https://orcid.org/0000-0001-7672-2948
Figures
Abstract
DNA barcoding is a supplementary tool in plant systematics that is extensively used to resolve species-level controversies. This study assesses the significance of using two DNA barcoding loci (e.g., psbA-trnH and trnC-petN) in distinguishing 33 plant samples of the genus Syringa. Results showed that the average genetic distance K2P of psbA-trnH DNA marker was 0.0521, which is much higher than that of trnC-petN, which is 0.0171. A neighbor-joining phylogenetic tree based on psbA-trnH and trnC-petN indicated that the identification rate of psbA-trnH and trnC-petN alone were 75% and 62.5%, respectively. The barcode combination of psbA-trnH+trnC-petN could identify 33 samples of the genus Syringa accurately and effectively with an identification rate of 87.5%. The 33 Syringa samples were divided into four groups: Group I is series Syringa represented by Syringa oblata; Group II is series Villosae represented by Syringa villosa; Group III is series Pubescentes represented by Syringa meyeri; and Group IV is section Ligustrina represented by Syringa reticulata subsp. pekinensis. These research results provided strong evidence that the combinatorial barcode of psbA-trnH+trnC-petN had high-efficiency identification ability and application prospects in species of the genus Syringa.
Citation: Yao R, Guo R, Liu Y, Kou Z, Shi B (2022) Identification and phylogenetic analysis of the genus Syringa based on chloroplast genomic DNA barcoding. PLoS ONE 17(7): e0271633. https://doi.org/10.1371/journal.pone.0271633
Editor: Branislav T. Šiler, Institute for Biological Research, University of Belgrade, SERBIA
Received: January 20, 2022; Accepted: July 5, 2022; Published: July 19, 2022
Copyright: © 2022 Yao 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: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the Key R & D projects of Hebei Province, China (No.19226367D). 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
DNA barcodes enable the rapid and accurate identification of species using short, standardized DNA regions as species tags [1]. In addition to assigning specimens to known species, DNA barcoding will accelerate the pace of species discovery by allowing taxonomists to sort specimens rapidly and by highlighting divergent taxa that may represent new species [2]. DNA barcoding had been widely used in various biological fields because of its advantages of high sensitivity, accuracy, and objectivity [3–6]. One of the major challenges faced by barcoding is the ability to resolve sister species within a large geographical range. Consortium for the Barcode of Life (CBOL) recommended the use of two plastid loci (e.g., matK and rbcL) as the standard plant DNA barcode loci [7]. A large number of experiments had been conducted using these two markers in different taxa and species. However, the identification results were unsatisfactory. Chase emphasized that the universality and identification effect of matK primers were not ideal [8]. Sass found that the rbcL often used for phylogenetic analysis across large groups of plants did not usually contain enough variability to identify individual species [9]. Increasing number of studies had shown that a system based on any one or small number of chloroplast genes will fail to differentiate taxonomic groups with extremely low amount of plastid variations while it will be effective in other groups [10,11]. Therefore, some scholars suggested that the screening of plant barcodes should not only focus on a single fragment but must be supplemented with additional fragment as required, and a combination of multiple fragment markers should be used [12]. Kress and Erickson combined the non-coding trnH-psbA spacer region, and the use of a portion of the coding rbcL gene as a two-locus global land plant barcode that provides the necessary universality and species discrimination is recommended [13]. Lahaye reported that the combination of matK to trnH-psbA and psbK-psbI could slightly increase its performance in identifying species [14]. Ho Viet identified 21 jewel orchids by rbcL+matK [15]. Meanwhile, Bhagya Chandrasekara found that rbcL+matK+trnH-psbA could still not completely solve the phylogenetic problem of Cinnamomum [16]. Therefore, for the species identification of different taxa, effective barcoding and their combination schemes, which can be used as supplementary markers for DNA barcoding, must be developed.
psbA-trnH and trnC-petN are chloroplast intergenic spacer sequences that are neither restricted by function nor affected by selection. Moreover, these two loci for the species level exhibited considerable genetic variability and divergence, ease of amplification, short sequence length, conserved flanking sites for developing universal primers, and ease of alignment and species relationship analysis [17]. Literature reported that psbA-trnH had successfully identified aquatic freshwater plants and the authenticity of herbal medicines accurately and effectively [18,19]. Niu sequenced psbA-trnH and 8 other chloroplast loci of 16 individuals of Triplostegia that represented the entire distribution range of both species recognized [20]. Similarly, trnC-petN showed high identification potential in Triticum plants [21], and Liu revealed the phylogenetic relationships and biogeographic diversification history of Cissus, which used trnH-psbA and trnC-petN markers [22].
