https://orcid.org/0000-0002-9355-3055
Figures
Abstract
Chaenomeles japonica (Thunb.) Lindl., an ornamental species belonging to the family Rosaceae, was molecularly identified from a cultivated population in the Hazara region, Khyber Pakhtunkhwa (KP), Pakistan. Fresh leaf samples were collected from a cultivated garden, and genomic DNA was extracted for molecular analysis. Four chloroplast DNA markers (matK, rbcLa, trnA, and ycf3) were amplified and sequenced. PCR amplification showed a 100% success rate, generating sequence lengths ranging from 400–1600 bp depending on the marker. BLAST analysis revealed 99–100% sequence identity, 100% query coverage, and E-values of 0.0 when compared with reference sequences available in GenBank, primarily from China, Japan, and the United States. Phylogenetic analysis using Neighbour Joining method demonstrated that the obtained sequences clustered with authenticated Chaenomeles japonica accessions, supported by bootstrap values. The results confirm the molecular identity of cultivated C. japonica in Pakistan and highlight the utility of chloroplast DNA barcoding markers for accurate species identification of ornamental plants.
Citation: Islam M, Sajjad S, Hazzazi Y, Sumayli M, Alruzayza S, Muhammad K, et al. (2026) Molecular identification and DNA barcoding of Chaenomeles japonica in Pakistan. PLoS One 21(5): e0348503. https://doi.org/10.1371/journal.pone.0348503
Editor: Tzen-Yuh Chiang, National Cheng Kung University, TAIWAN
Received: January 9, 2026; Accepted: April 15, 2026; Published: May 5, 2026
Copyright: © 2026 Islam 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 manuscript.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Chaenomeles japonica, commonly known as Japanese quince (Thunb.) Lindl., is a deciduous shrub belonging to the Rosaceae family [1]. Native to Japan, It is widely cultivated intemperate regions of the world due to its ornamental red to orange flowers, edible fruits, and adaptability to diverse environmental conditions [2]. The fruits, although astringent when raw, are rich in pectin and vitamin C and are commonly processed into jams, jellies, and beverages [3]. Beyond its culinary value, C. japonica has been used reported to possess antimicrobial, anti-inflammatory, and antioxidant properties, highligtening its ethnomedicinal importance [4]. These ecological, horticultural, and therapeutic attributed to its global distribution outside its native range [5].
The genus Chaenomeles comprises 4–5 species distributed primarily in East Asia, especially China and Japan. Among them, C. japonica is one of the most extensively cultivated species because of its hardness and ornamental appeal [2]. It has been introduced into Europe, North America, and other parts of Asia, where it occasionally naturalizes [6,7]. However, species delimitation within the genus remain challenging. Members of Chaenomeles share overlapping morphological traits, including similar leaf shapes, serration patterns, flower coloration, hypanthium structure, and fruit morphology [8]. Furthermore, phenotypic plasticity, environmental influences, horticultural selection, and possible interspecific hybridization complicate accurate identification based solely on morphology. Diagnostic characters may also be absent or incomplete during non-flowering or non-fruiting stages. These limitations highlight the need for integrative approaches combining molecular and morphological data to ensure precise taxonomic resolution [9].
Accurate identification is particularly critical for newly recorded or potentially invasive taxa [10]. Traditional morphological taxonomy can be constrained by environmental variability and developmental stages [11]. In this context, DNA barcoding has emerged as a reliable tool for species identification and phylogenetic inference [12]. Chloroplast markers are commonly used in plant barcoding due to their conserved structure and maternal inheritance. The rbcL gene is characterized by high universality and amplification success, although it typically exhibits relatively low interspecific variability [13]. In contrast, matK evolves more rapidly and provides greater discriminatory power at the species level but may show reduced amplification efficiency in some taxa. Non-coding regions such as trnA introns often display higher sequence variability, which enhances species resolution, although alignment challenges may arise due to insertions and deletions. Similarly, the chloroplast gene ycf3 has demonstrated moderate variability and phylogenetic informativeness in angiosperms, though it’s discriminatory capacity may vary among closely related species [14]. The combined use of these loci allows integration of conserved and variable genomic regions, thereby improving identification accuracy and phylogenetic robustness [15].
In Pakistan, the genus Chaenomeles has not previously been reported in floristic records, herbarium collections, or Rosaceae checklists. Therefore, the present study integrates morphological assessment with multi-locus DNA barcoding (matK, rbcL, trnA, and ycf3) to investigate a cultivated specimen suspected to belong to Chaenomeles. The specific aim of this study is to provide preliminary molecular identification and phylogenetic placement of the specimen and to confirm the presence of C. japonica as a new record for Pakistan. This work contributes to the documentation of non-native ornamental flora and underscores the importance of molecular tools in contemporary plant systematics and biodiversity assessment.
