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Article

DNA Barcoding and Intronic-ORF Structure Analyses of Cultivated Pyropia yezoensis in China: The Genetic Impact under Climate Change

by
Guihua Huang
1,2,
Cuicui Tian
3,
Liangdi Wei
2,
Chuanming Hu
2,
Guangping Xu
2,
Wei Zhou
2 and
Yinyin Deng
2,*
1
School of Marine Science and Fisheries, Jiangsu Ocean University, Lianyungang 222005, China
2
Jiangsu Marine Fisheries Research Institute, Nantong 226007, China
3
Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Nanjing 210014, China
*
Author to whom correspondence should be addressed.
J. Mar. Sci. Eng. 2024, 12(9), 1551; https://doi.org/10.3390/jmse12091551 (registering DOI)
Submission received: 8 August 2024 / Revised: 30 August 2024 / Accepted: 3 September 2024 / Published: 5 September 2024
(This article belongs to the Special Issue Marine Biota Distribution and Biodiversity)
Browse Figures
Versions Notes

Abstract

:
Pyropia yezoensis is the most widely cultivated and economically important alga. Affected by climate change, the cultivation of P. yezoensis has gradually migrated to the northern coast of China, increasing from 6.8% in 2019 to 19.5% in 2023. To date, the genetic impact of northern migration on cultivation resources has not been assessed and analyzed extensively. Here, DNA barcoding (rbcL and cox1) and the presence/absence of intronic-ORFs in mitochondrial regions (rnl and cox1) were applied to investigate genetic diversity in 44 P. yezoensis specimens from 17 aquaculture farms in China, with comparisons to Korean and Japanese cultivated resources. The lower intraspecific variation was 0.31% for the cox1 gene and 0.14% for the rbcL gene, with three haplotypes, indicating that intensive selection and breeding during cultivation had narrowed the germplasm genetic variation. The intron structure of mitochondrial regions showed that the cultivated resources had 17 phenotypes, and the northern specimens shared 35.3% of genotypes with the southern specimens, indicating that the cultivated P. yezoensis is expanding its cultivation ranges through north migration. Even with lower genetic diversity, the northern area of cultivation had already developed 17.6% site-specific specimens. The genetic diversity of cultivated P. yezoensis from the Northwest Pacific is also discussed. Our work provides a preliminary framework for P. yezoensis breeding and cultivation under climate change.

1. Introduction

The red algal genus Pyropia (formerly Porphyra) is a primitive photosynthetic eukaryote, which was classified in the new genus of Bangiales by Sutherland et al., in 2011 [1]. Although a recent study proposed a subdivision of Pyropia into six genera (Calidia, Neoporphyra, Neopyropia, Uedaea, Porphyrella and a redefined Pyropia) [2], the revision of Pyropia classification, with its potential complexity, remains ambiguous and requires more supporting genomic data [3,4]. Pyropia is an economically valuable red alga, which is commonly consumed as raw or processed food and is also a source of substances beneficial to human health [5]. Pyropia yezoensis is the most widely cultivated species in the aquaculture industry of the Northwest Pacific including Japan, Korea and China [6]. In China, the artificial cultivation of P. yezoensis was primarily developed on the coast of Nantong city (NT), Jiangsu Province (JS) and has become the main cultivation area of P. yezoensis since the 1970s. However, global warming in recent years has led to a continual increase in seawater temperature [7]. Affected by this, significant changes have taken place in the cultivation areas of P. yezoensis, and migration to the northern coast has gradually become a trend. Furthermore, other human factors, such as the prevention and control of green algae disasters and restrictions on sea area use, have also led P. yezoensis cultivation into a decline in JS and towards northern migration. Based on the data from Jiangsu Nori Associate, the northern coast of JS in Lianyungang city (LYG) has replaced Nantong as the main cultivation area (>50%) since 2018 and cultivation has increased year by year, accounting for 76.6% in 2022. Meanwhile, the cultivation areas have been migrating to the more northern coast of China, including Shandong and Liaoning Provinces (SD and LN). And the proportions of cultivation area in the north (SD and LN) have increased rapidly in recent years, from 6.8% in 2019 to 19.5% in 2023 (Figure 1a). However, according to the trading price, the quality of northern cultivation resources is still lower than those from JS (Figure 1b). Therefore, in order to cope with changes in cultivation environments, more diverse P. yezoensis varieties or cultivars need to be developed [8]. Strain identification and genetic impact at the intraspecific level of P. yezoensis is important to the aquaculture industry for the development of new varieties or cultivars and for the maintenance of excellent strains under climate change [9]. Instead of relying on morphological features with phenotypic plasticity, molecular approaches are necessary to assess the diversity of P. yezoensis at intraspecific level.
DNA barcoding is a rapid and efficient approach to studying the evolution, biogeography and systematics of red algae. The plastid RUBISCO large unit gene (rbcL) has been widely used to clarify the taxonomic position of Pyropia and to measure its intraspecific variation [10,11]. And the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) has been used as a supplementary marker to evaluate intra- and interspecific divergence and to assign Pyropia specimens [12,13,14]. The cox1 gene is an especailly reliable molecular marker for intraspecific study, revealing species relationships, population structure, and the hidden diversity of red algae [15]. With the development of genomics, red algal mitochondrial genomes (mtDNAs) can provide useful information for species identification. The mtDNAs of bladed Bangiales have shown diverse variation in their size and gene structures [16,17,18]. In particular, the presence or absence of introns and intronic open reading frames (intronic-ORFs) found in the ribosomal RNA large subunit gene (rnl) and cox1 significantly varied the mitochondrial genome size in Pyropia species, making it more effective in assessing genetic variation at the intraspecific level than using traditional DNA barcoding makers [9]. In P. yezoensis, the rnl and cox1 genes contained three and four introns with or without intronic-ORFs, separately [17]. Based on the present/absent intronic-ORFs structure of rnl and cox1, 12 genetic types were found in 40 cultivated strains of P. yezoensis from Korea and Japan, and only five haplotypes of the cox1 gene were revealed among these strains [9]. Therefore, the intronic-ORF structure of rnl and cox1 regions exhibited higher genetic resolution at the intraspecific level of P. yezoensis.
To respond and adapt to climate change, the species biodiversity, distribution, abundance, and population richness of seaweed resources will be affected [7]. With the north migration of Pyropia cultivation in China, the genetic diversity of current cultivation resources should be assessed and analyzed extensively to inform strategies for maintaining this economically important crop in the face of climate change. Here, 44 specimens of cultivated P. yezoensis, extensively collected from 17 different aquaculture farms in China, were investigated using DNA barcoding (rbcL and cox1) and the presence/absence of intronic-ORF structure of the rnl and cox1 regions to elucidate genetic diversity at the intraspecific level and the genetic impact of climate change. Our work provides a preliminary framework for selecting and breeding suitable strains for P. yezoensis cultivation under climate change in the future.

