Analysis of the genomic DNA of the harmful dinoflagellateProrocentrum minimum: a brief survey focused on the noncodingRNA gene sequences

Thangaraj Ponmani1 & Ruoyu Guo2 & Jang-Seu Ki1,2

Received: 21 October 2014 /Revised and accepted: 15 March 2015# Springer Science+Business Media Dordrecht 2015

Abstract Dinoflagellate algae are considered some of themost complicated organisms owing to their unusual genomiccharacteristics and novel gene regulatory mechanisms. Here,we extracted information from the genomic sequences of themarine dinoflagellate Prorocentrum minimum using GS FLXpyrosequencing technology. We obtained 473 Mb of se-quences from 1,379,588 reads, which generated 16,599contigs after assembly. Among the annotated sequences(4902 contigs, 30 %), BLAST analyses showed that 28.95 %(4731 contigs) of the genome fragments were homologouswith bacterial sequences and only 0.96 % (156 contigs) hadeukaryotic origins. However, analysis of bacterial 16S/23SrDNA sequences in the P. minimum genome revealed thatthe organism likely acquired this genetic material from sym-biotic bacteria, possibly through horizontal gene transfer, thusdemonstrating a definitive association between bacteria andP. minimum. Moreover, a specific consensus pattern was de-fined for dinoflagellate spliced leader (SL) sequences, a well-known genetic marker of dinoflagellates. Further, compari-sons of the various genomic proportions with respect to nu-clear, mitochondrial, and chloroplast genes revealed uniquegenomic features that include clusters of noncoding RNAgenes and tandem repeats. Taken together, this study detailsthe salient features of noncoding RNA genes in P. minimum

and provides further insights into the genome of this and otherdinoflagellates.

Keywords Dinoflagellate . Prorocentrumminimum .

Spliced leader . Genome . Noncoding RNA genes

Introduction

Dinoflagellate algae are unicellular eukaryotes and well-known primary producers in the aquatic ecosystem. Approx-imately half of dinoflagellates are photoautotrophs, with theremainder comprising mainly heterotrophs and severalmixotrophic species (Lin 2011). The study of these organismsis crucial, as they are widely distributed in aquatic environ-ments and can be responsible for harmful algal blooms(HABs) that lead to severe economic losses and environmen-tal impact (Hackett et al. 2004). According to the Tree of LifeWeb Project (http://tolweb.org/tree/), there are currentlyestimated to be more than 4500 species and 550 genera ofdinoflagellates based on morphology. In addition, unknowndinoflagellates continue to be discovered from naturalenvironments by means of molecular analyses (Stern et al.2010).

In recent years, next generation sequencing (NGS) hasbeen used to study the genomes of diverse marine eukaryotes,such as Tigriopus japonicus and Petromyzon marinus (Leeet al. 2010; Smith et al. 2013). However, these advanced tech-nologies have failed to provide the complete genomic se-quences of dinoflagellates, likely due to their complexity.Dinophyta usually have large genomes of 3–245 Gb, withthe number of chromosomes varying from several to morethan 100 (Lin 2011). In addition, dinoflagellates exhibit ex-cessive DNA methylation, high GC content, an absence oftypical eukaryotic transcriptional elements, and a lack of

* Jang-Seu Kikijs@smu.ac.kr

1 Institute of Natural Sciences, Sangmyung University, Seoul 110-743,Korea

2 Department of Life Science, Sangmyung University, Seoul 110-743,Korea

J Appl PhycolDOI 10.1007/s10811-015-0570-0

chromosomal decondensation during normal cell cycle pro-gression, as well as unusual gene regulatory mechanisms suchas mRNA editing, dinoflagellate spliced leader (dinoSL)trans-splicing, and polyuridylation. Moreover, transcriptionproceeds only on the peripheral and protruding DNA loops,while the permanently condensed chromosome remains silent(Lin 2011; Roy and Morse 2013). During evolution, the dino-flagellate genome was subjected to dramatic duplications(polyploidy) and gene transfer events (both lateral and hori-zontal) that resulted in the accumulation of noncoding tandemrepeats and multiple gene copies. These repetitive sequencespresent as a technical challenge in genome sequencing studies.

Dinoflagellate whole genome sequencing has been chal-lenging and little attempted due to their genomic complexity;thus, genetic information is limited. A previous attempt tosequence the nuclear genome of Heterocapsa triquetra re-vealed that only ~10 % of sequence reads exhibit similarityto other known sequences, with only half of these (5 %)depicting gene fragments, mobile genetic elements,pseudogenes, and low complexity sequences, while the re-mainder consists of repetitive sequences (McEwan et al.2008). In addition, characterization of 616-Mb draft genomeassemblies from the coral-dinoflagellate Symbiodiniumminutum led to the identification of ~42,000 protein-codinggenes and clusters of spliceosomal small nuclear RNA genes(U1–U6) (Shoguchi et al. 2013). Nevertheless, available dataon the genomic features of other dinoflagellates are insuffi-cient; thus, the sequencing of these genomes remains an on-going project. To date, no studies have been performed regard-ing the genetic information of Prorocentrum minimumwhere-as our previous studies characterized its transcriptome andconducted ecotoxicological assessments (Guo and Ki 2011,2012).

