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The First Symbiont-Free Genome Sequence of Marine Red Alga, Susabi-nori (Pyropia yezoensis)

The First Symbiont-Free Genome Sequence of Marine Red Alga, Susabi-nori (Pyropia yezoensis)

  • Yoji Nakamura, 
  • Naobumi Sasaki, 
  • Masahiro Kobayashi, 
  • Nobuhiko Ojima, 
  • Motoshige Yasuike, 
  • Yuya Shigenobu, 
  • Masataka Satomi, 
  • Yoshiya Fukuma, 
  • Koji Shiwaku, 
  • Atsumi Tsujimoto
PLOS
x

Abstract

Nori, a marine red alga, is one of the most profitable mariculture crops in the world. However, the biological properties of this macroalga are poorly understood at the molecular level. In this study, we determined the draft genome sequence of susabi-nori (Pyropia yezoensis) using next-generation sequencing platforms. For sequencing, thalli of P. yezoensis were washed to remove bacteria attached on the cell surface and enzymatically prepared as purified protoplasts. The assembled contig size of the P. yezoensis nuclear genome was approximately 43 megabases (Mb), which is an order of magnitude smaller than the previously estimated genome size. A total of 10,327 gene models were predicted and about 60% of the genes validated lack introns and the other genes have shorter introns compared to large-genome algae, which is consistent with the compact size of the P. yezoensis genome. A sequence homology search showed that 3,611 genes (35%) are functionally unknown and only 2,069 gene groups are in common with those of the unicellular red alga, Cyanidioschyzon merolae. As color trait determinants of red algae, light-harvesting genes involved in the phycobilisome were predicted from the P. yezoensis nuclear genome. In particular, we found a second homolog of phycobilisome-degradation gene, which is usually chloroplast-encoded, possibly providing a novel target for color fading of susabi-nori in aquaculture. These findings shed light on unexplained features of macroalgal genes and genomes, and suggest that the genome of P. yezoensis is a promising model genome of marine red algae.

Introduction

Marine red algae of the order Bangiales (Rhodophyta) such as Pyropia and Porphyra (laver) have been important seafoods in East and Southeast Asia for thousands of years [1]. Lavers are also harvested in New Zealand, Chile, Wales, and Pacific North America [2][6]. In Japan, the aquaculture of Bangiales seaweeds (so-called ‘nori’) started three hundred years ago, and many different species have been cultivated. Currently, susabi-nori (Pyropia yezoensis) is the highly valued seaweed crop in East Asia, as well as in Japan.

In terms of the morphogenesis of nori, their life history remained enigmatic, even after the aquaculture became popular. Bangiales seaweeds were usually harvested as macrophytes of thalli in winter, but little was known about their morphological characters in other seasons. The question was resolved by Drew’s works [7], [8], which showed that gametophytes alternate with a microscopic, filamentous, shell-boring sporophytic phase. This milestone accelerated research into the cell biology of nori, providing the basis for understanding how the organisms live in the seawater environment. Over the last decade, host-symbiont interaction has been a hot topic of research into the life history of seaweed. Many seaweeds contain the essential coenzyme vitamin B12 (cobalamin), and this metabolite is often supplied from environmental bacteria [9]. P. yezoensis also contains high levels of vitamin B12, and bacteria are considered the source. Although there are many kinds of marine bacteria on the cell surface of P. yezoensis [10], some of which are tightly attached to the cell wall, their biological significance is still uncertain. A recent study demonstrated that some of these bacteria play key roles in the normal development of P. yezoensis (unpublished data), suggesting that there is a symbiotic relationship between them.

In the field of aquaculture, cultivar improvement of P. yezoensis has been an important issue. Color fading, for example, is a disease of Pyropia caused by nutrient deficiency in water, and there have been efforts to clarify the mechanism and develop resistant cultivars. Climate change, such as global warming, has also raised concerns about nori aquaculture. Many of the current cultivars originate from the northern cold area, and their tolerance to higher temperatures has been examined. Recently, breeding and molecular cloning technologies have enabled the development of DNA markers for Pyropia. However, species of Bangiales have simple morphological features, making it difficult to distinguish them from each other. Therefore, the genome sequence of Pyropia will be a promising resource for the comprehensive development of high-resolution markers.

Among red algae, the 100%-complete genome sequence of a unicellular species, Cyanidioschyzon merolae, has been determined [11], [12]. This organism inhabits extreme environments, such as hot springs, and may be a target of special adaptation, though the genome is expected to provide basic information on the lifestyle of photosynthetic eukaryotes. As another special case, the nucleomorphs in cryptomonads cells, which are considered to be of red-algal symbiont origin, have been also sequenced [13][15]. The nucleomorph genomes are generally quite small (several hundred kilobase pairs) and have lost many genes, indicating that genome reduction or gene transfer to the host nucleus have occurred after symbiosis. The nuclear DNA contents of Bangiales species have been examined by fluorescence microscopy [16][18], and range from 260 to 500 Mb, but this has not been confirmed at the sequence level. P. yezoensis has been considered a good target for red algal genomics [19], and expressed sequence tag (EST) analyses have been conducted to explore the gene candidates related to the life cycle [20][22]. However, the whole genome sequencing of this marine alga has been difficult because of DNA contamination from symbiotic bacteria. Thus, for red algae in general, genomic information has been poor until now, and many of the molecular mechanisms related to their life cycle or other traits remained unsolved. In this study, we have prepared axenic protoplast culture of P. yezoensis, sequenced the nuclear genome, and report its genomic features.

