Genomic diversity of 39 samples of Pyropia species grown in Japan

Yukio Nagano; Kei Kimura; Genta Kobayashi; Yoshio Kawamura

doi:10.1371/journal.pone.0252207

. 2021 Jun 9;16(6):e0252207. doi: 10.1371/journal.pone.0252207

Genomic diversity of 39 samples of Pyropia species grown in Japan

Yukio Nagano ^1,^*,^#, Kei Kimura ^2,^#, Genta Kobayashi ², Yoshio Kawamura ²

Editor: Randall P Niedz³

PMCID: PMC8189503 PMID: 34106965

Abstract

Some Pyropia species, such as nori (P. yezoensis), are important marine crops. We conducted a phylogenetic analysis of 39 samples of Pyropia species grown in Japan using organellar genome sequences. A comparison of the chloroplast DNA sequences with those from China showed a clear genetic separation between Japanese and Chinese P. yezoensis. Conversely, comparing the mitochondrial DNA sequences did not separate Japanese and Chinese P. yezoensis. Analysis of organellar genomes showed that the genetic diversity of Japanese P. yezoensis used in this study is lower than that of Chinese wild P. yezoensis. To analyze the genetic relationships between samples of Japanese Pyropia, we used whole-genome resequencing to analyze their nuclear genomes. In the offspring resulting from cross-breeding between P. yezoensis and P. tenera, nearly 90% of the genotypes analyzed by mapping were explained by the presence of different chromosomes originating from two different parental species. Although the genetic diversity of Japanese P. yezoensis is low, analysis of nuclear genomes genetically separated each sample. Samples isolated from the sea were often genetically similar to those being farmed. Study of genetic heterogeneity of samples within a single aquaculture strain of P. yezoensis showed that samples were divided into two groups and the samples with frequent abnormal budding formed a single, genetically similar group. The results of this study will be useful for breeding and the conservation of Pyropia species.

Introduction

The genus Pyropia is a marine red alga belonging to the family Bangiaceae. Within the genus Pyropia, P. yezoensis Ueda (Susabi-nori in Japanese) and P. haitanensis Chang et Zheng (tan-zicai in Chinese) are economically important marine crops that are consumed in many countries [1]. These are farmed in Japan, China and Korea. In recent years, they have been eaten as ingredients of sushi and snacks around the world, and their consumption is increasing. Its production in 2018 was 2 million tonnes [2]. Other species are cultivated or wild, and some are occasionally used as edibles. For example, P. tenera Kjellman (Asakusa-nori in Japanese) was once cultivated in Japan but has now been replaced by P. yezoensis in many places. P. yezoensis has also been shown to have pharmacological and nutritional properties [reviewed in 3, 4]. For example, a peptide derived from this seaweed induces apoptosis in cancer cells [5,6]. The eicosapentaenoic acid-rich lipid has also been shown to be beneficial to health by alleviating hepatic steatosis [7].

Inter- and intraspecific genetic diversity of Pyropia has not been well studied genomically. Studying the genomic diversity of Pyropia species is necessary for planning the breeding and conservation of these seaweeds. This information is also important for the use of species that have not been utilized previously. Especially, utilization of underutilized species and breeding are important to address climate change. Prior to the genomic era, various studies analyzed the genetic diversity of Pyropia species. For example, studies used the methods based on simple sequence repeat [8,9], DNA sequencing of several genes [10–28], amplified fragment length polymorphism [29,30], and polymerase chain reaction-restriction fragment length polymorphism [31]. These previous studies analyzed small regions of the genome. In addition, many of these previous studies analyzed the genetic differences between species by phylogenetic analysis. In other words, few have analyzed population structure or admixture within species.

However, few genomic approaches were applied to study the genetic diversity of Pyropia species. For example, to study the genetic diversity of P. yezoensis in China, high-throughput DNA sequencing analyzed variations in the organellar genome [32]. This study divided wild P. yezoensis from Shandong Province, China, into three clusters. Several methods, such as restriction-site associated DNA sequencing [33], genotyping by sequencing [34], and whole-genome resequencing [35,36] use high-throughput DNA sequencing and can be applied to the analysis of the nuclear genome. Among them, whole-genome resequencing is the most effective way to get a comprehensive view of the entire nuclear genome. In general, whole-genome resequencing requires short reads generated by high-throughput DNA sequencing. The short reads obtained from whole-genome resequencing can be appropriate in another application, the de novo assembly of the organellar genome [37]. Hence, short reads are useful for research on both the nuclear and organellar genomes. Besides, whole-genome sequencing of the nuclear genome is suitable for analyzing the population structure and admixture within a species.

The Ariake sound is located in the south-west of Japan (Fig 1). It has an area of 1,700 km². It is characterized by a tidal range of up to 6 m. This tidal range is effectively used for the cultivation of seaweed. As a result, the Ariake sound is the most productive place for seaweed in Japan.

In this study, we conducted a phylogenetic analysis of 39 samples of Pyropia species grown in Japan using chloroplast DNA sequences assembled from short reads. To analyze the population structure and admixture, we also used whole-genome resequencing to analyze the nuclear genomes of 34 samples of P. yezoensis, one sample of the closely related P. tenera, and one sample of the offspring resulting from cross-breeding between P. yezoensis and P. tenera. Particularly, we focused on the analysis of some kinds of seaweed in the Ariake sound, Japan, where seaweed production is active.

Materials and methods

Materials

The collection sites were represented on a map generated with SimpleMappr [38] (Fig 1) and shown in Table 1. When the collection was performed, a single blade (a mixture of four types of haploid cell) was isolated. Next, carpospores (diploid spores) were collected from this blade and cultured. The culture of carpospores derived from single blade was defined as ‘culture’ in this study. Sometimes, one of the cultures was selected for aquaculture, which was defined as ‘strain’ in this study. A culture was also created from a strain, which is indicated in Table 1. Each culture was used as a sample in this study. In Japan, various cultures/strains are preserved in various institutions. Therefore, the acquisition of culture was not necessarily performed by the authors, so some cultures were provided by other researchers. From these cultures, this study focused on those grown in the Ariake sound.

Table 1. List of Pyropia samples.

