida although itis only partly suitable for a comprehensive description of allTAS3 genes in a particular organism [24] Previously therewere no other papers connected to phylogenetics aspectsof TAS3 genes in species outside flowering plants namelypines cycads ferns horsetails clubmosses and liverwortsIn mosses the only plant where TAS genes were studiedin details was Physcomitrella patens (see below) This papercombines our current data and new findings of other researchgroups to uncover a peculiar picture of an evolution of TAS3-like genes

2 Materials and Methods

21 Plant Material Plant material was taken from the col-lections of the NV Tsytsin Main Botanical Garden of theRussian Academy of Sciences and the Biological Faculty ofM V Lomonosov Moscow State University

22 Analysis of Nucleic Acids Genomic plant DNA wasisolated from 200mg of fresh or dried plant material by DNAextraction kit (Macherey-Nagel) according to the protocol ofthe manufacturer TAS3 genes were amplified and sequencedas described in [23 24] DNA sequences were deposited atthe NCBI data bank and the accession numbers are shownin Table 1

Total RNA was isolated from green parts of plantswith the Trizol reagent according to the manufacturerrsquosinstructions (Invitrogen) Digestion of any contaminatingDNA was achieved by treatment of samples with RQIRNase-free DNase (Promega) Reverse transcription wasperformed with 1 120583g of total RNA and oligo (dT)-primer t20-xho (attctcgaggccgaggcggccgacatgtttttttttttttttttttttttttv) usingthe RT system (Invitrogen) according to the protocolof the manufacturer Primers for dicotyledonous plantswere forward primer TAS-P (51015840-GGTGCTATCCTATCT-GAGCTT-31015840) and mixture of reverse primers TAS-Mcaa(51015840-AGCTCAGGAGGGATAGCAA-31015840) and TAS-Maca (51015840-AGCTCAGGAGGGATAGACA-31015840) For PCR 25ndash35 cycleswere used for amplification with a melting temperature of95∘C an annealing temperature of 58∘C and an extendingtemperature of 72∘C each for 30 seconds followed by a finalextension at 72∘C for 3min PCR products were separated byelectrophoresis of samples in 15 agarose gel and purified

using the GFXTM PCR DNA and Gel Band PurificationKit (AmershamBiosciences) For cloning the PCR-amplifiedDNA bands isolated from gel were ligated into pGEM-T (Promega) Cloned products were used as templates insequencing reactions with the ABI Prism BigDye TerminatorCycle Sequencing Ready Reaction Kit (Applied Biosystems)DNA and cDNA sequences were deposited at the NCBIdata bank the accession numbers are JN692262 JN692261JN692260 and JN692259

23 Computational Sequence Analysis TAS3-like sequencesidentified in this paper and found in the NCBI databasewere compared using multiple alignment tool at MAFFT(version 58) The phylogenetic tree was generated accordingMAFFT6 program (httpmafftcbrcjpalignmentserver)Sequences were additionally analysed at httpblastncbinlmnihgovBlastAligncgi

3 Results and Discussion

31 Diversity of miR390 and TAS3-Like Genes in Land PlantsSequence data for TAS3 genes of dicotyledonous [23 27]and monocotyledonous plants [28] have revealed two TAS3variants two- and one-tasiARF subfamilies respectivelyThefirst subfamily displays canonical structural features wellcharacterized for AtTAS3 in Arabidopsis thaliana [29] thetandem of conserved ta-siARF sequences are flanked by aconstant miR390-targeted site at the 31015840 end and a divergent(noncleavable) region at the 51015840 end In contrast transcriptsof the second subfamily encode only single copy of tasiARFImportantly there was no mismatch in the tenth positionof the 51015840 miR390 target site suggesting that the 51015840 miR390binding site may undergo cleavage for initiation of TAS3precursor RNA processing [14 15 27] Moreover theseshorter TAS3 genes despite the fact that they share thepresence of the dual miR390 with two-tasiARF subfamilyhave the lesser conserved regions flanking the tasiARF areain reverse orientation with a constant region at the 51015840 endand a divergent region at the 31015840 end [23 27]

It is well known that the species of miR390 function asactivators of a ta-siARF pathway [1 3 14 15] In plants mostmiRNA-encoding loci comprise independent nonprotein-coding transcription units miRNA genes are transcribed byRNA polymerase II (pol II) The primary miRNA transcripts(pri-miRNAs) contain cap structures as well as poly(A) tailsLike protein-coding genes promoters of miRNA loci containcanonical cis-promoter elements such as TATAbox and tran-scription initiator and various transcription factor responsiveelements [30] Plant pre-miRNAhairpins sometimes occur ingenomic clusters strongly suggesting expression of multiplehairpins from a single pri-miRNA Most clusters in thesespecies (61 to 90) contain hairpins encoding identicalmature miRNAs suggesting that they were the result of localtandem duplications and serve to increase the dosage of aparticular miRNA from a single promoter [1 9 31]

Gymnosperms and angiosperms are believed to divergefrom a common ancestor gt325 million years ago [32] ManymiRNAs are common between the two phyla [3 8 33] Thiscorrelates with an obvious conservation of the TAS3 and


Table 1 Description of sequenced TAS3-like loci in mosses

Classsubclass Order Species

aClonecluster

bLengthbp

cReference andoraccession number

dOrganization(one- or two-

tasiARFAP2TAS6)

BryopsidaDicranidae

Pottiales Bryoerythrophylluminaequalifolium

27-BinTAS3bce 236 KC812740 tasiARFAP2

Gremiales

Coscinodon humilis 20-ChuTAS3bce 234 Krasnikova et al 2011 [24]

HQ709423 mdashldquomdash

C humilis 16-ChuTAS3bce 228 Krasnikova et al 2011 [24]


Schistidium elegantulum 24-SceTAS3bce 229 Krasnikova et al 2011 [24]


Grimmia cribrosa 43-GcrTAS3bce 228 Krasnikova et al 2011 [24]


Dicranales

Fissidens taxifolius 17-FitTAS3bce 192 Krasnikova et al 2011 [24]


Ceratodon purpureus 48-CpuTAS3bce

231779e SRR07489011659772 tasiARFAP2

TAS6

Amphidium mougeotii 56-AmoTAS3bce 234 KC812745 tasiARFAP2

Leucobryum glaucum 41-LglTAS3bce 226 KC812750 mdashldquomdash

L glaucum 42-LglTAS3bce 192 KC812749 mdashldquomdash

Dicranum pacificum 44-DpaTAS3bce 232 KC812741 mdashldquomdash

BryopsidaBryidae

OrthotrichalesOrthotrichum pumilum 5-Opu

TAS3bce 199 Krasnikova et al 2011 [24]HQ709419 mdashldquomdash

O pumilum 24-OpuTAS3adf 246 Krasnikova et al 2011 [24]


Hypnales

Brachythecium latifolium 47-BrTAS3bce 199 Krasnikova et al 2011 [24]

FJ804748 mdashldquomdash

B latifolium 50-BrTAS3adf 255 Krasnikova et al 2011 [24]


B albicans 37-BalTAS3bce 199 Krasnikova et al 2011 [24]


B rivulare 11-BrarTAS3bce 199 KC812739 mdashldquomdash

B rivulare 36-BrarTAS3adf

2551444e

KC812738(Illumina sequence)

tasiARFAP2TAS6

Homalotheciumphilippeanum

7-HpTAS3bce 199 Krasnikova et al 2011 [24]

HQ709415 tasiARFAP2

Amblystegium sp 52-AmblyTAS3bce 191 KC812762 mdashldquomdash

Oxyrrhynchiumhians 25-OxyTAS3bce 199 Krasnikova et al 2011 [24]


O hians 32-OxyTAS3adf 253 KC812761 mdashldquomdash

Pylaisia polyantha 10-PypTAS3bce 199 Krasnikova et al 2011 [24]


P polyantha 11-PypTAS3adf 253 Krasnikova et al 2011 [24]


Hookeriales Hookeria lucens 49-HTAS3bce 190 Krasnikova et al 2011 [24]


Bryales

Pohlia nutans Pnu-30698TAS3adf

259920e GACA010231801 tasiARFAP2

TAS6

Plagiomnium medium 41-PmeTAS3adf 236 KC812756 tasiARFAP2

Ptychostomumpseudotriquetrum

72-PpsTAS3adf 246 KC812758 mdashldquomdash


Table 1 Continued


aClonecluster

bLengthbp



tasiARFAP2TAS6)

P pseudotriquetrum 16-PpsTAS3bce 193 KC812757 mdashldquomdash

Bryum argenteum 9-BarTAS3adf 233 KC812760 mdashldquomdash

B argenteum 4-BarTAS3bce 192 KC812759 mdashldquomdash

Bartramiales

Bartramia halleriana 32-BhaTAS3bce 228 KC812748 mdashldquomdash

B halleriana 28-BhaTAS3adf 272 KC812747 mdashldquomdash


BryopsidaFunariidae

Encalyptales

Encalypta rhaptocarpa 35-ErhTAS3adf 253 KC791767 mdashldquomdash

E rhaptocarpa 16-ErhTAS3bce 249 KC791768 mdashldquomdash

E rhaptocarpa 31-ErhTAS3adf 253 KC791769 mdashldquomdash

Funariales

Physcomitrella patens PpTAS3aTAS3adf

255895e Arif et al 2012 [20] tasiARFAP2

TAS6

P patens PpTAS3dTAS3adf

2561250e Arif et al 2012 [20] mdashldquomdash

P patens PpTAS3fTAS3adf


P patens PpTAS3cTAS3bce 259 Arif et al 2012 [20] tasiARFAP2

P patens PpTAS3bTAS3bce 192 Arif et al 2012 [20] mdashldquomdash

P patens PpTAS3eTAS3bce 192 Arif et al 2012 [20] mdashldquomdash

BryopsidaTimmiidae Timmiales Timmia austriaca 9-Tau

TAS3adf 254 KC812755 mdashldquomdash

Tetraphidopsida Tetraphidales Tetraphis pellucida 73-TpeTAS3bce 275 KC812754 mdashldquomdash