Materials and methods
Sample collection
The plant samples were collected in the form of flowers, fresh leaves and fruits from the cultivated garden of Hazara University (Table 1). The collected samples were properly tagged, pressed, dried, and pasted on the herbarium sheets for future study and record (Fig 1). The fresh leaves were collected in polythene zipper bags; the bags were tagged and stored in a genomic lab at −20°C deep freezer for molecular study.
DNA extraction
For genomic DNA extraction, fresh leaves were ground well with the help of a pestle and mortar in liquid nitrogen. CTAB method with minor changes was used to extracted total gDNA [16]. A pre-warmed CTAB of 800 µl was used for and incubated the ground tissue for 2 hours at 65°C, and samples were vortexed thoroughly [17]. After incubation, phenol chloroform isoamyl alcohol of 600 µl was added, and the homogenate was centrifuged for 20 minutes at 13,000 rpm. Then ice-cold isopropanol of 500 µl was added freeze the sample overnight. Again the samples were centrifuged for 20 minutes at 13,000 rpm. After centrifugation the final products in the form of pellet was washed with 70% ethanol [18]. The extracted genomic DNA was diluted, and checked on gel electrophoresis [19].
Primer selection and amplification
The most studied and well-known primers shown in Table 2, were selected, and amplified using the prescribed conditions. A 25 µL PCR reaction mixture was prepared in a 200 µL PCR tube, containing ddH2O 14 µL, 10 × PCR buffer 2.5 µL, MgCl2 2 µL, dNTPs 2 µL, tDNA 2 µL, Taq Polymerase 0.5 units (Catalogue K0171) and 2 µL of primers (forward and reverse). The PCR steps started with a pre-denaturation for 5 minutes at 94°C, followed by 35 cycles for 30 seconds at 94°C, 40 seconds at 52°C annealing for (matK), 56°C for (rbcla), 54°C for (trnA), and 58°C for (ycf3) and about 35 seconds at 72°C for extension. The amplified products were checked on a 1% agarose gel electrophoresis.
Nucleotide sequencing and analysis
The desired amplified regions were sequenced from Macrogn Inc., South Korea through Sanger sequencing. After positive sequencing, good-quality region were used for Basic local alignment (BLASTn) in online ncbi tools [24]. The closely related accessions were downloaded and arranged and analysed through a series of software via BioEdit [25], Multiple Sequence Alignment (MUSCLE) [26], and Clustal-W [27]. The resultant data was processed to compute the Kimura-2-parameter distances for each region by MEGA-11 [28].
Results
In the current study, four widely used and recommended chloroplast markers were used for initial investigation of experimental plant. The candidate barcode markers showed 100% with respect to amplification, sequencing and similarity with the reference database library sequence of C. japonica. The overall performance of the query barcode was higher than 90% in the current study.
Nucleotide BLAST of candidate barcodes
The query barcode sequences aligned in NCBI BLAST ensure alignment score and homology, and identified the closely related reference database library sequences for subsequent analysis. The nucleotide sequences set consists of sequence length, percent identity, query cover, and E. value of 0.0. After trimming of the targeted sequence along with reference sequences, generating scores in the final dataset as shown in (Table 3).
Molecular characterizations of DNA barcodes
For molecular characterization, a refined and well-developed FASTA file containing both the query and database accessions were uploaded in MEGA 1.5 software. After analysis, the total aligned sequence length, conserved, variable, parsimony informative and singletons were recorded (Table 4).
DNA barcode-based similarities indexes
Based on molecular characterisations, the monophyletic clade of query sequences was confirmed with database sequences using MEGA 11 software. The evolutionary history was inferred using different methods like blast similarities and percent identities. The optimal tree is shown in a different clade with bootstrap support. The distances were computed through Kimura 2-parameter method and are in the units of the number of base substitutions per site. All the missing positions were eliminated from the data. In the phylogenetic tree, the query sequence share clade with reference database sequences (Table 5).