The genus Syringa (family Oleaceae) are mainly distributed in southeast Europe, Japan, China, Afghanistan and North Korea. Approximately 27 wild species of the genus Syringa have been described, and most of which are native to China [23]. However, disputes about the infrageneric classification and relationships of the genus Syringa exist, and a comprehensive taxonomic system has not yet been established. The classification standard proposed by Zhang and Qiu that divided the genus Syringa into 2 sections and 4 series, including section Syringa and section Ligustrina is generally accepted. The section Syringa can be divided into series Syringa, series Pinnatifoliae, series Pubescentes, and series Villosae [24]. At present, some new varieties of the genus Syringa are constantly appearing in the market, but the classification standards are different, and the genetic relationship is uncertain. For example, on the question of species or subspecies of S. wolfii, it was classified as species in Flora Reipublicae Popularis Sinicae [24], but Chen pointed out that S. wolfii should be a subspecies of S. villosa [25]. In addition, no reports on the genetic relationship of S. ‘Si Ji Lan’, S. ‘Zhan Mu Shi’, and S. ‘Xiang Ya Duan’ are presented. Thus, solving these problems through morphological classification is challenging. Therefore, the main objective of this paper is to select gene fragments with multiple mutation loci according to the chloroplast genome sequence of the genus Syringa, identify various species of the genus Syringa by using sequence-specific markers, and develop DNA barcodes.
Materials and methods
Sample collection and DNA extraction
A total of 33 samples of the genus Syringa and 2 outgroup genera (Table 1) were collected from the garden nursery of Hebei Agricultural University and Beijing Botanical Garden in April–May 2021. The fresh leaves of the plant were placed in −80°C fridge. The genomic DNA was extracted from leaves by using PlantGen DNA Kit (CWBIO). The quantification and purity of the extracted DNA were measured using NanoDrop 2000 (Thermo Scientific) and 1.2% agarose gel electrophoresis.
PCR amplification
Based on the complete chloroplast genomes of five species of the genus Syringa in NCBI, intergenic spacer or intron regions with high variation were selected, and all the primers were designed by Primer primer 5.0 (Table 2). High-quality template DNA was used for PCR amplification (T100TM Thermal Cycler, BioRad). PCR reaction for psbA-trnH and trnC-petN was carried out in a total volume of 50 μL that contains 2 μL genomic DNA template, 3 μL of each primer, 25 μL 2 × Taq PCR MasterMix, 17 μL double distilled deionized water. The reaction conditions were initial denaturation at 94°C for 2 min, subsequently 32 cycles starting with 94°C denaturation for 30 s, annealing for 30 s, followed with a final extension at 72°C for 45 s, followed by 72°C for 8 min. The PCR products were detected by 1.2% agarose gel electrophoresis, and the bidirectional sequencing was completed by BGI (Beijing BGI Company).
Data analysis
The sequencing results were aligned and spliced by using SeqMan software (DNAStar). The sequence data were further utilized to analyze the AT and GC contents and SNPs for each species using BioEdit. The software MEGA X was used to compare the obtained sequences and analyze the loci of variation. Average interspecific and intraspecific distances were calculated by using a Kimura 2-parameter (K2P) distance model. A neighbor-joining (NJ) phylogenetic tree on the sequences was performed using the software MEGA X with 1000 bootstrap replications to check the support rate of each fulcrum. In addition, data were also used to develop DNA barcodes for each species by using online DNA Barcode Generator (QR barcode) software (http://biorad-ads.com/DNABarcodeWeb/), and the psbA-trnH and trnC-petN sequences of the genus Syringa were transformed into two-dimensional images using the QR barcode approach (https://www.the-qrcode-generator.com).
Results
Sequence characteristics
The specific DNA fragments of all tested species of the genus Syringa were successfully amplified by using psbA-trnH and trnC-petN primers, the lengths of the amplified products were in the ranges of 336–518 bp and 778–804 bp, and the average lengths were 465 bp and 785 bp (Fig 1). Similarly, DNA sequencing also suggested that psbA-trnH and trnC-petN generated high-quality amplicons. psbA-trnH and trnC-petN achieved amplifying and sequencing efficiencies of 100%. In the amplicons of psbA-trnH, the average nucleotide composition of AT and GC for species of the genus Syringa was 71.28% and 28.72%, respectively. In trnC-petN, the average AT and GC were 61.76% and 38.16%, respectively. Moreover, all the sequences of amplicons were aligned with those sequences published on NCBI. The consistency of psbA-trnH was 81.45%–84.81%, and that of trnC-petN was 96.67%– 97.23%.
