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

<div><p>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 (<i>Pyropia yezoensis</i>) using next-generation sequencing platforms. For sequencing, thalli of <i>P. yezoensis</i> were washed to remove bacteria attached on the cell surface and enzymatically prepared as purified protoplasts. The assembled contig size of the <i>P. yezoensis</i> 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 <i>P. yezoensis</i> 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, <i>Cyanidioschyzon merolae</i>. As color trait determinants of red algae, light-harvesting genes involved in the phycobilisome were predicted from the <i>P. yezoensis</i> 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 <i>P. yezoensis</i> is a promising model genome of marine red algae.</p></div

Characteristics of <i>P. yezoensis</i> genes.

<p>Characteristics of <i>P. yezoensis</i> genes.</p

Microsatellite distribution.

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

BLAST top hit distribution and gene set comparison.

<p>(A) Taxonomic distributions of BLASTP top hits of <i>P. yezoensis</i> genes. (left) Eukaryotes; (right) Prokaryotes; (B) A Venn diagram of gene sets among four species (<i>P. yezoensis</i>, <i>C. merolae</i>, <i>C. reinhardtii</i>, and <i>A. thaliana</i>). 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 <i>P. yezoensis</i>, <i>C. merolae</i> and <i>C. reinhardtii</i>.</p

Distribution of intron number.

<p>“All genes” and “Validated genes” correspond to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057122#pone-0057122-t002" target="_blank">Table 2</a>, respectively.</p

Correlation between gene statistics and algal genome sizes.

<p>The <i>y</i> value of <i>P. yezoensis</i> (plotted in red) indicates the contig size (43 Mb). The species and data that are not shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057122#pone-0057122-t002" target="_blank">Table 2</a> are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057122#pone.0057122.s006" target="_blank">Table S2</a>. (A) Intron density and genome size; (B) Average intron length and genome size; (C) Gene content and (logarithmic-transformed) genome size.</p

Assembly statistics of the <i>P. yezoensis</i> genome.

<p>Assembly statistics of the <i>P. yezoensis</i> genome.</p

Structure of the <i>NblA</i> locus and sequence analysis.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057122#pone.0057122.s008" target="_blank">Table S4</a>; (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 <i>P. yezoensis</i> nuclear/plastid NblA homologs, respectively; (C) Predicted genomic structure and PCR amplification of <i>NblA</i> 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 <i>P. yezoensis</i> as templates. The dotted line in the genomic structure represents undetermined nucleotide sequence.</p

Phylogenetic relationships of METH and METE.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057122#pone.0057122.s007" target="_blank">Table S3</a>.</p