Neighbour-joining tree based on trnA and ycf3
Construction of a phylogenetic tree based on the NJ method for the query sequence obtained through trnA and ycf3, and the reference sequences were used to inferred the evolutionary tree. In the phylogenetic tree, two major clades were formed. Based on ycf3 sequence, the query sequence Chaenomeles-LC919522 makes a close relationship with MN506261.1-C. japonica, NC035566.1-C. japonica, and MZ984211.1-C. japonica with bootstrap values of 26, 29, and 34 in clade B. Similarly, based on trnA the query sequence Chaenomeles-LC919521 makes a close relationship with MN506261.1-C. japonica, NC035566.1-C. japonica, and MZ984211.1-C. japonica with bootstrap values of 21, 22, 24 and 28 in clade B highlighted with green colour. While the remaining members in clade A of both candidate barcodes showed and shared a clade with NC056885.1-C. speciose, MT937182.1-C. speciose, MZ984212.1-C. speciose, KT932965.1-C. speciose, OL450372.1-C. speciose, MT561270.1-C. cathayensis, MN506260.1-C. cathayensis, NC045392.1-C. cathayensis, and NC049863.1-C. thibetica are highlighted with red colour (Figs 2 and 3).
Neighbour-joining tree based on matK and rbcLa
Similarly, based on the matK and rbcLa, the trees were classified into two clades, i.e., A and B. The query Chaenomeles, based on rbcLa sequence Chaenomeles-LC919520 makes a close relationship with MN506261.1-C. japonica, NC035566.1-C. japonica, MZ984211.1-C. japonica, and MN192833.1-C. japonica with bootstrap support of 35 and 58 in clade B highlighted with green (Figs 3 and 4). Similarly, based on the matK sequence, the query Chaenomeles-LC919519 makes a close relationship with MN506261.1-C. japonica, NC035566.1-C. japonica, MZ984211.1-C. japonica, and MN192833.1-C.japonica withbootstrap support of 54, 63, and 100 (Figs 4 and 5). While the rest of the members in clade A of both candidate barcodes showed and shared a clade with NC056885.1-C. speciose, MT937182.1-C. speciose, MZ984212.1-C. speciose, KT932965.1-C. speciose, OL450372.1-C. speciose, MT561270.1-C. cathayensis, MN506260.1-C. cathayensis, NC045392.1-C. cathayensis, and NC049863.1-C. thibetica highlighted with red colour (Figs 4 and 5).
Discussion
This study provides preliminary molecular evidence supporting the identification of a cultivated specimen as Chaenomeles japonica in Pakistan. Rather than constituting floristic confirmation of a wild population, the finding contribute to the molecular characterization of a horticulturally introduced ornamental taxon. The use of four chloroplast loci such as (matK, rbcLa, trnA, and ycf3) allowed assessment of multilocus barcode performance within the genus Chaenomeles and provided comparative insight into their discriminatory capacity.
In family Rosaceae, plastid loci often exhibit limited interspecific divergence, and species delimitation typically benefits from multilocus approaches combining plastid and nuclear markers [29,30]. In the present study, each chloroplast locus contributed partial resolution; however, none independently provided unequivocal species discrimination with strong statistical support.
Among the markers, matK exhibited the highest sequence variability and produced 100% BLAST identity with multiple C. japonica reference sequences from Japan, China, and the United States. Its greater number of variable and parsimony-informative sites compared with rbcla is consistent with previous studies identifying matK as one of the more informative plastid barcode in Rosaceae.
In contrast rbcLa showed lower variability and 98% identity, reflecting its conserved nature. While useful for higher-level taxonomic placement, rbcLa alone provided limited species-level resolution.. The additional loci trnA and ycf3, demonstrated moderate variability but did not substantially increase phylogenetic support when analyzed independently. Their contribution was complementary rather than decisive. When evaluated collectively, the multilocus dataset increased confidence in sequence similarity results, but phylogenetic support values remained low across several internal nodes [31,32].
Phylogenetic reconstruction using all markers placed the Pakistani specimen within a strongly supported C. japonica clade, clustering with reference sequences from China (MN506261.1), Japan (LC626016.1), and the United States (NC035566.1). The clear separation from C. speciosa, C. cathayensis, and C. thibetica in the sister clade confirms accurate species delimitation. These findings closely match global phylogenetic works on Chaenomeles, which consistently recognises C. japonica as a distinct lineage characterised by unique chloroplast haplotypes [29,33].
Comparable clustering patterns have been reported from Europe, where barcoding was used to authenticate horticulturally important Chaenomeles accessions and distinguish hybrids from pure species [7]. Similarly, North American studies have shown that plastid DNA reliably separates C. japonica from naturalised or misidentified specimens in ornamental landscapes [6]. The alignment of the Pakistani specimens with these international lineages confirms that the material introduced in the Hazara region corresponds to genetically validated C. japonica.
Morphological identification of Chaenomeles species can be challenging due to overlapping vegetative traits and reported -hybridisation among congeners [8,9]. In cultivated settings where reproductive characters may be absent, molecular barcoding provides an objective supplementary tool. In the present study, DNA sequence similarity offered supportive evidence for identification where morphology alone was inconclusive.