Sample name	Purification to became homozygous	Species	Collection site	Isolation year	Notes
Pyr_1	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2010	Culture derived from ‘Shin Saga 4 gou’ strain. Normal phenotype. Strain recommended by Saga Prefecture Fishery Cooperative Federation.
Pyr_2	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2010	Culture derived from ‘Shin Saga 4 gou’ strain. Normal phenotype. Strain recommended by Saga Prefecture Fishery Cooperative Federation. Preservation place is different from that of Pyr_1.
Pyr_3	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2010	Culture derived from ‘Shin Saga 4 gou’ strain, normal phenotype. Reisolated from Pyr_2 in 2017.
Pyr_4	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2010	Culture derived from ‘Shin Saga 4 gou’ strain, normal phenotype. Reisolated from Pyr_2 in 2017.
Pyr_7	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2010	Culture derived from ‘Shin Saga 4 gou’ strain, normal phenotype. Reisolated from Pyr_2 in 2017.
Pyr_8	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2010	Culture derived from ‘Shin Saga 4 gou’ strain, normal phenotype. Reisolated from Pyr_2 in 2017.
Pyr_9		P. yezoensis	Ariake sound, Saga Prefecture, Japan	2010	Culture derived from ‘Shin Saga 4 gou’ strain, normal phenotype. Reisolated from Pyr_2 in 2017.
Pyr_10	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2010	Culture derived from ‘Shin Saga 4 gou’ strain, abnormal phenotype. Reisolated from Pyr_2 in 2017.
Pyr_11	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2010	Culture derived from ‘Shin Saga 4 gou’ strain, abnormal phenotype. Reisolated from Pyr_2 in 2017.
Pyr_12	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2010	Culture derived from ‘Shin Saga 4 gou’ strain, abnormal phenotype. Reisolated from Pyr_2 in 2017.
Pyr_13	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2010	Culture derived from ‘Shin Saga 4 gou’ strain, abnormal phenotype. Reisolated from Pyr_2 in 2017.
Pyr_14	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2010	Culture derived from ‘Shin Saga 4 gou’ strain, abnormal phenotype. Reisolated from Pyr_2 in 2017.
Pyr_15	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2010	Culture derived from ‘Shin Saga 4 gou’ strain, abnormal phenotype. Reisolated from Pyr_2 in 2017.
Pyr_16	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2015	Isolated for research purposes. Isolated from the aquaculture farm of the Saga Prefectural Ariake Fisheries Research and Development Center.
Pyr_17	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	1999	Isolated for research purposes. Isolated as probable low-temperature resistant strain.
Pyr_18	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2017	Isolated for research purposes. Isolated from the aquaculture farm of the Saga Prefectural Ariake Fisheries Research and Development Center.
Pyr_19	+	P. tenera	Hiroshima Prefecture, Japan	1978	Isolated as P. tenera. Provided from Saga Prefecture Fishery Cooperative Federation.
Pyr_20	+	P. yezoensis	Hiroshima Prefecture, Japan	1978	Culture derived from marketed strain. Marketed as P. tenera, but morphologically similar to P. yezoensis.
Pyr_21	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2017	Isolated for research purposes. Isolated from the aquaculture farm of the Saga Prefectural Ariake Fisheries Research and Development Center.
Pyr_22	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	1997	Culture derived from ‘Shin Saga 1 gou’ strain, strain recommended by Saga Prefecture Fishery Cooperative Federation.
Pyr_23	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2014	Isolated for research purposes. Isolated from the aquaculture farm of the Saga Prefectural Ariake Fisheries Research and Development Center.
Pyr_24	+	P. yezoensis	Miyagi Prefecture, Japan	2018	Provided as P. tanegashimensis for research purposes, but morphologically similar to P. yezoensis.
Pyr_25	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	1984	Culture derived from ‘Hagakure’ strain. Strain recommended by Saga Prefecture Fishery Cooperative Federation.
Pyr_26	+	P. yezoensis	Chiba Prefecture, Japan	1983	Considered as P. yezoensis f. narawaensis.
Pyr_27	+	P. tenera × P. yezoensis	Japan	2005	Culture derived from ‘Gyoko strain. Crossed by National Federation of Nori & Shellfish-fishers cooperative Associations.
Pyr_28	+	P. yezoensis	Ehime Prefecture, Japan	1976	Culture derived from ‘Ariake 1 gou’ strain. Isolated by Nagasaki University. Used in Fukuoka Prefecture.
Pyr_29	+	P. yezoensis	Genkai Sea, Fukuoka Prefecture, Japan	1975	Culture derived from ‘Saga 1 gou’ strain. Strain recommended by Saga Prefecture Fishery Cooperative Federation.
Pyr_30	+	P. yezoensis	Kagoshima Prefecture, Japan	1978	Culture derived from ‘Saga 6 gou’ strain. Strain recommended by Saga Prefecture Fishery Cooperative Federation.
Pyr_33	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	1982	Culture derived from ‘Saga 10 gou’ strain. Strain recommended by Saga Prefecture Fishery Cooperative Federation named.
Pyr_34	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2017	Isolated for research purposes. Isolated from the aquaculture farm of the Saga Prefectural Ariake Fisheries Research and Development Center.
Pyr_35		P. dentata	Tsushima Island, Nagasaki Prefecture, Japan	2009	Isolated by the Saga Prefectural Ariake Fisheries Research and Development Center.
Pyr_36		P. yezoensis	Ariake sound, Kumamoto Prefecture, Japan	2004	Isolated for research purposes. Isolated from Amakusa district in Kumamoto Prefecture by Saga Prefectural Ariake Fisheries Research and Development Center.
Pyr_38	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2000	Isolated for research purposes. Green mutant. Isolated from the aquaculture farm of the Saga Prefectural Ariake Fisheries Research and Development Center.
Pyr_39	+	P. yezoensis	Ariake sound, Saga Prefecture, Japan	2009	Culture derived from ‘Shin Saga 3 gou’ strain. Strain recommended by Saga Prefecture fishery cooperative federation.
Pyr_40		P. yezoensis	Hiroshima Prefecture, Japan	1978	Culture derived from marketed strain. Marketed as P. tenera, but morphologically similar to P. yezoensis.
Pyr_41	+	P. yezoensis	Ariake sound, Fukuoka Prefecture, Japan	2002	Culture derived from ‘Fukuoka Ariake 1 gou’ strain.
Pyr_42	+	P. yezoensis	Ariake sound, Fukuoka Prefecture, Japan	1981	Culture derived from ‘Fukuoka 1 gou’ strain.
Pyr_44	+	P. tenuipedalis	Japan	2000	Provided by Daiichi Seimo Co., Ltd.
Pyr_45	+	P. haitanensis	China	2000	Provided from Daiichi Seimo Co., Ltd.

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Culture containing homozygous diploid cells derived from a single haploid cell was prepared. For the creation of this type of culture, blade was first cultured in the water of Ariake sound supplemented with modified nutrients SWM-III [39] at 17–22 °C under 100 μmol/m²/s (11–13:13–11 h LD). Then, a monospore (a single haploid cell) was obtained from each blade according to a previous method [26]. Finally, conchocelis (homozygous diploid cells) derived from a single monospore were cultured in the water of Ariake sound supplemented with modified nutrients SWM-III at 18 °C under 30 μmol/m²/s (11:13 h LD).

DNA purification and sequencing

DNA was extracted from the conchocelis of each sample using the DNAs-ici!-F (Rizo, Tsukuba, Japan) according to the instructions of the manufacturer, followed by RNase A (NIPPON GENE, Tokyo, Japan) treatment. The quality of the isolated genomic DNA was checked by 1% agarose gel electrophoresis. The concentration of DNA was determined by Qubit dsDNA BR Assay Kit (Thermo Fisher, Foster City, CA, USA).

Sequencing libraries of total DNA were generated using the NEBNext Ultra DNA library prep kit for Illumina (NEB, USA) by Novogene (Beijing, China). The libraries were sequenced with 150 bp paired-end reads using NovaSeq 6000 (Illumina, San Diego, CA, USA) by Novogene. Low-quality bases and adapter sequences from paired reads were trimmed using the Trimmomatic [40] (version 3.9) (ILLUMINACLIP:adapter_sequence:2:30:10 LEADING:20 TRAILING:20 SLIDINGWINDOW:5:20 MINLEN:50).

Assembly-based analysis of chloroplast and mitochondrial genomes

Chloroplast and mitochondrial DNA sequences were assembled from filtered reads or public reads using the GetOrganelle [37] program (version 1.6.4). As the seed sequences for chloroplast genome assembly, other_pt.fasta included in this program was used. As the seed sequences for mitochondrial genome assembly, the complete genome sequence of Pyropia yezoensis (NCBI accession number: NC_017837) was used. For visual confirmation of the assembled result, filtered reads were aligned with the assembled genome by using the short read alignment program bowtie2 [41] (version 2.3.5.1). Samtools [42,43] (version 1.9) was used to process aligned data. The aligned data were visually inspected by the Integrative Genomics Viewer [44] (version 2.5.0). Multiple alignments of the assembled chloroplast genome were performed by MAFFT [45] (version 7.455) to create FASTA files for phylogenetic analyses. A phylogenetic tree based on the maximum likelihood method was constructed by the program RAxML [46] (version 8.2.12). ModelTest-NG was used to select the model for each analysis [47]. The parameters used in the program RAxML were shown in the legends of each figure. S1 Fig is the multi-FASTA file of the large single copy sections of chloroplast genomes used to create the phylogenetic tree in Fig 2. S2 Fig is the multi-FASTA file of the large single copy sections of chloroplast genomes used to create the phylogenetic tree in S3 Fig. S4 Fig is the multi-FASTA file of the assembled complete sequences of mitochondrial genomes used to create the phylogenetic tree in S3 Fig.

Fig 2 — The numbers at the nodes indicate bootstrap values (% over 1000 replicates). The scale bar shows the number of substitutions per site. The sequence of *Porphyra umbilicalis* was used as a root. The large figure is a phylogram showing the relationships among all the data used in this analysis. The small figure in the upper left is a cladogram showing the relationships for P. *yezoensis* only. Colors were used to distinguish between Chinese and Japanese P. *yezoensis*. The parameters for RAxML were as follows: -f = a, -x = 12,345, -p = 12,345, -N (bootstrap value) = 1,000, and -m = GTRGAMMAIX).