2. Materials and Methods

2.1. The Collection and Culture of P. yezoensis

A total of 44 cultivated P. yezoensis specimens were collected from 17 aquaculture farms in Liaoning (LN), Shandong (SD) and Jiangsu (JS) provinces. These aquaculture farms were chosen to be in extensively different cities. The specimens from every farm were selected based on the different morphology of their blades, such as the length/width value, thickness or color. Three individuals with similar morphology from each specimen were chosen as three biological repeats. The letter of the specimen code referred to where it was collected from and the first number referred to the aquaculture farm, with farms arranged sequentially from the north to south. All specimens were preserved in the State Germplasm Bank for Porphyra at the Jiangsu Marine Fisheries Research Institute (Nantong, China). The blades of P. yezoensis were cultured in sterilized seawater. The growth conditions were 10 °C with a 12 h/12 h light/dark photoperiod under a light intensity of 100 μmol m−2 s−1. A small tissue from the middle of the blades was used for genetic analysis.

2.2. The Amplification of DNA Barcoding

The genomic DNA of P. yezoensis was extracted using a Hi-DNAsecure Plant Kit (Tiangen Biotech Co. Ltd., Beijing, China) according to the manufacturer’s protocol. The rbcL gene was amplified by using primers Kito-F1 (5′-ATGTCTCAATCCGTAGAATCA-3′) and JrSR (5′-AAGCCCCTTGTGTTAGTCTCAC-3′) [19]. And the amplification of the cox1 region was performed using the primer pairs GazF1 (5′-TCAACAAATCATAAAGATATTGG-3′) and GazR1 (5′-ACTTCTGGATGTCCAAAAAAYCA-3′) [20]. The PCR system was performed as previously described [21]. Amplification was under the following conditions: 3 min denaturation at 95 °C, 35 cycles of 30 s denaturation at 95 °C, 30 s annealing at 45 °C for the rbcL gene and 56 °C for the cox1 region, 1.5 min extension at 72 °C for the rbcL gene and 30 s for the cox1 gene, and a final extension of 10 min at 72 °C. PCR products were sequenced by Sangon Biotech (Shanghai, China). The sequences of different phenotypes of rbcL and cox1 were deposited into the NCBI database (National Centre for Biotechnology Information).

2.3. The Cloning and Analyses of Intronic-ORF Structure

In the mitochondrial genome, P. yezoensis had four exons and three introns in the rnl gene, and five exons and four introns in the cox1 gene. The presence/absence of intronic-ORFs in the rnl and cox1 regions of P. yezoensis were amplified with three and four primer pairs, which were designed in the binding sites on each exon region [9]. The PCR was carried out in a 25 µL volume containing 50 ng of total genomic DNA using a LATaq polymerase system (TaKaRa Co. Ltd., Shiga, Japan). The amplification system and conditions were performed as described by Hwang et al., 2018 [9]. Band patterns of PCR products were analyzed using 1% agarose gel electrophoresis. Based on the amplicon size, the present and absent intronic-ORFs of the rnl and cox1 genes were marked with 1 and 0, separately. The intron structural variation was represented as a code RXXXCXXXX (R = rnl, C = cox1, X = 0/1, 0 = intronic-ORFs absent, 1 = intronic-ORFs present).
The phylogenies for intron structure were generated using the unweighted paired group method based on arithmetic averages (UPGMA). The unrooted UPGMA trees were reconstructed using MEGA 7.0 after alignment with ClustalX [22].

2.4. The Haplotype Diversity and Network Analysis

The cox1 sequences of cultivated P. yezoensis obtained from the present study were trimmed to 659 bp, and the sequences of 132 Chinese specimens, 27 Korean specimens and 13 Japanese specimens from Hwang et al. (2018) [9] were used for the haplotype analysis. The rbcL sequences of cultivated P. yezoensis were trimmed to1392 bp for the haplotype analysis, with 132 Chinese specimens, 8 Korean specimens and 7 Japanese specimens from GenBank. The sequence divergence of different haplotypes was analyzed using MEGA 7.0 with 1000 bootstrap replicates after alignment with ClustalX [22]. The diversity indices including the number of haplotypes (Nh), the number of parsimony informative sites (Np), haplotype diversity and nucleotide diversity were analyzed using DnaSP 6 [23]. The haplotype diversity of the intron structures of the rnl and cox1 genes was calculated using the Simpson function from the vagan package in R (version 4.2.3). To evaluate genetic relationships, haplotype networks were generated using POPART 1.7 with the TCS method [24]. The map of haplotypes and distribution of cultivated P. yezoensis was also generated using POPART 1.7 software.

3. Results

3.1. Genetic Diversity of cox1 Gene

The cox1 sequences of 44 specimens from P. yezoensis cultivated in China were obtained. Except for the S11 specimen, three repeats of the other 43 specimens all had the same cox1 sequences. The S11 specimen with three biological repeats had two different cox1 sequences, with 2 bp differences. Therefore, a total of 45 cox1 sequences were used to analyze the diversity indices (Table 1). The length of the cox1 sequenced region was 659 bp, and three parsimony informative sites were identified. According to the mitochondrial genome of P. yezoensis (KF561997) [17], the amplified sequence of cox1 in the present study was located in the first exon of the cox1 gene, in total, at 1158 bp. And the three parsimony informative sites occurred at positions 435, 603, and 702 of exon of the cox1 gene. According to the results of DnaSP 6, the 45 sequences generated three haplotypes (C1: C435C603T702, C2: C435T603C702, C3: T435C603C702, GenBank accession numbers: OQ396573-OQ396575), with 0–3 bp intraspecific differences. Sequence divergence among the three haplotypes was from 0.20 to 0.31%. The amino acids of the three haplotypes were the same, indicating that the variable cox1 region differences of cultivated P. yezoensis were synonymous mutations (Table 2).
In cultivated P. yezoensis from China, haplotype C1 and C2 were shared among the LN, SD and JS specimens (Figure 2a). Haplotype C1 and C2 were found in the majority of the 45 specimens, accounting for 51.1% and 42.2% of all individuals. The haplotype C3 was infrequently present in LN (n = 1) and JS (n = 2). The haplotype and nucleotide diversity indices showed that specimens from JS had higher genetic diversity than those from the north (SD and LN). In Korea, four haplotypes (C01-C04) were present in 27 cultivated P. yezoensis specimens. The C03 was the same as C3 from the present study, whereas other haplotypes were specific for Korean specimens (Table 2). A total of 13 specimens from Japan showed one haplotype, C2. According to the haplotype and nucleotide diversity indices, specimens from China had higher genetic diversity than those from Korea, followed by those from Japan (Table 1).