Materials and Methods

Protoplast isolation from P. yezoensis Culture

Monospores of P. yezoensis strain U-51 were cultured in sterile modified half-strength SWM-III medium. The culture was incubated at 17°C under illumination (50 µmol·m−2·s−1, 10∶14 h light:dark cycle). The culture medium was replaced every week. For the isolation of protoplasts, samples of the formed thalli were harvested directly from the culture flasks. The isolation procedure was according to a previously modified method [23]. In brief, the thalli weighing about 50–100 mg were immersed in 0.5% citric acid (pH 2.0–2.3) for 90 s and rinsed with sterile 90% natural seawater (NSW). The cleaned thalli were cut with a microtome blade and shaken with a 2% papain solution for 30 min. After washing with 90% NSW containing 0.7 M mannitol, enzyme solutions of agarase, mannanase, and xylanase (1 unit/8 ml, Yakult pharmaceutical industry Co., LTD, Tokyo, Japan) were added to the thalli and shaken for 60–90 min to degrade the cell walls. The solution was filtered through 20-µm mesh filter to remove the undigested tissue debris. The filtrate fraction was collected as the cell wall sample. The filtered solution was washed with 90% NSW containing 0.7 M mannitol, and the protoplast solution was obtained.

Genome Sequencing and Assembly

From the protoplast DNA sample of P. yezoensis, whole-genome shotgun libraries were prepared for two platforms, a Roche 454 GS-FLX/FLX+ (Roche Diagnostics, Branford, CT) and an Illumina Genome Analyzer IIx (Illumina, Inc. San Diego, CA), respectively. The 454-pyrosequencing library for single-end reads was constructed from the sheared DNA by GS Titanium Rapid Library Preparation Kit (Roche Diagnostics). For the Illumina Genome Analyzer IIx, a 75-bp paired-end shotgun library (insert sizes of 500 bp) was prepared according to the manufacturer's protocols. The read data obtained have been deposited in DDBJ/EMBL/GenBank under accession number SRA061934. Both reads were assembled using CLC Assembly Cell™ version 4.06 beta (CLC bio, Aarhus N, Denmark). For the preliminary de novo assembly, the contigs obtained still contained the sequences of organelles (mitochondrion and chloroplast) and an unknown bacterium of the genus Agarivorans. To remove such non-nuclear sequences, reference sequences were prepared. For organelles, the sequences of the chloroplast (accession: NC_007932) and mitochondrion (NC_017837) genomes of P. yezoensis were downloaded from the GenBank. In addition, Agarivorans albus strain MKT 106 [24] was purchased from the National Institute of Technology and Evaluation, Japan (NBRC) and the genomic sequences were read with 454 GS FLX+ (Text S1). The sequences of the organelles and bacterium were then used to clean the assembly of P. yezoensis nuclear DNA sequences.

cDNA Sequencing

Total RNA was isolated from thalli of P. yezoensis U-51 and first-strand cDNA was synthesized to selectively enrich full-length one with a cap structure and polyA tail (Text S2). The cDNA library prepared was sequenced by Illumina Genome Analyzer IIx, and finally 12,570,945 read pairs were obtained.

Detection of Transposable Elements and Repetitive Sequences

Tandem repeats in the P. yezoensis nuclear genome were detected by Tandem Repeats Finder version 4.04 program [25] with default parameters, followed by analysis using Tandem Repeats Analysis program version 2.1 [26]. The resulting 6,300 consensus sequences were employed to build repeat library generated by RepeatScout version 1.0.5 [27]. Knowledge-based annotation was carried out by REPET pipeline (http://urgi.versailles.inra.fr/Tools/REPET) [28]. Transposons and SINEs (short interspersed nuclear elements) were identified using CENSOR software version 4.2 [29].

Gene Prediction

We downloaded the known protein sequences of red algae (Rhodophyta) from GenBank at NCBI. We mapped those to the genome contigs assembled and detected 547 coding regions using Exonerate [30]. To detect other coding regions in the contigs, we prepared a training model using the gene-finding program, AUGUSTUS [31]. The initial training model was constructed using the 547 nucleotide sequences, and 7,733 genes were predicted from the contigs. We then queried the predicted protein sequences against the NCBI nr database using BLASTP (E-value <1e-10) [32], and appended 382 well-matched queries to the training model. Thus, we reconstructed the training model with 929 nucleotide sequences in total. During gene prediction, 12,570,945 cDNA read pairs in this study and 28,465 EST sequences of Pyropia from GenBank were mapped to the contigs by BLAT [33] and utilized as hints of exon regions in AUGUSTUS. Algal and plant protein sequences from the public databases (the species and sources are listed in Table S1) were mapped by TBLASTN (E-value <1e-5) and also hinted for AUGUSTUS. Predicted gene sequences are available at http://nrifs.fra.affrc.go.jp/ResearchCenter/5_AG/genomes/nori/.