Mapping-based analysis of nuclear, chloroplast, and mitochondrial genomes

The reference nuclear genome data of P. yezoensis [48] were downloaded from the National Center for Biotechnology Information (NCBI; Assembly number: GCA_009829735.1 ASM982973v1). The reference genome sequence of chloroplast was the assembled sequence of Pyr_1, one of the P. yezoensis samples used in this study. The reference mitochondrial genome data of P. yezoensis were downloaded from the NCBI (accession number: MK695879) [32]. Filtered reads of our data were aligned with the reference genome by using the program bowtie2 [41]. For the analysis of the chloroplast and mitochondrial genomes, public data of wild samples in China (NCBI Sequence Read Archive under the BioProject: PRJNA55033) were also used. Samtools [42,43] was used to process aligned data. The quality of aligned data was analyzed using the Qualimap [49] program (version 2.2.1). From aligned data, DeepVariant [50] (version 0.9.0) was used to call the variants to make vcf files [51]. Vcf files were merged using GLnexus [52] (version 1.2.6) (—config DeepVariantWGS). To process and analyze vcf files, bcftools [43] (version 1.9), bgzip implemented in tabix [43] (version 0.2.5), vcffilter implemented in vcflib [53] (version 1.0.0_rc3), and “grep” and “awk” commands of Linux were used. Low-quality data (GQ (Conditional genotype quality) < 20) were filtered out by vcffilter. The aligned data were visually inspected by the Integrative Genomics Viewer [44]. FermiKit [54] (version 0.14.dev1) was used to detect the structural variations. Multidimensional scaling (MDS) analysis was conducted based on this vcf file using the SNPRelate [55] (version 1.20.1) program. For the admixture analysis, vcf file was processed by the program PLINK [56] (version 2–1.90b3.35) (—make-bed—allow-extra-chr—recode—geno 0.1). The resulting files were used to perform the admixture analysis using the program admixture [57] (version 1.3.0).

For the phylogenetic analysis of the chloroplast, the rRNA regions were removed from the vcf file. The vcf files were converted to the FASTA format file using the VCF-kit [58] (version 0.1.6). Using FASTA file, a phylogenetic tree based on the maximum likelihood method was constructed by the program RAxML [46] (version 8.2.12). ModelTest-NG was used to select the model for each analysis [47]. The parameters used in the program RAxML were shown in the legends of each figure. S5 Fig is the multi-FASTA file of variables sites in the chloroplast genomes used to create the phylogenetic tree in S6 Fig. S7 Fig is the multi-FASTA file of variables sites in mitochondrial genomes used to create the phylogenetic tree in Fig 3.

Fig 3 — The scale bar shows the number of substitutions per site. The sequences of Pyr_19 (P. *tenera*) and the hybrid (Pyr_27) between P. *yezoensis* and P. *tener*a were used as roots. Colors were used to distinguish between Japanese P. *yezoensis* and 3 clusters of Chinese P. *yezoensis*. The parameters for RAxML were as follows: -f = a, -x = 12,345, -p = 12,345, -N (bootstrap value) = 1,000, -c = 1, and -m = GTRCATX). Only variable sites were used in the analysis.

For the phylogenetic analysis of the nuclear genome, vcf files were converted to the FASTA format file using the VCF-kit [58] (version 0.1.6) and then were converted to NEXUS format file using MEGA X [59] (version 10.0.5). Phylogenetic tree analysis was performed using SVDquartets [60] implemented in the software PAUP [61,62] (version 4.0a) using a NEXUS file. The parameters used for SVDquartets were as follows: quartet evaluation; evaluate all possible quartets, tree inference; select trees using the QFM quartet assembly, tree model; multi-species coalescent; handling of ambiguities; and distribute. The number of bootstrap analyses was 1,000 replicates. Pyr_19 was used as a root.

Public RNA sequencing data (NCBI Sequence Read Archive under accession numbers: SRR5891397, SRR5891398, SRR5891399, SRR5891400, and SRR6015124) were analyzed using HISAT2 [63] (version 2.2.0) and StringTie [64] (version 2.1.1) to determine the transcribed regions of the nuclear genome.

Results

Samples used in this study

Table 1 summarizes the samples used in this study, and Fig 1 shows the isolation site of each sample. We used 34 samples of P. yezoensis, all of which were either cultivated or isolated from close to a nori farm. Among them, 24 samples were cultures derived from the strains, which are used for aquaculture. A characteristic feature is that the 15 samples originated from a single strain ‘Shin Saga 4 gou’. These 15 samples allowed for the study of genetic heterogeneity within a single strain. With seven exceptions (Pyr_20, Pyr_24, Pyr_26, Pyr_28, Pyr_29, Pyr_30, and Pyr_40), 27 of them originated from the Ariake sound. In addition to 34 samples of P. yezoensis, we used P. haitanensis (Pyr_45), P. dentata Kjellman (Pyr_35), P. tenuipedalis Miura (Pyr_44), P. tenera (Pyr_19), and the hybrid between P. yezoensis and P. tenera (Pyr_27) (the offspring resulting from cross-breeding between P. yezoensis and P. tenera). In this study, we prepared 35 cultures, each of which contains homozygous diploid cells derived from a single haploid cell.

Sequencing

The libraries from these samples were sequenced with 150 bp paired-end reads. The Number of reads ranged from 64 million to 110 million (S1 Table). These reads were used for two purposes: 1) de novo assembly of the chloroplast and mitochondrial genomes, and 2) mapping to a reference genome.

Chloroplast and mitochondrial genomes

To elucidate the relationships among the 39 samples, we attempted to determine the whole chloroplast DNA sequences. Of the 39 total cases, we successfully assembled the whole chloroplast genome of 30 samples, and 9 cases (Pyr_16, Pyr_18, Pyr_21, Pyr_29, Pyr_23, Pyr_25, Pyr_28, Pyr_33, and Pyr_34) were unsuccessful. The chloroplast genomes of Pyropia species contain two direct repeats carrying ribosomal RNA (rRNA) operon copies [32,65,66]. The circular chloroplast genome has a large single-copy section and a short single-copy section between two rRNA repeats. The presence of two non-identical direct repeats carrying two different rRNA operons are general features of Pyropia chloroplast genomes [32,65,66]. Reflecting the presence of two non-identical direct repeats, mapping of the short reads to the assembled chloroplast genome detected two types of reads in the rRNA operons (S8 Fig) in all 39 cases. The presence of two types of reads in the rRNA operons might cause the unsuccessful assembly of the whole chloroplast genome of the 9 samples. Indeed, we succeeded in assembling the long single section sequences of all 39 samples. We detected the presence of two chloroplast genomes, termed heteroplasmy, only in the sample Pyr_45 (P. haitanensis) (S9 Fig).

Previous research reported that wild samples of P. yezoensis from Shandong Province, China, can be classified into three clusters [32]. We also assembled the chloroplast genome of one sample of each cluster. By using the assembled sequences of large single copy sections and publicly available chloroplast sequences, we created a phylogenetic tree based on the maximum likelihood method (Fig 2). The number of parsimony informative sites was 24,914. The chloroplast sequences of all Japanese samples of P. yezoensis were very similar to each other. The Chinese wild samples of P. yezoensis were genetically similar to but clearly separated from the Japanese samples. The chloroplast sequence of a Chinese cultivar (MK695880) collected from the nori farm in Fujian province was identical to that of some Japanese samples but not identical to those of the Chinese wild samples. This suggests that this Chinese cultivar was introduced from Japan, where the cultivation of P. yezoensis was established.

We mapped short reads of P. yezoensis samples from Japan and China to the chloroplast genome and constructed the maximum likelihood tree using the mapped data (S6 Fig). As similar to Fig 2, Japanese and Chinese chloroplast genomes were clearly separated. In addition, chloroplast sequences of Chinese P. yezoensis did not clearly separate them into three clusters. Chloroplast sequences showed that the genetic diversity of Japanese P. yezoensis used in this study is slightly lower than that of the Chinese wild P. yezoensis. We applied a similar approach to the mitochondrial genome (Fig 3). Mitochondrial sequences separated Chinese P. yezoensis into three clusters as reported previously [32]. Thus, mitochondrial sequences, rather than chloroplast sequences, are responsible for clustering in the previous report [32]. The analysis did not separate Japanese from Chinese P. yezoensis. Rather, Japanese P. yezoensis is similar to cluster 2 of Chinese P. yezoensis. Importantly, mitochondrial sequences showed that the genetic diversity of Japanese P. yezoensis used in this study is significantly lower than that of Chinese wild P. yezoensis.

