https://orcid.org/0000-0002-0172-0021
Correction
27 Dec 2024: Lee M, Lee HY, Kang JS, Lee H, Park KJ, et al. (2024) Correction: Authentication of Allium ulleungense, A. microdictyon and A. ochotense based on super-barcoding of plastid genome and 45S nrDNA. PLOS ONE 19(12): e0316742. https://doi.org/10.1371/journal.pone.0316742 View correction
Figures
Abstract
Allium ulleungense (AU) and A. microdictyon (AM) are valuable medicinal and edible vegetables, referred to as mountain garlic in Korea. The identification of AU, AM and a neighboring species A. ochotense (AO) is difficult because of their morphological similarities. We collected samples from three species and 46 cultivated collections to understand the genetic diversity of these valuable Allium species. Among them, we sequenced six collections, including three species and three cultivating collections to obtain data from the plastid genome (plastome) and nuclear 45S ribosomal DNA (nrDNA) for super-barcoding. The AM and AO showed around 60 single nucleotide polymorphisms (SNPs) and 39 Insertion/Deletion (InDels) in the plastome but no variations in the nrDNA sequences. Conversely, the AU and AM showed more than 170 SNPs and 80 InDels in the plastomes, and 20 SNPs and 1 InDel were found in the 45S nrDNA sequences. Among the three cultivating collections, one TB collection was determined to be the AU type in both plastome and nrDNA sequences. However, the other two collections, JB and SA, showed the AM type plastome but were heterozygous in the 45S nrDNA sequences, indicating both AU and AM types (putative AM x AU hybrid). Ten molecular markers were developed based on sequence variations to identify these three species and assess their genetic diversity. A total of 49 collections were genotyped using the ten developed markers and classified into five groups: 14 AU, 22 AM, 1 AO, 3 putative AM x AU hybrids, and 9 putative AU x AM hybrid collections. Super-barcoding with plastomes and nrDNAs revealed the genetic diversity of the three Allium species and putative hybrids between species. The newly developed markers will facilitate species and hybrid identification, thereby benefiting marker-assisted molecular breeding of Allium species.
Citation: Lee M, Lee HY, Kang J-S, Lee H, Park K-J, Park JY, et al. (2023) Authentication of Allium ulleungense, A. microdictyon and A. ochotense based on super-barcoding of plastid genome and 45S nrDNA. PLoS ONE 18(11): e0294457. https://doi.org/10.1371/journal.pone.0294457
Editor: Yanbin Yin, University of Nebraska-Lincoln, UNITED STATES
Received: August 21, 2023; Accepted: October 31, 2023; Published: November 20, 2023
Copyright: © 2023 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The assembled plastome and 45S nrDNA sequences of six Allium samples were deposited in the NCBI GenBank under accession numbers OQ183591 to OQ182596 (plastomes), and OQ183920 to OQ183925 (45S nrDNAs), respectively.
Funding: TJY received a research grant from KIST ORP program (BlueBell Research program 2E32641-23-070). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Allium ulleungense (AU), A. ochotense (AO) and A. microdictyon (AM) that belong to subgenus Anguinum, are commonly known as ‘Siberian garlic’ or ‘Alpine leek’. They are widely used as a vegetable, called mountain garlic, due to their strong garlic-like odor and taste. These Allium species are known to have medicinal functions such as anti-cancer, anti-inflammatory and anti-oxidant [1–4]. Due to their morphological similarity, it is not easy to distinguish these three Allium species in the farm fields and agricultural market. However, the three Allium species can be distinguished by ploidy level, distribution, taste and growth. The AO is tetraploid (2n = 4x = 32) and is distributed in Japan, Northeast China, Southeast Russia and North Korea [5]. On the other hand, AM and AU are diploid (2n = 2x = 16) [5]. The AM is distributed in Siberia, the Southern Ural area, North Mongolia, Kazakhstan and Korea [5,6], and AU is endemic to the Ulleung Island of South Korea. The AU was recently treated as an independent species in South Korea [7]. The AU can be distinguished from AM by its broader leaves and larger whitish perianth [7]. In agricultural markets, AM is usually preferred by customers because of its flavor, taste and texture, compared with AU. However, the productivity of AM is lower than that of AU in farm fields. Therefore, these three Allium species need to be studied for species identification and for the molecular breeding of a new Allium cultivar with the better taste of AM and excellent productivity of AU.