T pellucida 80-Tpeoutside 251 KC812753 mdashldquomdash

Polytrichopsida Polytrichales Polytrichum commune 122-Pcooutside 227 KC812751 mdashldquomdash

P commune 124-PcoTAS3bce 262 KC812752 mdashldquomdash

Andreaeopsida Andreaeales Andreaea rupestris 13-Aruoutside 254 KC812744 mdashldquomdash

A rupestris 14-Aruoutside 272 KC812743 mdashldquomdash

Sphagnopsida Sphagnales Sphagnum squarrosum Not found mdash Krasnikova et al 2011 [24] mdashS girgensohnii Not found mdash Krasnikova et al 2011 [24] mdash

Marchantiopsida Marchantiales Marchantia polymorpha 1-Mpooutside 256 KC812742

SRR0721689978782 tasiAP2aThis column includes the names of all identified TAS3 clones (upper line) and clusterization with Physcomitrella patens loci on the dendrogram (lower line)bThis column indicates the length of all TAS3 species including both 51015840 and 31015840 miR390 target sequences on the borderscThis column includes the references and accession numbers (if known) for all identified TAS3 loci except Physcomitrella patens loci For loci 48-Cpu and 1-Mpo our sequence data are identical to the fragments of longer sequences found in NCBI SRA database For 36-Brar PCR-based sequence corresponds to thefragments of the longer Illumina reads of B rivulare genomic DNA (Goryunov et al unpublished)dThis column indicates internal organization of TAS3 loci which include tasiARF and tasiAP2 sequences TAS6 means the occurrence of the particular TAS3locus in close proximity to TAS6 locus (if known)eThis number indicates total length of TAS6-TAS3 complex element (from the 51015840 miR529 target site in TAS6 to 31015840 miR390 target site in TAS3)


miR390 loci between these plant groups [6 9 15] On theother hand evolutionary history of TAS3-like and miR390genes in more anciently diverged pteridophytes remainsenigmatic

Bryophytes sensu lato (liverworts mosses and horn-worts) are placed as a phylogenetic grade between the charo-phycean algae and pteridophytes [25] It is believed that theancestors of bryophytes and vascular plants separated shortlyafter the transition from water to land [34] Bryophytes likemoss Physcomitrella patens are thought to closely resemblethe first land plants in their gametophyte-dominated lifecycles lack of lignified vascular tissue and morphologySome abundant miRNAs are invariant between basal plantPhyscomitrella and more recently derived lineages Partic-ularly at least 11 miRNA families including miR390 areconserved between P patensA thaliana andO sativa [31 3536] In the case of miR390 three genomic loci were revealedin P patens [37] Recently this moss has been shown tocode for six precursor ta-siARF loci targeted by miR390 andreferred to as PpTAS3andashf [15 20 36 38] All six loci containtwo (51015840 and 31015840) miR390-target sites andmonomeric ta-siRNAsequences which regulate ARF genes and also target mRNAof AP2-related transcription factors [20]

311 Search for Novel miR390 Genes in Bryophyta andMarch-antiophyta Assuming that there is the only nonvascularplant (P patens) with miR390 genes identified and sequenced[31] we performed their search in liverworts and othermosses Identification of miRNA largely relies on two mainstrategies computer-based (bioinformatics) and experimen-tal approaches Search formiRNA genes using bioinformaticstools is one of the most widely used methods contributingconsiderably to the prediction of new miRNAs in differentsystems The main theory behind this approach is findingsignature sequences and secondary structures of knownmiRNAs both within a single genome and across genomesof related organisms [39] It is well known that miRNAsare well conserved from species to species within the sameplant taxa which allows researchers the ability to predictorthologues of previously known miRNAs by utilizing ESTand SRA NCBI databases (httpwwwncbinlmnihgov)Interestingly it was recently found that after the appearanceof the miR390 family the gene duplication divergence andneofunctionalization gave rise to at least seven families of newmiRNAs in two major superfamilies [15]

Availability of two moss cDNA databases (Pohlianutans and Ceratodon purpureus) in addition to P patenssuggested identifying new Bryopsida miRNA genes usingPhyscomitrella as a reference We used a method principallysimilar to that described by Jones-Rhoades and Bartel[39] The first step for identifying miR390 precursors inthe moss transcriptomes was detecting sequence readscontaining imperfect inverted repeats corresponding tosequences of miR390 and miR390lowast using the ldquoblastnrdquoImportantly we used all combination of somewhatdivergent miR390miR390lowast sequences found for P patens(httpwwwmirbaseorg) (Figure 1) Second the RNA-fold(httprnatbiunivieacatcgi-binRNAfoldcgi) programwas used to find potential miRNA hairpin structures Then

M 1 2 3 4 5 6 7

Figure 2 Analysis of PCR products in 15 agarose gel Amplifica-tion of genomic DNA sequences flanked by miR390 and miR390lowastsites PCR products were obtained on genomic DNAs with moss-specific primers [23 24] Brachythecium rivulare (1) Nicotianatabacum (control) (2) Marchantia polymorpha (3) Sphagnumsquarrosum (4) Selaginella kraussiana (control) (5) Polytrichumcommune (6) and primers with no template DNA (control) (7) (M)DNA size markers including bands ranging from 100 bp to 1000 bpwith 100 bp step and 1500 bp band (Sibenzyme)

strict criteria were adopted in the identification of pre-miR390-like sequences from hairpin structure sequences[40] namely (1) 60 nucleotides is the minimum length ofpre-miRNA sequences (2) the stem of the hairpin structure(including the GU wobble pairs) includes at least 22 basepairs (3) minus35 kcalmol should be the maximum free energyof the secondary structure and (4) the secondary structuremust not include multibranch loops Both Pohlia nutansand Ceratodon purpureus databases showed single copiesof the putative miR390 precursor genes capable of formingthe stem-loop structures with a minimum free energy ofminus5720 kcalmol (NCBI accession number GACA01009225)and approximately 64 kcalmol (NCBI accession numberSRR074894910234) respectively (Figure 1) The latterputative miR390 sequence had obvious similarity tomiR390b of P patens as it was revealed using software athttpwwwmirbaseorg (Figure 1 and data not shown)

Computational methods for identifying miRNAs inplants are rapid and less expensive However these bioin-formatic approaches can only identify conserved miRNAsamong organisms where DNA or RNA sequence informationis available Efficient and suitable miRNA detection areessential to reveal miRNA precursors in organisms wheresequence information is poorWe attempted to revealmiR390sequences using our PCR-based approach [23] previouslydeveloped for TAS3 genes PCR analysis was performedwith a pair of degenerate primers P-mir corresponded tothe miR390 sequence and M-mir complementary to themiR390lowast region of Physcomitrella pre-miR390 hairpin-loopstructure [8] First experiment represented PCR reactionusing Brachythecium rivulare (Bryopsida) DNA as templateand degenerate primers This reaction resulted in efficientsynthesis of a single PCR-fragment with the expected size of100 bp (Figure 2) that was in agreement with calculated dis-tance betweenmiR390 andmiR390lowast sites in P patensmiR390precursor RNAs (httpwwwmirbaseorg) Sequencing ofthis cloned DNA fragment showed amplification of at leastthree genome loci (data not shown) Application of strictcriteria that were used in the computer-based identificationof pre-miR390-like sequences (see above) allowed us to selectonly single sequence capable of forming typical miRNA-like stem-loop structure (Figure 1) The introduction of next


generation sequencing technology showed a powerful wayfor a more comprehensive exploration of miRNA generepertoire To confirm assignment of the revealed sequenceto miR390 we performed blast search against B rivulareIllumina genome sequence reads (to be published elsewhere)One of the reads showed 98 sequence similarity to thesequenced PCR fragment Moreover its foldback terminatesat 17 bp below the miRNAmiRNAlowast region (Figure 1) thatis typical for plant miRNAs and this characteristic appearsimportant for their optimal processing [8]

Further experiments were performed with moss speciesvery distantly related to P patens namely Timmia austriaca(class Bryopsida subclass Timmiidae) Tetraphis pellucida(class Tetraphidopsida) Bartramiopsis lescurii (class Poly-trichopsida) Polytrichum commune (class Polytrichopsida)Andreaea rupistris (class Andreaeopsida) Oedipodium grif-fithianum (class Oedipodiopsida) and Sphagnum squarro-sum (class Sphagnopsida) PCR amplification of chromo-somal DNA from all these species resulted in synthesis ofone major fragment of ca 100 bp (Figure 2 and data notshown) However S squarrosum showed additional minorfragment of 250 bp (Figure 2) Cloning and sequencing of theobtained DNA bands revealed that the one-third of ampli-fied sequences contained putative miR390 sequences com-posed of stem-loop structure starting frommiR390miR390lowastregions corresponding to PCR primers Moreover someamplified sequences showed obvious similarity to miR390from P patens (data not shown) Interestingly no one from9 clones of S squarrosum PCR products of 80ndash270 bp inlength fit strict criteria for identification of pre-miR390-likesequences (see above) It is in accordance with our previousfail to find TAS3-like sequences in this plant [24]