There were differences in topological structures between the chloroplast phylogenetic trees (Fig 2 and S6 Fig) and the mitochondrial phylogenetic tree (Fig 3); in the chloroplast phylogenetic trees, the Japanese and Chinese P. yezoensis were clearly separated, but in the mitochondrial phylogenetic tree, the two were not separated. This may be due to differences in the rate of evolution between the chloroplast and mitochondrial genomes. Comparisons of the phylogenetic trees showed that the rate of evolution of the mitochondrial genome was about 10 times faster than that of the chloroplast genome (S3 Fig).

Nuclear genomes

The chloroplast and mitochondrial sequences did not clearly discriminate each sample of Japanese P. yezoensis. The large size of the nuclear genome makes it easy to detect genetic differences. Therefore, comparisons based on the nuclear genome are important. We analyzed the nuclear genome of only P. yezoensis from Japan because the Chinese P. yezoensis have small amounts of published reads. We mapped the short reads from 39 samples to the reference genome of P. yezoensis. S1 Table summarizes the results. The mean coverage of Pyr_35 (P. dentata), Pyr_44 (P. tenuipedalis), and Pyr_45 (P. haitanensis) was 2.7 ×, 8.8 ×, and 4.2 ×, respectively. As described above, the analysis of the chloroplast genome showed that these three samples were not similar to P. yezoensis, which was reflected in the low mean coverage. Therefore, we did not use the data from these three samples for further analysis based on the mapping. In contrast, the mean coverage of Pyr_19 (P. tenera) and Pyr_27, the hybrid between P. yezoensis and P. tenera, was 47.4 × and 25.7 ×, respectively. Therefore, we used the data of these two samples for the subsequent analysis. The mean coverages for P. yezoensis ranged from 14.6 × to 83.4 ×. Before DNA extraction, we did not remove the bacteria co-cultured with seaweed. Differences in the extent of the range of mean coverage reflect the amount of these bacteria.

We called genetic variants from the mapping data. After merging the variant data of 36 samples, we filtered out low-quality data (GQ (Conditional genotype quality) < 20). The data included 697,892 variant sites. Subsequently, we analyzed variant information by MDS analysis (S10 Fig). Samples of P. yezoensis were clustered together. Observations of the mapping results suggests that there were very few regions that have been incorrectly mapped with bacterial sequences, but we cannot exclude the possibility that bacterial contamination may have influenced the results shown in S10 Fig.

The hybrid between P. yezoensis and P. tenera

The MDS analysis separated Pyr_27 from the Pyr_19 (P. tenera), although these two samples were similar in chloroplast sequences. The analysis located the hybrid at approximately the middle position between two species, which supports the record that the hybrid was created by an interspecific cross between two species.

The previous analysis using a small number of genes reported that the hybrid between P. yezoensis and P. tenera is allodiploid in the blade cell [67,68]. In other words, if the haploid genome of P. yezoensis and P. tenera is A and B, respectively, the blade cell of the hybrid has the allodiploid genome of AB, and the conchocelis cell of the hybrid has the allotetraploid genome of AABB. We reanalyzed this possibility using the whole genome data of Pyr_1 (P. yezoensis), Pyr_19 (P. tenera), and their hybrid Pyr_27. Both Pyr_1 and Pyr_19 samples should be homozygous diploid as they are derived from a single haploid cell. The sample Pyr_27 is also derived from a single haploid cell, but if it is an allotetraploid, the genotype will be observed as heterozygous in the genome viewer. Fig 4A shows a representative example of a comparison of genotypes among three samples. This figure showed that Pyr_27 has heterozygous-like data. To further analyze the tendency, we calculated the number of the types of loci in Pyr_27 when the Pyr_1 locus carries two reference alleles (homozygous locus) and when the Pyr_19 locus carries two alternate alleles (alternative homozygous locus) (Table 2). In principle, this calculation method effectively excludes the regions that have been mapped incorrectly with bacterial sequences. In about 90% of the cases, Pyr_27 had one reference allele and one alternate allele (heterozygous locus). Therefore, Pyr_27 must be an allotetraploid. Previously, nuclear rRNA genes have shown to become homozygous after conjugation [67,68]. Indeed, in this region, the genotypes of Pyr_27 were identical to those of Pyr_19 (P. tenera) (Fig 4B).

Fig 4 — (A) A representative example of allotetraploid formation. (B) Nuclear rRNA regions.

Table 2. Number of loci in Pyr_27, when Pyr_1 locus carries two reference alleles and Pyr_19 locus carries two alternate alleles.

Two reference alleles	One reference allele/one alternate allele	Two alternate alleles
18,484	139,259	141

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Genomic diversity of Japanese P. yezoensis

Of 34 samples of P. yezoensis, 31 samples were derived from a single haploid cell, so they should be homozygous diploid. Therefore, we analyzed these 31 samples. From the variant data, we removed loci containing potentially heterozygous loci, which may be created due to the presence of non-identical repeats. The removal of such loci can remove loci that have been mapped incorrectly with bacterial sequences. This data included 69,918 variant sites. We then performed an MDS analysis (Fig 5) and an admixture analysis (Fig 6 and S11 Fig) using this variant data. For the phylogenetic analysis, we created the tree using the data from 32 samples, including 31 samples of P. yezoensis and 1 sample of P. tenera as a root, all of which should be homozygous. After removal of heterozygous loci, the data included 681,115 variant sites, of which 33,985 were parsimony informative sites. We constructed a phylogenetic tree using the program SVDQuartet [60] (Fig 7). Under the coalescence model, this program assumes that intragenic recombination exists.

Fig 5 — Two-dimensional data were obtained in this analysis. Colors were used to show 3 clusters, cluster A, B, and C, found in this study.

Fig 6 — The number of populations (K) was set to 2. Colors of the sample names were used to show 3 clusters, cluster A, B, and C.

Fig 7 — The numbers at the nodes indicate bootstrap values (% over 1,000 replicates). The data of Pyr_19 (P. *tenera*) was used as a root. Colors of the sample names were used to show 3 clusters, cluster A, B, and C.

Although the analyses clearly separated each sample based on genetic distance, there were three closed clusters: cluster A containing 19 samples (Pyr_1, 2, 3, 4, 7, 8, 10, 11, 12, 13, 14, 15, 17, 22, 23, 25, 28, 39, and 41), cluster B containing 2 samples (Pyr_21 and 38), and cluster C containing 2 samples (Pyr_16 and 24) (Fig 5). The phylogenetic tree detected these closed clusters with high bootstrap supports (Fig 7).

Fig 6 and S11 Fig show the results of the admixture analysis. Among the possible values of K (the number of ancestral populations), K = 2 was the most likely because K = 2 had the smallest cross-validation error (S11 Fig). Thus, the number of ancestral populations could be 2, with the 19 samples shown in blue in Fig 6 belonging to one population and the six samples shown in red in Fig 6 belonging to a second population. The former 19 samples were cluster A, as described above. Six samples, Pyr_18, 20, 21, 29, 33, and 38 might belong to an admixture population formed by the hybridization between these two ancestral populations. However, it is also possible that K is 3 or 5 (S11 Fig). Because K = 1 had the highest cross-validation error, admixture has likely happened in these 39 samples.

Because 19 samples belonging to cluster A were closely related, it was difficult to distinguish these samples in the above MDS analysis shown in Fig 5. Therefore, we analyzed only these 19 samples. The variant data from these 19 samples included 36,459 variant sites. We used this data for MDS analysis (Fig 8). Twelve samples (Pyr_1, 2, 3, 4, 7, 8, 10, 11, 12, 13, 14, and 15) are samples isolated from the single strain ‘Shin Saga 4 gou’, which may contain heterogeneous cells. The ‘Shin Saga 4 gou’ strain has two subgroups: a subgroup containing seven samples of Pyr_1, 10, 11, 12, 13, 14, and 15 and a subgroup containing five samples of Pyr_2, 3, 4, 7, and 8. Phylogenetic analysis (Fig 7) supported this observation with high bootstrap values. Among the ‘Shin Saga 4 gou’ strain, Pyr_10, 11, 12, 13, 14, and 15 have an abnormal phenotype. In normal samples, the rate of abnormal budding is about 15%, whereas in abnormal samples it is about 40%. These 6 samples formed the closely related cluster and were separated from Pyr_1, although these 7 samples belonged to the former subgroup. In addition to the members of strain ‘Shin Saga 4 gou’, Pyr_17, 22, 23, 25, 28, 39, and 41 belonged to a clade containing 19 samples. Of these, Pyr_17, 22, 23, 25, 39, and 41 were cultivated or isolated in the Ariake sound. Pyr_28 was from the sound in Ehime Prefecture.