3.2. Genetic Diversity of rbcL Gene

PCR amplification of the rbcL gene of cultivated P. yezoensis generated a full ORF with 1392 bp in all 44 specimens. Except for S11, the three repeats for the 43 specimens had the same rbcL sequence. The S11 specimen with three repeats had two different rbcL sequences, with 2 bp differences. In cultivated P. yezoensis, a total of 45 rbcL sequences from China were used to analyze the diversity indices (Table 1). When fully aligned, two parsimony informative sites of rbcL gene were identified. The parsimony informative sites occurred at positions 900 and 1335 of the rbcL gene. The 45 sequences could be organized into three haplotypes (R1: C900C1335, R2: T900C1335, R3: T900T1335, GenBank accession numbers: OQ396570-OQ396572). Sequence divergence among haplotypes ranged from 0.07% to 0.14% (0–2 bp). And the amino acids of the three haplotypes were the same, indicating that the substitutions of rbcL gene in cultivated P. yezoensis were synonymous mutations (Table 2). In addition, the frequencies of haplotypes of the rbcL gene (R1, R2, R3) were in accordance with those of the cox1 gene (C1, C3, C2). And the incorporation of cox1 and rbcL genes presented three haplotypes R1C1, R2C3, R3C2 in the 44 specimens of cultivated P. yezoensis from China.
In cultivated P. yezoensis from China, haplotype R1 and R3 were shared among LN, SD and JS specimens (Figure 2b). The most abundant haplotypes, R1 and R3, accounted for 51.1% and 42.2% of all individuals. The haplotype R2 was infrequently present in LN (n = 1) and JS (n = 2). The haplotype and nucleotide diversity indices showed that specimens from JS had higher genetic diversity than the specimens from the north. The rbcL sequences of cultivated P. yezoensis analyzed in this study also included Korean and Japanese specimens. In cultivated P. yezoensis from Korea, three haplotypes (R1, R2, R3) were present in eight specimens. And seven specimens from Japan showed haplotypes R2 and R3. According to the haplotype and nucleotide diversity indices, specimens from Korea had a little greater genetic diversity than specimens from China, followed by those from Japan (Table 2).

3.3. The Intron Haplotype of Mitochondria

According to the presence/absence of the intronic-ORFs structure of the rnl and cox1 regions (Figure 3a), the cultivated P. yezoensis specimens showed four genotypes (R001, R011, R101, R111) in the rnl intron and nine genotypes (C0000, C0011, C0100, C0111, C1000, C1011, C1100, C1110, C1111) in the cox1 intron. Combined intron structural variations of rnl and cox1 exhibited 17 genotypes among 44 specimens, including H1–H17. Except S11 and S22, every specimen had unique intron structural variation. Two different structural variations of rnl and cox1 genes were shown in S11 (H3, H14) and S22 (H13, H17). Based on the presence and absence of intronic-ORF, the genotypes with close similarity matrices were clustered in groups with the UPGMA tree (Figure 3b).
In China, the three DL specimens exhibited three genotypes (H6, H13, H15). The SD specimens exhibited seven genotypes, among which three genotypes (H9, H10, H14) were site-specific, accounting for 17.6% of all genotypes. The diversity of JS specimens was the most, with fourteen genotypes; eight genotypes (H1, H2, H4, H5, H7, H8, H12, H16) were site-specific (47.1%), and six genotypes (H3, H6, H11, H13, H15, H17) were shared with the north specimens (35.3%) (Figure 4a). In JS, the genotype H6 was dominant (25.8%) and was broadly distributed in different aquaculture farms in Nantong City (Figure 5). In the 17 genotypes, the most abundant haplotype H13 was shared among DL, SD and JS, accounting for 10.9% of all individuals. The JS specimens shared H6 and H15 with DL and shared H3, H11 and H17 with SD (Figure 4a). Haplotype diversity indices showed that specimens from JS had high genetic diversity (0.884), followed by SD (0.833) and LN (0.667) (Table 3). The haplotype diversity of cultivated P. yezoensis from Korea and Japan were also analyzed, indicating that the genetic diversity of specimens from China (0.905) was slightly higher than those from Korea (0.842), followed by those from Japan (only 1 haplotype in 13 specimens). The UPGMA tree showed that China had two of the same haplotypes (H3 and H11) as the Korean and Japanese species (KH1 and KH12) (Figure 3c).
The haplotype of the cox1 gene showed that the cultivated P. yezoensis from China exhibited three types (C1, C2 and C3). The results on intron structure showed that the same cox1 haplotype of P. yezoensis yielded different intron structures (Table 4). The C1 haplotype had six intron structures (C0000, C0100, C1000, C1100, C1110, C1111), C2 haplotype presented five intron structures (C0011, C0111, C1011, C1100, C1111), and C3 had only one intron structure (C1000). With the combined rnl and cox1 genes, the most variable haplotype C1 was sub-divided into eight genotypes (H1-4, H6-9), with 50% of all specimens. The haplotype of C2 was sub-divided into eight genotypes (H5, H10-14, H16-17), with 43.5% of specimens. And the haplotype C3 had only H15 genotype (n = 3) (Figure 4b).

4. Discussion

4.1. The Intraspecific Variation of Cultivated P. yezoensis with Gene Barcoding

Intraspecific studies of red algae have relied on DNA barcoding to address questions of systematics, biogeography or population genetics. Knowledge of the extent of intraspecific variation in P. yezoensis is necessary to rearrange these cultivation resources on the basis of genetic polymorphisms. In the present study, 44 specimens of cultivated P. yezoensis in China presented three haplotypes (R1C1, R2C3, R3C2) based on the cox1 and rbcL genes. The intraspecific variation of cultivated P. yezoensis was 0.31% for the cox1 gene and 0.14% for the rbcL gene. In the genus Pyropia, the cox1 intraspecific nucleotide difference was 0–1.3%, and interspecific nucleotide differences ranged from 2.6 to 17.1% [12,20,25]. For the rbcL gene, the intraspecific divergence for Pyropia species was less than 1.0%, with a mean of 5.8% for interspecific divergence [19,26,27,28]. Our results for the cox1 and rbcL divergences of cultivated P. yezoensis in China were much lower than the intraspecific divergence of Pyropia from natural resources as previously reported [12,20,26,27]. The lower genetic variation of cultivated Pyropia was primarily due to seedling differences, and selection during cultivation had reduced the effective genetic size. Inbreeding and intensive selection during cultivation, which narrow the germplasm genetic base, reduce genetic diversity and promote adaptive divergence, have been reported in many plants and algae [29,30].
A comparison of cox1 with rbcL for Pyropia species revealed it was a more sensitive marker in revealing incipient speciation and cryptic diversity [25]. In the present study, analysis of the haplotype networks indicated that the haplotype number and diversity of cultivated P. yezoensis based on cox1 and rbcL genes were the same. However, higher nucleotide diversity was obtained from cox1 (0.00173) compared to the rbcL gene (0.00045). And the maximum intraspecific variation of cox1 (0.31%) in cultivated P. yezoensis was also higher than that of the rbcL gene (0.14%). The incorporation of the rbcL gene did not enhance the intraspecific genetic variation or the haplotype number. Hence, the cox1 marker was a more suitable marker for understanding the intraspecific variation and genetic diversity of cultivated P. yezoensis. Generally, the cox1 gene has revealed greater sequence divergence than the rbcL gene for identification of closely related red algal species, where the more conserved rbcL may be uninformative [31,32]. The evolution of the cox1 gene is sufficiently rapid to allow discrimination between closely related species and biogeographic subgroups within species of red algae [33]. However, relatively higher intraspecific variation of the cox1 gene may reduce accuracy for species identification [31,34]. In the present study, we recommend that cox1 is, overall, the best potential DNA barcode for cultivated P. yezoensis, whereas the combined mitochondrial-encoded cox1 and the plastid-encoded rbcL markers serve better as DNA barcodes encompassing the entire rhodophyte taxa [32,35].