In order to ensure accurate identification and efficient breeding of these species, analyzing molecular data is necessary to assess the genetic diversity of Allium species. In general, universal barcoding regions are commonly used for the molecular identification of plants. However, molecular identification using universal barcoding regions often provides insufficient data to identify the species, especially when two or more species are close and the barcoding region is conserved in the group [8]. Thus, super-barcoding, using complete plastid genomes (plastomes) and entire nuclear ribosomal DNA (nrDNA), is considered a powerful tool to identify the closely related species, by providing abundant genetic variation [9–11]. Due to the advances of next-generation sequencing, a large amount of plastome data in land plants has been reported and is available in NCBI GenBank [10], allowing us to assess the genetic diversity of the species from various locations.
In this study, we newly sequenced three Allium species (AU, AM, and AO) to obtain plastome and nrDNA data and then investigated variations among the three species to develop molecular markers for identifying the three Allium species and assessing the genetic diversity. The developed molecular markers were verified with 46 collections of the three species and demonstrated to be useful in identifying the species and assessing the genetic diversity. We believe that the developed markers in this study will be useful for the molecular breeding of the Allium species.
Materials and methods
Plant materials and genomic DNA extraction
Plant materials of six Allium samples were provided from the Gangwondo Agricultural Research & Extension Services, Wild Vegetable Research Institute, Republic of Korea (Table 1, S1 Fig), and no specific permission was required for collecting the species in this study, according to the national and local legislations. Specimens of the six Allium samples used in this study were deposited in the T.B. Lee Herbarium of Seoul National University under the voucher numbers SNUA00058899 to SNUA00058905 (Table 1). Leaf materials were ground with liquid nitrogen using a mortar and pestle, and total genomic DNA was extracted using the GeneAllⓇ ExgeneTM Plant SV Mini Kit (GeneAll Biotechnology LTD., South Korea) according to the manufacturer’s instructions. The quality and concentration of the extracted DNA were measured using a NanoDrop ND-1000 (Thermo Fisher Scientific, USA).
Next-generation sequencing and quality trimming
Paired-end library construction and next-generation sequencing (NGS) were conducted using the Illumina MiSeq platform (Illumina, USA) at Phyzen (www.phyzen.com, South Korea). About 1~2 Gbp of raw data were generated from six Allium samples (S1 Table). The obtained raw reads were trimmed based on the quality score, maintaining a minimum quality score ≥20, using the CLC quality trim (version 4.06beta.67189, CLC Inc., Denmark).
Plastome and 45S nuclear ribosomal DNA assembly and annotation
After quality trimming, plastome and 45S nrDNA sequences of six Allium samples were assembled using the de novo assembly of low-coverage whole-genome sequencing (dnaLCW) method [12,13], in the CLC genome assembler program (ver. 4.6 beta, CLC Inc., Denmark). Trimmed reads were assembled with parameters of overlapping distance ranging from 150 to 500 bp. Plastome contigs were extracted from the assembled sequences using MUMmer, and the Allium victorialis plastome sequence (NC_037240) was used as a reference. Extracted plastome contigs were assembled into a single draft sequence, and were manually curated based on mapping results of the NGS reads. The nrDNA contigs were retrieved and assembled in conjunction with the nrDNA sequence of Brassica oleracea (KM538957).
Complete plastome sequences were annotated using the GeSeq program (https://chlorobox.mpimp-golm.mpg.de/geseq.html) [14] and manually corrected using the Artemis program [15]. The circular map of plastomes was drawn using the OGDRAW program (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html) [16]. The 45S nrDNA region, including 18S, 5.8S, 26S and two internal transcribed spacer (ITS) regions, was annotated using a BLAST search. The assembled plastome and 45S nrDNA sequences of six Allium samples were deposited in the NCBI GenBank under accession numbers OQ183591 to OQ182596 (plastomes), and OQ183920 to OQ183925 (45S nrDNAs), respectively.