Fig 8 — Two-dimensional data were obtained in this analysis. Members of the strain ‘Shin Saga 4 gou’ were shown in red.

To detect variants specific to abnormal samples (Pyr_10, 11, 12, 13, 14, and 15), we extracted 69 candidate loci by analyzing variant call data and then further selecting the candidates by visual inspection (S2 Table). We also used the program FermiKit [54] to detect structural variants specific to abnormal samples but failed to detect them. Furthermore, Pyr_36 is a green mutant. The similar strategies detected 104 candidate loci (S3 Table). In both cases, some of the loci were located within the gene or close to the gene. For example, deletion is detected in the calmodulin gene in the green mutant.

Discussion

A comparison of Japanese and Chinese samples of P. yezoensis revealed that the diversity of chloroplast and mitochondrial sequences in Japan is lower than that in China (Figs 2 and 3 and S6 Fig). This is especially evident in the case of mitochondrial DNA, which may be because most of the Japanese P. yezoensis used in this analysis were either cultivated or isolated from close to a nori farm. Therefore, it is important to investigate Japanese wild samples, especially from sounds where farming is not performed, in the future. Since the farming of P. yezoensis has begun in Japan, studying their diversity is an attractive topic. The chloroplast and mitochondrial sequences did not clearly discriminate each sample of Japanese P. yezoensis, probably because the size of them is smaller than that of nuclear genome. Indeed, in this study, we were able to distinguish the Japanese samples by analyzing the nuclear genomes.

The phylogenetic tree of chloroplasts (Fig 2) is in good agreement with previous studies. Among Pyropia species, P. tenera is the closest relative to P. yezoensis at the DNA level [26–28]. In addition, they can be crossed with each other [69]. In fact, Pyr_19 (P. tenera) was present on a branch next to P. yezoensis. The nucleotide sequence of the hybrid (Pyr_27) between P. yezoensis and P. tenera was more similar to that of P. tenera than that of P. yezoensis. Examination of the sequence revealed that, as expected, Pyr_45 was P. haitanensis. The analysis showed that Pyr_35 (P. dentata) is similar to P. haitanensis, as in previous studies [28]. Pyr_44 (P. tenuipedalis) belonged to a clade containing P. yezoensis, which is also consistent with previous molecular analyses [28].

We examined the genetic changes that have occurred during an interspecific cross between P. yezoensis and P. tenera. Both chloroplast and mitochondrial genomes of the hybrid were similar to those of P. tenera (Figs 2 and 3 and S6 Fig). At least in this one conjugation event, a mechanism of maternal, paternal, or random DNA transmission might eliminate the chloroplast and mitochondrial genomes of P. yezoensis, one of two parental chloroplast genomes. However, there is the possibility that chloroplasts and mitochondria may have different inheritance mechanisms.

The changes that have occurred in the nuclear genome during an interspecific cross are more interesting. MDS analysis (S10 Fig) located the hybrid at approximately the middle position between two species. This is due to the allotetraploid formation and not due to the DNA recombination between two species. We analyzed the loci in the hybrid and found that when P. yezoensis had homozygous locus and the P. tenera had alternative homozygous locus, about 90% of the loci were allotetraploid (Table 2). Thus, to our knowledge, the current study demonstrated the allotetraploid formation at the genomic level for the first time, although the analysis of a small number of genes showed that the interspecific cross between P. yezoensis and P. tenera could produce an allotetraploid [67,68]. The creation of interspecific hybrids is an important method in the breeding of seaweeds. The methods used in this study will be useful in the analysis of interspecific hybridization after the conjugation because we can track changes in the nuclear genome. After the interspecific cross, the region of the nuclear rRNA became homozygous (Fig 4B). This was consistent with previous studies [67,68]. Thus, a mechanism to eliminate one of the two types of nuclear rRNA genes is present. In contrast, there were two types of rRNA genes in the chloroplast genome.

The hybrid is slightly more similar to P. yezoensis than to P. tenera in the MDS analysis (S10 Fig). Furthermore, we analyzed the loci in the hybrid and found that when the P. yezoensis had homozygous locus and the P. tenera had alternative homozygous locus, about 10% of the loci were identical to those of P. yezoensis (Table 2). In order to explain this observation, we must consider two possibilities. One possibility is that this study did not use the real parents of the hybrid. The P. tenera used in the cross might be a more genetically similar sample to P. yezoensis. Related to this possibility, the mitochondrial sequence of the hybrid was similar to but not identical to that of P. tenera used in this study (Fig 3). The second possibility is that recombination or other mechanisms eliminated the sequence of P. tenera drastically. Considering the two possibilities, it is important to analyze the genomes of the trio, the hybrid and its biological parents, immediately after performing an interspecific cross.

There were three closed cluster in MDS analysis (Fig 5) and phylogenetic analysis (Fig 7). The members of cluster B, Pyr_21 and 38, were independently isolated from an aquaculture farm of the Saga Prefectural Ariake Fisheries Research and Development Center, but they originated in the same sound. In cluster C, Pyr_16 was isolated from an aquaculture farm at the Saga Prefectural Ariake Fisheries Research and Development Center, and Pyr_24 was provided as P. tanegashimensis previously. However, Pyr_24 was morphologically similar to P. yezoensis. For this reason, it appeared that the sample had been misplaced previously. Most of the samples used in this study were cultured or isolated in the Ariake sound, which is surrounded by Fukuoka, Saga, Nagasaki, and Kumamoto Prefectures (Fig 1), whereas Pyr_20, Pyr_24, Pyr_26, and Pyr_30 were from the sound in Hiroshima, Miyagi, Chiba, and Kagoshima Prefectures, respectively. In addition, Pyr_29 was from the Genkai Sea in Fukuoka Prefecture. (The Genkai Sea in northern Fukuoka Prefecture and the Ariake sound in southern Fukuoka Prefecture are separated by land.) Thus, the analyses did not clearly separate the seaweeds of other places from those of the Ariake sound. Pyr_20 was once marketed as P. tenera, but morphologically it is more likely to be P. yezoensis. Our genetic analysis confirmed that Pyr_20 is P. yezoensis.

Our analysis revealed that genetically similar seaweeds were frequently used in the Ariake sound (Figs 5 and 8), although this fact had been unknown previously. Furthermore, it turns out that genetically similar seaweed had been repeatedly isolated by researchers. Cluster A is a typical example of the genomic similarity because cluster A contains samples that could not be considered an ‘Shin Saga 4 gou’ strain. Of the 19 ascensions of cluster A, all except one were cultivated in the Ariake sound or isolated from the Ariake sound. This may be a problem for the conservation of P. yezoensis. We need to examine whether the genetic diversity of P. yezoensis is maintained in sounds where farming is not performed.

Of the 19 ascensions of cluster A, Pyr_28 was isolated in the sound in Ehime Prefecture. It is possible that this was due to human activity. Seaweed farming began in the Ariake sound later than other areas. Therefore, ancestors of cluster A members may have been artificially brought into the Ariake sound from the other areas such as Ehime Prefecture. Seaweed farming is now very active in the Ariake sound. Therefore, we do not exclude the possibility that Pyr_28 was artificially brought into Ehime Prefecture from the Ariake sound and isolated in Ehime Prefecture. In any case, the actual situation will not be known until we analyze the genomes of various seaweeds in various parts of Japan.

The samples in the ‘Shin Saga 4 gou’ strain were very similar. Despite the similarities, we were able to detect genetic differences among these. In other words, we were able to detect heterogeneity in the strain successfully. It is an interesting finding, for example, that the samples having abnormal budding are in tight clusters. Six samples with frequent abnormal budding formed a single, genetically similar closed cluster. These abnormal samples probably diverged after an event in which a single mutation occurred. The heterogeneity of somatic cells has been studied in cancer research [70]. Similar studies are now possible in seaweed.

In this study, we detected variants specific to abnormal samples (S2 Table) or to a green mutant (S3 Table). However, a number of candidate loci were present. Further studies are needed to identify the responsible genes. Targeted deletion by genome editing is one of the ways to identify them.

In summary, we used high-throughput sequencing to examine the genomic diversity of Pyropia species. To our knowledge, this is the first study to examine the genomic diversity of Pyropia species at the genome level. The information obtained in this study could be used to develop a breeding and conservation plan. A variety of Pyropia species grow wild or are cultivated in Japan and around the world. It is essential to perform genomic studies of these seaweeds.