In the present study, we generated partial genomic se-quences of the toxic marine dinoflagellate P. minimum usingGS FLX pyrosequencing, performed a preliminary genomesurvey, and concentrated our analyses on assemblies thatshowed dinophyceaen sequence homology. After further clas-sification into nuclear and organelle genes (i.e., derived fromthe mitochondrial or chloroplast genomes), we analyzed thenoncoding RNA genes of the P. minimum genome extensive-ly. In this study, we concisely discussed the structural/functional features of the P. minimum genome.

Materials and methods

Cell culture and genomic DNA (gDNA) extraction

The D-127 strain of Prorocentrum minimum was obtainedfrom the Korea Marine Microalgae Culture Center (PukyongNational University, Busan, Korea). P. minimum cultures weremaintained in f/2 medium (Guillard and Ryther 1962) at

20 °C in a 12:12-h light/dark cycle with a photon flux densityof approximately 65 μmol photons m−2 s−1. Thecetyltrimethylammonium bromide (CTAB) method (Murrayand Thompson 1980) was used to isolate total genomic DNA(gDNA) from cells in prelogarithmic growth. The resultinggDNA was quantified as 0.91 μg μL−1 with a purity of 1.9(A260/280).

Genome library construction and assembly

A GS FLX Titanium sequencer was used to analyzeP. minimum gDNA (Roche; USA). For sequencing, adapter-ligated DNA fragments were prepared and clonally amplifiedvia emulsion PCR to generate template DNA beads. Beadswere then added to the wells of a PicoTiter-Plate (a fiber opticchip) that also contained DNA polymerase, ATP sulfurylase,and luciferase. During the sequencing reaction, the four nu-cleotides were added consecutively, and light signals wererecorded by the CCD camera when a complementary nucleo-tide was added to the template strand. GSDe Novo Assembler2.3 (Roche) was used for sequence assembly, and GlimmerM(www.tigr.org/softlab/glimmerm/) and NCBI were used forgene prediction with lowered expect values (E values). TheGS FLXlibrary was constructed from fragments of ~450–1200 nucleotides (nt), with an average fragment length of600–900 nt. Fragments smaller than 350 nt were excludedfrom the analysis. A total of 1,379,588 reads were used toobtain 473,480,387 nt of genome sequences, whichgenerated 16,599 contigs with an average length of 1770 nt(Table 1). This Whole Genome Shotgun project has been de-posited at DDBJ/EMBL/GenBank under the accessionJXLM00000000. The version described in this paper is ver-sion JXLM01000000.

Open reading frame (ORF) predictionand geneannotations

Genomic sequence data from Plasmodium falciparum wasused as a template for open reading frame (ORF) prediction(Gardner et al. 2002). The NCBI BLAST database (BLASTXand BLASTN) and Clustal X 1.83 were then used for ORFidentification and sequence alignment, respectively.

Results and discussion

Prorocentrum minimum is a “red tide”-forming marine dino-flagellate known to produce a potent biotoxin that causes thesevere diarrhetic shellfish poisoning (Cassis 2012). In recentyears, P. minimum has been used recurrently fortoxicogenomics and evolutionary studies in dinoflagellates(Zhang et al. 2007; Guo and Ki 2011; Guo and Ki 2012).Continued research on the P. minimum genome is necessary

J Appl Phycol

to understand the molecular processes underlying cellularbloom dynamics. Brief genome surveys using available liter-ature (LaJeunesse et al. 2005) as well as our genome sizemeasurements (data not shown) estimated the size of theP. minimum genome at ~6–7 Gb. Before this, there had beenno previous studies on the genome of P. minimum, despitetheir relatively smaller size compared to other dinoflagellates,such as Prorocentrum micans, which has a genome size of245 Gb. Here, we performed a preliminary survey to charac-terize the genomic features of P. minimum, including bacterialassociations, noncoding RNA genes, tandem repeats, and di-noflagellate spliced leader sequences, among others.

Proportion of identified contigs in the genomeof P. minimum

This study used 1,379,588 reads to generate sequences total-ing 473 Mb from the dinoflagellate P. minimum genome. Ofthe 16,599 contigs obtained, only 30 % (4902 contigs) hadsequence annotation and 70 % showed no hits in the BLASTsearch. Our analyses could not classify 70% of the sequences,since most of the dinoflagellate genome is not yet well char-acterized. Of the annotated sequences (4902 contigs, 30 %),28.95 % (4731 contigs) were found to have bacterial originsand only 0.96 % (156 contigs) showed homology with eu-karyotic taxa (Fig. 1a). Among the eukaryotic sequences,apicomplexa and dinophyceae occupied 1 and 46 %, respec-tively (Fig. 1b), the latter of which was the focus of the presentstudy.