4.2. The Intraspecific Variation of Cultivated P. yezoensis with Intronic-ORF Structure

In plants, mitochondrial genomes are highly dynamic, owing to gains and losses of repetitive noncoding DNA (intergenic spaces) and genetic elements (introns and transposable elements) throughout evolution, and they vary substantially among members of different genera or among species within the same genus [36]. In red algae, introns and intronic-ORFs are largely responsible for organellar genome expansion [37], leading to variation in the size of the mitochondrial genome of Pyropia species [17,38]. Based on the presence/absence of intronic-ORFs of rnl and cox1 genes, 27 cultivated Korean P. yezoensis strains exhibited 12 genetic types (H1-H12), and 13 Japanese strains showed 1 genotype (H12) [9]. In the present study, the intron structural variations of rnl and cox1 genes exhibited 17 genotypes among 44 specimens of cultivated P. yezoensis in China. And the same cox1 haplotype of P. yezoensis (C1-C3) yielded 1–6 different intron structures. Moreover, the haplotype diversity of the intron structure (0.905) was much more variable compared to DNA barcoding (0.569), which revealed higher variation for cultivated P. yezoensis. Therefore, the intron structure of the rnl and cox1 regions exhibited higher genetic resolution to discriminate P. yezoensis strains at the intraspecific level.
In the Bangiophycean species (Pyropia, Bangia, Wildemania), the rnl and cox1 introns both contain 1–3 copies, whereas they tend to be lost in most Florideophycean taxa, indicating that the rnl and cox1 introns may originate prior to the split of Bangiophyceae and Florideophyceae [16]. The mitochondrial rnl and cox1 genes of Bangiophyceae were extraordinarily similar to those found in the cyanobacteria and fungus, suggesting a recent lateral intron transfer from cyanobacteria or alpha-proteobacteria via mitochondrial primary endosymbiosis and targeted rnl and cox1 genes in red algae [39,40]. Because the intron did not reside in a vital gene, there would have been no selective pressure to conserve the RNA secondary structure that is required for proper splicing [39]. Therefore, the evolution of intron variation is rapid enough to identify the intraspecies relationships in red algae.

4.3. The Genetic Impact of P. yezoensis under Climate Change in China

For species of red algae, intraspecific variation, which may arise through the interplay between environmental heterogeneity and adaptive variation, has consequences for the resilience of species to climate change [41]. Due to climate change, the abundance and composition of seaweed resources have changed over the past decades. Some species showed a significant extension in their distribution to the north, whereas some species decreased or even became extinct, mostly likely due to the increased seawater temperature [42]. For example, P. tenera has gradually disappeared from both natural habitats and aquaculture farms, which was previously one of the main cultivation species in Japan and Korea [43].
Climate-driven changes in habitat have an immediate effect on mariculture and the industry of seaweed. The future cultivation center of Pyropia was predicted to exhibit poleward and offshore shifts in the 2050s under climate change, especially sea surface temperature change [7]. Expansion of Pyropia farming northward and offshore is not simply a prediction, but a current cultivation practice in China. In the present study, the haplotype diversity of DNA barcoding and intronic-ORF structure of mtDNA genes both showed that the cultivated P. yezoensis in JS had higher genetic diversity than those in the north. However, based on the intronic-ORF structure, the northern specimens shared 35.3% of genotypes with JS specimens, indicating that the cultivated P. yezoensis were expanding their cultivation ranges along with north migration. Even with lower genetic diversity, the northern coast of cultivation had already developed 17.6% of site-specific specimens. Although we did not assess the direct impact of global climatic change on intraspecific genetic variation using ecological methods [44,45], the genetic diversity of cultivated P. yezoensis was affected by changing the cultivation location and seedlings. However, the seedlings for the northern coast were mainly prepared using a traditional selective breeding method by seedling producers, which partly led to instability in quantification in the northern cultivation of P. yezoensis. Based on the data from Jiangsu Nori Associate, the trading prices of northern P. yezoensis were 16.4% and 10.7% lower than those of the south in 2021 and 2022. With the migration to the north, different sea surface temperatures and solar light intensity conditions for Pyropia cultivation were present, leading to phenomena such as photoinhibition which usually occurs in SD [46]. However, there were only two certified new varieties of P. yezoensis in China (Sutong No.1 and No.2), which were more suitable to those cultivated in JS. So, more cultivars or varieties of P. yezoensis need to be developed to adapt to the cultivation environments of the northern coast.