Supporting information

S1 Fig. The multi-FASTA file of the large single copy sections of chloroplast genomes used to create the phylogenetic tree in Fig 2.

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S2 Fig. The multi-FASTA file of the large single copy sections of chloroplast genomes used to create the phylogenetic tree in S3 Fig.

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Click here for additional data file.^{(2MB, pdf)}

S3 Fig. Comparison of phylogenetic trees between the chloroplast and mitochondrial DNA sequences.

Phylogenetic trees were constructed based on maximum likelihood method. The DNA sequences from the large single copy sections of chloroplast genomes were used to create a chloroplast phylogenetic tree. The DNA sequences from the assembled sequences of mitochondrial genomes were used to create a mitochondrial phylogenetic tree. The numbers at the nodes indicate bootstrap values (% over 1000 replicates). The scale bar shows the number of substitutions per site. In each analysis, the midpoint was used as a root. The parameters for RAxML in the analysis of chloroplast DNA sequences were as follows: -f = a, -x = 12,345, -p = 12,345, -N (bootstrap value) = 1,000, -c = 1 and -m = GTRCATX). The parameters for RAxML in the analysis of mitochondrial DNA sequences were as follows: -f = a, -x = 12,345, -p = 12,345, -N (bootstrap value) = 1,000, and -m = GTRGAMMAX).

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S4 Fig. The multi-FASTA file of the assembled complete sequences of mitochondrial genomes used to create the phylogenetic tree in S3 Fig.

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S5 Fig. The multi-FASTA file of variables sites in the chloroplast genomes used to create the phylogenetic tree in S6 Fig.

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S6 Fig. Phylogenetic tree of Pyropia yezoensis samples using the chloroplast DNA sequences based on maximum likelihood reconstruction.

The scale bar shows the number of substitutions per site. The sequences of Pyr_19 (P. tenera) and the hybrid (Pyr_27) between P. yezoensis and P. tenera were used as roots. Colors were used to distinguish between Japanese P. yezoensis and 3 clusters of Chinese P. yezoensis. The parameters for RAxML were as follows: -f = a, -x = 12,345, -p = 12,345, -N (bootstrap value) = 1,000, -c = 1, and -m = GTRCAT). Only variable sites were used in the analysis.

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S7 Fig. The multi-FASTA file of variables sites in mitochondrial genomes used to create the phylogenetic tree in Fig 3.

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S8 Fig. Genotypes of the rRNA repeats of the chloroplast genome of Pyr_1 (Pyropia yezoensis) visualized using the Integrative Genomics Viewer.

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S9 Fig. Genotypes of the chloroplast genome of Pyr_45 (Pyropia haitanensis) visualized using the Integrative Genomics Viewer.

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Click here for additional data file.^{(350.9KB, pdf)}

S10 Fig. Multidimensional scaling representation using nuclear DNA data of 34 Pyropia yezoensis samples, Pyr_19 (P. tenera), and Pyr_27 (the hybrid between P. yezoensis and P. tenera).

Two-dimensional data were obtained in this analysis. Colors were used to show P. yezoensis samples, P. tenera, and the hybrid between P. yezoensis and P. tenera.

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Click here for additional data file.^{(28KB, pdf)}

S11 Fig. The graph of K values vs cross-validation errors (upper) and admixture analysis using nuclear DNA data of 31 Pyropia yezoensis samples (K = 3, 4, 5).

Colors of the sample names were used to show 3 clusters, cluster A, B, and C.

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S1 Table. Summary of the quality of aligned data analyzed by the Qualimap program.

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Click here for additional data file.^{(11.1KB, xlsx)}

S2 Table. List of variants specific to abnormal samples.

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Click here for additional data file.^{(14.2KB, xlsx)}

S3 Table. List of variants specific to green mutant.

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Click here for additional data file.^{(15.1KB, xlsx)}

Acknowledgments

We would like to thank H. Matsuo for technical assistance with the experiments. We would also like to thank Daiichi Seimo Co. Ltd., Saga Prefectural Ariake Fisheries Research and Development Center, Ariake Regional Laboratory of Fukuoka Fisheries and Marine Technology Research Center, Saga Prefectural Ariake Fishery Cooperative, and the National Federation of Nori & Shellfish-fishers cooperative associations for providing us with the samples of Pyropia species used in this study. We would like to thank Editage (www.editage.com) for English language editing.

Data Availability

Sequences are available at the DNA Data Bank of Japan Sequence Read Archive (http://trace.ddbj.nig.ac.jp/DRASearch/submission?acc=DRA010178).

Funding Statement

This study was supported by the “Projects for sophistication of production and utilisation technology supporting local agriculture and marine industry” from Saga University (http://www.saga-u.ac.jp/) (to YN, KK, GK, YK) and KAKENHI (18K19235) from the Japan Society for the Promotion of Science (https://www.jsps.go.jp/) (to KK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0252207.r001

Decision Letter 0

Tzen-Yuh Chiang

15 Oct 2020

PONE-D-20-27527

Genomic diversity of 39 accessions of Pyropia species grown in Japan

PLOS ONE

Dear Dr. Nagano,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we have decided that your manuscript does not meet our criteria for publication and must therefore be rejected.

I am sorry that we cannot be more positive on this occasion, but hope that you appreciate the reasons for this decision.

Yours sincerely,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript was already published in “bioRxiv – the preprint server for biology” under the title of “Genomic diversity of 39 Pyropia species grown in Japan (doi: https://doi.org/10.1101/2020.05.15.099044). This case is the redundant publication and I wonder the guideline of “PLOS ONE” can allow this case. I think that the nature of “bioRxiv” is not the process of the pre-review system and it is just one of the publication. Therefore, I cannot accept this manuscript for the publication

Reviewer #2: This manuscript analyzed the phylogenetic relations of 39 accessions of Pyropia species grown in Japan by chloroplast, mitochondrial and nuclear genome using high-throughput sequencing. In addition, the author also analyzed the genetic background of hybrid between P. yezoensis and P. tenera. The results of this study will be useful for breeding and the conservation of Pyropia species in Japan.

However, there are some problems existing in the structure and contents of the whole manuscript. The author attempted to elucidate the phylogenetic relationship of the samples from Japan. This part is the focus of the whole manuscript. In addition, the author also confirmed the hybrid relationship between between P. yezoensis and P. tenera, which is not consistent with the first part and can be deleted.

About the phylogenetic analysis, the author should make model test using different softwares, such as PAUP、PYLIP and Bayesian. Otherwise, the author referred that the topological structure of phylogenetic trees based on chloroplast, mitrochondrial and nuclear genome sequences are not scientific. Furthermore, the conclusion about the evoltionary history of chloroplast and mitrochondrial genome is not identical is also not scientific. So the author should analyze the evolutionary speed of chloroplast and mitrochondrial genome.

The author should show the detailed information about the sequences from chloroplast and mitrochondrial genome used in constructing phylogenetic tree in the method section.

The author should explain the reasons why China cultivar MK695880 clustered with Japan samples in Fig2.

The taxonomy of some samples were confused by the author previously. They were corrected based this manuscript. Yet this is the mistake by the author in the previous research and is not similar with the wild sample identification using phylogenetic analysis. So these related analysis should be re-writen.

In the result section, I am not sure whether the author removed the sequences from the contaminant from bacterial nuclear genome. Or the author should clearly state the genome sequences used for constructing the phylogenetic tree were indeed mapped to the reference genome.

**********

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Reviewer #2: No

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For journal use only: PONEDEC3

PLoS One. 2021 Jun 9;16(6):e0252207. doi: 10.1371/journal.pone.0252207.r002

Author response to Decision Letter 0

1 Dec 2020

We thank the editorial team for reversing a decision that was made against the editorial policy of PLOS ONE.

We are grateful for the suggestions made by the reviewers to improve our manuscript. We have revised the manuscript according to these suggestions. Furthermore, we have provided a point-by-point response to the reviewers’ comments as attached file.

We trust that the manuscript has been improved satisfactorily and hope that it is now acceptable for publication in PLOS ONE.

