Rice (Oryza sativa L.) ranks as one of the most widely cultivated crops worldwide, serving as a staple food for over half of the global population (Mursyidin et al., 2021). Vietnam, a prominent rice-producing nation, is home to an extensive diversity of traditional rice varieties that have been cultivated for generations and exhibit remarkable adaptability to diverse local environmental conditions (Han et al., 2025). These landraces possess invaluable genetic traits, including pest resistance, tolerance to abiotic stresses and distinct nutritional characteristics, making them indispensable for rice breeding programs and genetic conservation initiatives (Jekkaral et al., 2025). However, the increasing reliance on high-yielding hybrid varieties and modern agricultural practices has contributed to a decline in the cultivation of traditional landraces, thereby placing their genetic diversity at significant risk (Bonnin et al., 2014). Accurate identification and classification of these traditional varieties are vital for their conservation, utilization in breeding programs and safeguarding under intellectual property laws.
       
Historically, rice varieties have been identified based on morphological and agronomic traits, such as grain shape, plant height, flowering time and resistance to biotic and abiotic stresses (Akter et al., 2025). However, these traits are frequently influenced by environmental factors, resulting in inconsistencies in classification (Patindol et al., 2015). Biochemical markers, including isozymes and protein profiles, have also been employed for varietal discrimination but generally lack the resolution required to distinguish closely related genotypes (Eevera and Vanangamudi, 2009). In contrast, DNA-based methodologies offer a precise and objective approach to varietal identification, enabling the detection of genetic differences at the molecular level (Azizi et al., 2021). Among these, DNA barcoding has emerged as an efficient tool for species authentication and genetic diversity assessment. This technique employs short, standardized DNA sequences to differentiate species and infer phylogenetic relationships, providing a rapid and reliable method for plant identification (Hebert et al., 2003).
       
In plants, the matK (maturase K) and rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit) genes have been recommended by the Consortium for the Barcode of Life (CBOL) as core DNA barcode markers for land plants (CBOL Plant Working Group, 2009). While the rbcL gene is relatively conserved and suitable for broad taxonomic classification, the matK gene demonstrates higher sequence variability, offering superior resolution for distinguishing closely related taxa (Mursyidin et al., 2021). These markers have been successfully applied in various plant barcoding studies, including research on economically significant crops such as leguminosae, grass forages and rice (Li et al., 2022; Singh et al., 2017).
       
Despite the growing application of DNA barcoding in plant systematics and crop research, limited efforts have systematically utilized this technique to characterize the genetic diversity of traditional rice varieties in Vietnam. For giving the extensive genetic wealth of Vietnamese rice landraces, establishing a barcode reference dataset based on matK and rbcL sequences would provide critical support for species authentication, germplasm management and biodiversity conservation.
       
This study analyzed 90 traditional rice accessions collected from Northern, Central and Southern regions of Vietnam using matK and rbcL barcode markers. The key objectives were to (1) Evaluate the effectiveness of these chloroplast genes for species-level discrimination, (2) Assess genetic diversity and phylogenetic relationships among the accessions and (3) Develop a molecular framework for classifying and conserving traditional Vietnamese rice varieties. By employing DNA barcoding for varietal authentication, this research contributes to the conservation of rice genetic resources, enhances understanding of genetic diversity within traditional Vietnamese landraces and supports breeding programs aimed at developing improved rice varieties with desirable traits. Furthermore, this study holds broader implications for agricultural biodiversity conservation, food security and safeguarding indigenous genetic resources under international intellectual property frameworks.

Plant materials
 
A total of 90 traditional rice accessions from Vietnam were analyzed in this study. These accessions were collected in 1994 and 2015, representing three major geographic regions: Northern, Central and Southern Vietnam. All seed samples were conserved at the plant resource center, where each accession was assigned a unique voucher code for reference in data presentation (Table 1).