Assuming our promising results with mosses (see above)which are evolutionary distant from P patens we attemptedto reveal miR390 sequences in liverworts Marchantia poly-morpha (class Marchantiopsida) and Harpanthus flotovianus(class Jungermanniopsida) where these genes were notreported PCR-based approach (see above) revealed miR390-like locuses in both species (Figures 1 and 2)These sequenceswere validated as miR390 precursor genes by MaturePredweb server (httpnclabhiteducnmaturepred) and sec-ondary structure prediction at RNAfold web server (httprnatbiunivieacatcgi-binRNAfoldcgi)These potential liv-erwort miR390 genes fit strict criteria adopted for theprediction of pre-miRNA-like species from hairpin struc-tures (see above) Using BLAST we also revealed severalsequence reads containing the PCR-tagged potential miR390sequence in NCBI SRA database ofMarchantia genome (seeie NCBI accession number SRR072169449069) Takinginto account basal position of Marchantiopsida (particularlygenusMarchantia) among land plants [25 32 34] (Figure 3)it can be proposed that the ancient miR390-dependent TASmolecular machinery firstly evolved soon after the transitionof plants from water to land Importantly using A thalianaprotein mRNAs that are involved in different ta-siRNA mat-uration steps as queries for BLAST searches in Marchantiapolymorpha random whole genome shotgun library at NCBIidentified (as it was previously found for P patens [36])presence of sequences coding for fragments of some proteins

HookerialesHypnales

PtychomnialesHypnodendralesRhizobialesSplachnaceaeBryales

Bartramiales

OrthotrichalesHedwigialesGrimmiales

Dicranidae

Bryidae

DicranalesTimmiidae

Funariidae

DiphysciidaeBuxbaumiidaeTetraphidopsida

Bryopsida

Bryophyta

Polytrichopsida

OedipodipsidaAndreaeobryopsidaAndreaeopsidaSphagnopsida

Takakiopsida

Marchantiophyta

Figure 3 Synoptic consensus view on phylogeny of major Bry-ophyta and Marchantiophyta taxa based on recent molecular anal-yses adopted from Figure 3 of Shaw et al [25] with modificationsTaxa discussed in the current study are underlined

involved in small RNA pathways guided by miR390 (datanot shown) namely AtDCL4 (eg NCBI accession numberSRR0721693697522) and AtRDR6 (eg SRR0721652615932and SRR0721673667912)

312 Search for Novel TAS3 Genes in Bryophyta andMarchan-tiophyta In a previous paperwe usedPCRamplificationwithdesigned degenerated primers BryoTAS-P and BryoTAS-M as a comparative profiling tool to probe genomic DNAsamples derived from 13 moss species [24] Our data onmiR390 detection in mosses and liverworts (see above)suggested further search for TAS3 genes in lower land plantsParticularly we tested moss species from Timmia austriaca(class Bryopsida subclass Timmiidae) Tetraphis pellucida(Tetraphidopsida) Polytrichum commune (Polytrichopsida)and Andreaea rupestris (Andreaeopsida) In all these speciesone or more potential TAS3 loci were detected (Figures 3 and4 Table 1) The detection approach was principally identicalto that described in our previous paper [24] In addition wesequenced a dozen of newTAS3 species in the representativesof class Bryopsida (Table 1) In general identification ofaround 40 new TAS3 species in four classes of mosses includ-ing the early diverged Andreaeopsida [25 34] showed thatall sequenced TAS3 loci fit general structural organization of


Marchantia polymorpha1-Mpo(Marchantiopsida)

Andreaea rupestris13-Aru(Andreaeopsida)

Polytrichum commune122-Pco(Polytrichopsida)

Tetraphis pellucida80-Tpe(Tetraphidopsida)

Timmia austriaca9-Tau(Bryopsida)

target targetta-siR-AP2

1 25623618216221ta-siR-AP2 ta-siR-ARF

1 25423420218215713721

ta-siR-AP2 ta-siR-ARF

1 25423420218215713721


1 25123119917915413421


1 22720717415413011021

5998400 miR390 3998400 miR390

(a)

target targetta-siR-ARF

1 155135997921

ta-siR-ARF

1 164144725221

ta-siR-ARF

1 25223213711721

ta-siR-ARF

1 22620613111121ta-siR-ARF

1 2001801068621

ta-siR-ARF

1 189169957521

Equisetum arvenseEqu 83(Equisetopsida)

Angiopteris angustifoliaAng-T16(Marattiopsida)Angiopteris angustifoliaAng-24(Marattiopsida)

Angiopteris angustifoliaAng-T24(Marattiopsida)

ta-siR-ARF ta-siR-ARF

1 2962761591399575212 times ta-siR-ARF

1 38936925221121

2 times ta-siR-ARF

1 37735724220121

2 times ta-siR-ARF

1 2342141409921

2 times ta-siR-ARF

1 21219316111921

Dicksonia fibrosaDif-410(Polypodiopsida)


Pteridium aquilinum(Polypodiopsida)

Gnetum gnemonone-tasi(Gnetopsida)

Gnetum gnemontwo-tasi(Gnetopsida)Pinus taedactg7180052440390(Coniferophyta)

Pinus taedactg7180050584048(Coniferophyta)

5998400 miR390 3998400 miR390

(b)

Figure 4 Comparison of TAS3 internal organization between nonflowering plants Numbers below boxes show relative nucleotide positionsof miR390 target sites and ta-siRNAs (a) Organization of TAS3 loci in Bryophyta and Marchantiophyta (b) Organization of TAS3 loci inferns and gymnosperms For other details see text


Physcomitrella genes namely dual miR390 target sites on theborders andmonomeric tasiARFtasiAP2 sequences betweenthem (Figure 4(a) and Table 1) Interestingly recent studiesonPhyscomitrellaTAS3 loci showed that tasiAP2 sequences inPpTAS3c and PpTAS3e do not appear functional because ofa number of substitutions preventing Watson-Crick pairingwith target mRNA (see Table S3 in [20]) This observationallows us to put forward the hypothesis that evolution of TAS3loci toward the tasiARF only species typical for floweringplants involved selection of those diverged moss TAS3 genesthat have lost functional tasiAP2 sequences

Our data on miR390 detection in Marchantiophyta (seeabove) allowed us to propose that miR390-dependent molec-ular machinery should act in these basal land plants It isof great importance to reveal the role of miR390 in earlyland plant evolution It seems this role is more complicatedthan simple triggering of the TAS3 precursor processing ifwe assume potential involvement of miR390 in the directtargeting and cleavage of protein-coding mRNAs [41 42]This potential complicated role is seemingly illustrated byour data showing that despite the finding of miR390-likesequence inHarpanthus flotovianus andOedipodium griffithi-anum (Figure 1) we failed to detect TAS3 species in theseplants (data not shown) In contrast PCR-based approachwith BryoTAS-PBryoTAS-M primers and Marchantia totalgenomic DNA revealed a weak but reproducible amplifiedDNA fragment of 250 bp (data not shown) Cloning andsequencing of this PCR product showed TAS3-like sequencewhich was also found among genome reads of Marchantiapolymorpha genome by BLAST search of NCBI SRA database(NCBI accession number SRR072168997878) (Table 1) Strik-ingly the TAS3-like liverwort locus contains onlymonomerictasiAP2 site and no tasiARF sequences (Figure 4(a)) Assum-ing basal position of Marchantiopsida among land plants[25 32 34] it can be proposed that the ancient plant miR390-dependent TASmolecular machinery firstly evolved to targetAP2-like mRNAs and only then both ARF- and AP2-specificmRNAs

Recently analysis of the Physcomitrella patens RNAdegradome revealed the novel moss TAS loci TAS6 whichare dependent on the activity of dual sites complementaryto miR156miR529 [20] All three revealed TAS6 loci arepositioned in close genomic proximity to PpTAS3 loci (vizPpTAS3a PpTAS3d and PpTAS3f) (Table 1) and expressed ascommon RNA precursors with these TAS3 species (Table 1)[15 20 38] Moreover miR156 influences accumulationof tasiRNAs specific for PpTAS3a [38] We found thatproximity of TAS6 loci to TAS3 genes is not unique forPhyscomitrella patens (subclass Funariidae) These TAS geneclusters are present also in the mosses of subclasses Bryidaeand Dicranidae (Table 1) Importantly most TAS3 sequencespotentially expressed as common precursors with TAS6species form common cluster on themoss TAS3 phylogeneticdendrogram with PpTAS3a PpTAS3d and PpTAS3f (TAS3-like locus of Marchantia polymorpha was used as out-group) (Figure 5 and Table 1) This dendrogram also showedthat TAS3 sequences from representatives of Tetraphi-dopsida Polytrichopsida and Andreaeopsida positioned

mostly as basal molecular species in two main moss TAS3clusters (Figure 5)

