An Efficient PCR-RFLP Method for the Rapid Identification of Korean Pyropia Species

Abstract

The present study utilizes polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using partial plastid rbcL and mitochondrial trnC–trnP gene sequences to distinguish the six representative Pyropia species produced via mariculture in Korea. The rbcL, trnC, and trnP sequences of 15 Pyropia species from the NCBI database were aligned to determine specific restriction enzyme sites of the six Pyropia species. To confirm the presence of restriction sites of eight enzymes, PCR amplicons were digested as follows: a 556 bp fragment within the rbcL region of chloroplast DNA was confirmed in P. yezoensis using BglI, whereas Tth111I, AvaII, BsrI, and BsaAI enzymes produced fragments of 664, 271, 600, and 510 bp, respectively, from the rps11–trnG region of mitochondrial DNA in P. seriata, P. dentata, P. suborbiculata, and P. haitanensis. In the case of P. pseudolinearis, HindIII, SacII, and SphI enzymes each had two cleavage sites, at positions 174 and 825, 788 and 211, and 397 and 602 bp, respectively. All six species were successfully distinguished using these eight restriction enzymes. Therefore, we propose that PCR-RFLP analysis is an efficient tool for the potential use of distinguishing between the six Pyropia species cultivated via mariculture in Korea.

1. Introduction

Red algae (Bangiales, Rhodophyta) of the genus Pyropia, including more than 75 species, are economically important mariculture crops and are available in China, Japan, and South Korea [1]. This genus is widely used as a dried sheet product, “zicai” in China, “nori” in Japan, and “gim” in South Korea, and is commonly consumed as a food source (laver) in Asian countries [2]. Pyropia is becoming increasingly popular worldwide. The economic value of Pyropia products exported in 2016 from South Korea was 353.3 million in US dollars [3,4]. Twelve species within the genus Pyropia have been recorded in South Korea [5]. Among them, four species, P. yezoensis, P. seriata, P. dentata, and P. suborbiculata, are widely distributed and extensively cultured in the South Jeolla Province, whereas P. pseudolinearis is distributed in Ulreung Island and its export accounts for about 80% of the total production of Pyropia in South Korea [3]. P. haitanensis is widely distributed and extensively cultured in the Fujian, Zhejiang, and Guangdong Provinces of China, and its export accounts for about 75% of the total production of Pyropia in China [6]. Recently, P. haitanensis has been utilized in Korean mariculture industries to develop improved cultivars that are optimized for the warm temperature zones typically used in Pyropia breeding.

The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method is based on the digestion of PCR amplicons with appropriate restriction enzymes to produce distinct polymorphic fragments used as markers for species identification [7,8]. This method is beginning to be used for identifying different Pyropia species or breeding cultivars that could presumably be applied for mariculture in warming ocean waters. Japanese researchers have revealed clear differences between native Pyropia species based on PCR-RFLP analysis of plastid, internal transcribed spacer (ITS), and mitochondrial DNA (mtDNA) [9,10,11]. Chinese and Korean researchers have mainly used this method to identify and discriminate among seaweed diseases and pest species [12,13]. In South Korea, Pyropia species are identified using morphological and anatomical features. However, morphological identification of Pyropia taxa is limited because of the temporal variation in morphological characteristics and surface contamination from symbiotic seaweed and bacteria. Therefore, a better method is required for efficient and rapid identification to distinguish among these Pyropia species in natural environments.

In this study, we utilized PCR-RFLP analysis to successfully develop species-specific markers for five Pyropia species collected around the south-west coastal regions of South Korea, and P. haitanenesis collected from sites in China.