However, it is important to emphasize that DNA barcoding functions most effectively when supported by comprehensive reference sampling and multilocus data, ideally including nuclear markers such as ITS or other single-copy nuclear genes.
The confirmation of C. japonica as a new record for Pakistan is biogeographically significant. Despite the species widespread cultivation in temperate regions of the world, such as Europe, Central Asia, and North America, its existence in Pakistan has never before been documented in botanical literature, herbarium collections, or natural checklists. Because C. japonica is prized for its flowers, fruits, and adaptability, its presence in the Hazara region is consistent with global patterns of horticultural importation [5].
Although C. japonica is not widely acknowledge as an aggressive invader, research from Latvia, Romania, and some regions of the United States of America suggest that it can naturalise along riverbanks, forest edges, and semi-natural habitats [6,7]. This highlights the need to monitor its ecological behaviour in Pakistan, especially in sensitive Himalayan foothill ecosystems. Documenting new species is essential for early detection, management planning, and accurate updates to national biodiversity databases [13].
This study illustrates the usefulness of combining DNA barcoding with ongoing horticultural species monitoring and highlights the significance of molecular identification in identifying neglected taxa by confirming C. japonica using several independent molecular markers and phylogenetic analysis.
Conclusion
This study provides preliminary chloroplast DNA evidence supporting the identification–of a cultivated specimen as C. japonica within the genus Chaenomeles in Pakistan. The analysis of four plastid loci (matK, rbcLa, trnA, and ycf3) demonstrated that multilocus chloroplast barcoding can assist in the molecular characterization of ornamental taxa when morphological identification is uncertain or limited.
Among the markers examined, matK showed comparatively higher variability, whereas rbcLa displayed lower discriminatory capacity, consistent with its conserved nature. When interpreted collectively, the four loci provided sequence similarity patterns consistent with reference accessions of C. japonica. However, phylogenetic reconstruction based solely on plastid data yielded generally low bootstrap support values, limiting confidence in species level resolution.
Because the analysis was conducted on a single cultivated individual and did not incorporate nuclear markers or population-level sampling, the finding should be regarded as tentative molecular evidence rather than definitive taxonomic confirmation. Importantly, this study documents the molecular identity of a cultivated ornamental specimens and does not confirm the occurrence of a naturalized or wild population in Pakistan’s flora.
References
- 1. Moneva K, Gancheva S, Valcheva-Kuzmanova S. Chemical composition and biologic activities of different preparations of Japanese quince (Chaenomeles japonica). Acta Sci Nat. 2023;10(2):39–54.
- 2. Samatova S, Berdiyev M, Baysunov B, Zafar M, Majeed S, Ramadan MF, et al. Phenological and morphological adaptations of Japanese quince (Chaenomeles japonica—(Thunb.) Lindl. ex Spach.) [Rosaceae]: insights from palynology and leaf histology. Genet Resour Crop Evol. 2025:1–13.
- 3. Marat N, Danowska-Oziewicz M, Narwojsz A. Chaenomeles species-characteristics of plant, fruit and processed products: a review. Plants (Basel). 2022;11(22):3036. pmid:36432767
- 4. Kostecka-Gugała A. Quinces (Cydonia oblonga, Chaenomeles sp., and Pseudocydonia sinensis) as medicinal fruits of the Rosaceae family: current state of knowledge on properties and use. Antioxidants. 2024;13(1):71.
- 5. Gailis J, Jakobija I, Jakobsone E, Ozoliņa-Pole L, Rancāne R, Salmane I. Studies on potential pests of Japanese quince (Chaenomeles japonica (Thunb.) Lindl. ex Spach) in Latvia. Rural Sustain Res. 2023;50(345):16–30.
- 6. Serviss BE, Tumlison R. Guide to the naturalized, escaped, and adventive woody flora of Arkansas. Phytoneuron. 2021;29:1–193.
- 7. Cristea AM, Smeu A, Cîmpeanu I-A, Iftode A, Liga S, Tchiakpe-Antal D-S, et al. Biological effects of rosaceae species in skin disorders-an up-to-date overview. Plants (Basel). 2025;14(11):1605. pmid:40508280
- 8. Argiropoulos A, Spanakis E, Rhizopoulou S. Functional micromorphology of petals of Chaenomeles japonica exposed to humid and cold season. Acta Physiol Plant. 2017;39(11):246.
- 9. Wang C, Zhang L, Liu Y, Sun G. Hybridization and morphological plasticity in Chaenomeles (Rosaceae): implications for taxonomy. Pl Syst Evol. 2023;309(2):27.