313 TAS3Genes in Ferns andGymnosperms Gymnospermsare different from angiosperms in peculiarities of seedformation and flowering Recent works on gymnospermmiR390-based gene regulation have focused mainly ondiscovery and functional analysis of miRNA [6 9] UnlikeTAS3 of flowering plants it seems that only the 51015840miR390-target site is cleaved in conifer TAS3-like RNA precursor[15 29] Our screening of NCBI and other databases forTAS3 sequences showed two TAS3 subfamiles (one-tasiARFand two-tasiARF) in many representatives Coniferophyta(our unpublished data) Particularly the Pinus taeda genomecontains at least 4 one-tasiARF TAS3 loci (sequencesfrom clones deg7180055903842 ctg7180052440390ctg7180045727317 and ctg7180039236567) and 7 two-tasiARFTAS3 loci (sequences from clones ctg7180050584048ctg7180047037037 ctg7180045493386 gtctg7180064976704ctg7180063936100 ctg7180044268127 andctg7180058353378) (httpdendromeucdaviseduresourcesblast) (Figure 4(b) and data not shown) Likewise the plantsfrom Gnetophyta (viz Welwitschia mirabilis and Gnetumgnemon) code for TAS3 loci belonging to one-tasiARF (NCBIaccession number CK744773 and SRR064399239159) andtwo-tasiARF (SRR064399294117 and SRR06439972564)(Figure 4(b)) To further reveal the conservation of TAS3-likeloci in lower seed plants we performed PCR amplificationof total DNA from Cycadophyta (Cycas revoluta Strangeriaeriupus Zamia pumila and Macrozamia miqnolii) usingdicot-specific primers P-Tas3 M-Tas3caa and M-Tas3aca[23] Sequencing and BLAST analysis of the obtained PCRproducts revealed that they corresponded to the monomericand dimeric ta-siARF precursors of flowering plants (datanot shown) Thus the available data on sequencing theTAS3 genes from gymnosperms unambiguously showed twoTAS3 subfamiles related to those found in flowering plants(Figure 4(b))

In general first tracheophytes are proposed to haveevolved gt420 million years ago and a major innovationin the evolution of tracheophytes from their bryophyte-likeancestors was the ability to form supporting and conductingtissues that contain cells with lignified cell walls [32 34] Theferns comprise one of the most ancient tracheophytic plantlineages and occupy habitats ranging from tundra to desertsand the equatorial tropics Like their nearest relatives (vizconifers) extant ferns possess tracheid-based xylem Little isknown about themiR390TAS3-based formation of tasiRNAsin ferns [43] and the selection pressures that influencedthe structure of TAS3 genes in diverse taxonomic groupsof ferns and fern allies (particularly classes LycopodiopsidaEquisetopsida Marattiopsida and Polypodiopsida) remainobscure For example it was unexpectedly found that thegenome of Selaginella moellendorffii from Lycopodiopsidalacks DCL4 RDR6 TAS3 and MIR390 loci which arerequired for the biogenesis of ta-siARF RNAs [44]

To achieve an understanding on the evolutionary historyof TAS3 genes in ferns we focused on two aims (1) identifying


P patens TAS3a

P patens TAS3d

B rivulare TAS454B rivulare 36-BrarO hians 32-Oxy

P medium 41-Pme

P nutans Pnu-30698P patens TAS3f

B rivulare 11-BrarB albicans 37-Bal

O hians 25-Oxy

B rivulare 11-BrarH lucens 49-H

P patens TAS3bC humilis 20-Chu

L glaucum 41-Lgl

G cribrosa 43-GcrC humilis 16-ChuL glaucum 42-LglP patens TAS3c

P patens TAS3e

T austriaca 9-TauB halleriana 29-BhaE rhaptocarpa 31-Erh

O pumilum 24-OpuB latifolium 50-Br

P polyantha 11-Pyp B argenteum 9-BarP pseudotriquetrum 72-Pps

B halleriana 28-Bha

E rhaptocarpa 35-ErhT pellucida 80-TpeA rupestris 13-AruP commune 122-Pco

H philippeanum 7-HpP polyantha 10-Pyp

B latifolium 47-BrO pumilum 5-Opu

B halleriana 32-Bha

A mougeotii 56-AmoF taxifolius 17-FitD pacificum 44-DpaC purpureus 48-Cpu

Amblystegium 52-AmblyP pseudotriquetrum 16-PpsB argenteum 4-BarT pellucida 73-TpeP commune 124-PcoS elegantulum 24-Sce

B inaequalifolium 27-Bin

E rhaptocarpa 16-ErhA rupestris 14-AruM polymorpha 1-Mpo

Figure 5 The minimal evolution phylogenetic tree based on analysis of the aligned TAS3 genes from mosses This tree was generatedaccording MAFFT6 program For full plant names and accession numbers see Table 1

possible TAS3 genes in 4 fern orders and (2) unraveling thepattern of TAS3 sequence organization among fern lineagesWe choose two earliest diverged orders (Marattiales andEquisetales) which positioned relatively close to the commonancestor of ferns and seed plants as well as two core leptospo-rangiates orders (Polypodiales and Cyatheales) (Figure 6)[26 45] Using dicot-specific primers P-Tas3 M-Tas3caaand M-Tas3aca (see above) allowed us to find that plantsof class Polypodiopsida (Polypodiales) encode both one-and two-tasiARF TAS3 subfamilies like gymnosperms and

angiosperms (Figure 4(b))However sequencing of amplifiedTAS3 loci from Equisetopsida (Equisetales) revealed onlyone-tasiARF TAS3 species (Figure 4(b)) Angiopteris angusti-folia (Marattiopsida) showed single two-tasiARF TAS3 locuswhich included the tandem of conserved tasiARF sequencescorresponding to the nearby phased segments defined bythe 31015840 miR390 processing site as observed in Arabidopsisand some other plant species [14 27] Strikingly anotherpeculiar two-tasi locus also encoded two tasiARFrsquos which areseparated by the spacer sequence (Figure 4(b)) It is tempting


Polypodiales

Cyatheales

Salviniales

Schizaeles

Gleicheniales

Hymenophyllales

Marattiales

Osmundales

Equisetales

Ophioglossales

Psilotales

Isoetales

Figure 6 Synoptic consensus view on phylogeny of major fern taxabased on recent molecular analyses adopted from Figure 2 of Senet al [26] with modifications Note that Isoetales species are outsideferns

to speculate that two-tasiARF subfamily possessing spacerbetween tasiARF sequences may represent an intermediateevolutionary stage from one-tasiARF to tandem tasiARForganization of TAS3 loci

314 Peculiar Evolution of TAS3 Genes in Land Plants Sim-ilarity of miR390-triggered siRNA regulation of angiospermand bryophyte ARF transcripts could be regarded as evidencethat all modern TAS3 genes might have descended from acommon ancestor [46] Importantly it was recently revealedthat all TAS3 genes may have important and complex rolesin the evolution of plant developmental processes and bioticstress response [5 15 33 38] However a vast majorityof higher plant ARF3 and ARF4 mRNAs possessed dualcomplementary sites toTAS3-derived ta-siRNAswhereas theP patensARFmRNA possessed only one site complementaryto TAS3 ta-siRNAs As it was stated by Axtell et al [46]ldquoThese observations add to the examples of convergentevolutionary origins for similarly functioning small RNAswhile illustrating that cleavage of a homologous target at ananalogous location does not always indicate descent from acommon ancestor Moreover the ARF-targeting ta-siRNAsare derived from the (+) strand of the angiosperm TAS3 lociwhereas they derived from the (ndash) strands of the moss TAS3loci suggesting a more complex evolutionary path for plantTAS3 loci than simple sequence divergence from a commonancestor Data from additional plant lineages might provideimportant clues for distinguishing between these interestingpossibilitiesrdquo

Our data on additional plant lineages indicate that themain mechanism of TAS3 gene evolution that could generate

Table 2 Summary of the diversity in the organization of TAS3-likeloci

Organization of TAS3(One- or two-tasiARFAP2TAS6) Classsubclass

One-tasiAP2 only Marchantiopsida

One-tasiAP2-one-tasiARF

AndreaeopsidaPolytrichopsidaTetraphidopsidaBryopsidaTimmiidaeBryopsidaFunariidaeBryopsidaBryidaeBryopsidaDicranidae

TAS6-TAS3BryopsidaFunariidaeBryopsidaBryidaeBryopsidaDicranidae

One-tasiARF only EquisetopsidaOne-tasiARF and two-tasiARF species MarattiopsidaTwo-tasiARF (no tandem repeat) MarattiopsidaOne-tasiARF and two-tasiARF species PolypodiopsidaOne-tasiARF and two-tasiARF species Spermatophytalowast