Genetic distance between interspecific and intraspecific
The interspecific and intraspecific genetic distances of all samples were calculated by MEGA X software, and the results were shown in Fig 2. For psbA-trnH, the maximum and mean of K2P genetic distance in tested species of the genus Syringa were calculated as 0.1359 and 0.0521±0.0013, respectively. Similarly, for trnC-petN, the maximum and means of K2P genetic distance in the tested species were 0.0438 and 0.0171±0.0005, respectively. The distance of the psbA-trnH marker has been increased because it is considered a high potential barcoding region for the systematic study in plant evolution.
Analysis of variant sites and barcodes
The results showed 91 variable sites (V), 73 parsimony-informative sites (Pi), 18 singleton sites (S) in psbA-trnH, and 45 variable sites (V), 41 parsimony-informative sites (Pi), and 4 singleton sites (S) in trnC-petN (S1 Table).
Unique barcodes with psbA-trnH 508 bp and trnC-petN 182 bp and 330 bp were highly conserved in series Syringa. Other species in the series Syringa had their own unique barcodes, except for sharing one barcode of S. × hyacinthiflora ‘Asessippi’, S. × hyacinthiflora ‘Blanche Sweet’, and S. × hyacinthiflora ‘Mount Baker’.
Series Villosa generated a highly conserved unique barcode in psbA-trnH 424 bp. S. josikaea, S. × prestoniae ‘James Macfarlane’ and S. × prestoniae ‘Minuet’ shared a single set of barcodes, whereas all others had shown characteristic barcode.
Series Pubescentes was identified by the absence of the barcode at psbA-trnH 162, 165, 166, 170–172, 176, 178, 182, 189, 231, 236, 260, 263, 272, 277, 286, 304, and 319 bp. Meanwhile, the presence of the highly conserved barcode in trnC-petN 381, 403, 431, 570 bp. S. meyeri shared barcodes with S. meyeri ‘Palibin’. The others had unique DNA barcodes.
The highly conserved barcodes that identify section Ligustrina were psbA-trnH 172 bp and trnC-petN 401, 495, 497–499, 503, 505, 507, 511–513, 514, 535, and 802 bp. Each species of section Ligustrina had unique SNPs.
Phylogenetic analysis for the psbA-trnH and trnC-petN
Phylogenetic tree based on psbA-trnH showed that S. × chinensis and S. × chinensis ‘Saugeana’, which belongs to series Syringa, was clustered inside the series Villosae, and the success rate of identification was 75% (Fig 3). However, in trnC-petN, a crossover between series Villosae (S. tomentella) and series Pubescentes was observed, and the identification success rate was 62.5% (Fig 4). The results showed that the two markers used alone could not distinguish all samples of the genus Syringa. Therefore, the phylogenetic tree was established by the two marker combinations. Meanwhile, series Syringa, series Villosa, series Pubescentes, and section Ligustrina formed independent branches, and the success rate of identification was 87.5% (Fig 5). Phylogenetic tree based on psbA-trnH and trnC-petN indicated that the 33 samples of the genus Syringa were divided into four groups: Group I is series Syringa represented by S. oblata; Group II is series Villosae represented by S. villosa; Group III is series Pubescentes represented by S.meyeri; and Group IV is section Ligustrina represented by S.reticulata subsp. pekinensis. The DNA barcodes of species of the genus Syringa were established using psbA-trnH+trnC-petN variable sites. The combination of two barcodes can distinguish the genus Syringa, and species of the genus Syringa information was captured by scanning the QR code image using a mobile terminal. Fig 6 only showed the QR code information of four representative the genus Syringa group based on psbA-trnH+trnC-petN sequence. The results showed that the combination of the barcodes psbA-trnH and trnC-petN were sufficient for classifying Syringa species.
(A) S. oblata; (B) S. villosa; (C) S. meyeri; (D) S. reticulata subsp. pekinensis. In the center-colored DNA image, the different colors represent various nucleotides (A T C G) and the numbers represent the lengths of the sequences that can be used in obtaining clear sequence information.
Discussion
Traditional morphological markers are greatly influenced by environmental factors, as well as the developmental stages of the plant. These markers failed to effectively distinguish some morphologically consistent species, which consist of S. reticulata subsp. pekinensis and S. reticulata. However, molecular markers had been extensively employed in species classification and identification because of their abundance and high polymorphism. Specifically, AFLP, SSR, ISSR, and other polymorphisms are identified by complying with changes in DNA length [26–28]. As indicated from the previous study of the authors, ISSR molecular markers were adopted to identify plants of the genus Syringa [29]. According to the results, gene exchange was reported between series Pubescentes and series Villosae, which was consistent with the results in Gao who used the germplasm characterization of different plants of the genus Syringa by applying AFLP markers [30]. Thus, neither of the two markers could accurately distinguish the two groups. As the sequencing technology had been leaping forward, the method of exploiting DNA sequence was recognized to be reliable and accurate in identifying species. DNA barcoding can accurately identify species by marking the sequence variation site. In this study, the built chloroplast DNA barcodes could identify 33 samples of the genus Syringa accurately.