Bacterial sequences related with the genomeof P. minimum

Extensive efforts were taken to avoid bacterial contaminationin the P. minimum cultures used for gDNA isolations andincluded exponential phase cells and density gradient separa-tions. Unexpectedly, the majority of the sequences (4731contigs, 28.95 %) were attributed to bacterial origins (seeFig. 1a). For this reason, we tried to determine whether thosesequences belong to P. minimum genome or associated bacte-ria. First, we searched the P. minimum genome for the pres-ence of 16S/23S rDNA sequences, since these could be usedto indicate the presence of endosymbiotic/ectosymbiotic bac-teria in the P. minimum culture. As such, the absence of 16S/23S rDNA sequences could signify the integration of thesesequences into the P. minimum genome. As a result, only3.9 % (4731 contigs) of sequences with bacterial origin werefound to be derived from the associated bacteria (Fig. 1a). Ofthese sequences, a major proportion (3.73 %) was attributed toRoseobacter denitrificansOCh 114 (244/638 contigs, 1.49 %)andMarinobacter aquaeolei VT8 (367/638 contigs, 2.24 %),sugges t ing that these bacter ia may have s t rongecto/endosymbiotic associations with P. minimum.T

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J Appl Phycol

In addition, 25.05 % (4,093/4,731 contigs) of the bacterialsequences identified lacked corresponding 16S/23S rRNA se-quences, indicating that a majority of the sequences with bac-terial origin might be integrated in the P. minimum genome.This data is supported by a previous study that shows that66.6 % of chromosome condensation protein coding genesexhibited bacterial origin in the S. minutum genome(Shoguchi et al. 2013). Furthermore, it is also possible thatthese bacterial sequences might be derived directly from bac-teria or indirectly from other algae with integrated bacterialgenes, which were later transferred to the P. minimum genome(Wisecaver et al. 2013). These results are largely supported bythe mixotrophic behavior of dinoflagellates (Doolittle 1998;Stoecker 1999; Johnson 2014). Altogether, these findings in-dicate that ecto/endosymbiotic associations between bacteriaand marine dinoflagellates, and likely influence the genomicarchitecture of dinoflagellates through horizontal gene transferevents (Mackiewicz et al. 2013; Mungpakdee et al. 2014).Subsequently, these events may further complicate the ge-nome structure of marine dinoflagellates. Based on our se-quence analyses, we explored the possible association be-tween bacteria and P. minimum; however, further studies areneeded to confirm these findings. In addition, further analysesrevealed that proteobacteria-derived sequences predominatedthose observed in the P. minimum genome, including 60 %alphaproteobacteria, 26 % gammaproteobacteria, 5 %betaproteobacteria, and 2 % deltaproteobacteria.

Analyses of dinophyceaen sequences in the P. minimumgenome

We evaluated the dinophyceaen-contigs (dino-contigs) of theP. minimum genome using the public databases BLASTN andBLASTX (Table 2). E values for each analysis supported theputative functions of these sequences (i.e., homology inverse-ly correlated with E values). Among the dino-contigs ana-lyzed, 39, 7, and 54% contigs encoded nuclear, mitochondrial(mt), and chloroplast (Chl) genes, respectively (Fig. 1c).When we analyzed the nuclear gene sequences, most showed

similarity with the mRNA/clones of P. minimum and Kareniabrevis, another dinoflagellate (Zhang et al. 2009; Guo and Ki2011). Notably, 21 % of the nuclear sequences accounted forspliced leader (SL) RNA genes (see below and Table 2), withthe remainder encoding cell surface protein p43 (cp43), nitratetransporter, heat shock protein 90 (Hsp90), two conservedproteins, two variants of ribulose-1,5-bisphosphate carboxyl-ase oxygenase (RubisCO) form II, rRNA (18S, 5.8S, 28S, and5S), and snRNA (U5 and U6) (see Table 2).

The analysis of mt-genes revealed the presence of cyto-chrome oxidase subunit 1 (cox1) and cytochrome b (cob)genes. Among the mt-contigs, two contigs showed homologywith pseudogenes of large subunit rRNA, and few of the mt-sequences (S12 gene and unknown mt-sequence) exhibitedhomology with stramenopile algal sequences (Table 2). Thisis supported by a study describing gene transfer betweenstramenopiles and the dinoflagellate Lepidodiniumchlorophorum (Minge et al. 2010). It was surprising that mt-genes accounted for only <10 % of dino-contigs despite oftheir active contribution in harmful algal blooms.

