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Edited by

Idrees Ahmed NasirUniversity of the Punjab, Lahore, Pakistan

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Aiman ZahraVirtual University of Pakistan
Muhammad Azam AliVirtual University of Pakistan

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  • Full Length Research Article
  • Date Received: 20-03-2024
  • Date Revised: 01-12-2025
  • Date Accepted: 24-01-2026
  • Date Published: 31-03-2026
Sequence analysis of rbcL and matK regions for a comparison study of Senna species
Patcharawarin Ruanto1, Somruthai Tunma2, Natthiya Chaichana3
  1. Biology program, Faculty of Education, Chiang Rai Rajabhat University, Thailand
  2. Chemistry program, Faculty of Education, Chiang Rai Rajabhat University, Thailand
  3. Science program, Faculty of Education, Chiang Rai Rajabhat University, Thailand

Abstract

Background: DNA barcoding is an efficient molecular biology technique that utilizes a short genetic locus with sufficient variability to enable precise organism identification. Typically, regions such as ribulose-bisphosphate carboxylase (rbcL) and maturase K (matK) in plants are widely used due to their balance between interspecific divergence and intraspecific conservation. These standardized genomic fragments are amplified by Polymerase Chain Reaction (PCR) and subsequently sequenced for comparative analysis. Every species has its own characteristic DNA barcode, which can be matched against curated reference libraries, enabling accurate identification even when morphological traits are ambiguous, damaged, or insufficient for taxonomic classification. This approach has been particularly transformative in biodiversity monitoring, ecological studies, and the detection of cryptic or invasive species. Presenting DNA barcodes in a graphical form provides an alternative and powerful way to store and display sequence information as it facilitates cross-comparison. The generated graphical outputs can be incorporated into machine learning algorithms for species recognition in further large-scale ecological and conservation research.

Methods: The matK and rbcL genes of the collected five senna species were selected as DNA barcodes to confirm and distinguish these plant samples. An alternative method was also presented to extract the characteristics of the DNA sequences with graphical operation based on Chaos Game Representation (CGR).

Results: It was found that the power to discriminate between species was high enough when a two-locus barcode approach was applied with 90% successful amplification using the provided protocol optimization. The similarity/dissimilarity comparison of the collected plant samples was also achieved by the obtained CGR.

Conclusion: Grouping of individuals based on genetic relationship was consistent with morphology and taxonomy, particularly when the primers for matK were used, whereas the rbcL barcode was less effective in distinguishing species.

Keywords

DNA barcode, matK, rbcL, Senna species, Chaos Game Representation

Introduction

The uniqueness of plants is studied through a number of methods including examination of the morphological characteristics, which is the most common way.  However, plants in the same or related family can appear to have similar characteristics and essential substances [1].  Therefore, molecular biology technique is used in this study for conducting DNA barcoding studies and providing guidelines of plant identification. DNA barcoding is an effective molecular biology technique that is gaining a lot of attention. The technique uses short standardized genomic sequences to identify species through PCR amplification. It can be applied in evolutionary biology field, biodiversity, as well as conservation genetics. A short genetic locus used as DNA barcode must be highly variable enough to be able to quickly identify each organism. To effectively identify the organism, more than one location (locus) of DNA barcode is used. At present, there are several DNA loci that are agreed to be used as plant DNA barcodes, such as the internal transcribed spacer (ITS) [2], rbcL, trnL-F intergenic spacer[3], matK [4], ndhF, atpB [5,6]. There are a number of research studies that confirm the ability of matK as a global plant DNA barcode; however, the success is still limited in gymnosperms [7]. Currently, we can say that more than one location of standard DNA barcode is required in order to effectively identify the organism.  CBOL (the Consortium for the Barcode of Life) has proposed using a combination of matK and rbcL, gene loci found in chloroplasts, as a DNA barcode [8]. The matK gene (maturaseK gene) is located in the intron section II within the chloroplast DNA, encoding maturase enzyme which is related to RNA splicing process [9]. The rbcL gene (ribulose bisphosphate carboxylase gene) is an extremely important gene as the encoded enzyme is a subunit of ribulose bisphosphate carboxylase (RUBISCO), which is used in the process of CO2 fixation during photosynthesis. It is also popularly used as a marker in studies of evolutionary relationships.

Presenting DNA barcode in a graphical form is a technique to collect information and show accomplished results in a more beneficial way. A graphic display can be easily understood and its numerical data can be further used for DNA sequence comparison analysis. Until now, there are many formats of multidimensional sequence of DNA, each of which has its advantages and limitations. Chaos Game Representation (CGR) is a two-dimensional graphical representation of DNA sequence. It uses a simple walk model to locate coordinates in the orthogonal x y system[10]. Different biological data represented by different nucleotide sequences are taken into account in the model to conduct the result that is relevant to the chemical structures and uncomplicated to understand. The particular numerical characterization of DNA sequences is also a proper data form for the further study of similarities and dissimilarities of gene structures.