2. Results and Discussion

Currently, identification and discrimination between Pyropia species is primarily conducted based on classical taxonomic methods using microscopic analysis, which is limited by a lack of sufficient morphological features, time constraints, and inconsistencies between different taxonomists in South Korea [5]. Many phylogenetic analyses in other families have been based on the trnC–trnP region of mtDNA and the plastid rbcL intergenic spacer, as these two regions have high rates of variation between species [14,15]. Based on the partial sequences of rbcL and trnC–trnP for 15 Pyropia species available in the GenBank public database (National Center for Biotechnology Information (NCBI), Rockville, MD, USA), we found that six restriction enzymes were predicted to show species-specific RFLP patterns and could be used to identify the six Pyropia species using rbcL in P. yezoensis and rps11–sdh3 in P. seriata, P. pseudolinearis, P. dentate, P. suborbiculata, and P. haitanensis. As shown in Figure 1, the predicted fragment sizes for the six Pyropia species were as follows: BglI produced a 556 bp fragment from rbcL of chloroplast DNA in P. yezoensis, whereas Tth111I produced a 664 bp fragment from P. seriata, AvaII produced a 271 bp fragment from P. dentata, BsrI produced a 600 bp fragment from P. suborbiculata, and BsaAI produced a 510 bp fragment from P. haitanensis in the rps11–trnG region of mtDNA. In the case of P. pseudolinearis, HindIII, SacI, and SphI produced one cleavage site in rps11–trnG at positions 174, 788 and 397 bp, respectively.

The consistency of the presence of each enzyme restriction site was confirmed by analyzing PCR-RFLPs of 11 Pyropia samples collected at coastal areas in Korea and dried laver production in China. The samples were analyzed with the above restriction enzymes. Figure 2 illustrates the band sizes obtained by digestion using BglI for rbcL and Tth111I, AvaII, BsaAI, BsrI, HindIII, SacI, and SphI for rps11–trnG. BglI produced a 556 bp fragment in the P. yezoensis samples and was used for their identification (Figure 2A). The following enzymes were used to identify P. seriata, P. dentata, and P. haitanensis: Tth111I produced two fragments (664 bp and 333 bp) in P. seriata (Figure 2B), AvaII produced two fragments (271 bp and 732 bp) in P. dentata (Figure 2C), and BsaAI produced one fragment (510 bp) in P. haitanensis (Figure 2E). BsrI was used to identify P. suborbiculata samples and produced two fragments (600 bp and 401 bp) in this species, whereas BsrI produced two fragments (119 bp and 844 bp) in both P. seriata and P. dentata (Figure 2D). Among four Pyropia samples that were origianlly identified as P. dentata using morphological and anatomical features, two samples were confirmed as P. haitanensis using our PCR-RFLP method (Figure 2E). We confirmed the ability of this PCR-RFLP assay to differentiate between P. dentata and P. haitanensis. Furthermore, three enzymes of HindIII, SacII, and SphI were used to identify P. pseudolinearis samples and produced two fragments of the 174 and 825 bp, 788 and 211 bp, and 397 and 602 bp, respectively (Figure 2F–H,

Table 1. The fragment size (bps) for restriction digests of partial rbcL and rps11–trnG region in six Pyropia species.

	Restriction Enzymes
Species	BglI	Tth111I	AvaII	BsrI	BsaAI	HindIII	SacII	SphI
P. yezoensis	556	-	-	-	-	-	-	-
P. seriata	-	664 333	-	119 844	-	-	-	-
P. dentata	-	-	271 732	119 844	-	-	-	-
P. suborbiculata	-	-	-	600 401	-	-	-	-
P. haitanenesis	-	-	-	-	510	-	-	-
P. pseudolinearis	-	-	-	-	-	174 825	788 211	397 602

).

The different restriction fragment patterns obtained for each species were analyzed to develop a discrimination method using PCR-RFLP analysis without the need for sequencing. Futhermore, our PCR-RFLP method showed that a higher sensitiviy and specificity to six Pyropia species found in Korean mariculture industires can be determined within a short time (approximately 2.5 h) compared to traditional morphological techiniques.