Sequence comparison and molecular marker development
Plastome and nrDNA sequences were aligned using MAFFT (www.mafft.cbrc.jp) [17] with six Allium samples. After the alignment, single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) were counted and categorized into inter- and intraspecies polymorphisms. To develop molecular markers, InDel regions among the Allium plastomes longer than 10 bp were selected to design the codominant marker, and one SNP region was chosen for designing the dominant marker. The SNPs in the 45S nrDNA regions were selected and used for the high-resolution melting curve (HRM) analysis. Primers for codominant, dominant markers and HRM analysis were designed using Primer-BLAST [18] with default parameters.
PCR amplification was conducted in 25-μL reactions containing 1 unit of Taq DNA polymerase (Inclone Biotech, South Korea), 2.5 μL of 10x reaction buffer, 0.2 mM dNTPs, 20 ng genomic DNA, and 10 pM of each primer. The thermal cycling conditions were as follows: pre-amplification at 95°C for 5 min; 35 cycles of denaturation at 95°C for 20 sec, annealing at 58–63°C for 20 sec, and elongation at 72°C for 20 sec; followed by a final elongation at 72°C for 5 min. PCR products were confirmed by electrophoresis using a 3% (w/v) agarose gel.
The nrDNA-based HRM analysis was performed using a LightCycler 96 Real-time PCR Machine (Roche Applied Science, Indianapolis, IN, United States) under the following conditions: pre-amplification at 95°C for 5 min; 45 cycles of 95°C for 20 sec, 60°C for 20 sec, and 72°C for 20 sec; 1 cycle of 95°C for 1 min, 40°C for 1 min, 60°C for 5 sec, 95°C for 5 sec; followed by a cycle of 40°C for 30 sec. The reaction was conducted in 20-μL reactions containing 1 unit of Taq DNA polymerase (Inclone Biotech, South Korea), 2 μL of 10x reaction buffer, 0.2 mM dNTPs, 20 ng genomic DNA, 10 pM of each primer, and 2.5μM SYTO 9 (Thermo Fisher Scientific, USA).
Phylogenetic analysis
Plastome sequences of six Allium samples and 19 previously reported Allium plastomes that were downloaded from NCBI GenBank were aligned using MAFFT [17]. For the nrDNA phylogeny, the ITS1-5.8S-ITS2 region of 45S nrDNA of six Allium samples and those from 64 previously reported Allium individuals downloaded from NCBI were aligned using MAFFT. Phylogenetic analyses based on whole plastome sequences (excluding one copy of IR region) and 45S nrDNA (18S, 5.8S, 26S and two ITS regions) sequences were conducted using RAxML [19] with 1000 bootstrap replicates and a substitution model of GTRGAMMA, respectively.
Results
Plastome and nrDNA assemblies
Through the Illumina Miseq platform, approximately 1 gbp of high quality reads were used for simultaneous de novo assembly of plastomes and nrDNAs (S1 Table). Complete plastomes and 45S nrDNAs of six Allium samples were assembled. Total length of the assembled plastomes ranged from 153,125 to 154,051 bp (Table 1). The plastomes of AU and Farm-TB were relatively larger than those of AM, AO, Farm-JB, and Farm-SA. Plastomes consisted of 82,183 to 83,147 bp of large single copy region (LSC), 26,542 to 26,526 bp of the IR, and 17,852 to 17,858 bp of the SSC (S2 Table). All the six Allium plastomes contained 86 protein-coding genes, 38 tRNA genes and eight rRNA genes (S2 Table, Fig 1A). No difference in the number of genes among six Allium plastomes was found. Total length of 45S nrDNAs ranged from 5,878 to 5,879 bp with coverage of 451.61 to 812.71x (Table 1, Fig 1B). The assembled 45S nrDNAs of AU, Farm-TB, Farm-JB and Farm-SA were identical in length with 5,878 bp, and those of AM and AO were 5,879 bp. A one bp InDel between AU and AM was found at the 1,936th nucleotide of the 45S nrDNA sequence.