The CGR-Walk applies a nonlinear dynamical system that is based on chaos theory to CGR mapping [11]. Representation in this graphical form can be used to display patterns of gene sequences. Moreover, the algorithm allows fractal structure pictures to be viewed as continuous reference systems of which all four possible nucleotides occupy unique positions corresponding to vertices of a binary square. The binary CGR vertices are assigned to four nucleotides; A = (0, 0), G= (1, 1), C= (0, 1), T = (1, 0). The CGR coordinates are calculated iteratively by moving the plot to half the distance between the previous and the current positions of base corner. The function can be given by

CGR𝑖 = CGR𝑖−1 − 0.5 (CGR𝑖−1 − 𝑔𝑖),                                     (1)

Where;

𝑖 = 1, . . . , 𝑛G; CGR0 = (0.5, 0.5); 𝑔𝑖 ∈ {𝐴, 𝐺, 𝐶, 𝑇}.             (2)

The given formula represents a mathematical expression that maps DNA by starting in the center (0.5,0.5) and progressing halfway toward each nucleotide’s designated corner (A, T, G, and C). DNA sequences are transformed in this manner to produce a fractal-like image that encodes information about the DNA sequence’s structure.

Methods

Plant samples were collected from the Chiang Saen Lake (20°15’N., 100°2’E.), a wetland located in Yonok district, Chiang Saen, Chiang Rai province, Thailand.  Genomic DNA Extraction was accomplished using Viviantis GF-1 Plant DNA Extraction Kit. The DNA fragments of the targeted gene of the collected plants were amplified by Polymerase Chain Reaction (PCR), using primers and protocols for the matK and rbcL barcodes [12] as shown in Table 1. Then, the purified PCR fragments were sent to Macrogen Inc. (Korea) for sequencing. The sequence analysis of each sample was further performed using finchTV 1.4.0 to remove all ambiguous sequences, and MAFFT 7.0 as well as NTSYS (2.02pc)  were used for multiple sequence alignment and analysis of molecular phylogeny.

Then, the DNA sequence of each sample was converted into the pattern of CGR-RY walk, illustrated by virtual graph paper application for further DNA analysis.

Results

DNA amplification and sequencing

The collected 5 species of plants belonged to Leguminosae family as shown in Table 2. DNA amplification for PCR products of matK was successful for 5 out of 5 species, whereas the rbcL amplification failed to give a good quality PCR band for Senna siamea as shown in Table 2.

The size of PCR products at the regions of matK and rbcL were approximately 900 and 600 base pairs, respectively. The successful purified PCR samples were sent to Macrogen Inc. (Korea) for sequencing. The obtained nucleotide sequences of matK and rbcL genes were further used for multiple sequence alignment and phylogenetic analysis.

Phylogeny analysis of matK and rbcL barcodes

To study genetic relationship and classification of plant species, the CLUSTAL format alignment and molecular phylogeny analysis based on Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) of partial matK and rbcL barcode loci from 5 taxa were performed using NTSYS (2.02pc) to create the dendrogram as shown in Figure 1 and 2, respectively. For the matK barcode, S. tora, S. alata and S. siamea were closely related, as they were clustered together in one group, then joined together with S. occidentalis , while S. hirsuta was further isolated from the rest. The result indicated that nucleotide substitution rate in matK region was higher than rbcL. The successful amplified rbcL sequences of 4 species were used for constructing the molecular phylogenetic tree. The result was found to be compatible with the prior matK dendrogram, with S. tora and S. alata being more connected to one another than S. occidentalis, and S. hirsuta being the species most distant from the others.”

Two-dimensional graphical representation

The previous branching diagrams showed evolutionary relationship based on DNA sequences and allowed highly related species to cluster together. DNA sequence differences of S. alata, S. tora and S. siamea were very small (3% and 2% differences among 3 species for matK and rbcL respectively), therefore, they were grouped together and separated from S. occidentalis and S. hirsuta. Apart from a dendrogram, we proposed another method to compare clear visual similarities/dissimilarities of these 5 models of Senna species. In this research, the sequences of matK and rbcL barcoding regions as indicated in table 3 were used for all 5 species to create a two-dimensional (2D) graph of CGR walk that represented partial matK and rbcL sequences.

The 2D graphical techniques were used to analyze and visualize overall characteristics of DNA sequences and even for thorough consideration of specific positions. In addition, all data were obtained in order to convert the base sequences to a numerical form which still contained the same data without any elimination. The sizes and directions of vectors representing nucleotide sequences of different species that made up the graph were unique. Different genes generated various different 2D patterns. The repetitive patterns with same bases among species were also represented at the same time. However, the graphs clearly showed different leaps at positions containing different bases. The relationship between the 2D graph and DNA sequence of any species or any gene was one to one. Since N represented the length of the studied DNA, any i = 1, 2,…, N would be considered a vector with 4 possible directions depending on the sequence of the next base. As a result, DNA dissimilarity could be clearly expressed in the 2D graph even if there was only one base difference of the compared DNA as shown in figure 3.