Most studies on PCR-RFLP analysis of Pyropia species have been conducted in Japan. Touhata et al. [16] demonstrated that the dried Pyropia products produed in China, Japan, and South Korea could be identified using PCR-RFLP analysis of the ITS-1 region. Abe et al. [9] distinguished 18 Pyropia species from Japan using restriction enzymes on mtDNA related to the atp6 gene and partial mtDNA including the trnC, rps11, sdh3, trnG, trnN, trnP, and rns genes. However, these methods are hampered by the need for interpretation of complex RFLP patterns as well as the inter-laboratory differences and are relatively difficult to apply in the field.

The six species used in this study were successfully distinguished using a combination of eight restriction enzymes (BglI, Tth111I, AvaII, BsrI, BsaAI, HindIII, SacII, and SphI ) and two plastid and mitochodrial primer sets. These primer sets were designed based on the partial plastid and mtDNA sequences of 15 Pyropia species. As our study used only one or two samples per species, further studies should use a larger sample size as well as refined Pyropia markers based on a comparison with East Asian Pyropia species.

3. Materials and Methods

3.1. Sequence Analysis

To rapidly identify the six Pyropia species, a PCR-RFLP method was designed base on the rbcL and trnC–trnP gene sequences from all the publicly available Pyropia species in the NCBI GenBank database (

Table 2. DNA sequences of 15 Pyropia species used for RFLP analysis.

No.	Species	Source	Accession Number
			rbcL	trnC–trnP
1	P. dentata	National Fisheries University, Shimonoseki, Japan	AB118579	AB525393
2	P. yezoensis	National Research Institute of Fisheries Science, Yokohama, Japan	AB818918
		National Fisheries University, Shimonoseki, Japan		AB525218
3	P. tenera	Fisheries Technology Institute, Hyogo Prefectural Technology Center for Agriculture, Japan	AB366148
		National Fisheries University, Shimonoseki, Japan		AB525217
4	P. seriata	Department of Biochemistry, University of Otago, New Zealand	HQ687533
		National Fisheries University, Shimonoseki, Japan		AB525391
5	P. haitanensis	National Fisheries University, Shimonoseki, Japan	AB118585	AB525402
6	P. pseudolinearis	National Fisheries University, Shimonoseki, Japan	AB118581	AB525220
7	P. katadae	Department of Biology and Food Engineering, Changshu Institute of Technology, China	GQ427228
		National Fisheries University, Shimonoseki, Japan		AB525395
8	P. suborbiculata	National Fisheries Research Institute, Nagasaki, Japan	LC277175
		National Fisheries University, Shimonoseki, Japan		AB525392
9	P. koreana	Environmental Sciences, Botany, University of Girona, Girona, Spain	KC347610
10	P. vietnamesis	Department of Biochemistry, University of Otago, New Zealand	HQ687544
		National Fisheries University, Shimonoseki, Japan		AB525403
11	P. kurogii	Department of Biochemistry, University of Otago, New Zealand	HQ687526
		National Fisheries University, Shimonoseki, Japan		AB525398
12	P. lacerata	Department of Biochemistry, University of Otago, New Zealand	HQ687527
		National Fisheries University, Shimonoseki, Japan		AB525394
13	P. yamadae	National Fisheries Research Institute, Nagasaki, Japan	LC277182
		National Fisheries University, Shimonoseki, Japan		AB525401
14	P. ishigecola	Department of Biology and Food Engineering, Changshu Institute of Technology, China	GQ427225
		National Fisheries University, Shimonoseki, Japan		AB525397
15	P. tanegashimensis	National Fisheries Research Institute, Nagasaki, Japan	AB671541
		National Fisheries University, Shimonoseki, Japan		AB525400

). These sequences were aligned by multiple sequence alignment (MSA) (Clustal Omega tool, EMBL-EBI, www.ebi.ac.uk) [17] and MEGA version 7.0 [18]. Scanning for the various restriction enzyme sites in the reference sequences was carried out using Restriction Mapper, version 3 (http://restrictionmapper.org). Restriction enzymes were selected based on the potential of showing variation between the reference sequences.