4.4. The Genetic Diversity of P. yezoensis in Northwest Pacific

In the present study, the haplotype and nucleotide diversity index of the cox1 gene showed that the cultivated P. yezoensis in China had higher genetic diversity than in Korea and Japan. However, the haplotype and nucleotide diversity index of the rbcL gene had greater diversity in Korea, followed by China and Japan. Based on the intronic-ORF structure of the rnl and cox1 regions, the haplotype diversity of Chinese and Korean specimens had much higher genetic diversity than those from Japan, which had only one haplotype. These results indicate that cultivated P. yezoensis from China and Korea have more genetic diversity than those from Japan. The cultivation resources of P. yezoensis had the same haplotype as Korean specimens (H3), and also Japanese specimens (H11). The Korean strain (H3) was widely cultivated in SD and JS (LYG, YC, NT). And the Japanese strain (H11), P. yezoensis f. narawaensis, was also cultivated in SD and the north of JS (LYG). These results show that the cultivation resource in China is genetically diverse. In China, the fisherman bought conchocelis germplasm of P. yezoensis from different seedling producers, including some imported from Korea and Japan. Niwa et al. (2008, 2009) reported the presence of three genotypes in 13 Japanese aquaculture strains of P. yezoensis, 11 of which strains had one genotype in common with P. yezoensis f. narawaensis [10,47]. Moreover, the diversity of chloroplast and mitochondrial genomes of P. yezoensis was lower in Japan than in China [48]. However, Japan had more registered cultivars of P. yezoensis than Korea and China. A total of 13 cultivars of P. yezoensis have been registered in Japan, and many cultivars are managed by seedling producers, which have already been in application [49]. Most of the Japanese cultivars were P. yezoensis f. narawaensis, which was selected in the 1960s and then subjected to selective breeding and provided large-sized, high-yielding cultivars for Japanese Pyropia aquaculture [49]. This explains why cultivated P. yezoensis in Japan had the lowest genetic diversity. Although the expansion of geographical sampling significantly increased intraspecific variation and genetic impact, the data from the present study offer a preliminary framework for future strain breeding of Pyropia in the Northwest Pacific.
However, the molecular markers for genetic diversity and genetic structure inferences still have limitations. Rapid progress in high-throughput sequencing technologies has provided an opportunity to infer genome-wide information, which provides thousands of genetic markers. Therefore, the detection of genome-wide markers needs to be analyzed, which can contribute to more targeted breeding efforts for Pyropia cultivation under climate change.

Author Contributions

Conceptualization, Y.D.; methodology, G.H. and L.W.; validation, G.X.; formal analysis, C.H.; data curation, G.H. and C.T.; writing—original draft preparation, Y.D.; writing—review and editing, Y.D. and W.Z.; funding acquisition, Y.D. and C.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Jiangsu Seed Industry Revitalization Project [grant number JBGS [2021]033], the National Natural Science Foundation of China [grant number 42206134], the National Key R&D Program of China [grant number 2023YFD2400102], and the Nantong Science and Technology Projects [grant number JC2023030, JC12022085].