Chl genes comprised the largest fraction of dino-contigs(54 %), with most exhibiting homology with mRNA of pho-tosystem II (PSII) D1 subunit gene psbA of P. minimum(GenBankNo. JX573313) (Table 2). In addition, we identifiedsequences encoding ATP synthase subunit alpha (atpA), andthe PSII proteins CP47 (psbB), CP43 (psbC), D2 (psbD) cy-tochrome B559 subunit alpha (pbsE), and light harvestingprotein (Table 2). Furthermore, Chl-genes also exhibited ho-mology with the mRNA of P. minimum and with other dino-flagellates, including H. triquetra, P. micans, P.dentatum,Lingulodinium polyedrum, S. minutum, and H.niei.

Next, we compared both genome and cDNA sequences ofP. minimum (Guo and Ki 2011) to analyze mRNA editing,since this is essential for the gene function in dinoflagellates.These analyses revealed that a >90 % identity between gDNAand mRNA of P. minimum with respect to nuclear genes andmt genes, whereas Chl genes showed only 58–100 %. Thissupports the possibility of extensive mRNA editing in theplastid genome of P. minimum as reported by Lin et al.

Fig. 1 Composition ofassembled sequences ofProrocentrum minimum genome:a bacterial sequences predicted tobe integrated with P. minimumgenome (25.05 %), concernedbacterial sequences identifiedwith 16S/23S rRNA (3.85 %),and other Eukaryota (0.96 %); bproportions of contigs showedhomology with Eukaryota; cproportions of dinophyceaengenes in P. minimum genome

J Appl Phycol

Table 2 Blast analyses of dinophyceaen genes obtained from P. minimum gDNA database

Contigs BLASTN Species Accession no. E value

SL-contigs

contig02085 5S rRNA ; trans-spliced leader sequenceSL-g2-2 gene; vesicle transport protein;SymkaFL1061064217570(5′-), partialsequence;U5snRNA

P. minimum; K. micrum;Babesia divergens;Symbiodiniumkawagutii; C.cohnii

FJ434802; EF143079;LK934711; KC947473;

K01189

0; 8.00E-14;2.00E-18;4.00E-57;2.00E-18

contig14648; contig10359;contig12174;contig12239;contig13103;contig16212;contig04866; contig10639

SL sequence from mRNA clone librarySymkaFL

S. kawagutii KC940689; KC945796;KC951037;KC949275;KC940440;KC950389;KC939539; KC943935

3.00E-04; 0.2; 0.015;0.014; 0.004; 0.44;0.008; 2.4

contig03590 trans-spliced leader sequence SL-g2 gene P. minimum EF143084 6.00E-36

contig06179 trans-spliced leader sequence SLs and 5SrRNA-like genes

Polarella glacialis FJ434776 3.00E-22

contig07293 trans-spliced leader sequence SL-like genes P. minimum FJ434912 0

contig08675 SL sequence of heat shock protein 70(Hsp70) mRNA, partial

P. minimum JN560150 0.009

contig08642; contig14305 No hit; but showed SL sequence in ouranalyses

Nuclear-contigs

contig09817 unknown protein P. minimum ACU45142 2.00E-75

contig10727 cell surface protein p43 P. minimum FJ600121.1 9.00E-46

contig11268 nitrate transporter P. minimum ABI14400 2.00E-98

contig10663 heat shock protein 90 P. minimum JN831315 0

contig07256 conserved hypothetical protein P. minimum ABX80192 7.00E-122

contig13963 conserved hypothetical protein P. minimum ABX80192 0.17

contig08565; contig15855 ribulose 1,5-bisphosphate carboxylaseoxygenase form II

P. minimum AY169179; AAO13048 0; 4.00E-86

contig14300 internal transcribed spacer of the cloneshowed trans-spliced leader sequenceSL, U6 snRNA, 5S rRNA

K. brevis FJ434722 2.00E-27

contig02789 U6 snRNA gene K. brevis FJ434729 2.00E-46

contig00489 18S rRNA gene, internal transcribed spacer(ITS) 1, 5.8S ribosomal RNA gene,ITS2, 28S rRNA gene, and intergenicspacer (IGS)

P. minimum JX402086 0

contig02893 5S rRNA; tRNA ; tRNA P. minimum; B. microti;Neospora caninum

FJ434802; FO082874;FR823393

1.00E-52; 4.00E-23;1.00E-18

contig00402 18S rRNA; ITS1, 5.8S rRNA, ITS2 and28S rRNA

Spumella-like flagellateJBM/S11;Chrysolepidomonasdendrolepidota

EF043285; AF409121 0

Contig01490 iron superoxide dismutase Symbiodinium sp. PF-2005 AAX99422 1E-63

Mitochondrial-contigs

Contig09482 ribosomal protein S12 (mt) Chrysodidymus synuroideus(stramenopile alga)

NP_038194 6.00E-39

Contig03402 unknown mRNA sequence (mt) K. micrum EF443052 1.00E-07

Contig13467 mtDNA Ochromonas danica(stramenopile alga)

AF287134.1 0.00E+00

Contig02833 cytochrome oxidase subunit 1 (cox1)gene (mt)

P. minimum AF463414 0

Contig11482 cytochrome b gene (mt) P. minimum AY030286 0

Contig05891 large subunit rRNA pseudogene (mt) D. baltica JX001589 1.00E-65

Contig08246 large subunit rRNA pseudogene (mt) D. baltica JX001594.1 9.00E-27

Chloroplast-contigs

contig06975 H. triquetra; P. micans AAD44703; BAD90040 5E-28; 3.00E-12

J Appl Phycol

(2008), as well as a study by Mungpakdee et al. (2014), inwhich high frequency of mRNA editing was observed withChl genes in S. minutum.