(A) Circular map of six Allium plastomes. The species and total length are written in the center of the circular map. Annotated genes are marked on the outer circle and functions of these genes are categorized based on the legend placed in the bottom left. Red bars of the inner circle indicate SNP positions and black bars of the inner circle indicate InDel positions. (B) Map and variable sites within the 45S nrDNA sequence of six Allium samples. Gray boxes indicate transcription unit and red bars indicate variable sites among six Allium samples.
Intra- and interspecific variations in the plastome and 45S nrDNA
We aligned the plastome and 45S nrDNA sequences of six Allium samples, and identified sequence variations among the six Allium samples. A total of 14 SNPs and six InDels were identified between the AU and Farm-TB plastomes, and less than six SNPs and less than two InDels were detected among the plastomes of AM, Farm-JB, and Farm-SA (Fig 2A). The AO plastome was distinct from the other five plastomes, containing about 180 SNPs and 80 InDels with the AU and Farm-TB plastomes, and about 60 SNPs and 40 InDels with the plastomes of AM, Farm-JB, and Farm-SA (Fig 2A). In the plastome-based phylogeny, both AU and Farm-TB formed their own clade, which was sister to the clade of AM, AO, Farm-JB, and Farm-SA (Fig 2C). Within the clade of the four Allium samples, the former three formed a subclade, and AO was sister to the subclade of three Allium samples (Fig 2C).
(A) Variations observed among six Allium plastomes. (B) Variations observed among six Allium 45S nrDNA sequences. (C) Phylogenetic relationships among six Allium samples based on whole plastome sequences (excluding one copy of IR region) and 45S nrDNA sequences. The plastome and 45S nrDNA sequences of A. cepa were included as the outgroup. The phylogenetic tree on the left is based on plastomes, while the one on the right is based on 45S nrDNA sequences. Bootstrap values greater than 50% are shown near the branches. The colored lines in the middle highlight comparisons between the two trees.
The nrDNA sequence comparison showed that the nrDNA sequences of AU and three farm collections (TB, JB and SA) were identical, and those of AM and AO were identical (Fig 2B). We found 20 SNPs and one InDel between nrDNA sequences of the two groups (AU, Farm-TB, Farm-JB, and Farm-SA vs. AM and AO) (Fig 2B). Unlike the plastome-based phylogeny, the nrDNA phylogeny showed that AO and AM formed a subclade, and AU and the three farm collections (TB, SA, and JB) formed a subclade (Fig 2C). We found phylogenetic discordance on the phylogenetic positions of farm collections JB and SA in the plastome and nrDNA phylogenies. The two samples formed a clade with AM and AO in the plastome-based phylogeny, but they formed a clade with AU and Farm-TB in the nrDNA phylogeny (Fig 2C).
Furthermore, heterozygous sites in the 45S nrDNA sequences of the six Allium samples were investigated. Among the variations in the 45S nrDNA sequences, 21 heterozygous sites were observed in the 45S nrDNA sequences of farm collections JB and SA, containing two nucleotides at the same site. Each nucleotide of the two nucleotides in farm collections JB and SA was consistent with the nrDNA sequences of AU and AM, respectively, and depth ratio of each nucleotide type was about 7:3 in Farm-JB and 6:4 in Farm-SA overall (Fig 2B, Table 2).
Phylogenetic relationships of Allium species
We downloaded 19 previously reported plastome sequences of 12 Allium species from the NCBI GenBank. These included sequences from A. microdictyon, A. ochotense, A. ullengense, A. victorialis, A. listera, A. ovalifolium, A. nanodes, A. prattii, A. tricoccum, A. neriniflorum, A. karataviense and A. cepa (KM088013). Using these 19 downloaded plastomes and the six newly assembled plastomes from this study, we conducted a phylogenetic analysis to elucidate the phylogenetic relationships of the subgenus Anguinum of the genus Allium. The plastome-based phylogeny revealed that AU and Farm-TB formed a clade with three previously reported A. ullengense, and the clade was sister to the clade of A. listera and one A. victorialis (NC_037240). The other two A. victorialis were clustered with A. microdictyon and A. ochotense. Within the clade of A. microdictyon and A. ochotense, two farm collections, JB and SA, formed a clade with AM, previously reported A. microdictyon (OP743936) and A. ochotense (OP743938), and then the clade was sister to the clade of two A. ochotense (OP743947 and OP743948) (S2 Fig).