No TAS3 loci revealed LycopodiopsidaSphagnopsida

See text for details lowastSpermatophyta species form a group which includesseveral classes

diversity is the duplication and divergence of TAS3 lociAfter appearance of the putative primitive (AP2-specific)TAS3-like locus this growing gene family must have under-gone copy number gains upon evolutionary flow towardmosses and one of the duplicates may be subjected toneofunctionalization resulted in evolving tasiARF sequencesin addition to tasiAP2 site (Figure 4(a) Table 2) Our analysissuggests that moss TAS3 families may undergone losses oftasiAP2 sites during evolution toward ferns As a resulthorsetails seem to preserve only one-tasiARF TAS3 lociImportantly unlike mosses ARF-targeting ta-siRNAs arederived from the (+) strand of the fern TAS3 precursor RNAsuggesting complex evolutionary events toward tracheophyteTAS3 species [46] resulted particularly in complete deletingmiR390-based machinery from lycophyte genomes [44]Finally the evolutionary process to build up angiosperm-typeTAS3 sequences may be related to step-by-step duplicationsof monomeric tasiARF sites first observed in Angiopterisangustifolia (Figure 4 Table 2) Interestingly ldquocore consen-susrdquo in one-tasiARF species was found to be different fromconsensus in two-tasi TAS3 [23] Although the significanceof this variation remains obscure we observed the samephenomenon in fern TAS3 species (data not shown)

32 Differential Expression of TAS3-Like Genes from the TribeSenecioneae Global transcriptome nextgeneration sequenc-ing studies in many plant species have shown shifts inthe pools of the different ta-siRNAs and miRNA classes invarious tissues and organs [47] (httpsmallrnaudeledu) Ingeneral plant cells have a small number of unique and highlyabundant 21 nt miRNAsta-siRNAs and a large number of


ta-siR-ARF

1 28726717315321ta-siR-ARF

1 25223213711721ta-siR-ARF

1 192172785821ta-siR-ARF

1 185165705021ta-siR-ARF

1 164144725221

2 times ta-siR-ARF

1 2712511369521TAS3-Sen1

TAS3-Sen2

TAS3-Sen3

TAS3-Sen4

TAS3-Sen5

target target

TAS3-Sen6

5998400 miR390 3998400 miR390

Figure 7 Comparison of TAS3 internal organization in plants belonging to subtribe Senecioneae Numbers below boxes show relativenucleotide positions of miR390 target sites and ta-siRNAs For other details see text

diverse siRNAs mainly 24 nt in size [1] A comprehensiveanalysis can be compiled on variation in the miRNAs presentin a large population of dozens of millions sequences derivedfrom several libraries representing multiple tissuesorgans ofthe plant [48] Around several thousand unique signaturescorresponding to miRNAs were detailed for some plantsExamination of the number of reads of each signature mayreveal all the length or sequence variations found in thehigh throughput populations for all miRNA family mem-bers Thus the flux of expression patterns for each of theunique signatures in the libraries representing both seedand vegetative tissues are currently detailed in many speciesSome of the miRNAs were very abundant in certain tissuessuch as members of the miR167 family which were highlyprevalent in the seed coats from multiple samples Someother miRNAs showed preferential expression in either ofthe flowers germinating cotyledons stems or leaves Insummary it is clear that tissue differences in normalized readcounts are apparent in many of the known miRNAs and ta-siRNAs that are conserved widely in plant systems [1 48 49]

Senecioneae is the largest tribe of family Asteraceaecomprised of ca 150 genera and 3000 species which includemany common succulents of greenhouses Approximatelyone-third of species are placed in genus Seneciomaking it oneof the largest genera of flowering plants [50] However therewas no information on the structure of TAS genes in theseplants Recently we revealed that the conserved TAS3 locus(TAS3-Sen1) in Senecio talinoides Curio repens and Curioarticulatus was actively transcribed and its close relativesare distributed among many Asteraceae plants and found tobe similar to Arabidopsis AtTAS3a gene [51] To reveal thepossible novel TAS3-like loci in these plants we performedPCR amplification of total DNA using dicot-specific primersP-Tas3 M-Tas3caa and M-Tas3aca As it was found fortobacco [23] PCR on Senecioneae DNA gave rise to one

major DNA fragment of 250 bp and in addition smallerminor bands including those of 170ndash190 bp in length (data notshown) Sequencing of these PCR DNA fragments revealedthat they corresponded to five new one-tasiARF and two-tasiARFTAS3 precursors (Figure 7) BLAST analysis ofNCBIdatabases revealed their distant relation to the sequencedTAS3-Sen1 ta-siARF precursors from Asteracea (data notshown)

We have studied the transcription of TAS3-Sen1 genesin three above mentioned Senecioneae species by sequenceanalysis of multiple cDNA clones from vegetative organs(roots true leaves and succulent leaves) Since reversetranscription priming with the oligo (dT) at the poly A tailsof the TAS3 genes analyzed may not occur with the sameefficiency we used correct priming with six-specific primersduring PCR on the total cDNA preparation Thus the cDNAclones obtained should represent true qualitative measureof the specific TAS3 RNAs Unexpectedly we found eightcDNA clones for C repens TAS3-Sen1 seven clones for Carticulatus TAS3-Sen1 and five clones for Senecio talinoidesTAS3-Sen1 but no TAS3 clones for five other TAS3 species(TAS3-Sen2 to TAS3-Sen6) (data not shown) In accordancewith our experimental data bioinformatic analysis of SRAdatabase of Senecio madagascariensis transcriptome at NCBIrevealed only TAS3-Sen1 read (GenBank accession numberSRR006592) (data not shown) Thus despite the presenceof at least six TAS3 genes in representatives of subtribeSenecioneae a single locus is transcribed in the vegetativeorgans

Disclosure

The authors do not have a direct financial relation with thecommercial identities mentioned in this paper that mightlead to a conflict of interests


Acknowledgment

The authors are grateful to E A Ignatova (Department ofBiology Moscow State University) for providing some plantsamples

References

[1] M J Axtell ldquoClassification and comparison of small RNAs fromplantsrdquo Annual Review of Plant Biology vol 64 pp 137ndash1592013

[2] E Gottwein ldquoKaposirsquos sarcoma-associated herpesvirusmicroR-NAsrdquo Frontiers in Microbiology vol 3 article 165 2012

[3] K Rogers and X Chen ldquoBiogenesis turnover and mode ofaction of plant microRNAsrdquo Plant Cell vol 25 pp 2383ndash23992013

[4] X Chen ldquoSmall RNAsmdashsecrets and surprises of the genomerdquoPlant Journal vol 61 no 6 pp 941ndash958 2010

[5] D S Skopelitis A Y Husbands andM C Timmermans ldquoPlantsmall RNAs as morphogensrdquo Current Opinion in Cell Biologyvol 24 no 2 pp 217ndash224 2011

[6] G Sun ldquoMicroRNAs and their diverse functions in plantsrdquoPlant Molecular Biology vol 80 pp 17ndash36 2012

[7] J E Tarver P C Donoghue and K J Peterson ldquoDo miRNAshave a deep evolutionary historyrdquo BioEssays vol 34 pp 857ndash866 2012

[8] J T Cuperus N Fahlgren and J C Carrington ldquoEvolution andfunctional diversification of MIRNA genesrdquo Plant Cell vol 23no 2 pp 431ndash442 2011

[9] M W Jones-Rhoades ldquoConservation and divergence in plantmicroRNAsrdquo Plant Molecular Biology vol 80 no 1 pp 3ndash162012

[10] M Nozawa S Miura and M Nei ldquoOrigins and evolutionof microRNA genes in plant speciesrdquo Genome Biology andEvolution vol 4 pp 230ndash239 2012

[11] G Le Trionnaire R T Grant-Downton S Kourmpetli H GDickinson and D Twell ldquoSmall RNA activity and function inangiospermgametophytesrdquo Journal of Experimental Botany vol62 no 5 pp 1601ndash1610 2011

[12] F Vazquez S Legrand and D Windels ldquoThe biosyntheticpathways and biological scopes of plant small RNAsrdquo Trends inPlant Science vol 15 no 6 pp 337ndash345 2010

[13] Y Endo H O Iwakawa and Y Tomari ldquoArabidopsis ARG-ONAUTE7 selects miR390 through multiple checkpoints dur-ing RISC assemblyrdquo EMBO Report 2013

[14] E Allen and M D Howell ldquomiRNAs in the biogenesis oftrans-acting siRNAs in higher plantsrdquo Seminars in Cell andDevelopmental Biology vol 21 no 8 pp 798ndash804 2010

[15] Q Fei R Xia and B C Meyers ldquoPhased secondary smallinterfering RNAs in posttranscriptional regulatory networksrdquoPlant Cell vol 25 pp 2400ndash2415 2013

[16] R Rajeswaran and M M Pooggin ldquoRDR6-mediated synthesisof complementary RNA is terminated by miRNA stably boundto template RNArdquoNucleic Acids Research vol 40 no 2 pp 594ndash599 2012

[17] N Fahlgren T AMontgomeryMDHowell et al ldquoRegulationof AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affectsdevelopmental timing and patterning in Arabidopsisrdquo CurrentBiology vol 16 no 9 pp 939ndash944 2006

[18] E Marin V Jouannet A Herz et al ldquomir390 arabidopsis TAS3tasiRNAs and theirAUXIN RESPONSE FACTOR targets define

an autoregulatory network quantitatively regulating lateral rootgrowthrdquo Plant Cell vol 22 no 4 pp 1104ndash1117 2010

[19] T Yifhar I Pekker D Peled et al ldquoFailure of the tomatotrans-acting short interfering RNA program to regulateAUXINRESPONSE FACTOR3 and ARF4 underlies the wiry leaf syn-dromerdquo Plant Cell vol 24 pp 3575ndash3589 2012