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

We are grateful to Jiangsu Nori Associate for the support valuable data on Pyropia cultivation. We also would like to thank the editors of JMSE and the anonymous reviewers for their feedback on this submission, which have helped to improve the quality of this manuscript.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Sutherland, J.E.; Lindstrom, S.C.; Nelson, W.A.; Brodie, J.; Lynch, M.D.; Hwang, M.S.; Choi, H.G.; Miyata, M.; Kikuchi, N.; Oliveira, M.C.; et al. A new look at an ancient order: Generic revision of the Bangiales (Rhodophyta). J. Phycol. 2011, 47, 1131–1151. [Google Scholar] [CrossRef] [PubMed]
  2. Yang, L.-E.; Deng, Y.-Y.; Xu, G.-P.; Russell, S.; Lu, Q.-Q.; Brodie, J. Redefining Pyropia (Bangiales, Rhodophyta): Four new genera, resurrection of Porphyrella and description of Calidia pseudolobata sp. nov. from China. J. Phycol. 2020, 56, 862–879. [Google Scholar] [CrossRef]
  3. Zuccarello, G.C.; Wen, X.; Kim, G.H. Splitting blades: Why genera need to be more carefully defined; the case for Pyropia (Bangiales, Rhodophyta). Algae 2022, 37, 205–211. [Google Scholar] [CrossRef]
  4. San, M.H.; Kawamura, Y.; Kimura, K.; Witharana, E.P.; Shimogiri, T.; Aye, S.S.; Min, T.T.; Aung, C.; Khaing, M.M.; Nagano, Y. Characterization and organelle genome sequencing of Pyropia species from Myanmar. Sci. Rep. 2023, 13, 15677. [Google Scholar] [CrossRef] [PubMed]
  5. Cho, T.J.; Rhee, M.S. Health functionality and quality control of laver (Porphyra, Pyropia): Current issues and future perspectives as an edible seaweed. Mar. Drugs 2019, 18, 14. [Google Scholar] [CrossRef] [PubMed]
  6. Yang, L.; Lu, Q.; Brodie, J. A review of the bladed Bangiales (Rhodophyta) in China: History, culture and taxonomy. Euro J. Phycol. 2017, 52, 251–263. [Google Scholar] [CrossRef]
  7. Zhou, W.; Li, B.; Xu, H.; Liang, Z.; Lu, X.; Yang, L.; Wang, W. Potential distribution of two economic laver species-Neoporphyra haitanensis and Neopyropia yezoensis under climate change based on MaxEnt prediction and phylogeographic profiling. Ecol. Indic. 2023, 150, 110219. [Google Scholar] [CrossRef]
  8. Kim, M.; Wi, J.; Lee, J.; Cho, W.-B.; Park, E.-J.; Hwang, M.-S.; Choi, S.-J.; Jeong, W.-J.; Kim, G.H.; Choi, D.-W. Development of genomic simple sequence repeat (SSR) markers of Pyropia yezoensis (Bangiales, Rhodophyta) and evaluation of genetic diversity of Korean cultivars. J. Appl. Phycol. 2021, 33, 3277–3285. [Google Scholar] [CrossRef]
  9. Hwang, I.K.; Kim, S.-O.; Hwang, M.S.; Park, E.-J.; Ha, D.-S.; Lee, S.-R. Intraspecific variation of gene structure in the mitochondrial large subunit ribosomal RNA and cytochrome c oxidase subunit 1 of Pyropia yezoensis (Bangiales, Rhodophyta). Algae 2018, 33, 49–54. [Google Scholar] [CrossRef]
  10. Niwa, K.; Iida, S.; Kato, A.; Kawai, H.; Kikuchi, N.; Kobiyama, A.; Aruga, Y. Genetic diversity and introgression in two cultivated Species (Porphyra yezoensis and Porphyra Tenera) and closely related wild species of Porphyra (Bangiales, Rhodophyta). J. Phycol. 2009, 45, 493–502. [Google Scholar] [CrossRef]
  11. Park, E.-J.; Fukuda, S.; Endo, H.; Kitade, Y.; Saga, N. Genetic polymorphism within Porphyra yezoensis (Bangiales, Rhodophyta) and related species from Japan and Korea detected by cleaved amplified polymorphic sequence analysis. Eur. J. Phycol. 2007, 42, 29–40. [Google Scholar] [CrossRef]
  12. Koh, Y.H.; Kim, M.S. DNA barcoding reveals cryptic diversity of economic red algae, Pyropia (Bangiales, Rhodophyta): Description of novel species from Korea. J. Appl. Phycol. 2018, 30, 3425–3434. [Google Scholar] [CrossRef]
  13. Koh, Y.H.; Kim, M.S. Genetic diversity and distribution pattern of economic seaweeds Pyropia yezoensis and Py. suborbiculata (Bangiales, Rhodophyta) in the northwest Pacific. J. Appl. Phycol. 2020, 32, 2495–2504. [Google Scholar] [CrossRef]
  14. Milstein, D.; Medeiros, A.S.; Oliveira, E.C.; Oliveira, M.C. Native or introduced? A re-evaluation of Pyropia species (Bangiales, Rhodophyta) from Brazil based on molecular analyses. Euro J. Phycol. 2015, 50, 37–45. [Google Scholar] [CrossRef]
  15. Yow, Y.-Y.; Lim, P.-E.; Phang, S.-M. Assessing the use of mitochondrial cox1 gene and cox2-3 spacer for genetic diversity study of Malaysian Gracilaria changii (Gracilariaceae, Rhodophyta) from Peninsular Malaysia. J. Appl. Phycol. 2012, 25, 831–838. [Google Scholar] [CrossRef]
  16. Yang, E.C.; Kim, K.M.; Kim, S.Y.; Lee, J.; Boo, G.H.; Lee, J.H.; Nelson, W.A.; Yi, G.; Schmidt, W.E.; Fredericq, S.; et al. Highly conserved mitochondrial genomes among multicellular red algae of the Florideophyceae. Genome Biol. Evol. 2015, 7, 2394–2406. [Google Scholar] [CrossRef]
  17. Hwang, M.S.; Kim, S.-O.; Ha, D.-S.; Lee, J.E.; Lee, S.-R. Complete mitochondrial genome sequence of Pyropia yezoensis (Bangiales, Rhodophyta) from Korea. Plant Biotechnol. Rep. 2014, 8, 221–227. [Google Scholar] [CrossRef]
  18. Hughey, J.R.; Gabrielson, P.W.; Rohmer, L.; Tortolani, J.; Silva, M.; Miller, K.A.; Young, J.D.; Martell, C.; Ruediger, E. Minimally destructive sampling of type specimens of Pyropia (Bangiales, Rhodophyta) recovers complete plastid and mitochondrial genomes. Sci. Rep. 2014, 4, 5113. [Google Scholar] [CrossRef]
  19. Broom, J.E.S.; Nelson, W.A.; Farr, T.J.; Phillips, L.E.; Clayton, M. Relationships of the Porphyra (Bangiales, Rhodophyta) flora of the Falkland Island a molecular survey using rbcL and nSSU sequence data. Aust. Syst. Bot. 2010, 23, 27–37. [Google Scholar] [CrossRef]
  20. Saunders, G.W. Applying DNA barcoding to red macroalgae: A preliminary appraisal holds promise for future applications. Philos. Trans. R. Soc. Lond. B 2005, 360, 1879–1888. [Google Scholar] [CrossRef]
  21. Deng, Y.; Lu, Q.; Shen, S.; Shen, Z.; Tian, C.; Zhu, J. Cytological observations and rbcL, nrSSU gene sequence analyses of filamentous Bangiales (Rhodophyta) from China. J. Appl. Phycol. 2014, 27, 1355–1363. [Google Scholar] [CrossRef]
  22. Tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.; Nei, M.; Kumar, S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 2011, 28, 2731–2739. [Google Scholar] [CrossRef]
  23. Rozas, J.; Ferrer-Mata, A.; Sánchez-DelBarrio, J.C.; Guirao-Rico, S.; Librado, P.; Ramos-Onsins, S.E.; Sánchez-Gracia, A. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol. Biol. Evol. 2017, 34, 3299–3302. [Google Scholar] [CrossRef] [PubMed]
  24. Leigh, J.W.; Bryant, D.; Nakagawa, S. POPART: Full-feature software for haplotype network construction. Methods Ecol. Evol. 2015, 6, 1110–1116. [Google Scholar] [CrossRef]
  25. Robba, L.; Russell, S.J.; Barker, G.L.; Brodie, J. Assessing the use of the mitochondrial cox1 marker for use in DNA barcoding of red algae (Rhodophyta). Am. J. Bot. 2006, 93, 1101–1108. [Google Scholar] [CrossRef] [PubMed]