       
For germination, seeds were wrapped in cotton cloth, soaked in water overnight and incubated at 37°C for two days under moist conditions to promote sprouting. Germinated seeds were subsequently sown in soil trays. Young leaves were harvested from seedlings at a height of 15–20 cm for DNA extraction. Molecular experiments were conducted at the Agricultural Genetics Institute in 2024.
 
DNA extraction and amplification
 
Genomic DNA was extracted from fresh leaf tissue using the TopPURE® Food DNA Extraction Kit (ABT, Vietnam), following the manufacturer’s instructions. DNA quantity and quality were assessed using a Nanodrop 2000 UV-VIS Spectrophotometer (Thermo Scientific, USA). The matK and rbcL regions of the chloroplast genome were amplified using universal primers (sequences shown in Table 2). PCR was performed in a 25 µL reaction volume containing 20 ng of genomic DNA, 0.2 µmol of each primer and MyTaq HS Red Mix (Bioline, UK). The PCR cycling conditions were as follows: Initial denaturation at 94°C for 3 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 40 seconds, extension at 72°C for 45 seconds, final extension at 72°C for 7 minutes. PCR products were separated by electrophoresis on a 2% agarose gel in 1X TBE buffer, stained with GelRed (ABT, Vietnam) and visualized under UV transillumination. Successfully amplified products were purified and sequenced bidirectionally using the Sanger method. DNA sequence data were obtained in ABI, FASTA and chromatogram formats.


 
Data analysis
 
Forward and reverse reads were processed with trimming by using BioEdit software. Species identification was performed utilizing the BLAST tool on the NCBI platform. Multiple sequence alignments were conducted with CLUSTAL W. Phylogenetic analysis and genetic diversity evaluation were executed using MEGA 11, employing the UPGMA method with the Tamura-Nei model. The reliability of tree topology was assessed through bootstrap analysis with 1,000 replicates (Tamura et al., 2021).

Analysis of MatK sequences in 90 local rice varieties collected from Vietnam
 
PCR amplification of the matK gene was successful in 77 out of 90 rice varieties, achieving a success rate of 85.56%. Each amplified sample exhibited a distinct, single band of approximately 900 bp, consistent with the expected amplicon size of the matK region, with no evidence of non-specific amplification. This high amplification efficiency underscores the reliability of the selected primers for analyzing Vietnamese local rice germplasm.
       
The nucleotide composition of the matK sequences averaged 29.4% adenine (A), 35.8% thymine (T), 16.2% guanine (G) and 18.6% cytosine (C).
       
Multiple sequence alignment of matK gene sequences from 77 Vietnamese local rice varieties, performed using MEGA software, identified 11 single nucleotide polymorphisms (SNPs) at positions 218, 231, 792, 843, 844, 845, 847, 848, 849, 867 and 875 (data not shown).
       
Most of the varieties shared conserved nucleotides at these positions; however, a few accessions showed unique SNP profiles. Hai bong (sample 32) exhibited a unique T substitution at position 218, where all other samples had C. Nep bo (sample 5) showed two SNPs at positions 231 (A) and 792 (T), in contrast to the conserved G allele. Ran trang (sample 55) presented a deletion (gap) at position 875. Ba la (sample 78) had substitutions at positions 843 (T) and 847 (T). Beo but bua (sample 112) displayed the most divergence, with unique alleles at four consecutive positions: 844 (G), 845 (A), 848 (C) and 849 (T). These SNP patterns provide clear molecular distinctions among certain rice accessions.
       
A phylogenetic tree was constructed based on matK sequences using the UPGMA method under the Tamura-Nei model, supported by 1,000 bootstrap replicates (Fig 1). The resulting tree had a total branch length of 0.009 and pairwise genetic distances ranged from 0.000 to 0.007, with an average of 0.0033. The phylogenetic tree revealed two major clades. Clade I consisted exclusively of Beo but bua (sample 112), indicating its strong genetic divergence from the remaining accessions. Clade II included the remaining 76 samples, which were further divided into smaller subclusters. Notably, Nep bo (5) formed a distinct branch. Hai bong (32) and Ba la (78) also branched separately from the core group.