Gene Annotation and Comparison

Each gene function was inferred by BLASTP (E-value <1e-5) to the GenBank database and mapping to the Gene Ontology (GO) was conducted by Blast2GO [34]. Protein sequences from red/green algal and plant genomes, C. merolae, Chlamydomonas reinhardtii and Arabidopsis thaliana (Table S1), were used for gene set comparison. Orthologous gene pairs were defined as reciprocal best hit pairs by BLASTP (E-value <1e-5). The proportions of GO categories annotated by Blast2GO among species were compared using WEGO [35].

Analyses of Methionine Synthase Genes and Light-harvesting Genes

Multiple sequence alignment and phylogenetic analysis were conducted by MAFFT [36] and MEGA5 [37], respectively. Phylogenetic trees were constructed by the Neighbor-Joining method [38]. The subcellular localization of light-harvesting proteins to chloroplast were predicted by WoLF PSORT [39] and ChloroP 1.1 [40]. For an NblA homolog of P. yezoensis, the cDNA was synthesized from total RNA described above and PCR experiment was conducted to confirm the gene structure and expression (Text S3).

Results

Assembly and Genome Statistics

The Roche 454 GS FLX and the Illumina GA IIx platforms generated 1,810,613 reads with 9,474,994,049 base pairs (bp) and 508,033,476 reads with 254,016,738 base pairs, respectively. Based on read coverage analysis, the whole sequence of nearly 510 million reads was estimated to represent an approximately 166-fold coverage of the P. yezoensis nuclear genome (Figure S1). During the assembly, we removed non-nuclear contigs such as organellar and bacterial sequences, finally obtaining 46,634 contigs (Table 1). The contigs totaled 43 Mb with a high GC content (63.6%), while the organellar genomes have a low GC% (mitochondrion; 32.7% and chloroplast; 33.1%). The average contig length and N50 were 932 bp and 1,669 bp, respectively, indicating that the genome sequences are still fragmented. About 98% (12,309,453 read pairs) of cDNA read pairs were mapped on the contigs. In addition to the nuclear genome, we found two closed-circular plasmids similar to those of Pyropia tenera (Text S4).

Repetitive Sequences and Telomere Structure

In the nuclear genome, there are only 392 microsatellite (2 to 5-bp) repeats, most of which are of 3-bp (326 repeats) (Figure 1). In particular, the majority of triplet repeats (233/326) are CCG (i.e., CCG/CGC/CGG/GCC/GCG/GGC) and are generally located in intergenic regions. The paucity of short tandem repeats is comparable to the C. merolae genome, which has 569 repeats, mostly AGC triplets (Figure S2). Non-redundant repetitive sequences make up 1.4% of all the contigs (Table1). We did not find telomere sequences in the assembled contigs, but did in the paired-end reads produced by Illumina. The unit of the telomere was estimated to be 5′-TTAGGG-3′, similar to those of land plants and green algae (5′-TTTAGGG-3′) [41].

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Figure 1. Microsatellite distribution.

Repeats with frequency > = 3 are shown. Locations of microsatellite repeats are classified by color: exon (green), intron (red) and intergenic (blue) regions.

http://dx.doi.org/10.1371/journal.pone.0057122.g001

Gene Statistics

We predicted 10,327 protein-coding gene models from the nuclear genome (Table 2), which is about twice as many as C. merolae [11]. In comparison with other algal genes, the average CDS length was short (849 bp), probably because many of the genes are partially predicted from fragmented contigs. Therefore, to obtain robust statistics of P. yezoensis genes, we selected 1,314 genes as validated, each protein sequence of which covered more than 95% of the homologous sequence in an alignment. This produced an increased average CDS length (1,247 bp), which is comparable to those of other algae. In particular, we found that the genes of P. yezoensis had few introns: about 60% of the validated genes lacked introns (Figure 2). As for all predicted genes, the genes lacking introns made up nearly three quarters of those. The average number of introns per gene (0.7/gene) is similar to those of small-genome algae (Figure 3A), and is 10-fold lower than that of Chlorella variabilis NC64A, a green alga with a similar genome size (46 Mb) [42]. The average intron size was also small (300 bp) compared with large-genome algae, such as a green alga C. reinhardtii [43] and a brown alga Ectocarpus siliculosus [44]. We observed a correlation between average intron length and algal genome/contig size (Figure 3B), as well as between gene number and genome/contig size (Figure 3C).

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Figure 2. Distribution of intron number.

“All genes” and “Validated genes” correspond to those shown in Table 2, respectively.

http://dx.doi.org/10.1371/journal.pone.0057122.g002

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Figure 3. Correlation between gene statistics and algal genome sizes.

The y value of P. yezoensis (plotted in red) indicates the contig size (43 Mb). The species and data that are not shown in Table 2 are summarized in Table S2. (A) Intron density and genome size; (B) Average intron length and genome size; (C) Gene content and (logarithmic-transformed) genome size.

http://dx.doi.org/10.1371/journal.pone.0057122.g003

Gene Annotation and Comparison

Of the 10,327 genes predicted in the P. yezoensis nuclear genome, 6,716 genes matched known genes in GenBank and 3,611 were novel. About 80% of the BLAST top hits were to other eukaryotes: the Streptophyta (mainly land plants) genes account for the majority (Figure 4A, left), followed by Chlorophyta (green algae), Chordata (e.g. vertebrates), and Rhodophyta (red algae). This distribution probably reflects the proportion of entries in GenBank, because many land plant and vertebrate genomes have been sequenced. The remaining 20% of the top hits were to bacteria (Figure 4A, right), many of which were from cyanobacteria, which may have been transferred from the chloroplast genome. Similarly, α-proteobacterial hits might be accounted for by the transfer from an ancestral genome of the mitochondrion. The other hits might be examples of gene transfer from environmental species, or represent artifacts from deeply diverged sequences. In this study, a case of horizontal gene transfer from γ-proteobacteria was suggested by Sanger resequencing (Figure S3).