Attachment

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PLoS One. doi: 10.1371/journal.pone.0252207.r003

Decision Letter 1

Arun Kumar Jugran

25 Jan 2021

PONE-D-20-27527R1

Genomic diversity of 39 accessions of Pyropia species grown in Japan

PLOS ONE

Dear Dr. Nagano,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The MS entitled Genomic diversity of 39 accessions of Pyropia species grown in Japan describes scientific advances in its findings. The research is up to date and suggests the further improvement of the species. Data is analyzed properly and findings from the study are fact-based. However, as all the reviewers have raised concern about the novelty of the work (as a preprint is available on a website), we suggest to address the issue (as suggested below) along with addressing all the comments. Based on the reviewers' comments we strongly recommend the MS for Major revision.

Please submit your revised manuscript by Mar 08 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Arun Kumar Jugran and Berthold Heinze

Academic Editors

PLOS ONE

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Additional Editor Comments (if provided):

1 - The authors are right in saying that submitting a pre-print to bioRxiv is actually encouraged by PLOS ONE. The best way forward (or the prefered and suggested one) from there, however, is to upload a revised version to bioRxiv and use the "direct transfer to journal" possibility there. In this way, the various versions of the manuscript stay tightly connected to each other. bioRxiv points later readers to the journal article, if the material gets published. So a bioRxiv item is not an independent publication on its own, and that's why PLOS ONE encourages it.

2 - The style of the manuscript is not according to PLOS ONE criteria in that Results and Discussion come before Material and Methods here. So this must be corrected by the authors.

3 - I found the abstract not as informative about what was actually done in this study, as opposed to the last paragraph of the introduction, which gives a far better overview of the study. So I would like to encourage the authors to re-write their abstract accordingly, so that it gives a clear story of what was done in this study.

4 - It would help for the understanding of the sampling strategy to categorize/summarize the samples as, e.g. numbers of varieties, hybrids, related species, et cetera, and to also summarize the geographical sampling sites. Readers not familiar with the geography of Japan currently have bit of a hard time following the text, even when refering to the geographical map. This should be addressed and the text improved, in my opinion.

5 - Moreover, for the international and diverse readership of PLOS ONE, it would help I think if the authors briefly give a definition of these terms - varieties, hybrids, pure accessions et cetera - for non-seaweed specialists (as myself). This is important because of the implications for genomic data, i.e., whether samples are homozygous or heterozygous - what is the expectation in the various categories (varieties, hybrids, wild accessions, ...)? I found this a bit confusing when the 'allodiploid' nature of the hybrid is mentioned and discussed. My understanding after reading the text is that normally, these species are diploid; while the hybrid must then be allotretraploid, and by sampling an originally haploid cell line (which, in this case, must be allodiploid, correct?) which then undergoes diploidization (correct?), in this case we arrive at an allotetraploid? So when they write "allodiploid" here, they really mean an F1 hybrid cell lineage?

Please clarify all this; maybe my assumptions are wrong, or different terminology is used in the seaweed world; but a clarification would greatly improve the manuscript.

6 - further, I have the following minor editorial remarks:

L 109 - ... addition, they can be crossed WITH each other[32]

L 122 - Thus, mitochondrial sequences are determinant for clustering in the previous report[26] - ? I do not understand the meaning of this sentence.

L 147 - The mean coverage of Pyr_35 (P. dentata), Pyr_44 (P. tenuipedalis), and Pyr_45 (P. haitanensis) was 2.7, 8.8, and 4.2, respectively. - Use the X sign to indicate coverage, which is a convention that is useful I think: 2.7 X et cetera.

L 173 - The Pyr_27 is also a pure accession,... - define what "pure accession" means. See remark 5 just above.

L 194 - Of 34 accessions of P. yezoensis, 31 accessions were pure,so they should be homozygous. - define "pure" as opposed to (or identical to ?) "homozygous". Again, see remark 5 above.

L 300 - In contrast, there were two types of rRNA genes in the chloroplast genome. - Have the auhors considered the possibility of nuclear and/or mitochondrial copies of chloroplast rRNA genes? There are many publications on this phenomenon in the plant world. Or is this another peculiarity in the seaweed world that chloroplasts are "added" when species hybridize? Or is it just simply unknown (as they also point out that the mode of inheritance of organelles is not known in this group of seaweeds, at another location in the manuscript)?

L 367 - GetOrganelle - which reference genomes or 'baits' (if any?) were used to extract and map (assemble) the chloropalsts and mitochondria to?

"target genome(s) or sequence(s) as the seed" are used in GetOrganelle, when I look at the description of this programme.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Partly

Reviewer #4: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

Reviewer #4: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: No

Reviewer #4: Yes

**********

6. Review Comments to the Author

Reviewer #3: This research paper is already available in public database/repository, so in the current scenario this unethical to publish. Paper doesn’t have authenticity and novelty. It may resubmit to the journal only after withdrawal from bioRxiv preprint server (Cold spring harbor laboratory) https://www.biorxiv.org/content/10.1101/2020.05.15.099044v1.full.

Nevertheless, the manuscript submitted to the Journal entitled “Genomic diversity of 39 accessions of Pyropia species grown in Japan" is an informative piece of work and seems useful in view of harnessing the potential of these important marine algae in breeding and genetics. Although, authors have included limited (39) number of accessions in the study, work may help to breeders and researcher to generate the information for identification of best planting material.

Based on my observations I recommend the manuscripts for major revision. To improve the manuscript quality, the following comments and suggestions are required to be addressed;

� The economic importance and medicinal values of the Pyropia species is lacking in the introduction section of the manuscript.

� Why the assessment of the genetic diversity is important within the the Pyropia species. Explain in introduction in brief.

� What is Ariake sound, mention clearly in M&M of MS.

� In material and methods part of the MS, the construction of DNA library is missing, include some lines about that.

� How organellar (mitochondrial and chloroplast) DNA was extracted..?

� What type of data was used to construct a phylogenetic tree, need to clearly mention in materials and methods.

� Discuss the results that mitochondrial DNA sequences couldn’t revealed genetic variability among the of 39 accessions of Pyropia species; What may be the probable cause of that.

� And also explain how about the chloroplast based genetic variations showed in gbPyropia species.

� Nuclear DNA based genetic analysis was significant in compared to that of organellar genetic diversity, what may be the possible reasons for this distinctive nature of between organellar and nuclear genetics.

� Line 403 is the repeat of 402; these can be clubbed in a single sentence.

� In results section, lines 73 -75 should be part of materials methods.

� There are discussions parts mingled with results which enlarged the size of results, check thoroughly and trim the results section.

Reviewer #4: Dear authors,

The research work presented in the manuscript detailed about genetic diversity of 39 accessions of Pyropia species grown in Japan.The study seems important. In my view the work will be of more significance if the authors could make out correlation of phenotypic differences with their genotypes. The results are organized into headings which does not go well, the results of sequencing are not mentioned in the results section. In the M&M section the authors wrote that DNA and RNA was isolated but in the heading they have mentioned DNA sequencing only. No clarity about what was sequenced. Details need to be incorporated.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: Yes: Ram Baran Singh

Reviewer #4: No

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

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PLoS One. 2021 Jun 9;16(6):e0252207. doi: 10.1371/journal.pone.0252207.r004

Author response to Decision Letter 1

7 Mar 2021

Please check "Response to reviewers", because we have many responses.

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PLoS One. doi: 10.1371/journal.pone.0252207.r005

Decision Letter 2

Randall P Niedz

4 May 2021

PONE-D-20-27527R2

Genomic diversity of 39 samples of Pyropia species grown in Japan

PLOS ONE

Dear Dr. Nagano,

Address the minor edit suggested by Reviewer #4 to "merge the DNA purification and DNA sequencing sub-sections" and to rewrite/reword the DNA purification step as described by the reviewer (i.e., the procedure is for purifying DNA - no need to indicate RNA other than the procedure includes RNAase to get rid of the RNA).

Please submit your revised manuscript by Jun 18 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Randall P. Niedz

Academic Editor

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #4: All comments have been addressed

Reviewer #5: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #4: Partly

Reviewer #5: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #4: I Don't Know

Reviewer #5: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #4: Yes

Reviewer #5: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #4: Yes

Reviewer #5: Yes

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6. Review Comments to the Author

Reviewer #4: Authors should merge the DNA purification and DNA sequencing sub-sections and I recommend to rewrite the DNA purification step in a proper way. When DNA is isolated RNAase treatment is done to get rid of RNA contamination, but that need not be written as DNA and RNA is isolated, when your aim is to isolate or purify only DNA.