 
Analysis of rbcL sequences in 90 local rice varieties from Vietnam
 
All 90 local rice accessions produced successful PCR amplification of the rbcL gene, with 100% amplification efficiency. Amplicons were approximately 600 base pairs in length and showed sharp, single bands on agarose gels, confirming both high DNA quality and primer specificity.
       
Nucleotide composition analysis of the aligned rbcL sequences revealed an average of 26.7% adenine (A), 29.1% thymine (T), 23.2% guanine (G) and 20.9% cytosine (C). The sequence lengths varied from 552 bp (sample 83 -Tam thom ap be) to 571 bp (sample 20-Nep do).
       
Alignment and polymorphism analysis using MEGA software identified seven SNP positions at nucleotide sites 27, 28, 29, 30, 31, 42 and 543 (data not shown). The majority of accessions shared the consensus nucleotide pattern A - (–) - (–)-C-T - (–) - C, but eight samples displayed clear sequence divergence. Samples 6, 11, 43, 44, 45, 52, 93 and 99 exhibited unique combinations of substitutions at positions 27 (T), 28 (A), 29 (C) and 30 (T). Several of these also had distinct alleles at positions 31 and 42, particularly showing the presence of an “A” nucleotide at position 42 instead of a deletion found in most accessions. These variants indicate possible evolutionary divergence or environmental adaptation among these accessions.
       
A phylogenetic tree was constructed using the UPGMA method and the Tamura-Nei model, with 1,000 bootstrap replicates (Fig 2). The tree revealed a total branch length of 0.003 and pairwise genetic distances among accessions ranged from 0.000 to 0.004, with an average of 0.00053, indicating relatively low overall genetic diversity.The tree clearly separated the 90 rice accessions into two major clades. Group I included the eight genetically divergent samples listed above. These varieties occupied peripheral branches and showed multiple unique SNPs. Group II comprised the remaining 82 accessions, which were tightly clustered and displayed minimal sequence divergence, reflecting high genetic similarity.


       
This study provides the first comprehensive evaluation of genetic diversity among 90 local Vietnamese rice varieties using two chloroplast DNA barcoding markers, matK and rbcL. By integrating both markers, we were able to assess sequence variation and infer phylogenetic relationships across landraces collected from northern, central and southern Vietnam.
       
The amplification efficiency for matK reached 85.56%, higher than previously reported by Singh et al. (2017), who achieved 66.2% amplification across diverse rice genotypes using multiple primer sets. A consistent about 900 bp amplicon was observed in 77 accessions, confirming the reliability of the selected matK primers for Vietnamese rice germplasm. Meanwhile, rbcL demonstrated a 100% amplification success rate, producing clean 600 bp bands in all 90 accessions, reaffirming its high conservation and primer robustness. These findings support the utility of both matK and rbcL as effective tools for initial genetic diversity assessments in rice.
       
The matK region showed moderate variability, with eleven informative SNPs across 77 accessions, while rbcL revealed only seven SNPs across 90 accessions- consistent with the low mutation rate typically observed in rbcL for angiosperms (Clegg, 1993). Despite its low variability, rbcL analysis revealed meaningful divergence in a small subset of accessions, highlighting its complementary value in diversity studies.
       
Notably, the rbcL gene sequence analysis revealed a generally low level of genetic diversity among Vietnamese local rice varieties, with an average genetic distance of just 0.00053. This narrow genetic base likely stems from traditional farming practices, prolonged local adaptation and limited seed exchange, all of which have contributed to the high genetic homogeneity observed across most accessions.
       
However, eight accessions - Vang nghe (sample 6), Tam thom ap be (sample 11), Te hat dai (sample 43), Xe dang (sample 44), Nep luong (sample 45), Cam (sample 52), Chong ba la (sample 93) and Khau cai ca (sample 99) - were clearly differentiated from the remaining samples, forming a distinct phylogenetic clade (Group I) in the rbcL-based tree. These accessions were genetically distinct and carried unique allelic combinations at multiple SNP positions, most notably at positions 27, 28, 29, 30 and 42. These polymorphisms included base substitutions (e.g., T replacing A or C) and an insertion at position 42. Such SNPs serve as potential diagnostic markers for varietal grouping and molecular identification, underscoring their utility in rice germplasm characterization and genetic resource management.
       