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Figure 4. BLAST top hit distribution and gene set comparison.

(A) Taxonomic distributions of BLASTP top hits of P. yezoensis genes. (left) Eukaryotes; (right) Prokaryotes; (B) A Venn diagram of gene sets among four species (P. yezoensis, C. merolae, C. reinhardtii, and A. thaliana). Numbers of gene groups are shown on the diagram. Each gene group is defined as a singleton or a cluster of paralogs; (C) GO category comparison among P. yezoensis, C. merolae and C. reinhardtii.

http://dx.doi.org/10.1371/journal.pone.0057122.g004

Rhodophyta are phylogenetically close to Viridiplantae (Chlorophyta and Streptophyta) [45]; therefore, we selected the representative model species, C. merolae, C. reinhardtii and A. thaliana, and compared the gene sets among those three and P. yezoensis. As a result, 1,509 gene groups were common to C. merolae, C. reinhardtii and A. thaliana (Figure 4B). These groups are composed of 1,813 genes of P. yezoensis, 1,610 genes of C. merolae, 1,762 genes of C. reinhardtii, and 3,237 genes of A. thaliana, respectively (Figure S4), indicating that many of the algal core genes are singletons. We detected 3,946 ( = 1,252+301+2,393) Rhodophyta-specific groups, and 2,393 of these were found in only P. yezoensis, but not in C. merolae. There were 2,069 ( = 301+171+1,509+88) orthologous gene groups between the two red algae: 2,059 between P. yezoensis and C. reinhardtii, and 2,141 between P. yezoensis and A. thaliana. There are slightly more gene groups missing only from P. yezoensis (673 groups) than the other lineages, but the proportion of GO categories does not show marked differences from other algae (Figure 4C).

We identified two types of genes encoding methionine synthase: vitamin B12-dependent (METH) and independent (METE), in the P. yezoensis genome. To the best of our knowledge, this is the first identification of a red algal METH. Phylogenetic analysis showed that each of the proteins formed a sister clade to those of green algae, such as Chlamydomonas (Figure 5). In the tree of METE, the P. yezoensis protein was positioned near to that of another red alga Galdieria sulphuraria, but distant from that of C. merolae. Thus, it is likely that both of the P. yezoensis methionine synthase genes have originated in the lineage of red algae and green algae. We did not found any vitamin B12 biosynthesis genes, except for cobalamin adenosyltransferase and cobalamin 5′-phosphate synthase, among the current contigs, implying that P. yezoensis utilizes the vitamin via symbiotic bacteria, like many algae [9].

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Figure 5. Phylogenetic relationships of METH and METE.

Nodes with bootstrap probabilities > = 90% (1000 replicates) are shown. (A) Phylogenetic tree for METH and (B) Phylogenetic tree for METE. The accession numbers of sequences compared are summarized in Table S3.

http://dx.doi.org/10.1371/journal.pone.0057122.g005

Photosynthesis Genes in the Nuclear Genome

From the lineage-specific genes detected above, we surveyed those involved in photosynthesis, which may control color traits of P. yezoensis. From the nuclear genome, we indentified light-harvesting genes that were unreported in P. yezoensis (Table 3). All of the genes have high GC content (average 66.5%), which is close to the average of nuclear genes but not plastid genes. Most of these proteins were predicted to be localized in the chloroplast, suggesting that they actually function in photosynthesis. In this study, phycobilisome-related genes, such as an R-phycoerythrin γ-subunit gene, were mainly predicted rather than chlorophyll-related genes. In particular, we found two types of genes that have never been reported in red algae: phycobilisome linker proteins and phycobilisome-degradation protein (NblA) that match to cyanobacterial homologs. Previously, an NblA-like gene, Ycf18, was identified near to phycoerythrin α- and β-subunit genes in the chloroplast genome of P. yezoensis [46], but another NblA homolog in the nuclear genome was not as similar to Ycf18 as it was to cyanobacterial homologs. In the phylogenetic analysis, we did not obtain a robust tree because the NblA sequences are too short (∼60 aa) (Figure 6A). However, the topology apparently shows that the nuclear NblA homolog of P. yezoensis has evolved in a different manner from the red-algal-plastid NblA homologs. The nuclear NblA homolog is predicted to comprise two exons, both of which have conserved regions with the known homologs (Figure 6B). This gene structure, mRNA expression and splicing were confirmed experimentally (Figure 6C). Therefore, we now propose that the nuclear NblA is active in P. yezoensis cells, although the subcellular localization is not predicted to be the chloroplast.

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Figure 6. Structure of the NblA locus and sequence analysis.