To establish the DNA barcode suitable for the identification of the plants of the genus Syringa, the complete chloroplast genomes of five species of the genus Syringa were first found on the NCBI. Then, sequence alignment was performed to screen the DNA fragments suitable for the barcode. Eventually, eight fragments with larger variations were determined as DNA barcode candidates. From the experience of other scholars, this study was carried out sequentially from the fragments with large variability [3,12], and psbA-trnH and trnC-petN had high and reliable identification abilities for the genus Syringa. The success rate of amplification and sequencing of psbA-trnH and trnC-petN fragment was 100%, and the identification rate of two marker combinations was 87.5%. The PCR amplification and sequencing success rates for psbA-trnH in 122 plant samples of Apocynaceae were 100% and 61%, and the identification efficiency at the species level is 82% [31]. A study used trnC-petN and other markers to construct the relationships and biogeographic diversification history of Cissus [22]. psbA-trnH and trnC-petN fragments can be used as DNA barcode options.
PCR amplification and sequencing results showed that psbA-trnH spans a large gene length (336–518 bp) because of the role of insertions/deletions in the evolution of the intergenic region in psbA-trnH, even among sister species [32]. As a result, the fragment length varied greatly among different plants. In this study, the psbA-trnH DNA length of series Pubescentes was significantly lower than those of other species. The length of trnC-petN ranged from 778 bp to 804 bp, and the number of S. reticulata and S. ‘Xiang Ya Duan’ was 803 bp, which was significantly higher than the 778 bp of S. reticulata subsp. pekinensis and S. reticulata subsp. amurensis. The results also indicated that the degree of base variation was positively correlated with the distance of genetic relationship between species.
In this study, 33 samples were divided into four groups, namely, series Syringa, series Villosae, series Pubescentes, and section Ligustrina. In traditional morphological markers, section was divided according to the length of the corolla tube. Generally, the genus Syringa could be divided into two types: section Syringa and section Ligustrina [24]. However, differences were not observed in section at the chloroplast genome level. This finding was consistent with our results using ISSR molecular markers to examine the relationships of the genus Syringa [29]. This finding may be due to the weak linkage among these sequences or the molecular markers and corolla tube length traits used in the experiment. Yang conducted a correlation analysis between SSR markers and corolla traits and discovered that SO649 markers were linked to the length of the corolla tube. The transcriptome sequence of the SO649 marker was annotated as E3 ubiquitin-protein ligase, which was a B3 domain-containing protein. The B3 domain-containing protein is essential for stress responses and plant growth and development [23]. Therefore, the corolla tube length related genes were assumed to be located in the nuclear genome rather than in the chloroplast intergenic spacers. The anthers of S. emodii are longer than the corolla tube, which is consistent with the morphological classification of the section Ligustrina [33]. The results of Ki-Joong and Robert’s cpDNA tree analysis revealed that S. emodii clustered in the series Villosae but not in the section Ligustrina [33], indicating a weak association between the corolla tube length and the chloroplast genes. In addition, the IPlant (http://www.IPlant.cn) and Chen proposed that S. wolfii was a subspecies of S. villosa [25]. In this study, S. wolfii was closely related to S. villosa, forming sister relationship. S. wolfii was identified as an independent species in the Flora Reipublicae Popularis Sinicae [24], and our study supported their view. Furthermore, the three unknown genetic relationship species were successfully identified by using psbA-trnH and trnC-petN fragments. The S. ‘Si Ji Lan’ was closely related to S. meyeri, the S. ‘Zhan Mu Shi’ was closely associated with S. josikaea, and the S. ‘Xiang Ya Duan’ was near S. reticulata. These two chloroplast genomic primers may provide sufficient molecular data for identifying closely related Syringa species.
The current study tested the effectiveness of these two fragments and their combination markers using a large number of experimental samples, and the identification efficiency of the combination markers below the species level was 85%. The result had shown that the chloroplast fragments psbA-trnH and trnC-petN could be used as identification barcodes of Syringa plants. Moreover, we developed QR codes based on the DNA sequence and established characteristic barcodes for each species.
Supporting information
S1 Table. Barcode of selected species of the genus Syringa based on variable regions of psbA-trnH and trnC-petN markers.
https://doi.org/10.1371/journal.pone.0271633.s001
(PDF)
Acknowledgments
The authors would like to thank Dr. Mengxin, Beijing Botanical Garden for her guidance throughout Syringa sample collection.
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