Mining of dinoflagellate spliced leader (dinoSL) sequencesin gDNA of P. minimum

Dinoflagellates utilize dinoSL trans-splicing events for post-transcriptional regulation, which was originally discovered inparasitic kinetoplastids (Zhang et al. 2007; Roy and Morse2013). During dinoSL trans-splicing, a 22-nt spliced leader(SL) sequence is transferred to the 5′-end of mRNA mole-cules, from the 5′-end of a small noncoding SL RNA. Trans-splicing only occurs on genes encoded in the nucleus and isabsent in organelle-encoded transcripts. Since dinoSL se-quences are considered important markers of dinoflagellates(Zhang et al. 2013), we screened the P. minimum genome fordinoSL sequences (see Table 2). In total, 15 contigs werefound to contain dinoSL sequences, of which 13 exhibiteddinophyceaen homology during our BLAST analyses. In this

study, we have showed eight distinct SL sequences (Fig. 2).The accepted dinoSL sequence is 5′-DCC GTA GCC ATTTTG GCT CAA G-3′ (D = U, A, or G) (Zhang et al. 2007);however, based on the eight different SL sequences identifiedin our study, we propose the following consensus dinoSL:DKK M|TA GCC ATT TTG GCT C|KB G (Fig. 2), where15 nt in the middle indicate a conserved region (separated bylines) in all dinoSL sequences analyzed in this study andothers (with the exception of SL3 in Polarella glacialis;Zhang et al. 2009). This conserved region is flanked on bothsides with variable regions in which D = A, T, G, or C; K = C,A, or T; M = G or T; B = A or T. Among the dinoSL-contigs,33 and 17% carried TCCG|TAGCCATTTTGGCTC|AAG(named as PmiSL-1) and CCC G|TA GCC ATT TTG GCTC|A AG SL (named as PmiSL-2) sequences, respectively.

Furthermore, we also assessed the expression of these eightSL RNA genes in our P. minimum expressed sequence tag (EST)data base (Guo and Ki 2011). Surprisingly, only PmiSL-1(TCCGTAGCCATTTTGGCTCAAG; showed in 5 singletonsand 1 isotig) and SL 7 (ACCGTAGCCATTTTGGCTCAAG;

Table 2 (continued)

Contigs BLASTN Species Accession no. E value

ATP synthase CF1 subunit alpha;photosystem II CP43 protein (Chl)

contig07590 plastid psbC mRNA for photosystem IICP43 protein (Chl)

P. dentatum; H. triquetra AB158776; ACS34802 0; 0.15

contig11087 photosystem II chlorophyll-binding proteinCP-47 (Chl)

L. polyedrum ABB89027 8.00E-67

contig12701 photosystem II chlorophyll-binding proteinCP47 (Chl)

Symbiodinium sp. C3 CDH98074 4.00E-25

contig12998; contig14114;contig16055; contig09626;contig10311; contig11129;contig15003; contig15565;contig15685; contig16361;contig14530; contig14250;contig13391; contig11047;contig14579; contig16647;contig14681

photosystem II D1 subunit (psbA) mRNA(Chl)

P. minimum JX573313 3.00E-20; 7.00E-716.00E-36; 0; 0; 8.00E-

81; 8.00E-45;1.00E-47; 0; 2.00E-06;

1.00E-42; 9.00E-40; 5.00E-23;9.00E-46; 2.00E-30; 6.00E-21;1.00E-33

contig13907; contig14250;contig14280; contig15113

photosystem II D1 subunit (Chl) P. minimum AGO03484 3.00E-08; 2.00E-08;5.00E-07; 9.00E-10

contig16329; contig06511 cytochrome b559 alpha subunit (Chl) H. triquetra ABC24987; ACS34804 2.00E-16; 1.00E-35

contig05785 light-harvesting protein (Chl) Symbiodinium sp. C3 CBI83419 1.00E-10

contig09167 ATP synthase beta chain (atpB) gene (Chl) P. minimum JX573312 8.00E-77

contig10392 ATP synthase CF1 subunit alpha (Chl) H. triquetra ACS34795 3.00E-106

contig14181 ATP synthase CF1 subunit alpha (Chl) H. triquetra AAD44703 1.00E-87

contig08052 photosystem II protein D2 (Chl) H. niei AAX82964 2.00E-145

contig05460 andcontig14545;contig12970 andcontig13574;

cytochrome b6/f complex subunit IV H. triquetra ACS34797;ABC24988 2.00E-42; 7.00E-636.00E-11; 1.00E-11

contig07073 PSII CP47 apoprotein H. triquetra AAD44701 5.00E-130

contig09405 cytochrome b6 H. triquetra Q9XQU7 3.00E-96

contig12421 photosystem I P700 Chl a apoprotein A1 Karenia mikimotoi AFX73784 2.00E-9