[20] M A Arif I Fattash Z Ma et al ldquoDICER-LIKE3 activityin Physcomitrella patens DICER-LIKE4 mutants causes severedevelopmental dysfunction and sterilityrdquo Molecular Plant vol5 pp 1281ndash1294 2012

[21] O Saleh N Issman G I Seumel et al ldquoMicroRNA534a controlof BLADE-ON-PETIOLE 1 and 2 mediates juvenile-to-adultgametophyte transition inPhyscomitrella patensrdquoPlant Journalvol 65 no 4 pp 661ndash674 2011

[22] W Arthur ldquoThe emerging conceptual framework of evolution-ary developmental biologyrdquo Nature vol 415 no 6873 pp 757ndash764 2002

[23] M S Krasnikova I A Milyutina V K Bobrova et al ldquoNovelmiR390-dependent transacting siRNA precursors in plantsrevealed by a PCR-based experimental approach and databaseanalysisrdquo Journal of Biomedicine and Biotechnology vol 2009Article ID 952304 9 pages 2009

[24] M S Krasnikova I A Milyutina V K Bobrova A V TroitskyA G Solovyev and S Y Morozov ldquoMolecular diversity ofmir390-guided trans-acting siRNA precursor genes in lowerland plants experimental approach and bioinformatics analy-sisrdquo Sequencing vol 2011 Article ID 703683 6 pages 2011

[25] A J Shaw P Szovenyi and B Shaw ldquoBryophyte diversity andevolution windows into the early evolution of land plantsrdquoAmerican Journal of Botany vol 98 no 3 pp 352ndash369 2011

[26] L Sen M Fares Y J Su and T Wang ldquoMolecular evolutionof psbA gene in ferns unraveling selective pressure and co-evolutionary patternrdquo BMC Evolutionary Biology vol 12 article145 2012

[27] R Xia H Zhu Y Q An E P Beers and Z Liu ldquoApple miRNAsand tasiRNAswith novel regulatory networksrdquoGenomeBiologyvol 13 p R47 2012

[28] D Shen S Wang H Chen Q-H Zhu C Helliwell andL Fan ldquoMolecular phylogeny of miR390-guided trans-actingsiRNA genes (TAS

3) in the grass familyrdquo Plant Systematics and

Evolution vol 283 no 1-2 pp 125ndash132 2009[29] M J Axtell C Jan R Rajagopalan and D P Bartel ldquoA two-hit

trigger for siRNA biogenesis in plantsrdquo Cell vol 127 no 3 pp565ndash577 2006

[30] M Megraw V Baev V Rusinov S T Jensen K Kalantidis andA G Hatzigeorgiou ldquoMicroRNA promoter element discoveryin Arabidopsisrdquo RNA vol 12 no 9 pp 1612ndash1619 2006

[31] T Arazi ldquoMicroRNAs in themoss Physcomitrella patensrdquo PlantMolecular Biology vol 80 no 1 pp 55ndash56 2012

[32] W S Judd C S Campbell E A Kellog P F Stevens and M JDonoghue Plant Systematics A Phylogenetic Approach SinauerAssociatec Sunderland Mass USA 2008

[33] C Lelandais-Briere C Sorin M Declerck A Benslimane MCrespi and C Hartmann ldquoSmall RNA diversity in plants andits impact in developmentrdquo Current Genomics vol 11 no 1 pp14ndash23 2010

[34] J L Bowman ldquoWalkabout on the long branches of plantevolutionrdquo Current Opinion in Plant Biology vol 16 pp 70ndash772013

[35] M J Axtell and J L Bowman ldquoEvolution of plant microRNAsand their targetsrdquo Trends in Plant Science vol 13 no 7 pp 343ndash349 2008


[36] M A Arif W Frank and B Khraiwesh ldquoRole of RNA interfer-ence (RNAi) in the moss Physcomitrella patensrdquo InternationalJournal of Molecular Sciences vol 14 pp 1516ndash1540 2013

[37] M Talmor-Neiman R Stav W Frank B Voss and T ArazildquoNovel micro-RNAs and intermediates of micro-RNA biogene-sis from mossrdquo Plant Journal vol 47 no 1 pp 25ndash37 2006

[38] S H Cho C Coruh and M J Axtell ldquomiR156 and miR390regulate tasiRNA accumulation and developmental timing inPhyscomitrella patensrdquo Plant Cell vol 24 pp 4837ndash4849 2012

[39] MW Jones-Rhoades and D P Bartel ldquoComputational identifi-cation of plant MicroRNAs and their targets including a stress-induced miRNArdquo Molecular Cell vol 14 no 6 pp 787ndash7992004

[40] W Jin N Li B Zhang et al ldquoIdentification and verificationof microRNA in wheat (Triticum aestivum)rdquo Journal of PlantResearch vol 121 no 3 pp 351ndash355 2008

[41] C Addo-Quaye J A Snyder Y B Park Y-F Li R Sunkarand M J Axtell ldquoSliced microRNA targets and precise loop-first processing of MIR319 hairpins revealed by analysis of thePhyscomitrella patens degradomerdquo RNA vol 15 no 12 pp2112ndash2121 2009

[42] R Karlova J C van Haars C Maliepaard et al ldquoIdentificationof microRNA targets in tomato fruit development using high-throughput sequencing and degradome analysisrdquo Journal ofExperimental Botany vol 64 no 7 pp 1863ndash1878 2013

[43] M J Axtell and D P Bartel ldquoAntiquity of microRNAs and theirtargets in land plantsrdquo Plant Cell vol 17 no 6 pp 1658ndash16732005

[44] J A Banks T Nishiyama M Hasebe et al ldquoThe selaginellagenome identifies genetic changes associated with the evolutionof vascular plantsrdquo Science vol 332 no 6032 pp 960ndash963 2011

[45] S Lehtonen ldquoTowards resolving the complete fern tree of liferdquoPLoS ONE vol 6 no 10 Article ID e24851 2011

[46] M J Axtell J A Snyder and D P Bartel ldquoCommon functionsfor diverse small RNAs of land plantsrdquo Plant Cell vol 19 no 6pp 1750ndash1769 2007

[47] A N Egan J Schlueter and D M Spooner ldquoApplications ofnext-generation sequencing in plant biologyrdquoAmerican Journalof Botany vol 99 no 2 pp 175ndash185 2012

[48] S R Strickler A Bombarely and L A Mueller ldquoDesigning atranscriptome next-generation sequencing project for a non-model plant speciesrdquo American Journal of Botany vol 99 no2 pp 257ndash266 2012

[49] G Jagadeeswaran P Nimmakayala Y Zheng K Gowdu UK Reddy and R Sunkar ldquoCharacterization of the small RNAcomponent of leaves and fruits from four different cucurbitspeciesrdquo BMC Genomics vol 13 article 329 2012

[50] L V Ozerova and A C Timonin ldquoOn the evidence ofsubunifacial and unifacial leaves developmental studies in leaf-succulent Senecio L species (Asteraceae)rdquoWulfenia vol 16 pp61ndash77 2009

[51] L V Ozerova M S Krasnikova A V Troitsky A G Solovyevand S Y Morozov ldquoTAS

3genes for small ta-siARF RNAs

in plants belonging to subtribe Senecioninae occurrence ofprematurely terminated RNA precursorsrdquo Molecular GeneticsMicrobiology and Virology vol 28 pp 79ndash84 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


5998400 3998400

5998400-a

rm

3998400-a

rm

(a)

P patens ppt-MIR390a (minimum free energy of minus6440 kcalmol)CAUGAUACAAUUACGAAGCUCAGGAGGGAUAGCGCCAUUCUCUGUUCUUCUCUAUCGACACUUGGUAAACCAGCUUGUGCUAGCUAUGGGCAGAGAUAUGGCGUUAUCCAUUCUGAGCUUUGCAAGUGUCUCGUG

(((((((((((((((((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))))))))))))))))

ppt-MIR390b (MFE of minus6400 kcalmol)UGCUGGGAGAUACAAUUACGAAGCUCAGGAGGGAUAGCGCCCAGCCCUGCUUUUACUGUUCAAUCAGUUAGAGUGCUUCUGAUAGUAAGAUGCAGAUGCAUGACGCUAUCCAUUCUGAGCUUUGCAACUGUGUCCCCCUAAUUUGGGCA

((((((((((((((((((((((((((((((((((((((((((((((((((((((((()))))))))))))))))))))))))))))))))))))))))))))))))))))))))

ppt-MIR390c (MFE of minus6230 kcalmol)GGUAUAAUUACAGAGCUCAGGAGGGAUAGCGCCCAUCUCAGUCUUUUUUAGCAUCCAAUUGAAAUGGUUAACAUUCCUUUGGAUAGCUAGACGGCGAAGCAUGGCGCUGUCCAUUCUGAGCAUUGCAUUUGUACC

(((((((((((((((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))))))))))))))

Pohlia nutans (MFE of minus5720 kcalmol)GGAUGAUACCAUUACGAAGCUCAGGAGGGAUAGCGCCUGCCUCUGUUUCUUCCUUGCAGCAGUCGUCGCAUGGCGCUAUCCAUUCUGAGCUUUGCAACUGUGUCGUCC

(((((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))))