- 10. Pysek P, Hulme PE, Meyerson LA, Smith GF, Boatwright JS, Crouch NR, et al. Hitting the right target: taxonomic challenges for, and of, plant invasions. AoB PLANTS. 2013;5(0):plt042–plt042.
- 11. Sommer RJ. Phenotypic plasticity: from theory and genetics to current and future challenges. Genetics. 2020;215(1):1–13. pmid:32371438
- 12. Yang F, Ding F, Chen H, He M, Zhu S, Ma X, et al. DNA barcoding for the identification and authentication of animal species in traditional medicine. Evid Based Complement Altern Med. 2018;2018:5160254. pmid:29849709
- 13. Sajjad S, Islam M, Muhammad K, Hazzazi Y, Sumayli M, El-Shabasy A. Carya illinoinensis (Juglandaceae): a new genus and species for the flora of Pakistan. Ann Appl Biol. 2025:1–9.
- 14. Al-Juhani W. Evaluation of the capacity of the DNA barcode ITS2 for identifying and discriminating dryland plants. Genet Mol Res. 2019;18(1):32–46.
- 15. Ahmed SS. DNA barcoding in plants and animals: a critical review. 2022.
- 16. Sheth BP, Thaker VS. DNA barcoding and traditional taxonomy: an integrated approach for biodiversity conservation. Genome. 2017;60(7):618–28. pmid:28431212
- 17. Gill K, Negi S, Kumar P, Irfan M. Improved genomic DNA extraction from citrus species using a modified CTAB method. Mol Biol Rep. 2025;52(1):638. pmid:40569330
- 18. Nihad SAI, Kabir A, Rashid MM, Honey O, Khan MAI, Latif MA. Comparative assessment of quick and cost-efficient quality DNA extraction methods from rice leaves. 2022.
- 19. Sajjad S, Islam M, Muhammad K, Ghafoor S-U, Ullah I, Khan A, et al. Comprehensive evaluation of cryptic Juglans genotypes: insight from molecular markers and phylogenetic analysis. Genes (Basel). 2024;15(11):1417. pmid:39596617
- 20.
Javaid A, Wani S, Iralu N, Javaid L, Meghanath D, Ali G, et al. Extraction of high-quality genomic DNA from plants using modified CTAB-based method. In: Detection of plant viruses: advanced techniques. Springer; 2025. p. 69–75.
- 21. Nigmatullina NV, Kuluev AR, Kuluev BR. Molecular markers used to determine the genetic diversity and species identification of wild plants. Biomics. 2018;10(3):290–318.
- 22. Dong W, Cheng T, Li C, Xu C, Long P, Chen C, et al. Discriminating plants using the DNA barcode rbcLb: an appraisal based on a large data set. Mol Ecol Resour. 2014;14(2):336–43. pmid:24119263
- 23. Shaw J, Lickey EB, Schilling EE, Small RL. Comparison of whole chloroplast genome sequences to choose loci for plant DNA barcoding. Syst Bot. 2007;32(2):354–65.
- 24. Dong W, Xu C, Li C, Sun J, Zuo Y, Shi S, et al. ycf1, the most promising plastid DNA barcode of land plants. Sci Rep. 2015;5:8348. pmid:25672218
- 25. Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL. NCBI BLAST: a better web interface. Nucleic Acids Res. 2008;36(Web Server issue):W5-9. pmid:18440982
- 26. Hall T, Biosciences I, Carlsbad C. BioEdit: an important software for molecular biology. GERF Bull Biosci. 2011;2(1):60–1.
- 27. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23(21):2947–8.
- 28. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 2007;24(8):1596–9. pmid:17488738
- 29. Hall BG. Building phylogenetic trees from molecular data with MEGA. Mol Biol Evol. 2013;30(5):1229–35. pmid:23486614
- 30. Li Q, Fan S, Zhang H, Liu X, Zhang X. Phylogenetic analysis of Chaenomeles species based on chloroplast DNA sequences. Biochem Syst Ecol. 2017;70:243–50.
- 31. Yan H, Liu Y, Li Q, Zhang X. Performance of matK and rbcL in discriminating Rosaceae species. Mitochondrial DNA Part A. 2018;29(7):1107–15.
- 32. Liu J, Li X, Yang L, Zhao Y. Evaluation of chloroplast markers trnA and ycf3 for DNA barcoding in Rosaceae. Plant Divers. 2020;42(2):65–74.
- 33. Zhao Y, Yang L, Liu J, Wang S. Assessment of ycf3 and trnA regions for species identification in Rosaceae. Pl Ecol Evol. 2021;154(3):329–39.