J Appl Phycol

showed in 1 isotig) sequences were observed in EST (Guo and Ki2011) and SL 8 (TCC GTA GCC ATT TTG GCT CCA G) wasalready reported in gene sequence of P. minimum (Zhang et al.2007). While we observed eight different SL sequences,their expression pattern showed 27 different SL RNA se-quences in ESTof P. minimum. Of these 27 types of SL, twoEST sequences were found to have additional relict SL se-quences (data not shown). Moreover, the SL sequencesshowed different consensus patterns in genome and EST(genome, TCC GTA GCC ATT TTG GCT CAA G; andEST, TCC GTA GCC ATT TTG GCT CAA G; conservedsequences are highlighted in bold); however, further studiesare needed to classify conclusively these as canonical/relictSL sequences, as various conserved patterns were observedin the SL sequences from the genome and transcriptome ofP. minimum. Also, relict SL sequences are found to be poorlyconserved and are likely to be present at the downstream of thecanonical SL sequences (Slamovits and Keeling 2008).

In addition, our analyses revealed novel SL-5S rRNA geneclusters in three contigs: SL-5S rRNA-U5 small nuclear RNA(snRNA) cluster, SL-5S rRNA cluster, and SL-U6 snRNA-5SL cluster (see Table 2). The SL-5S rRNA cluster has not yetbeen reported in P. minimum, though SL-5S clusters havebeen identified in other dinoflagellates (Zhang et al. 2009;Drouin and Tsang 2012). This is the first report of this kindin P. minimum. In dinoflagellates, conserved sequencesamong SL RNA coding regions have been reported by Zhanget al. (2009); however, our Clustal X 1.83 analyses in the

P. minimum genome failed to show any conserved regionsoutside of SL sequences (Fig. 2). These results emphasizethe complex nature of SL genes in dinoflagellates.

Organization of ribosomal DNA (rDNA) unitsin P. minimum genome

In the present study, we obtained seven contigs that showedsimilarities to the rDNA genes of P. minimum and other dino-flagellates (Durinskia baltica and Karenia brevis) (Table 2),including the genes encoding 5S, 18S, 5.8S, 28S rRNA, andsnRNA (U5 and U6). In addition, we identified sequences thatencode the 18S rRNA, internal transcribed spacer (1 and 2),5.8S rRNA, and 28S rRNA in two different contigs, whichexhibited homology with dinoflagellate and stramenopiles al-gae, respectively (Table 2). While 5S rRNA genes are oftenclustered with other rDNA units (e.g., 18S, 5.8S, 28S rRNAgenes) in protists including few dinoflagellates (Drouin andTsang 2012), we did not find this cluster in our analyses.However, we identified three 5S rRNA clusters, includingthe 5S-SL, 5S-SL-snRNA (U5 or U6), and 5S-tRNA clusters.This is supported by previous reports identifying the 5S-SLand 5S-SL-U6 snRNA clusters in other dinoflagellates (Zhanget al. 2009). To date, the 5S-tRNA cluster has only been re-ported in the Entamoeba species (Tawari et al. 2008). Thepresent study is the first report that describes the 5S-SL-U5and 5S-tRNA clusters in dinoflagellates. Among these, twocontigs (contig02085 and contig02893) contained promoter

Fig. 2 Different type of SL RNA coding region identified in this study. SL1-SL4 RNA genes possess PmiSL-1 (shaded box); SL5 and SL6 showedPmiSL-2 (open box)

J Appl Phycol

elements for the 5S rRNA gene (122 nt) as reported by Zhanget al. (2009) (highlighted in bold below with 5S rRNA se-quence: GCT GAC GGC CAT ACC GCG TCG AAT GCACCGGATCTCTTC TGACCTCCGAAGTTAAGCGGCGCA GGG CCC GGT TAG TAC TGG GGT GGG GGACCG CCT GGG AAG ACC TCG GGT GCT GTC AGC T).Herein, the underlined bases were different from the al-ready reported P. minimum 5S rRNA sequence but havebeen reported in that of other dinoflagellates (Karlodiniumveneficum and Pfiesteria piscicida) and in Perkinsusatlanticus, a close relative of P. minimum (Zhang et al.2009). In P. minimum, different gene linkages were foundto be associated with 5S rRNA, suggesting that the mobil-ity of the 5S rRNA gene could underlie this rearrangement.As reported by Drouin and Tsang (2012), the presence ofinternal promoter elements in 5S rRNA gene (see above)might aid in its transposition. Furthermore, the various 5SrRNA gene organizations can be observed from yeast tohigher eukaryotes, suggesting that they might have someevolutionary significance but little functional relevance.Nevertheless, the definitive reasons behind this clusteringarrangement are yet to be identified.