Ceratodon purpureus (MFE of minus6400 kcalmol) GGACGAUACAAUUACCAAGCUCAGGAGGGAUAGCGCCUAGCUCUGUUCUUCUGUCACACUGUGAUAACAGAGUGCCAGACGUAUGGCGCUAUCCAUUCUGAGCUUUGCAAGUGUGUCGUCC

(((((((((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))))))))

Brachitecium rivulare (MFE of minus4980 kcalmol) GGACGACACAGUUGCAAAGCUCAGAAUGGAUAGCGCCGUGCUACUGUCCCAAACUUGAUUUCAACAGAGGUAGGCGCUAUCCCUCCUGAGCUUCGUAACGGUAUCAUCC

(((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))

Marchantia polymorpha (MFE of minus6960 kcalmol) AGGUUGGUCAGAGCGACAAAGCUCAGGAGGGAUAGCGUCUGCUUUUCCGUCAGGCUCGUGUGGGGCACGGGUACUUGUUCCUGGCUGUGGAGUUUACGCUAUCCAUUGUGAGCUCUGUCGCUCUCCCUUCCU

((((((((((((((((((((((((((((((((((((((((((((((((()))))))))))))))))))))))))))))))))))))))))))))))))

Timmia austriaca Tau-miR390a (MFE of minus5010 kcalmol)AGCUCAGGAGGGAUAGCGCCCAGCUCUGUUCUUCUGUAUCAUUGCUUGGUAAACAAGUAUUUGACAGUGAAGCGCAGAUGUAUGGCGCUCUCCAUUCUGAGCA

((((((((((((((((((((((((((((((((((((()))))))))))))))))))))))))))))))))))))

Timmia austriaca Tau-miR390b (MFE of minus5610 kcalmol)AGCUCAGGAGGGAUAGCGCCUAUGUUUUCUGUUACUCUGUUACAUUGCUUAUUGUUUAAGCUUGUGACAGUUAAGUGCAGAAGUAAGGCGUUAUCCAUUCUGAGCU

((((((((((((((((((((((((((((((((((((((((((()))))))))))))))))))))))))))))))))))))))))))

Tetraphis pellucida (MFE of minus4810 kcalmol)AGCUCAGGAGGGAUAGCGCCUAGCUCUUGGUUUCUGUGGCAUUGCUGUCAUUUGAAGCUCUGUAGCAGGAAAGGCUUUGAAGCAAUGCGCUAUCCAUUCUGAGCU

((((((((((((((((((((((((((((((((((((((()))))))))))))))))))))))))))))))))))))))

Polytrichum commune (MFE of minus6380 kcalmol)AGCUCAGGAGGGAUAGCGCCUAGCUCUGGAGCCACUCUGCUGUUUCACCAGUUGUGGAAAACUGGUUGUUCACCAGUCAGUCCCAGACGUACAGCGCUAUCCCUCCUGAGCU

(((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))

Oedipodium griffithianum (MFE of minus4950 kcalmol)AGCUCAGGAGGGAUAGCGCCUAGCCCUGGUCUCUUUGUGGCAUUGCUUUUCAUCGGAAGCUGUGUACCAAUUAGGCUCAGAAGGAAAGCGCUAUCCAUUCUGAGCU

(((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))

Andreaea rupestris (MFE of minus4910 kcalmol)AGCUCAGGAGGGAUAGCGCCCAACUCUGUACUAGAACUUCUGUAACUAUAUCUCAUUGUUUGGUAAACAGCCUGCAUUUGACAGUUCUUUGCAGAAGUGUGGCGUUAUCCAUUCUGAGCU

(((((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))))

Harpanthus flotovianus (MFE of minus4710 kcalmol) AGCUCAGGAGGGAUAGCGCCAAAUUCUGGCCUUUUUGUGGGAUCGCUUUCGAUGAAAGCUUGCACAGUUUGGUUCAGAUGCAAAUCGCUAUCCAUUCUGAGCU

((((((((((((((((((((((((((((((((((((()))))))))))))))))))))))))))))))))))))

(b)

Figure 1 (a) Schematic representation of a typicalmiRNAhairpin showing the position of the lowerDCL processing site (filled and open darkgray arrowheads) upper DCL processing site (filled and open gray arrowheads) and the positions of potential mature miRNA or miRNAlowast(filled and open arrowheads resp) (b) Primary and predicted secondary structures of stem-loop elements in Bryophyta andMarchantiophytamiR390 precursors Sequences of the highly conserved AGCU tetranucleotides at the lower DCL processing sites of miRNA or miRNAlowast areunderlined Names of plant species with sequenced portions limited by conserved AGCU tetranucleotides are underlined

















aClonecluster

bLengthbp



tasiARFAP2TAS6)

BryopsidaDicranidae



Gremiales









Dicranales




231779e SRR07489011659772 tasiARFAP2

TAS6





BryopsidaBryidae





Hypnales









2551444e


tasiARFAP2TAS6



HQ709415 tasiARFAP2











Bryales



TAS6





Table 1 Continued


aClonecluster

bLengthbp



tasiARFAP2TAS6)




Bartramiales




BryopsidaFunariidae

Encalyptales




Funariales



TAS6
























M 1 2 3 4 5 6 7








HookerialesHypnales


Bartramiales


Dicranidae

Bryidae

DicranalesTimmiidae

Funariidae


Bryopsida

Bryophyta

Polytrichopsida


Takakiopsida

Marchantiophyta












1 25423420218215713721


1 25423420218215713721


1 25123119917915413421


1 22720717415413011021

5998400 miR390 3998400 miR390

(a)


1 155135997921

ta-siR-ARF

1 164144725221

ta-siR-ARF

1 25223213711721

ta-siR-ARF

1 22620613111121ta-siR-ARF

1 2001801068621

ta-siR-ARF

1 189169957521





1 2962761591399575212 times ta-siR-ARF

1 38936925221121

2 times ta-siR-ARF

1 37735724220121

2 times ta-siR-ARF

1 2342141409921

2 times ta-siR-ARF

1 21219316111921







5998400 miR390 3998400 miR390

(b)











P patens TAS3a

P patens TAS3d


P medium 41-Pme



O hians 25-Oxy



L glaucum 41-Lgl


P patens TAS3e




B halleriana 28-Bha




B halleriana 32-Bha









Polypodiales

Cyatheales

Salviniales

Schizaeles

Gleicheniales

Hymenophyllales

Marattiales

Osmundales

Equisetales

Ophioglossales

Psilotales

Isoetales

















ta-siR-ARF

1 28726717315321ta-siR-ARF

1 25223213711721ta-siR-ARF

1 192172785821ta-siR-ARF

1 185165705021ta-siR-ARF

1 164144725221

2 times ta-siR-ARF

1 2712511369521TAS3-Sen1

TAS3-Sen2

TAS3-Sen3

TAS3-Sen4

TAS3-Sen5

target target

TAS3-Sen6

5998400 miR390 3998400 miR390






Disclosure



Acknowledgment


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















aClonecluster

bLengthbp



tasiARFAP2TAS6)

BryopsidaDicranidae



Gremiales









Dicranales




231779e SRR07489011659772 tasiARFAP2

TAS6





BryopsidaBryidae





Hypnales









2551444e


tasiARFAP2TAS6



HQ709415 tasiARFAP2











Bryales



TAS6





Table 1 Continued


aClonecluster

bLengthbp



tasiARFAP2TAS6)




Bartramiales




BryopsidaFunariidae

Encalyptales




Funariales



TAS6
























M 1 2 3 4 5 6 7








HookerialesHypnales


Bartramiales


Dicranidae

Bryidae

DicranalesTimmiidae

Funariidae


Bryopsida

Bryophyta

Polytrichopsida


Takakiopsida

Marchantiophyta












1 25423420218215713721


1 25423420218215713721


1 25123119917915413421


1 22720717415413011021

5998400 miR390 3998400 miR390

(a)


1 155135997921

ta-siR-ARF

1 164144725221

ta-siR-ARF

1 25223213711721

ta-siR-ARF

1 22620613111121ta-siR-ARF

1 2001801068621

ta-siR-ARF

1 189169957521





1 2962761591399575212 times ta-siR-ARF

1 38936925221121

2 times ta-siR-ARF

1 37735724220121

2 times ta-siR-ARF

1 2342141409921

2 times ta-siR-ARF

1 21219316111921







5998400 miR390 3998400 miR390

(b)











P patens TAS3a

P patens TAS3d


P medium 41-Pme



O hians 25-Oxy



L glaucum 41-Lgl


P patens TAS3e




B halleriana 28-Bha




B halleriana 32-Bha









Polypodiales

Cyatheales

Salviniales

Schizaeles

Gleicheniales

Hymenophyllales

Marattiales

Osmundales

Equisetales

Ophioglossales

Psilotales

Isoetales

















ta-siR-ARF

1 28726717315321ta-siR-ARF

1 25223213711721ta-siR-ARF

1 192172785821ta-siR-ARF

1 185165705021ta-siR-ARF

1 164144725221

2 times ta-siR-ARF

1 2712511369521TAS3-Sen1

TAS3-Sen2

TAS3-Sen3

TAS3-Sen4

TAS3-Sen5

target target

TAS3-Sen6

5998400 miR390 3998400 miR390






Disclosure



Acknowledgment


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 1 Continued


aClonecluster

bLengthbp



tasiARFAP2TAS6)