(A) A phylogenetic tree of NblA proteins. Nodes with bootstrap probabilities > = 90% (1000 replicates) are shown. Red algae are indicated in red and cyanobacteria are indicated in black. The accession numbers of sequences compared are summarized in Table S4; (B) A partial alignment of NblA proteins. More highly conserved residues are shown in deeper blue. The numbers in the corners indicate alignment start/end positions of amino acid residues in the P. yezoensis nuclear/plastid NblA homologs, respectively; (C) Predicted genomic structure and PCR amplification of NblA locus. The position of forward (F) and reverse (R) primers used is indicated by arrows on the predicted genomic structure. PCR amplification of the gene was performed using genomic DNA (gDNA) from protoplasts and complementary DNA (cDNA) from thalli of P. yezoensis as templates. The dotted line in the genomic structure represents undetermined nucleotide sequence.

http://dx.doi.org/10.1371/journal.pone.0057122.g006

Discussion

During the sequencing of the P. yezoensis genome, a potentially serious problem was DNA sampling. As reported previously, many bacterial species are deeply attached to P. yezoensis cells and form a sort of biofilm, which could result in contamination during genome sequencing. In this study, we cultured axenic protoplasts of P. yezoensis to remove such symbiotic or attached bacteria and prepared the purified nuclear DNA. Since we found that some of the contigs were still of organellar (chloroplast and mitochondrion) and bacterial sequences in the pilot genome assembly, we removed these contigs by reference sequecnes. After removing non-nuclear contigs, we finally obtained 46,634 contigs for the P. yezoensis genome, spanning approximately 43 Mb. A plausible explanation of the contig’s fragmentation is that the nuclear genome has a high GC content, whereby next-generation sequencers read through chromosomal regions [47]. The contig coverage of 166-fold redundancy is nonetheless enough to sketch the genome features of P. yezoensis. One of the implications is that the total contig size was unexpectedly compact. It is commonly argued that nuclear DNA contents of Pyropia species range from 260 Mb of P. yezoensis [18] to 500 Mb [17], and our estimate is an order of magnitude smaller than those. This discrepancy does not seem to be explained only by genomic GC content bias. In a previous study, GC-rich sequences uncovered by Illumina did not exceed 10% of the whole genome [48]. Therefore, we propose that the genome of P. yezoensis is actually smaller than previously imagined. The evidence for a compact genome may also be provided by comparison of introns among algal genomes. While exon regions may be influenced by gene functional constraints, introns are considered relatively neutral and their lengths probably reflect genome expansion/reduction tendencies [49]. Actually, the average intron length of P. yezoensis was smaller than those of C. reinhardtii and E. siliculosus. In particular, we observed a linear correlation between the average intron length and genome size, which may be applicable for estimating the DNA contents of unfinished algal genomes. Although the intron density is not linearly correlated with genome size, that of P. yezoensis (0.7 per gene) is a feature of small-genome algae. In addition, a lower frequency of repetitive sequences including microsatellite repeats might be involved in the compact genome structure of P. yezoensis.

In this study, we predicted 10,327 gene models from the nuclear genome of P. yezoensis. One caveat to this estimate is that many of the genes are partially predicted because of short contigs: it is possible that exons of a single gene are predicted on separate contigs; thus, many genes may be double-counted. Nevertheless, the GO categories of predicted genes are comparable to those of other algae, suggesting that major functional genes are contained in the current gene set of P. yezoensis. Concerning the rough estimate of gene number, a positive correlation with genome size may be a clue to the argument. If the draft sequences cover most of the P. yezoensis genome, the total number of genes is around 10,000 according to the correlation. Sequence homology searching showed that about 35% of the predicted genes have no known function, and around 2,400 gene groups are P. yezoensis-specific compared to C. merolae, C. reinhardtii and A. thaliana. Some of those may provide clues about the uncharacterized molecular basis of the lifecycle of P. yezoensis or other red algae. Moreover, only 2,069 gene groups are common to C. merolae, which is consistent with that these red algae are deeply divergent from each other [50]. Considering that C. merolae has adapted to extreme environmental conditions, this unicellular eukaryote may have undergone specific gene gain or loss. In particular, we identified two types of methionine synthases (METH and METE) in P. yezoensis: C. merolae does not have a METH gene and its METE gene is of uncertain origin [51]. In hot springs, where vitamin B12-producing bacteria are rare, C. merolae may not require vitamin B12-dependent metabolic pathways. The P. yezoensis METH gene will be a target for understanding vitamin B12-dependent metabolism and the symbiotic relationship in red algae.