Investigation of tandem repeats (TR) in P. minimum

Repeat sequences seem to play a vital role in maintaining thestructural integrity of the genome and gene regulatory mech-anisms in dinoflagellates (McEwan et al. 2008). Gene dupli-cation is reported to have occurred in a vast majority of thedinoflagellates (Shoguchi et al. 2013). To examine the pres-ence of tandem repeats (TRs) in the P. minimum genome,sequences were analyzed with the online tool tandem repeatsfinder (TRF) to locate TRs (Benson 1999). These analysesrevealed that 6 % of the P. minimum genome (Fig. 3) and39 % of the dino-contig sequences contained TRs (insertFig. 3). This finding corroborated the previous genomic

studies in other dinoflagellates (Allen et al. 1975; Jaeckischet al. 2011; Shoguchi et al. 2013). For example, theCrypthecodinium cohnii, Alexandrium ostenfeldii, andS. minutum genomes comprised ~50–60, ~51, and <10 % ofTRs, respectively (Allen et al. 1975; Jaeckisch et al. 2011;Shoguchi et al. 2013). In H. triquetra, only 5 % of sequencesanalyzed showed repetition (McEwan et al. 2008). The pres-ence of TRs often correlates with genome size, suggesting thatthe fraction of TR might be directly proportional to the size ofthe cell/genome. To date, the actual function of TR in thegenome has not been elucidated fully; however, previous stud-ies indicate that TRs might participate in the maintenance ofgenomic architecture and the adaptive response to environ-mental stress in dinoflagellates and bacteria (Zhou et al. 2014).

In the present study, we found that most of the SL contigscontain TRs in their upstream regions (Table 3), suggestingthat these TR sequences may play a role in gene regulation.Moreover, we examined the consensus patterns of tandemrepeats in dino-contigs and were unable to find any sharedconserved sequences in nuclear and Chl-genomes (data notshown). Based on these findings, we could infer that TRsmay facilitate genome stability in dinoflagellates in responseto environmental stressors in the marine environment.

In conclusions, this study provides novel insights into theP. minimum genome with particular relevance to SL se-quences, 5S rRNA-SL gene cluster organization, and TR se-quences. These analyses demonstrate the complex nature ofthe dinoflagellate genome that is maintained throughout theirlife cycle by various genetic events. In addition, our resultssuggest that many of the bacterial sequences identified in theP. minimum genome could be the product of gene transferevents. These stable bacterial associations with P. minimumfurther increase the complexity of an already complicated ge-nome. Overall, these results improve our understanding of thegenome of the toxic dinoflagellate P. minimum as well as thatof other Dinophyta.

Fig. 3 Number of tandem repeats(TR) showed in P. minimumgenome (1-19 TR) anddinophyceaen sequences (1-5 TR;insert figure)

J Appl Phycol

Table 3 Analyses of tandem repeats (TR) in SL-contigs of P. minimum genome

SL contigs Tandem repeats (TR)

Consensus pattern Sequence description

contig04866 CCCCAAGAGA ....564CCCCAAGAGA|CCCCAAGAGA|GCCCAAGAGG|CCCCCTAGAGGCCTCCTAGAGGCTCCACGAGGCCCCACAGGAGCCACCAAAGACGCCCCACGAGCATCCGTAGCCATTTTGGCTCAAG681….

contig08675 CCCAAGAGGCC ….77CCCAAGAGGCC|CCCAAGAGGGC|CCCAAGACGGCC|CGAGAGAGCCCGGAGACGGCCCCAGAGCCGCCCAAGACGGCGCAAGAGGGTCCCAAGAAGGCCCCAACAAGAGGGGTCCGTAATTAGCCATTTTGGCTCAAG212….

GCCCAAGAGGGCCCCAAGACG

….43GCCCAAGAGGCTCCCCAAAACC|GCTCCAAGACGGCCCAAGAG|GCCCCCAAGAGGGCCCCAAGACG|GCCCGAGAGAGCCCGGAGACG|GCCCCAGAGCCGCCCAAGACG|GCGCAAGAGGGTCCCAAGAAG|GCCCCAACAAGAGGGGTCCGTAATTAGCCATTTTGGCTCAAG212….