Bartramiales




BryopsidaFunariidae

Encalyptales




Funariales



TAS6
























M 1 2 3 4 5 6 7








HookerialesHypnales


Bartramiales


Dicranidae

Bryidae

DicranalesTimmiidae

Funariidae


Bryopsida

Bryophyta

Polytrichopsida


Takakiopsida

Marchantiophyta












1 25423420218215713721


1 25423420218215713721


1 25123119917915413421


1 22720717415413011021

5998400 miR390 3998400 miR390

(a)


1 155135997921

ta-siR-ARF

1 164144725221

ta-siR-ARF

1 25223213711721

ta-siR-ARF

1 22620613111121ta-siR-ARF

1 2001801068621

ta-siR-ARF

1 189169957521





1 2962761591399575212 times ta-siR-ARF

1 38936925221121

2 times ta-siR-ARF

1 37735724220121

2 times ta-siR-ARF

1 2342141409921

2 times ta-siR-ARF

1 21219316111921







5998400 miR390 3998400 miR390

(b)











P patens TAS3a

P patens TAS3d


P medium 41-Pme



O hians 25-Oxy



L glaucum 41-Lgl


P patens TAS3e




B halleriana 28-Bha




B halleriana 32-Bha









Polypodiales

Cyatheales

Salviniales

Schizaeles

Gleicheniales

Hymenophyllales

Marattiales

Osmundales

Equisetales

Ophioglossales

Psilotales

Isoetales

















ta-siR-ARF

1 28726717315321ta-siR-ARF

1 25223213711721ta-siR-ARF

1 192172785821ta-siR-ARF

1 185165705021ta-siR-ARF

1 164144725221

2 times ta-siR-ARF

1 2712511369521TAS3-Sen1

TAS3-Sen2

TAS3-Sen3

TAS3-Sen4

TAS3-Sen5

target target

TAS3-Sen6

5998400 miR390 3998400 miR390






Disclosure



Acknowledgment


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






M 1 2 3 4 5 6 7








HookerialesHypnales


Bartramiales


Dicranidae

Bryidae

DicranalesTimmiidae

Funariidae


Bryopsida

Bryophyta

Polytrichopsida


Takakiopsida

Marchantiophyta












1 25423420218215713721


1 25423420218215713721


1 25123119917915413421


1 22720717415413011021

5998400 miR390 3998400 miR390

(a)


1 155135997921

ta-siR-ARF

1 164144725221

ta-siR-ARF

1 25223213711721

ta-siR-ARF

1 22620613111121ta-siR-ARF

1 2001801068621

ta-siR-ARF

1 189169957521





1 2962761591399575212 times ta-siR-ARF

1 38936925221121

2 times ta-siR-ARF

1 37735724220121

2 times ta-siR-ARF

1 2342141409921

2 times ta-siR-ARF

1 21219316111921







5998400 miR390 3998400 miR390

(b)











P patens TAS3a

P patens TAS3d


P medium 41-Pme



O hians 25-Oxy



L glaucum 41-Lgl


P patens TAS3e




B halleriana 28-Bha




B halleriana 32-Bha









Polypodiales

Cyatheales

Salviniales

Schizaeles

Gleicheniales

Hymenophyllales

Marattiales

Osmundales

Equisetales

Ophioglossales

Psilotales

Isoetales

















ta-siR-ARF

1 28726717315321ta-siR-ARF

1 25223213711721ta-siR-ARF

1 192172785821ta-siR-ARF

1 185165705021ta-siR-ARF

1 164144725221

2 times ta-siR-ARF

1 2712511369521TAS3-Sen1

TAS3-Sen2

TAS3-Sen3

TAS3-Sen4

TAS3-Sen5

target target

TAS3-Sen6

5998400 miR390 3998400 miR390






Disclosure



Acknowledgment


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





HookerialesHypnales


Bartramiales


Dicranidae

Bryidae

DicranalesTimmiidae

Funariidae


Bryopsida

Bryophyta

Polytrichopsida


Takakiopsida

Marchantiophyta












1 25423420218215713721


1 25423420218215713721


1 25123119917915413421


1 22720717415413011021

5998400 miR390 3998400 miR390

(a)


1 155135997921

ta-siR-ARF

1 164144725221

ta-siR-ARF

1 25223213711721

ta-siR-ARF

1 22620613111121ta-siR-ARF

1 2001801068621

ta-siR-ARF

1 189169957521





1 2962761591399575212 times ta-siR-ARF

1 38936925221121

2 times ta-siR-ARF

1 37735724220121

2 times ta-siR-ARF

1 2342141409921

2 times ta-siR-ARF

1 21219316111921







5998400 miR390 3998400 miR390

(b)











P patens TAS3a

P patens TAS3d


P medium 41-Pme



O hians 25-Oxy



L glaucum 41-Lgl


P patens TAS3e




B halleriana 28-Bha




B halleriana 32-Bha









Polypodiales

Cyatheales

Salviniales

Schizaeles

Gleicheniales

Hymenophyllales

Marattiales

Osmundales

Equisetales

Ophioglossales

Psilotales

Isoetales

















ta-siR-ARF

1 28726717315321ta-siR-ARF

1 25223213711721ta-siR-ARF

1 192172785821ta-siR-ARF

1 185165705021ta-siR-ARF

1 164144725221

2 times ta-siR-ARF

1 2712511369521TAS3-Sen1

TAS3-Sen2

TAS3-Sen3

TAS3-Sen4

TAS3-Sen5

target target

TAS3-Sen6

5998400 miR390 3998400 miR390






Disclosure



Acknowledgment


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









1 25423420218215713721


1 25423420218215713721


1 25123119917915413421


1 22720717415413011021

5998400 miR390 3998400 miR390

(a)


1 155135997921

ta-siR-ARF

1 164144725221

ta-siR-ARF

1 25223213711721

ta-siR-ARF

1 22620613111121ta-siR-ARF

1 2001801068621

ta-siR-ARF

1 189169957521





1 2962761591399575212 times ta-siR-ARF

1 38936925221121

2 times ta-siR-ARF

1 37735724220121

2 times ta-siR-ARF

1 2342141409921

2 times ta-siR-ARF

1 21219316111921







5998400 miR390 3998400 miR390

(b)











P patens TAS3a

P patens TAS3d


P medium 41-Pme



O hians 25-Oxy



L glaucum 41-Lgl


P patens TAS3e




B halleriana 28-Bha




B halleriana 32-Bha









Polypodiales

Cyatheales

Salviniales

Schizaeles

Gleicheniales

Hymenophyllales

Marattiales

Osmundales

Equisetales

Ophioglossales

Psilotales

Isoetales

















ta-siR-ARF

1 28726717315321ta-siR-ARF

1 25223213711721ta-siR-ARF

1 192172785821ta-siR-ARF

1 185165705021ta-siR-ARF

1 164144725221

2 times ta-siR-ARF

1 2712511369521TAS3-Sen1

TAS3-Sen2

TAS3-Sen3

TAS3-Sen4

TAS3-Sen5

target target

TAS3-Sen6

5998400 miR390 3998400 miR390






Disclosure



Acknowledgment


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










P patens TAS3a

P patens TAS3d


P medium 41-Pme



O hians 25-Oxy



L glaucum 41-Lgl


P patens TAS3e




B halleriana 28-Bha




B halleriana 32-Bha









Polypodiales

Cyatheales

Salviniales

Schizaeles

Gleicheniales

Hymenophyllales

Marattiales

Osmundales

Equisetales

Ophioglossales

Psilotales

Isoetales

















ta-siR-ARF

1 28726717315321ta-siR-ARF

1 25223213711721ta-siR-ARF

1 192172785821ta-siR-ARF

1 185165705021ta-siR-ARF

1 164144725221

2 times ta-siR-ARF

1 2712511369521TAS3-Sen1

TAS3-Sen2

TAS3-Sen3

TAS3-Sen4

TAS3-Sen5

target target

TAS3-Sen6

5998400 miR390 3998400 miR390






Disclosure



Acknowledgment


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Polypodiales

Cyatheales

Salviniales

Schizaeles

Gleicheniales

Hymenophyllales

Marattiales

Osmundales

Equisetales

Ophioglossales

Psilotales

Isoetales

















ta-siR-ARF

1 28726717315321ta-siR-ARF

1 25223213711721ta-siR-ARF

1 192172785821ta-siR-ARF

1 185165705021ta-siR-ARF

1 164144725221

2 times ta-siR-ARF

1 2712511369521TAS3-Sen1

TAS3-Sen2

TAS3-Sen3

TAS3-Sen4

TAS3-Sen5

target target

TAS3-Sen6

5998400 miR390 3998400 miR390






Disclosure



Acknowledgment


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


ta-siR-ARF

1 28726717315321ta-siR-ARF

1 25223213711721ta-siR-ARF

1 192172785821ta-siR-ARF

1 185165705021ta-siR-ARF

1 164144725221

2 times ta-siR-ARF

1 2712511369521TAS3-Sen1

TAS3-Sen2

TAS3-Sen3

TAS3-Sen4

TAS3-Sen5

target target

TAS3-Sen6

5998400 miR390 3998400 miR390






Disclosure



Acknowledgment


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgment


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article peculiar evolutionary history of mir390...

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