For the nrDNA-based analysis, we utilized the ITS1-5.8S-ITS2 sequences of 45S nrDNA, including the six Allium samples and 64 accessions from eight closely related Allium species (A. microdictyon, A. ochotense, A. neriniflorum, A. ovalifolium, A. prattii, A. tricoccum, A. victorialis and A. listera) obtained from NCBI. The ITS1-5.8S-ITS2 sequence of A. cepa (KM117265) was included as the outgroup. The AM and AO were located in the same clade with other sequences from A. microdictyon, A. ochotense, A. listera and A. victorialis. This clade was observed to be a sister group to the A. tricoccum clade. The AU and Farm-TB along with farm collections JB and SA, and two previously reported A. ulleungense accessions, formed a distinct clade (S2 Fig). A comparison of the plastome and nrDNA phylogenies showed that A. ulleungense was distinctly separated from other Allium species in the subgenus Anguinum. In addition, while the two farm collections, JB and SA, aligned closely with AM in the plastome phylogeny, they were closer to AU and Farm-TB in the nrDNA phylogeny (S2 Fig). As a result, among the three farm collections, Farm-TB was identified as A. ulleungense. However, Farm-JB and Farm-SA presented conflicting results between the plastome and nrDNA phylogenies (Figs 2C and S2). This result, combined with the results from heterozygous sites of 45S nrDNA in the two farm collections (Table 2), suggests that these two farm collections likely originated by hybridization between A. ulleungense as the paternal and A. microdictyon as the maternal species.
Molecular marker design and validation
Based on sequence variations among six Allium accessions, ten molecular markers were designed to identify three Allium species: AU, AM, and AO. Six InDels located in the rpoC2, rpoB–trnC, rps18–rpl20, trnV–rps12, and rps16–trnQ regions, and one SNP located in the psbC–trnS region of the Allium plastomes were selected to design six codominant markers (AL_1, AL_2, AL_3, AL_4, AL_5, and AL_6) and one dominant marker (AL_7) (Table 3). Moreover, SNPs in the nrITS region of the 45S nrDNA, were used for the HRM analysis (Table 3). The ten designed markers were applied initially to verification with the six Allium samples. As the results, three Allium species, AU, AM, and AO could be identified using the seven designed markers from the plastome data (Fig 3A); AO (including Farm-TB) and AM could be identified using AL_1, AL_2, AL_3, AL_5, and AL_6, while AO could be distinguished using AL_4, AL_6, and AL_7 (Fig 3A). Furthermore, the two farm collections, JB and SA, which contained heterozygous sites in the nrDNA sequences, were identified using the melting peaks from three HRM analyses (Fig 3B). The melting peaks of the six Allium samples were categorized into three types: 1) AU and Farm-TB are denoted by red-colored peaks (AU), 2) AM and AO are denoted by blue-colored peaks (AM), and 3) the farm collections, JB and SA, are represented by green-colored peaks (Hybrid) (Fig 3B).
(A) Gel electrophoresis result for PCR products derived from the molecular markers. (B) Melting peak results from HRM analysis using the HRM markers.
The ten developed markers were validated with extensive 49 samples of Allium species (S3 Table, S3 Fig). By Combining the results of these ten markers, the 49 samples were classified into five genotypes: AU-type, AM-type, AO-type, and two hybrid-estimated types (Tables 4 and S3). The two hybrid-estimated types were determined by their plastome type, and exhibited distinct melting peaks in the HRM analyses of AL_8 and AL_9 (S3 Table, S3 Fig). Among the 49 samples, fourteen samples, including AU and Farm-TB, were identified as AU-type, 22 individuals, including AM, were identified as AM-type; and only AO was categorized as AO-type (Tables 4 and S3, S3 Fig). The remaining 12 samples were identified as two hybrid-estimated types. Specifically, three samples (HJ1, HJ12, and HJ13) were identified as hybrids with the AU-type plastome (AM x AU hybrid), while nine samples (HC_OD1, HC_OD2, HC_OD3, HC_OD4, HC_OD5, JBds1, IJga, Farm-JB, and Farm-SA) were classified as hybrids with the AM-type plastome (AU x AM hybrid). The two farm collections, JB and SA, which had heterozygous sites in the nrDNA, were included in the AU x AM hybrid type (Tables 4 and S3, S3 Fig).