P. yezoensis is an important seafood in East Asia and the molecular information is expected to provide a basis for aquaculture. Microsatellite repeats found in this study, mostly CCG triplets, may be useful as polymorphic markers for identifying cultivars. In addition, the photosynthetic system has a critical role in the color of nori, and the related genes may be targets for breeding cultivars resistant to color fading. The R-phycoerythrin γ-subunit gene was already known in other red algae, such as Aglaothamnion neglectum and Corallina officinalis, but was unreported in P. yezoensis. As a whole, we identified phycobilisome-related genes rather than chlorophyll-related genes, suggesting a potential importance in color traits of red algae. In relation to nori aquaculture, a disease of P. yezoensis, color fading, is caused by the deficiency of nitrogen and phosphorus essential for the normal growth. In cyanobacteria, nutrient starvation triggers phycobilisome degradation and a small protein plays a key role by being upregulated during the process. The protein, named NblA (non-bleaching A) [52], can bind the phycobilisome complex, though its detailed function is not known. In the case of P. yezoensis, an NblA homolog (Ycf18) was identified from the plastid genome and its expression pattern was investigated, but no induction occurred during the phycobilisome degradation in nitrogen-deficient medium [46]. The same study showed that the upregulation of Ycf18 was induced in ammonium medium, but not in nitrate medium. In the P. yezoensis nuclear genome, we found a second homolog of NblA, which is evolutionary distinct from the plastid-encoded version. Interestingly, the nuclear NblA-like gene has a high GC content (65.0%) and has an intron that interrupts two conserved regions. Therefore, it is not likely that this gene originated through recent horizontal transfer from the chloroplast or cyanobacteria. If this gene has been maintained in the red algal nuclear genome for a long time, it must have an important role in the lifecycle of P. yezoensis. Thus, we propose a hypothesis in which the nuclear NblA-like gene may be involved in phycobilisome-dependent light harvesting and color fading in nori farms. Otherwise, this gene might affect the growth of P. yezoensis in nitrate medium. This hypothesis will be tested by further genome sequencing of other red algae and by expression analysis.

Conclusions

We determined, for the first time, the symbiont-free genome sequence of P. yezoensis. The result suggests that the nuclear genome of this alga is much smaller than previously estimated. The paucity and shortness of the introns within genes support this estimate: intron length is linearly correlated with genome size among algal genomes. This report will call for a rethink of the estimation of red algal genome sizes. From the draft sequence, we obtained important findings about P. yezoensis that may be applicable to other red algae. We identified a cobalamin-dependent methionine synthase that has never been reported in red algae. This gene is a promising target for analyzing the interaction with symbiotic bacteria in marine environments. We further detected phycobilisome-related genes in the nuclear genome, some of which may be candidates DNA marker for cultivar improvement. Among these, we report a second homolog of NblA in the nuclear genome, and hypothesize that the gene is involved in a color trait of P. yezoensis. These findings will shed light on unexplained but probably characteristic features of marine red algae. The P. yezoensis genome could represent a model genome for examining red algal life history, and will provide insights into nori aquaculture in the near future.

Supporting Information

Figure S1.

Read coverage for each contigs. The dots represent the distribution of contig length with contig coverage (average depth of mapped reads), and histogram shows the frequency of the coverage of contigs. Black and open arrowheads indicate the points of average coverage and the mode of contigs respectively. Black arrow indicates the contig derived from two plasmids (pPY1-U51 and pPY2-U51) (Text S4).

doi:10.1371/journal.pone.0057122.s001

(EPS)

Figure S2.

Microsatellite distribution in the C. merolae genome. Microsatellite repeats of 2–5 bp unit were detected by Tandem repeats finder, with the same parameters as those for the P. yezoensis genome. Repeats with frequency > = 3 are shown.

doi:10.1371/journal.pone.0057122.s002

(EPS)

Figure S3.

Structure of a genomic contig containing a putative gene from γ-proteobacteria. An 1874-bp contig, which contained a gene similar to the 2-octaprenyl-6-methoxyphenol hydroxylase (ubiH) gene of γ-proteobacteria (e.g., GenBank accession number EGU43500), was obtained from the assembled P. yezoensis genome data. This contig also contained a transcription elongation factor 1 (ELF1) gene of P. yezoensis (GenBank accession number; AB480827). The accuracy of the contig assembly was verified by inverse PCR and Sanger resequencing. Arrows indicate gene orientation.

doi:10.1371/journal.pone.0057122.s003

(EPS)

Figure S4.

A detailed Venn diagram of gene sets among P. yezoensis, C. merolae, C. reinhardtii, and A. thaliana. Numbers of gene groups are shown in black on the diagram. Each gene group is defined as a singleton or a cluster of paralogs. Total numbers of genes included in the groups are shown in red (P, P. yezoensis; Cm, C. merolae; Cr, C. reinhardtii; A, A. thaliana).

doi:10.1371/journal.pone.0057122.s004

(EPS)

Table S1.

Species used for gene prediction and comparison.

doi:10.1371/journal.pone.0057122.s005

(XLS)

Table S2.

Characteristics of algal genes.

doi:10.1371/journal.pone.0057122.s006

(XLS)

Table S3.

List of public sequences used for the METH tree.

doi:10.1371/journal.pone.0057122.s007

(XLSX)

Table S4.

List of public sequences used for the NblA tree.

doi:10.1371/journal.pone.0057122.s008

(XLS)

Text S1.

Treatment of bacterial sequences in protoplast culture.

doi:10.1371/journal.pone.0057122.s009

(DOC)

Text S2.

Preparation of cDNA library.

doi:10.1371/journal.pone.0057122.s010

(DOC)

Text S3.

Analysis of gene structure and expression of the P. yezoensis nuclear NblA.

doi:10.1371/journal.pone.0057122.s011

(DOC)

Text S4.

Plasmids of Pyropia yezoensis.

doi:10.1371/journal.pone.0057122.s012

(DOC)

Acknowledgments

We thank Mahiko Abe, National Fisheries University, Japan, for help in preparing axenic protoplast of P. yezoensis U-51. We also thank Kazuki Mori and Kosuke Tashiro, Kyusyu University, Japan, for preliminary experiment and data analysis, and Aki Namba and Youhei Fukui for information on symbiotic bacteria of P. yezoensis. A part of analysis done in this study was conducted by using the computational facility of the Research Program of Innovative Cell Biology by Innovative Technology (Cell Innovation).