  26. Guillemin, M.L.; Contreras-Porcia, L.; Ramirez, M.E.; Macaya, E.C.; Contador, C.B.; Woods, H.; Wyatt, C.; Brodie, J. The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity. Mol. Phylogenetics Evol. 2016, 94, 814–826. [Google Scholar] [CrossRef] [PubMed]
  27. Lindstrom, S.C.; Fredericq, S. rbcL gene sequences reveal relationships among north-east Pacific species of Porphyra (Bangiales, Rhodophyta) and a new species, P. aestivalis. Phycol. Res. 2003, 51, 211–224. [Google Scholar] [CrossRef]
  28. Kucera, H.; Saunders, G.W. A survey of Bangiales (Rhodophyta) based on multiple molecular markers reveals cryptic diversity. J. Phycol. 2012, 48, 869–882. [Google Scholar] [CrossRef]
  29. Doebley, J.F.; Gaut, B.S.; Smith, B.D. The molecular genetics of crop domestication. Cell 2006, 127, 1309–1321. [Google Scholar] [CrossRef]
  30. Voisin, M.; Engel, C.R.; Viard, F. Differential shuffling of native genetic diversity across introduced regions in a brown alga: Aquaculture vs. maritime traffic effects. Proc. Natl. Acad. Sci. USA 2005, 102, 5432–5437. [Google Scholar] [CrossRef]
  31. Yang, E.C.; Kim, M.S.; Geraldino, P.J.L.; Sahoo, D.; Shin, J.-A.; Boo, S.M. Mitochondrial cox1 and plastid rbcL genes of Gracilaria vermiculophylla (Gracilariaceae, Rhodophyta). J. Appl. Phycol. 2008, 20, 161–168. [Google Scholar] [CrossRef]
  32. Yang, M.Y.; Kim, M.S. Phylogeography of the economic seaweeds Chondrus (Gigartinales, Rhodophyta) in the northwest Pacific based on rbcL and COI-5P genes. Algae 2022, 37, 135–147. [Google Scholar] [CrossRef]
  33. Yang, M.Y.; Geraldino, P.J.L.; Kim, M.S. DNA barcode assessment of Gracilaria salicornia (Gracilariaceae, Rhodophyta) from Southeast Asia. Bot. Stud. 2013, 54, 27. [Google Scholar] [CrossRef]
  34. Geraldino, P.J.L.; Yang, E.C.; Kim, M.S.; Boo, S.M. Systematics of Hypnea asiatica sp. nov. (Hypneaceae, Rhodophyta) based on morphology and nrDNA SSU, plastid rbcL, and mitochondrial cox1. Taxonomy 2009, 58, 606–616. [Google Scholar] [CrossRef]
  35. Tan, J.; Lim, P.-E.; Phang, S.-M.; Hong, D.D.; Sunarpi, H.; Hurtado, A.Q. Assessment of four molecular markers as potential DNA barcodes for red algae Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae, Rhodophyta). PLoS ONE 2012, 7, e52905. [Google Scholar] [CrossRef] [PubMed]
  36. Mukhopadhyay, J.; Hausner, G. Organellar introns in fungi, algae, and plants. Cells 2021, 10, 2001. [Google Scholar] [CrossRef]
  37. van Beveren, F.; Eme, L.; López-García, P.; Ciobanu, M.; Moreira, D.; Sloan, D. Independent size expansions and intron proliferation in red algal plastid and mitochondrial genomes. Genome Biol. Evol. 2022, 14, evac037. [Google Scholar] [CrossRef]
  38. Hwang, M.S.; Kim, S.-O.; Ha, D.-S.; Lee, J.E.; Lee, S.-R. Complete sequence and genetic features of the mitochondrial genome of Pyropia tenera (Rhodophyta). Plant Biotechnol. Rep. 2013, 7, 435–443. [Google Scholar] [CrossRef]
  39. Burger, G.; Saint-Louis, D.; Gray, M.W.; Lang, B.F. Complete sequence of the mitochondrial DNA of the red alga Porphyra purpurea: Cyanobacterial introns and shared ancestry of red and green algae. Plant Cell 1999, 11, 1675–1694. [Google Scholar] [CrossRef]
  40. Kim, D.; Lee, J.; Cho, C.H.; Kim, E.J.; Bhattacharya, D.; Yoon, H.S. Group II intron and repeat-rich red algal mitochondrial genomes demonstrate the dynamic recent history of autocatalytic RNAs. BMC Biol. 2022, 20, 2. [Google Scholar] [CrossRef]
  41. Li, J.J.; Liu, Z.Y.; Song, W.H.; Qin, S. The contribution of intraspecific variation to future climate responses of brown algae. Limnol. Oceanogr. 2023, 69, 53–66. [Google Scholar] [CrossRef]
  42. Hwang, E.K.; Park, C.S. Seaweed cultivation and utilization of Korea. Algae 2020, 35, 107–121. [Google Scholar] [CrossRef]
  43. Kim, H.S.; Choi, H.G.; Hwang, M.S.; Jeon, Y.J.; Yarish, C.; Kim, J.K. Concise review of the genus Neopyropia (Rhodophyta: Bangiales). J. Appl. Phycol. 2022, 34, 1805–1824. [Google Scholar] [CrossRef]
  44. Pauls, S.U.; Nowak, C.; Bálint, M.; Pfenninger, M. The impact of global climate change on genetic diversity within populations and species. Mol. Ecol. 2013, 22, 925–946. [Google Scholar] [CrossRef] [PubMed]
  45. Yannic, G.; Pellissier, L.; Ortego, J.; Lecomte, N.; Couturier, S.; Cuyler, C.; Dussault, C.; Hundertmark, K.J.; Irvine, R.J.; Jenkins, D.A.; et al. Genetic diversity in caribou linked to past and future climate change. Nat. Clim. Chang. 2014, 4, 132–137. [Google Scholar] [CrossRef]
  46. Feng, Z.; Wu, L.; Sun, Z.; Yang, J.; Liu, G.; Niu, J.; Wang, G. Control of reactive oxygen species through antioxidant enzymes plays a pivotal role during the cultivation of Neopyropia yezoensis. J. Mar. Sci. Eng. 2022, 10, 109. [Google Scholar] [CrossRef]
  47. Niwa, K.; Kato, A.; Kobiyama, A.; Kawai, H.; Aruga, Y. Comparative study of wild and cultivated Porphyra yezoensis (Bangiales, Rhodophyta) based on molecular and morphological data. J. Appl. Phycol. 2008, 20, 261–270. [Google Scholar] [CrossRef]
  48. Nagano, Y.; Kimura, K.; Kobayashi, G.; Kawamura, Y. Genomic diversity of 39 samples of Pyropia species grown in Japan. PLoS ONE 2021, 16, e0252207. [Google Scholar] [CrossRef]
  49. Hwang, E.K.; Yotsukura, N.; Pang, S.J.; Su, L.; Shan, T.F. Seaweed breeding programs and progress in eastern Asian countries. Phycologia 2019, 58, 484–495. [Google Scholar] [CrossRef]
Jmse 12 01551 g001
Figure 1. The cultivation area (a) and value (b) of P. yezoensis in China. The data were obtained from Jiangsu Nori Associate.
Figure 1. The cultivation area (a) and value (b) of P. yezoensis in China. The data were obtained from Jiangsu Nori Associate.
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Figure 2. Haplotype network of cultivated P. yezoensis with cox1 gene (a) and rbcL gene (b).
Figure 2. Haplotype network of cultivated P. yezoensis with cox1 gene (a) and rbcL gene (b).
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Figure 3. The UPGMA trees of cultivated P. yezoensis based on intronic-ORF structures. (a) The gene structure of exon (e), intron (i) and intronic-ORF (orf) in rnl and cox1 genes. (b) The UPGMA tree constructed with 17 haplotypes (H1-17) in the present study. (c) The UPGMA tree constructed with 17 haplotypes in the present study and 12 Korean and Japanese haplotypes (KH1-12).
Figure 3. The UPGMA trees of cultivated P. yezoensis based on intronic-ORF structures. (a) The gene structure of exon (e), intron (i) and intronic-ORF (orf) in rnl and cox1 genes. (b) The UPGMA tree constructed with 17 haplotypes (H1-17) in the present study. (c) The UPGMA tree constructed with 17 haplotypes in the present study and 12 Korean and Japanese haplotypes (KH1-12).