Discussion
Genetic diversity of plastome and nrDNA among three Allium species
The AU (A. ulleungense) was previously recognized as AO (A. ochotense). However, Herdan et al. [5] and Prokhanov [20] argued that AO should be treated as two independent species based on their ploidy levels; diploid AO (= A. latissimum) and tetraploid AO [5,20]. Recently, the diploid AO was treated as a new species, endemic to Ulleung Island in South Korea [7]. According to Choi et al. [7], the AU (A. ulleungense) was clearly distinguished from AM (A. microdictyon) and AO (A. ochotense) by its morphological characters, such as relatively broader leaves and larger whitish perianth, chromosome number, and molecular evidence from nrITS and plastome data [7,21]. In this study, significant genetic diversity among AU, AM and AO was discovered through sequence variations and phylogenetic results based on the plastome and 45S nrDNA sequences. In plastomes of the three Allium species, about 180 SNPs and 80 InDels were found between AU and the other two species (AM and AO), and nearly 60 SNPs and 40 InDels were found between AM and AO, and the relationships among the three species were supported by 100% branch supports in the plastome phylogeny (Fig 2C). These results imply that the three Allium species are genetically differentiated from each other, and the taxonomic treatment of the AU is genetically supported.
However, two farm collections, JB and SA, showed conflicting results between plastome and 45S nrDNA data in sequence comparisons and phylogenetic analyses. The two farm collections were closer to AM in plastome data (Fig 2A and 2C), whereas they were identical with AU and Farm-TB in nrDNA data (Fig 2B and 2C). The discrepancy between plastome and nrDNA data is likely caused by a hybridization between AU and AM. While the plastome is maternally inherited in most angiosperms, 45S nrDNA is inherited from both parents [22,23]. The 45S nrDNA sequences have been used to study hybrids in various plants [24–26]. In this study, nucleotide type and read mapping depth of the 45S nrDNAs were analyzed to verify the possibility of crossing. The results indicated that two farm collections, JB and SA, displayed heterozygous sites containing both AU- and AM-types (Table 2). According to the heterozygous sites in the nrDNA sequences and both phylogenies based on plastome and nrDNA sequences, the farm collections JB and SA are likely to have originated from the hybridization between AU (paternally) and AM (maternally), but they may not be the F1 hybrids, having an uneven genetic proportion inherited from the two parental species (Table 2).
Phylogenetic relationships of three Allium species
In a previous study of the subgenus Anguinum using sequences of the nrITS region and three plastid markers (rps16 intron, rbcL-atpB, and rpl32-trnL), the phylogenetic relationships of three Allium species, AM, AO, and A. victorialis, were revealed [5]. In this study, we performed phylogenetic analyses using complete plastome and 45S nrDNA sequences of six Allium samples with other related species and showed that the AU, including AU, Farm-TB, and three previously reported AU, were clearly distinguished from AM and AO. These samples were sister to the clade of one A. victorialis and A. listera in the plastome phylogeny (S2 Fig), which was consistent with the previously constructed plastome phylogeny [21]. The sister relationship between AU and the clade of A. victorialis, A. listera, AM, and AO was supported in the nrITS tree (S2 Fig).
Based on a few DNA barcoding regions, the divergence time of the AU from other Allium species was previously estimated at approximately 2.54 million years ago (Ma) [5]. However, a recent study using plastome data indicated that the split between AU and other species, such as A. listera, A. victorialis, AM, and AO, occurred around 1.6 Ma [21]. The estimated time was almost consistent with the formation of Ulleung Island (1.5 Ma) [27,28]. We speculate that the genetic differentiation between AU and other Allium species shown in the phylogenetic analyses may be related to the geographical factor of Ulleung Island, where AU is distributed. Ulleung Island in South Korea is a volcanic island located far from the Korean peninsula [27,28], and several species originating from anagenetic speciation have been reported [29–31]. The conflicting positions of AU observed between the plastome and nrDNA phylogenies were similar to those observed in Phedimus takesimensis (Crassulaceae), where the origin of anagenetic speciation was revealed through a previous study [30]. Therefore, we speculate that AU may have undergone anagenetic speciation in the isolated environment of Ulleung Island like other endemic species on the island [29–31].