From the 2D graphs of partial matK sequences of 5 senna species, it was found that the CGR patterns of S. hirsuta and S. occidentalis  were clearly different from other species, while S. alata, S. tora and S. siamea had more similar patterns. However, these three closely related species still had some different degrees of vectors as all 3 graphs had their own characteristics. For rbcL sequences, it was clear that S. hirsuta diverged the most from the remaining three species of which similarities were very high so that it was harder to differentiate S. alata, S. tora and S. occidentalis from each other using CGR graphs of rbcL sequences.

Discussion

All collected plant samples used in this study are medicinal species in the Leguminosae family commonly found in Thailand. A range of DNA sequence that is suitable to be used as a barcode must be sufficiently variable to discriminate among species, have appropriate length of DNA sequence for PCR and sequence analysis techniques and contain a conserved sequence located at both ends that are suitable for universal PCR primer design for a variety of species [6]. From the study, it was found that the prepared primers and conditions as mentioned in the procedure section showed good amplification of the matK and rbcL gene regions of all samples except the rbcL fragment of Senna siamea. The single DNA band of matK and rbcL during electrophoresis had the size as expected, approximately 900 and 600 base pairs, respectively.

DNA barcode could be used to group individuals based on genetic relationship, particularly when the primers for matK were used. The results were in agreement with the report of DNA barcoding in flowering plants that the rbcL genes appeared to have low effectiveness of evolutionary separation [5] and it was less effective in identification of some genera such as Dendrobium [13]. It was also found that a sequence of rbcL gene had lower rate of evolution when compared with the nucleotide sequences of other genes, therefore the information it provided alone to create a dendrogram of genetic relationship might not be enough [14]. Therefore, a two-locus global DNA barcode was more effective. Moreover, the altogether analysis of the matK gene and the rbcL gene showed that discrimination power between species was high enough for identification of 5 species of the collected Senna plants.

In addition, applying the Chaos Game Representation (CGR) to CGR-walk of the DNA sequences could be effectively used for comparison of similarities and dissimilarities between species. The 2D graph patterns of matK and rbcL barcoding regions, especially matK, of all five Senna species, were unique. It provided a more complete overview of biological information apart from a dendrogram. From the results, S. alata, S. tora and S. siamea were the closest in terms of DNA sequence. This indicated that 2D graphical representation was consistent with the evolutionary data as well. Although phylogenetic tree was sufficient to provide evolutionary relationship, CGR walk had more advantages as it was a graphical representation method that clearly displayed data of any length of DNA and easily detected differences between species, even with only slight sequence dissimilarities. Moreover, each base coordinate on the 2D graph could be converted into various digital forms for further downstream analysis.

While phylogenetic analysis requires the maximum amount of sequence information, CGR walk relies on characteristic patterns that can be representative of the sequence’s overall features without a detailed analysis of the entire sequence. The selected representative regions for CGR should capture the essential sequence (e.g., coding regions, conserved motifs, or functionally important domains). Results from phylogenetic trees and CGR may differ due to the use of alternative data summaries and assumptions. CGR provides rapid screening, while phylogenetic analysis provides detailed evolutionary relationships and fine-scale resolution. Therefore, both methods should be used together. When results diverge, the differences themselves serve as an informative aspect requiring further investigation.

DNA sequences play an important role in modern biological research because all the information of the hereditary and species evolution is contained in these macromolecules. This research is capable of providing useful guidelines to optimize procedures for DNA amplification in combination with DNA barcoding which can be applied as a fundamental protocol to construct the DNA database of native plants based on a two-locus global DNA barcode consisting of matK and rbcL. The research also presents a novel method to extract the characteristic of the DNA sequence with the graphical operation which can effectively achieve the similarity/dissimilarity comparison of different species.

Conclusion

Statement & Declarations

Author Contributions

Patcharawarin Ruanto presented the idea, designed the methodology, planned and conducted the experiments, performed the computations and data analysis, and wrote the manuscript.

Somruthai Tunma conducted field sampling and assisted in performing the experiments.

Natthiya Chaichana investigated plant morphology and identification, and assisted with DNA experiments and data analysis.

Acknowledgment

We wish to thank for financial support from Chiang Rai Rajabhat University and we would like to thank staff from Nong Bong Kai restricted hunting area, Chiang Saen District, Chiang Rai and the Institute of biodiversity and environment for local and ASEAN development for the support.

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Article Sections

Edited by

Idrees Ahmed NasirUniversity of the Punjab, Lahore, Pakistan

Reviewed by

Aiman ZahraVirtual University of Pakistan
Muhammad Azam AliVirtual University of Pakistan

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Tables

Editors & Reviewers

Edited by

Idrees Ahmed NasirUniversity of the Punjab, Lahore, Pakistan

Reviewed by

Aiman ZahraVirtual University of Pakistan
Muhammad Azam AliVirtual University of Pakistan

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Tables

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