Author Contributions

Conceived and designed the experiments: TK IN FI KN M. Sano TW SK K. Inouye TG K. Ikeo. Performed the experiments: MK NO MY YS. Analyzed the data: YN NS NO M. Sano. Contributed reagents/materials/analysis tools: M. Satomi YF KS AT. Wrote the paper: YN NS MK NO MY M. Sano TG K. Ikeo.

References

  1. 1. Mumford TF, Miura A (1988) Porphyra as food : cultivation and economics. In: Lembi CA, Waaland JR, editors. Algae and Human Affairs: Cambridge University Press. 87–117.
  2. 2. Colenso W (1880) On the vegetable food of the ancient New Zealanders before Cook's visit. Christchurch: Kiwi Publishers. 35 p.
  3. 3. Hoffmann A, Santelices B (1997) Marine Flora of Central Chile. Santiago de Chile: Univesidad Católica de Chile. 434 p.
  4. 4. Brodie J, Irvine LM (2003) Seaweeds of the British Isles : Vol. 1, Rodophyta, Part 3B, Bangiophycidae: The Natural History Museum (London). 167 p.
  5. 5. Williams MD (1979) The harvesting of sluckus (Porphyra-Perforata) by the Straits Salish Indians of Vancouver Island, British-Columbia. Syesis 12: 63–69.
  6. 6. Turner NJ (2003) The ethnobotany of edible seaweed (Porphyra abbottae and related species; Rhodophyta : Bangiales) and its use by First Nations on the Pacific Coast of Canada. Canadian Journal of Botany-Revue Canadienne De Botanique 81: 283–293.
  7. 7. Drew KM (1949) Conchocelis-phase in the life-history of Porphyra-Umbilicalis (L) Kutz. Nature 164: 748–749.
  8. 8. Drew KM (1954) Life-history of Porphyra. Nature 173: 1243–1244.
  9. 9. Croft M, Lawrence A, Raux-Deery E, Warren M, Smith A (2005) Algae acquire vitamin B12 through a symbiotic relationship with bacteria. Nature 438: 90–93.
  10. 10. Namba A, Shigenobu Y, Kobayashi M, Kobayashi T, Oohara I (2010) A new primer for 16S rDNA analysis of microbial communities associated with Porphyra yezoensis. Fisheries Science 76.
  11. 11. Matsuzaki M, Misumi O, Shin-I T, Maruyama S, Takahara M, et al. (2004) Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D. Nature 428: 653–657.
  12. 12. Nozaki H, Takano H, Misumi O, Terasawa K, Matsuzaki M, et al. (2007) A 100%-complete sequence reveals unusually simple genomic features in the hot-spring red alga Cyanidioschyzon merolae. BMC biology 5: 28.
  13. 13. Douglas S, Zauner S, Fraunholz M, Beaton M, Penny S, et al. (2001) The highly reduced genome of an enslaved algal nucleus. Nature 410: 1091–1096.
  14. 14. Lane CE, van den Heuvel K, Kozera C, Curtis BA, Parsons BJ, et al. (2007) Nucleomorph genome of Hemiselmis andersenii reveals complete intron loss and compaction as a driver of protein structure and function. Proc Natl Acad Sci U S A 104: 19908–19913.
  15. 15. Tanifuji G, Onodera N, Wheeler T, Dlutek M, Donaher N, et al. (2011) Complete nucleomorph genome sequence of the nonphotosynthetic alga Cryptomonas paramecium reveals a core nucleomorph gene set. Genome biology and evolution 3: 44–54.
  16. 16. Kapraun DF, Freshwater DW (1987) Karyological studies of 5 species of Porphyra (Bangiales, Rhodophyta) from the North-Atlantic and Mediterranean. Phycologia 26: 82–87.
  17. 17. Kapraun DF, Hinson TK, Lemus AJ (1991) Karyology and cytophotometric estimation of interspecific and intraspecific nuclear-DNA variation in 4 species of Porphyra (Rhodophyta). Phycologia 30: 458–466.
  18. 18. Matsuyama-Serisawa K, Yamamoto M, Fujishita M, Endo H, Serisawa Y, et al.. (2007) DNA content of the cell nucleus in the macroalga Porphyra yezoensis (Rhodophyta). Fisheries Science 73.
  19. 19. Waaland JR, Stiller JW, Cheney DP (2004) Macroalgal candidates for genomics. Journal of Phycology 40: 26–33.
  20. 20. Nikaido I, Asamizu E, Nakajima M, Nakamura Y, Saga N, et al. (2000) Generation of 10,154 expressed sequence tags from a leafy gametophyte of a marine red alga, Porphyra yezoensis. DNA Res 7: 223–227.
  21. 21. Asamizu E, Nakajima M, Kitade Y, Saga N, Nakamura Y, et al. (2003) Comparison of RNA expression profiles between the two generations of Porphyra yezoensis (Rhodophyta), based on expressed sequence tag frequency analysis. Journal of Phycology 39: 923–930.
  22. 22. Shen S, Zhang G, Li Y, Wang L, Xu P, et al. (2011) Comparison of RNA expression profiles on generations of Porphyra yezoensis (Rhodophyta), based on suppression subtractive hybridization (SSH). BMC Res Notes 4: 428.
  23. 23. Araki T, Hayakawa M, Tamaru Y, Yoshimatsu K, Morishita T (1994) Isolation and regeneration of haploid protoplasts from Bangia-Atropurpurea (Rhodophyta) with marine bacterial enzymes. Journal of Phycology 30: 1040–1046.