Species identification and marker-assisted breeding of Allium species using molecular markers
Species identification in plants has predominantly relied on morphological characters, due to their highly polymorphic and easily observable nature. However, this traditional approach to species identification comes with limitations [32]. One major challenge is the presence of variation in plant morphology within the same species, which can lead to discrepancies in classification and create difficulties in precisely differentiating between closely related species [32]. To address these shortcomings and enhance the accuracy of species identification, a combination of molecular markers and morphological characters has become imperative [32]. In this study, we sought to overcome these challenges by applying molecular markers for species identification of Allium species. We developed ten molecular markers based on sequence variations in the plastome and nrDNA sequences of six Allium samples, providing a robust toolset to identify and distinguish three Allium species: AU, AM, and AO (Table 3). Moreover, three HRM markers developed in this study demonstrated their capability to detect hetero-genotypes of hybrid-estimated species (Table 3, Figs 3 and S3).
In the contemporary context of plant breeding, marker-assisted breeding (MAB) has emerged as a cutting-edge methodology with considerable advantages over conventional breeding approaches [33]. The utilization of molecular markers streamlines the breeding process, making it less time-consuming and reducing the influence of environmental factors on breeding outcomes [33]. Notably, MAB also permits the effective selection of recessive alleles and traits that are controlled by multiple genes, further enhancing breeding efficiency and precision [33]. Given the advantages of MAB and the performance of the newly developed molecular markers in this study, we believe that these markers will be instrumental for species identification and genotype assessment in Allium species.
Conclusions
In this study, we investigated the genetic diversity among three Allium species by analyzing data from the plastome and 45S nrDNA. Our findings revealed substantial genetic variation among the species, leading to the classification of six individuals into three distinct types: AU (A. ulleungense), AM (A. microdictyon), and AO (A. ochotense). This observation underscores the significant genetic differentiation present among three Allium species. Consequently, we developed ten molecular markers based on sequence variations to facilitate the identification of these three species. To validate the efficacy of the developed markers, we examined a total of 49 individuals from the three species, categorizing them into five types: AU, AM, AO, and two hybrid-estimated types. The newly developed markers are expected to be valuable tools for both species identification and marker-assisted breeding of these species in the future.
Supporting information
S1 Table. Information of generated NGS data from six Allium samples.
https://doi.org/10.1371/journal.pone.0294457.s002
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S2 Table. Detail information of plastomes of six Allium samples.
https://doi.org/10.1371/journal.pone.0294457.s003
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S3 Table. Genotyping results using ten molecular markers on 49 individuals of Allium species.
https://doi.org/10.1371/journal.pone.0294457.s004
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S1 Fig. Allium samples collected from various locations.
(A-F) Leaf samples of UL, TB, JB, SA, OD and JP respectively. (G) Green stem color of UL. (H) Purple stem color of OD.
https://doi.org/10.1371/journal.pone.0294457.s005
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S2 Fig. Phylogenetic analyses of Allium species including other related Allium species based on plastome and 45S nrDNA sequences.
Bootstrap values are indicated near the branches (indicated only if the bootstrap value is greater than 50). (A) Phylogenetic tree based on whole plastome sequences. (B) Phylogenetic tree based on ITS1-5.8S-ITS2 region of 45S nrDNA sequences.
https://doi.org/10.1371/journal.pone.0294457.s006
(TIF)
S3 Fig. Application of ten molecular markers on 49 individuals of Allium species.
(A) Gel electrophoresis result of PCR product derived from molecular markers. (B) Melting peak results of HRM analysis using HRM markers.
https://doi.org/10.1371/journal.pone.0294457.s007
(TIF)
Acknowledgments
We thank to all the staff members of the Wild Vegetable Research Institute, Gangwondo Agricultural Research & Extension Service, for providing the materials of Allium species used in this study.
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