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Figure 4. The haplotype network of cultivated P. yezoensis with intronic-ORF structures of rnl and cox1 genes. (a) The haplotype network constructed with 17 haplotypes based on intronic-ORF structures according to different cultivation locations (LN, SD and JS). (b) The haplotype network constructed with 17 haplotypes based on intronic-ORF structures according to different cox1 gene haplotypes (C1, C2 and C3).
Figure 4. The haplotype network of cultivated P. yezoensis with intronic-ORF structures of rnl and cox1 genes. (a) The haplotype network constructed with 17 haplotypes based on intronic-ORF structures according to different cultivation locations (LN, SD and JS). (b) The haplotype network constructed with 17 haplotypes based on intronic-ORF structures according to different cox1 gene haplotypes (C1, C2 and C3).
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Figure 5. Cultivated P. yezoensis with different haplotypes in China. The red squares indicate the 17 aquaculture farms of P. yezoensis. The circles indicate three haplotypes based the DNA barcoding. The following squares indicate 17 haplotypes based on the intronic-ORF structure of rnl and cox1 regions.
Figure 5. Cultivated P. yezoensis with different haplotypes in China. The red squares indicate the 17 aquaculture farms of P. yezoensis. The circles indicate three haplotypes based the DNA barcoding. The following squares indicate 17 haplotypes based on the intronic-ORF structure of rnl and cox1 regions.
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Table 1. Diversity indices of cultivated Pyropia yezoensis inferred from mitochondrial cox1 and plastid rbcL genes.
Table 1. Diversity indices of cultivated Pyropia yezoensis inferred from mitochondrial cox1 and plastid rbcL genes.
LocationHaplotypeNhNpHaplotype DiversityNucleotide DiversityReferences
cox1
ChinaNorth (14)C1/2/3330.4730.00144This study
JS (31)C1/2/3330.5120.00156
All (45)C1/2/3330.5690.00173
Korea27C01/02/03/04460.4390.00165Hwang et al., 2018 [9]
Japan13C2----
rbcL
ChinaNorth (14)R1/2/3320.4730.00042This study
JS (31)R1/2/3320.5120.00040
All (45)R1/2/3320.5690.00045
Korea8R1/2/3320.6070.00049GenBank
Japan7R2/3210.4760.00034
Nh: Number of haplotypes. Np: Number of polymorphic sites. The rbcL sequences of Korean specimens were AB818919, DQ227860, DQ227861, DQ227862, DQ227863, DQ227864, DQ227865, DQ227866. The rbcL sequences of Japanese specimens were AB243204, AB243205, AB118587, AB118588, AB118589, AB118590, AB818917.
Table 2. The variation sites of cox1 and rbcL genes in different haplotypes of P. yezoensis.
Table 2. The variation sites of cox1 and rbcL genes in different haplotypes of P. yezoensis.
GeneHaplotypesVariation Sites
cox1KF561997TTT258CCT309AGT435GCT462ACG522TTC603GGC702
ChinaC1 AGC GGT
C2 AGC TTT
C3
KoreaC01 AGC
C02TTCCCCAGC ACT
C03
C04 AGCGCC TTT
Amino acid *PheValSerAlaThrPheGly
rbcL GGT900TTC1335
R1GGC
R2
R3 TTT
Amino acidGlyPhe
* Mitochondrial code was used with transl table = 4.
Table 3. Diversity indices of cultivated Pyropia yezoensis inferred from the intron structures of rnl and cox1 regions.
Table 3. Diversity indices of cultivated Pyropia yezoensis inferred from the intron structures of rnl and cox1 regions.
LocationHaplotypeNhSite-Specific NhHaplotype DiversityReferences
ChinaLN (3)H6, 13, 15300.667This study
SD (12)H3, 9, 10, 11, 13, 14, 17730.833
JS (31)H1-8 11-13 15-171480.884
All (46)H1-1717-0.905
Korea27H1-1212-0.842Hwang et al., 2018 [9]
Japan13H121--
Table 4. Details of the specimens analyzed in this study.
Table 4. Details of the specimens analyzed in this study.
CodeCollection LocationDataBarcode HaplotypeIntron Haplotype
L11Dalian, LN31 December 2021R3C2R101C1111H13
L12R2C3R111C1000H15
L13R1C1R011C1100H6
S11Wendeng, SD22 January 2022R1C1
R3C2		R011C0100
R111C0111		H3
H14
S12R3C2R101C0011H10
S13R3C2R101C0011H10
S14R3C2R101C0111H11
S21Rongcheng, SD9 December 2020R3C2R101C1111H13
S22R3C2R111C1111 R101C1111 H17
H13
S3Rushan, SD8 December 2020R3C2R111C1111H17
S4Jimo, Qingdao, SD10 December 2020R3C2R111C1111H17
S51Laoshan, Qingdao, SD13 January 2021R3C2R111C0111H14
S52R1C1R101C0000H9
J11Ganyu, Lianyungang, JS7 December 2020R3C2R111C1111H17
J1216 November 2020R3C2R101C0111H11
J1316 November 2020R3C2R011C1011H5
J1416 November 2020R2C3R111C1000H15
J21Liandao, Lianyungang, JS16 November 2020R3C2R101C1011H12
J2223 December 2021R3C2R101C1111H13
J31Gaogong Island, Lianyungang, JS26 January 2021R3C2R111C1100H16
J3223 December 2021R3C2R111C1111H17
J3323 December 2021R3C2R111C1111H17
J4Haibing Avenue, Lianyungang, JS18 March 2021R1C1R011C0100H3
J5Dafeng-1, Yancheng, JS27 February 2019R3C2R101C1111H13
J6Dafeng-2, Yancheng, JS8 March 2022R1C1R011C0100H3
J71Rudong-1, Nantong, JS8 January 2022R1C1R011C0100H3
J72R1C1R001C0100H1
J73R1C1R011C1111H8
J81Rudong-2, Nantong, JS20 February 2022R1C1R011C1110H7
J8220 January 2022R1C1R011C1111H8
J91Rudong-3, Nantong, JS20 January 2022R1C1R011C1100H6
J9221 February 2022R1C1R011C1110H7
J10-1Haimen, Nantong, JS10 March 2020R2C3R111C1000H15
J10-216 January 2020R1C1R011C1100H6
J10-316 January 2020R1C1R011C1100H6
J10-416 January 2020R1C1R011C1100H6
J10-519 March 2020R1C1R011C1100H6
J10-627 February 2018R1C1R011C1100H6
J11-1Qidong, Nantong, JS15 March 2021R1C1R011C1100H6
J11-2R1C1R011C1000H4
J11-3R1C1R011C1000H4
J11-4R1C1R011C0000H2
J11-5R1C1R011C1100H6
J11-6R1C1R011C0000H2
The barcode haplotype was represented as a combination of rbcL gene (R) and cox1 gene (C). The haplotypes of intron structural variation were represented as a code (R = rnl, C = cox1, 0 = intron absent, 1 = intron present).
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Huang, G.; Tian, C.; Wei, L.; Hu, C.; Xu, G.; Zhou, W.; Deng, Y. DNA Barcoding and Intronic-ORF Structure Analyses of Cultivated Pyropia yezoensis in China: The Genetic Impact under Climate Change. J. Mar. Sci. Eng. 2024, 12, 1551. https://doi.org/10.3390/jmse12091551

AMA Style

Huang G, Tian C, Wei L, Hu C, Xu G, Zhou W, Deng Y. DNA Barcoding and Intronic-ORF Structure Analyses of Cultivated Pyropia yezoensis in China: The Genetic Impact under Climate Change. Journal of Marine Science and Engineering. 2024; 12(9):1551. https://doi.org/10.3390/jmse12091551

Chicago/Turabian Style

Huang, Guihua, Cuicui Tian, Liangdi Wei, Chuanming Hu, Guangping Xu, Wei Zhou, and Yinyin Deng. 2024. "DNA Barcoding and Intronic-ORF Structure Analyses of Cultivated Pyropia yezoensis in China: The Genetic Impact under Climate Change" Journal of Marine Science and Engineering 12, no. 9: 1551. https://doi.org/10.3390/jmse12091551

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