Reviewer #5: Review of the manuscript PONE-D-20-27527 entitled: “Genomic diversity of 39 samples of Pyropia species grown in Japan”

The submitted manuscript is an article that presents a phylogenetic analysis of 39 samples of Pyropia yezoensis, an important marine crop that is used as ingredients of sushi and snacks around the world. The Authors using HTS methods were able to perform a deep study of molecular variability of P. yezoensis isolated from different localities.

In my opinion, the revised paper after several corrections is well written and introduces the reader to the subject matter presented here. The methodology has been selected and presented correctly. The description of the results is presented clearly. The discussion section is revised according to reviewers' comments.

The main complaint relating to an earlier publication of the text in the bioRxiv repository has already been explained.

Therefore, I believe that it is a very valuable text which in revised form can be published in PLOS ONE.

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Reviewer #4: No

Reviewer #5: No

PLoS One. 2021 Jun 9;16(6):e0252207. doi: 10.1371/journal.pone.0252207.r006

Author response to Decision Letter 2

6 May 2021

We appreciated your helpful comments.

We have merged the two sections and changed the title to "DNA purification and sequencing".

In addition, we have changed the sentence about DNA purification as follows: “DNA was extracted from the conchocelis of each sample using the DNAs-ici!-F (Rizo, Tsukuba, Japan) according to the instructions of the manufacturer, followed by RNase A (NIPPON GENE, Tokyo, Japan) treatment.”

Reviewer #5: Review of the manuscript PONE-D-20-27527 entitled: “Genomic diversity of 39 samples of Pyropia species grown in Japan”

The main complaint relating to an earlier publication of the text in the bioRxiv repository has already been explained.

We are encouraged by your comments as we have made repeated revisions (*^-^*)

Additional Changes

In addition to the changes mentioned above, we have made the following changes. We apologize for these corrections.

1. We made some minor changes, such as spelling errors.

2. We have moved the legend in Figure 3 to the appropriate position.

3. The internal title of the PDF file of S9 Fig was S2 Fig, so we changed it to S9 Fig.

4. There was a mistake in Figure 8 that we failed to fix in the previous revision. In order to fit the figure to the manuscript, we changed the strain name from "S-18" to "Shin Saga 4 gou" in Figure 8.

Attachment

Submitted filename: Response_to_Reviewers.docx

Click here for additional data file.^{(17.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0252207.r007

Decision Letter 3

Randall P Niedz

12 May 2021

Genomic diversity of 39 samples of Pyropia species grown in Japan

PONE-D-20-27527R3

Dear Dr. Nagano,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Randall P. Niedz

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0252207.r008

Acceptance letter

Randall P Niedz

17 May 2021

PONE-D-20-27527R3

Genomic diversity of 39 samples of Pyropia species grown in Japan

Dear Dr. Nagano:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Randall P. Niedz

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. The multi-FASTA file of the large single copy sections of chloroplast genomes used to create the phylogenetic tree in Fig 2.

(PDF)

Click here for additional data file.^{(9.3MB, pdf)}

S2 Fig. The multi-FASTA file of the large single copy sections of chloroplast genomes used to create the phylogenetic tree in S3 Fig.

(PDF)

Click here for additional data file.^{(2MB, pdf)}

S3 Fig. Comparison of phylogenetic trees between the chloroplast and mitochondrial DNA sequences.

(PDF)

Click here for additional data file.^{(18.5KB, pdf)}

S4 Fig. The multi-FASTA file of the assembled complete sequences of mitochondrial genomes used to create the phylogenetic tree in S3 Fig.

(PDF)

Click here for additional data file.^{(636.1KB, pdf)}

S5 Fig. The multi-FASTA file of variables sites in the chloroplast genomes used to create the phylogenetic tree in S6 Fig.

(PDF)

Click here for additional data file.^{(1,010.6KB, pdf)}

S6 Fig. Phylogenetic tree of Pyropia yezoensis samples using the chloroplast DNA sequences based on maximum likelihood reconstruction.

(PDF)

Click here for additional data file.^{(20KB, pdf)}

S7 Fig. The multi-FASTA file of variables sites in mitochondrial genomes used to create the phylogenetic tree in Fig 3.

(PDF)

Click here for additional data file.^{(471.4KB, pdf)}

S8 Fig. Genotypes of the rRNA repeats of the chloroplast genome of Pyr_1 (Pyropia yezoensis) visualized using the Integrative Genomics Viewer.

(PDF)

Click here for additional data file.^{(128.2KB, pdf)}

S9 Fig. Genotypes of the chloroplast genome of Pyr_45 (Pyropia haitanensis) visualized using the Integrative Genomics Viewer.

(PDF)

Click here for additional data file.^{(350.9KB, pdf)}

S10 Fig. Multidimensional scaling representation using nuclear DNA data of 34 Pyropia yezoensis samples, Pyr_19 (P. tenera), and Pyr_27 (the hybrid between P. yezoensis and P. tenera).

Two-dimensional data were obtained in this analysis. Colors were used to show P. yezoensis samples, P. tenera, and the hybrid between P. yezoensis and P. tenera.

(PDF)

Click here for additional data file.^{(28KB, pdf)}

S11 Fig. The graph of K values vs cross-validation errors (upper) and admixture analysis using nuclear DNA data of 31 Pyropia yezoensis samples (K = 3, 4, 5).

Colors of the sample names were used to show 3 clusters, cluster A, B, and C.

(PDF)

Click here for additional data file.^{(34.2KB, pdf)}

S1 Table. Summary of the quality of aligned data analyzed by the Qualimap program.

(XLSX)

Click here for additional data file.^{(11.1KB, xlsx)}

S2 Table. List of variants specific to abnormal samples.

(XLSX)

Click here for additional data file.^{(14.2KB, xlsx)}

S3 Table. List of variants specific to green mutant.

(XLSX)

Click here for additional data file.^{(15.1KB, xlsx)}

Attachment

Submitted filename: Point-by-point_response _to_the_reviewers.docx

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Submitted filename: Reviwers comments#.docx.doc

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Attachment

Submitted filename: Response to Reviewers.docx

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Submitted filename: Response_to_Reviewers.docx

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Data Availability Statement

Sequences are available at the DNA Data Bank of Japan Sequence Read Archive (http://trace.ddbj.nig.ac.jp/DRASearch/submission?acc=DRA010178).

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PERMALINK

Genomic diversity of 39 samples of Pyropia species grown in Japan

Yukio Nagano

Kei Kimura

Genta Kobayashi

Yoshio Kawamura

Roles

Abstract

Introduction

Fig 1. Positions of the isolation sites.

Materials and methods

Materials

Table 1. List of Pyropia samples.

DNA purification and sequencing

Assembly-based analysis of chloroplast and mitochondrial genomes

Fig 2. Phylogenetic tree of Pyropia species using long single section sequences of chloroplast DNA based on maximum likelihood reconstruction.

Mapping-based analysis of nuclear, chloroplast, and mitochondrial genomes

Fig 3. Phylogenetic tree of Pyropia yezoensis samples using the mitochondrial DNA sequences based on maximum likelihood reconstruction.

Results

Samples used in this study

Sequencing

Chloroplast and mitochondrial genomes

Nuclear genomes

The hybrid between P. yezoensis and P. tenera

Fig 4. Comparison of the nuclear genotypes of Pyr_1 (Pyropia yezoensis), Pyr_27 (the hybrid between P. yezoensis and P. tenera), and Pyr_19 (P. tenera) visualized by the Integrative Genomics Viewer.

Table 2. Number of loci in Pyr_27, when Pyr_1 locus carries two reference alleles and Pyr_19 locus carries two alternate alleles.

Genomic diversity of Japanese P. yezoensis

Fig 5. Multidimensional scaling representation using nuclear DNA data of 31 Pyropia yezoensis samples.

Fig 6. Admixture analysis using nuclear DNA data of 31 Pyropia yezoensis samples.

Fig 7. Phylogenetic tree constructed using the SVDquartets with PAUP using nuclear DNA data of 31 Pyropia yezoensis samples and 1 P. tenera.

Fig 8. Multidimensional scaling representation using nuclear DNA data of 19 Pyropia yezoensis samples belonging to the cluster A.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Tzen-Yuh Chiang

Roles

Author response to Decision Letter 0

Decision Letter 1

Arun Kumar Jugran

Roles

Author response to Decision Letter 1

Decision Letter 2

Randall P Niedz

Roles

Author response to Decision Letter 2

Decision Letter 3

Randall P Niedz

Roles

Acceptance letter

Randall P Niedz

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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