  24. 24. Kurahashi M, Yokota A (2004) Agarivorans albus gen. nov., sp. nov., a gamma-proteobacterium isolated from marine animals. Int J Syst Evol Microbiol 54: 693–697.
  25. 25. Benson G (1999) Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res 27: 573–580.
  26. 26. Sobreira TJ, Durham AM, Gruber A (2006) TRAP: automated classification, quantification and annotation of tandemly repeated sequences. Bioinformatics 22: 361–362.
  27. 27. Price AL, Jones NC, Pevzner PA (2005) De novo identification of repeat families in large genomes. Bioinformatics 21 Suppl 1i351–358.
  28. 28. Flutre T, Duprat E, Feuillet C, Quesneville H (2011) Considering transposable element diversification in de novo annotation approaches. PLoS One 6: e16526.
  29. 29. Kohany O, Gentles AJ, Hankus L, Jurka J (2006) Annotation, submission and screening of repetitive elements in Repbase: RepbaseSubmitter and Censor. BMC Bioinformatics 7: 474.
  30. 30. Slater GS, Birney E (2005) Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics 6: 31.
  31. 31. Stanke M, Diekhans M, Baertsch R, Haussler D (2008) Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics 24: 637–644.
  32. 32. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402.
  33. 33. Kent WJ (2002) BLAT–the BLAST-like alignment tool. Genome Res 12: 656–664.
  34. 34. Conesa A, Gotz S (2008) Blast2GO: A comprehensive suite for functional analysis in plant genomics. Int J Plant Genomics 2008: 619832.
  35. 35. Ye J, Fang L, Zheng H, Zhang Y, Chen J, et al. (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34: W293–297.
  36. 36. Katoh K, Toh H (2008) Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 9: 286–298.
  37. 37. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, et al. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739.
  38. 38. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406–425.
  39. 39. Horton P, Park KJ, Obayashi T, Fujita N, Harada H, et al. (2007) WoLF PSORT: protein localization predictor. Nucleic Acids Res 35: W585–587.
  40. 40. Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8: 978–984.
  41. 41. Fulneckova J, Hasikova T, Fajkus J, Lukesova A, Elias M, et al. (2012) Dynamic evolution of telomeric sequences in the green algal order Chlamydomonadales. Genome Biol Evol 4: 248–264.
  42. 42. Blanc G, Duncan G, Agarkova I, Borodovsky M, Gurnon J, et al. (2010) The Chlorella variabilis NC64A genome reveals adaptation to photosymbiosis, coevolution with viruses, and cryptic sex. Plant Cell 22: 2943–2955.
  43. 43. Merchant S, Prochnik S, Vallon O, Harris E, Karpowicz S, et al. (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science (New York, NY) 318: 245–250.
  44. 44. Cock JM, Sterck L, Rouze P, Scornet D, Allen AE, et al. (2010) The Ectocarpus genome and the independent evolution of multicellularity in brown algae. Nature 465: 617–621.
  45. 45. Bhattacharya D, Medlin L (1995) The phylogeny of plastids: a review based on comparisons of small-subunit ribosomal RNA coding regions. Journal of Phycology 31: 489–498.
  46. 46. Kawakami T, Sakaguchi K, Takechi K, Takano H, Takio S (2009) Ammonium induced expression of the red algal chloroplast gene Ycf18, a putative homolog of the cyanobacterial NblA gene involved in nitrogen deficiency-induced phycobilisome degradation. Biosci Biotechnol Biochem 73: 740–743.
  47. 47. Dohm JC, Lottaz C, Borodina T, Himmelbauer H (2008) Substantial biases in ultra-short read data sets from high-throughput DNA sequencing. Nucleic Acids Res 36: e105.
  48. 48. Kozarewa I, Ning Z, Quail MA, Sanders MJ, Berriman M, et al. (2009) Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes. Nat Methods 6: 291–295.
  49. 49. Nakamura Y, Itoh T, Martin W (2007) Rate and polarity of gene fusion and fission in Oryza sativa and Arabidopsis thaliana. Molecular biology and evolution 24: 110–121.
  50. 50. Yoon HS, Hackett JD, Ciniglia C, Pinto G, Bhattacharya D (2004) A molecular timeline for the origin of photosynthetic eukaryotes. Mol Biol Evol 21: 809–818.
  51. 51. Helliwell KE, Wheeler GL, Leptos KC, Goldstein RE, Smith AG (2011) Insights into the evolution of vitamin B12 auxotrophy from sequenced algal genomes. Mol Biol Evol 28: 2921–2933.
  52. 52. Collier JL, Grossman AR (1994) A small polypeptide triggers complete degradation of light-harvesting phycobiliproteins in nutrient-deprived cyanobacteria. EMBO J 13: 1039–1047.