GCCCAAGAGGCCCCCAAGACGGCCCCAAGACG

….43GCCCAAGAGGCTCCCCAAAACCGCTCCAAGACG|GCCCAAGAGGCCCCCAAGAGGGCCCCAAGACG|GCCCGAGAGAGCCCGGAGACGGCCCCAGAGCC|GCCCAAGACGGCGCAAGAGGGTCCCAAGAAGGCCCCAACAAGAGGGGTCCGTAATTAGCCATTTTGGCTCAAG212….

contig02085 GGCGCGGCATCGGGGGC ….1050GGCGCGCATCTGGGGC|GGCGCGGCATCGGGGGC|GCTGGGTCTGGGGCGGCGCAGGGGCGGGGCGCGCCAACTGTGCGTTTTGCGTGCTGGGGCGCAGTGGCGCGCATCTGGCGCGGCGCAGGGGTGGTCCACCTCAGTCCTGGGACTGGGATGGGCCCCTCGGGGCGCCGAAGCCTCGAGCCAAATGCCCCCAGGGAGGAGTCCCCCCCTGGGGGGGGGAGGGTATTTTGTAGCTGCCGCGGCGGCGCTGGTGGCGGCCGCGCGATTTGGAGCATACCTTCTCGGCCTTTTGGCTATGATCAAGTGTAGTATCTGTTCTTATCAGTGTGACAACTGATATGTCTCCAACTGGAGACTTGCTTGTCGCATTTATTTTTTCTGGGGAGGTACATCCTTGAGCTTGCTCAGGATTCCTCAAGCACTGCTCTGGCTTGGCACACCGCCAGATGTGGCGCACCCTTCGGGGTAACAGTTGACAGTTGACATGTGTGAGTGTGTGTGTGTGTGCGTGTGTGTGCGTTCCTTGTTGTGTCTACTGCGTAGCGCGCCTGACTGCACCGCACGGCGGAGCGGCGCAAAACAACTTCGCGCCGCGACCCTGGGCTGCGCCAAAGCTGACAGCACCCGAAGGTCTTCCCAGGCGGTCCCCCACCCCAGTACTAACCGGGCCCTGCGCCGCTTAACTTCGGAGGTCAGAAGAGATCCGGTGCATTCGACGCGGTATGGCCGTCAGCGAAAGCCATCGCGCGGCTGAAATAGCGCCCCGTCGGCTGGGGCGCGGCGGCTGCTCGGTCGGTCGGGCTTACATTCGCCCCTGCACCTCAGAACCCTTCGCCCGCGCAAGCCGCCGTCGGGGAGCGGGGCGGCCGAGGTCTTCGGCTGAGAGTCGCAGCTGCGCTCGCGCCCTGCAGCCGCCGCGCCCACCGCCAACTTGCATCTGACTGGTTGGGCCGAAGCCCGCGTAAAAATTGCAAGAGTGACCTTGACGATCAAACTTGACGGGAATTGGAGTAGTTTTCAACTAGCAAACAGCGCACCTCCGCTAAGCGTCACTGATAGCGCTATTGCCCCTTCCGCAAGGATAAATAAAGAAAGGGCGCCGCGAAGTTTGGCGAACGAATGAAACGCGCGCGAGGGATTTGGCGCAGGGCGCGGTTGCGCGGCACCAGTGACAAAGGCAAAGTGCGCGCGGTCTACACGAAGCACCGTAGCCATTTTGGCTCAAG2275….

contig10359 TCTTGGGGGCC ….1TCTTGGGGGCC|TCTTGGGGGCC|TCTTGGGGGCC|TCTTGGGGCC|TCCCGTAGCCATTTTGGCTCAAG66….

contig12239 CCT ….1CCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCT|CCTCCTCCTCCTCCTCTCCGTAGCCATTTTGGCTCAAG86….

contig14648 CA ….1CACACAACACACACAACACACAACACACAACACACAA|CACACAACACACACACACACACCCGTAGCCATTTTGGCTCAAG80….

ACACACA ….9ACACACA|ACACACA|ACACACA|ACACACA|ACACACA|ACACACA|CACACACA|CCCGTAGCCATTTTGGCTCAAG80….

contig16212 TCCTCCTTCC ….424TCCTCCTTCC|TCCTCCTTCC|TCCTCCTCCC|CTTCCTCCCCCCTCTGTAGCCATTTTGGCTCAAG487….

contig06179 TTTG ….756TTTGTGTTGTTTGTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGCTTG807….

contig14300 CACACAAACACGC ….494CACACAAACACGC|CACACAAACACGC|GACACA525….

CA ….539CACACACGCAAACACACACACACACACACACACACACACACACACACACACACACACA596

TR are indicated by bold with underlines and each TR are indicated by line separation; SL sequences are indicated by bold highlighted letters

J Appl Phycol

Acknowledgments This work was supported by a National ResearchFoundation of Korea (NRF) grant funded by the Korea government (Nos.NRF-M1A5A1-2013-044476, and 2013R1A1A2013596), and a grant fromtheNational Fisheries Research andDevelopment (NFRDI) funded to J.-S.Ki.

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