3.2. Sample Collection and DNA Isolation

Table 3. Pyropia samples used in this study.

No.	Scientific Name	Common Name	Collection Site	Location
1	P. yezoensis	Bangsamunuigim	Songji-myeon, Haenam-gun, Jeollanam-do	34°21′05.92′′ N 126°27′40.76′′ E
2			Soan-myeon, Wando-gun, Jeollanam-do	34°08′53.12′′ N 126°41′10.12′′ E
3	P. dentata	Itbadidolgim	Yuldo-dong, Mokpo-si, Jeollanam-do	34°48′13.22′′ N 126°18′34.88′′ E
4			Palgeum-myeon, Sinan-gun, Jeollanam-do	34°46′13.04′′ N 126°10′22.44′′ E
5			Uisin-myeon, Jindo-gun, Jeollanam-do	34°19′07.66′′ N 126°17′31.87′′ E
6			Songji-myeon, Haenam-gun, Jeollanam-do	34°23′35.54′′ N 126°28′23.45′′ E
7	P. seriata	Momunuidolgim	Anjwa-myeon, Sinan-gun, Jeollanam-do	34°45′49.37′′ N 126°07′50.54′′ E
8			Songji-myeon, Haenam-gun, Jeollanam-do	34°45′49.37′′ N 126°07′50.54′′ E
9	P. suborbiculata	Dunggeundolgim	Nam-myeon, Yeosu-si, Jeollanam-do	34°25′32.73′′ N 127°47′31.33′′ E
10	P. pseudolinearis	Ginipdolgim	Ulleung-gun, Gyeongsangbuk-do	37°27′31.55′′ N 130°54′14.98′′ E
11	P. haitanensis	Haitanensisgim	Dried laver product from China

lists the six Pyropia species collected from China and South Korea used in this study. All samples were identified based on morphological characteristics. Cultures of the conchocelis stage of each specimen were maintained at the Ocean & Fisheries Science Institute, Haenam Branch, Jeollanamdo, Korea.

Prior to DNA extraction, conchocelis-stage samples of all strains (~300 mg wet weight) were washed twice with distilled water and frozen using liquid nitrogen, and then mechanically homogenized using a pestle tissue grinder. Genomic DNA was extracted from ground conchocelis-stage samples using the Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions.

3.3. PCR Amplification and Restriction Digestion

PCR was performed using two 18S rDNA internal transcribed regions within rbcL; forward primer 5′-GCGAACGTTACGAATCTGGAGT-3′ and reverse primer 5′-ATGCTACTGGTACAACTTTACGTA-3′ (product size: 1107 bp), trnC–P; forward primer 5′-GTCAGTTCGAATCTGGCCCTAGTTT-3′ and reverse primer 5′-ATTCCCTTGCACCCAAAGCAAGTAC-3′ (approximately product size: 1005 bp). The PCR reactions were conducted in a 50 μL mixture containing 25 μL premix (Ex Taq Version 2.0, TaKaRa, Otsu, Japan), 50 ng genomic DNA templates, and 1 μL (10 pM) forward and reverse primers. The mixtures were denatured at 95 °C for 5 min and amplified with 40 cycles of 95 °C for 30 s, 55 °C for 20 s, and 72 °C for 30 s, with a final extension at 72 °C for 5 min. The PCR amplified products were electrophoresed on 1.5% agarose gels for 30 to 40 min.

Two μL of each PCR amplified rbcL and trnC–P product(concentration ranging from 0.6 to 1 μg/μL) was digested in 2 μL of 10× buffer, 1 unit of restriction enzyme, and 15.8 μL of distilled water in a final volume of 20 μL, followed by incubation at 37 °C for 1 h. The digested fragments were separated by electrophoresis on 1.5% agarose gels stained with ethidium bromide, and fragment patterns were visualized under UV light.