The identification of this genetically distinct Group I suggests that these accessions may harbor valuable agronomic traits or unique ecological adaptations. As such, they should be prioritized in conservation efforts-both in situ and ex situ-and considered as important genetic materials for future breeding programs aimed at improving stress tolerance, yield stability, or local adaptation.
       
Phylogenetic reconstruction using the UPGMA method and Tamura-Nei model revealed clear and consistent clustering patterns. The matK-based tree grouped 76 accessions into a large, homogeneous cluster (Group II), while Beo but bua (sample 112) formed a separate clade (Group I), supported by its unique SNP profile and longer branch length. Similarly, the rbcL tree divided accessions into two main groups: Group I, composed of the eight genetically distinct accessions mentioned above and Group II, comprising the remaining 82 samples with highly conserved sequences. Although the total branch length in the rbcL tree was relatively short (0.003), compared to matK (0.009), it still captured meaningful variation, particularly among outlier accessions. These findings align with previous studies by Mursyidin et al. (2021) and Dang et al., (2021) which reported that matK generally offers greater resolution in phylogenetic analyses, though rbcL can still detect key divergences, particularly when combined with other markers.
       
Despite these observations of divergent lineages, overall nucleotide diversity remained low across both markers-0.0033 for matK and 0.00053 for rbcL. This genetic uniformity likely reflects the effects of domestication bottlenecks, local seed-saving traditions and limited gene flow across geographical regions. Such trends have also been observed in other crops subjected to similar cultural and ecological pressures (Johnston-Monje and Raizada, 2011).
       
Nonetheless, accessions identified as outliers in both SNP and phylogenetic analyses -particularly Beo but bua (sample 112), Chong ba la (sample 93) and Khau cai ca (sample 99) -warrant further attention. These varieties may harbor rare alleles associated with adaptive traits and should be prioritized for in-depth molecular characterization, agronomic evaluation and conservation. As noted by Wilberg (2015), peripheral clustering in phylogenies often corresponds to ancestral or underutilized lineages, which may hold untapped potential for crop improvement.
       
In summary, this study demonstrates that while rbcL exhibits a lower level of polymorphism compared to matK, it remains a valuable complementary marker for detecting genetic differentiation among closely related rice varieties. The identification of eight distinct accessions within Group I and their unique SNP profiles-especially at positions 27-30 and 42-highlights the marker’s applicability in varietal classification and conservation. These findings reinforce the role of chloroplast DNA markers in supporting sustainable rice breeding and the preservation of traditional germplasm in Vietnam.

This study presents the first comprehensive assessment of chloroplast DNA diversity in 90 Vietnamese rice landraces using matK and rbcL markers. A total of 18 informative polymorphic sites were identified, revealing both conserved and divergent maternal lineages across agro-ecological regions. The matK marker demonstrated higher resolution for detecting intraspecific variation, while rbcL offered complementary support for broader phylogenetic relationships. Notably, genetically distinct landraces such as Beo but bua, Chong ba la and Khau cai ca were consistently differentiated, representing valuable genetic resources for future breeding and conservation efforts. The narrow range of chloroplast variation observed among most accessions highlights the risk of genetic erosion and the need for urgent conservation strategies. This DNA barcode dataset, combined with SNP profiling, provides an effective and cost-efficient tool for varietal identification, germplasm management and monitoring of genetic integrity. Integration of these molecular findings into national rice conservation strategies is recommended to enhance sustainable utilization and long-term preservation of Vietnam’s traditional rice germplasm.

The present study was supported by the program: The Project for the Development of Biotechnology in the Agricultural Sector by 2030, Ministry of Agriculture and Environment  of Vietnam.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.