Edubiotik : Jurnal Pendidikan.
Biologi dan Terapan ISSN 2528-679X .
ISSN 2597-9833 .
Vol.
No.
November 2025, pp.
398 Ae 411 Available online at:
https://ejurnal.
id/index.
php/edubiotik Research Article Development of primers amplifying DNA barcoding genes matK and rbcL of Legundi (Vitex trifoli.
Nadhifah Riski Hartanto 1,a.
Banuwati Kartika Sari 1,b.
Triasuti Rahayu 1,c.
Rima Munawaroh 2,d.
Muammar Fawwaz 3,e.
Bambang Purwono 4,f.
Dzarifah Zulperi 5,g.
Trio Ageng Prayitno 6,h.
Yasir Sidiq 1,i,* 1 Department of Biology Education.
Universitas Muhammadiyah Surakarta.
Surakarta.
Indonesia 2 Department of Pharmaceutical.
Universitas Muhammadiyah Surakarta.
Surakarta.
Indonesia 3 Department of Pharmaceutical Science.
Universitas Muslim Indonesia.
Makassar.
Indonesia 4 Laboratory of Organic Chemistry.
Universitas Gadjah Mada.
Yogyakarta.
Indonesia 5 Department of Plant Protection.
Universiti Putra Malaysia.
Selangor.
Malaysia 6 Department of Biology Education.
Universitas Insan Budi Utomo.
Malang.
Indonesia Email: a420210038@student.
id 1,a, a420210037@student.
id 1,b, tr124@ums.
id 1,c, munawaroh@ums.
id 2,d, muammar.
fawwaz@umi.
id 3,e, purwono.
bambang@ugm.
id 4,f, dzarifah@upm.
my 5,g, trioagengprayitno@uibu.
id 6,h, ys120@ums.
id 1,i,* * Corresponding author Article Information Article History:
Submitted: 2025-04-21 Revised: 2025-12-28 Accepted: 2025-12-30 Published: 2025-12-31 Keywords:
DNA Barcoding.
matK gene.
rbcL gene.
PCR.
Publisher Biology Education Department Universitas Insan Budi Utomo.
Malang.
Indonesia
ABSTRACT
Legundi (Vitex trifoli.
is one of the crucial ethno-pharmacological plants.
However, the genetic exploration of these plants in Indonesia remains limited.
Moreover, a set of primers can be an initial important step to explore the genetics of V.
trifolia as well as to molecularly identify the species of Legundi.
This study aimed to develop the two pairs of primers of maturase-K .
atK) and ribulose bisphosphate carboxylase large subunit .
bcL) genes.
This type of research is exploratory research using a quantitative approach.
The research sampling are the leaves of the V.
trifolia species, which were collected from Makassar.
Indonesia, and the purposive sampling method.
The data were obtained through DNA barcoding, and the data were analyzed using bioinformatics analysis.
This study found two pairs of developed primers, matK and rbcL, successfully amplified both matK and rbcL target genes of V.
The newly developed species-specific primers successfully amplified the matK and rbcL genes of V.
trifolia, and sequence analysis revealed high similarity values in BLAST and BOLD databases ranging from 99.
2 to 100%, with PCR amplification of the matK marker showing particularly high DNA concentration and specificity for species-level identification.
This study supports the genetic exploration and identification of useful ethno-pharmacy plants.
The conclusion of the study shows that two primer pairs of maturase-K .
atK) and ribulose bisphosphate carboxylase large subunit .
bcL) genes have been developed from V.
This study supports the genetic exploration and identification of ethnopharmaceutical plants of V.
trifolia and the role of bioinformatics tools in molecular studies of medicinal plants.
How to Cite Hartanto.
Sari .
Rahayu.
Munawaroh .
Fawwaz .
Purwono .
A Sidiq, .
Development of primers amplifying DNA barcoding genes matK and rbcL of Legundi (Vitex trifoli.
Edubiotik : Jurnal Pendidikan.
Biologi Dan Terapan, 10.
, 398Ae411.
https://doi.
org/10.
33503/ebio.
Copyright A 2025.
Hartantoet al.
This is an open-access article under the CC-BY-SA license edubiotik@uibu.
: https://doi.
org/10.
33503/ebio.
Edubiotik : Jurnal Pendidikan.
Biologi dan Terapan Vol.
No.
, pp.
398 Ae 411
INTRODUCTION
Vitex trifolia, commonly known as legundi plant, is one of the species of the Vitex genus (Verbenacea.
whose active compounds are commonly used as traditional medicine.
Vitex trifolia is widely used by various countries in Asia, such as China (Wee et al.
, 2.
Malaysia (Abas et al.
, 2.
India (Naikwad & Jeedi, 2.
, and Indonesia (Arpiwi et al.
, 2020.
Ikawati et al.
, 2.
to reduce fever.
reduce pain, reduce migraines, treat wounds .
nti-bacteria.
, asthma, and eye pain (Goh, 2024.
Nisa et , 2.
All parts of this plant can be used as medicine, including seeds, roots, stems, flowers, and leaves (Zulkifli et al.
, 2.
Recently, many studies have been interested in discovering new pharmacological activities of Vitex plants (Kamal et al.
, 2.
From studies related to the Vitex genus, extracts and fractions from the leaves and fruits of the Vitex genus contain various types of secondary metabolites, including flavonoids, terpenoids, steroids, and iridoids (Bello et al.
, 2018.
Gupta et al.
, 2022.
Mottaghipisheh et al.
, 2024.
Purwitasari et al.
, 2.
that significantly exhibit pharmacological activities such as anti-cancer, anti-oxidant, and anti-inflammatory (Bao et al.
, 2018.
Ghafari et al.
, 2022.
Liou et al.
Vitex trifolia is distributed and sold as herbal medicine in Asia under various local names, but the genus Vitex L.
is difficult to identify and delineate because it has a high morphology that is unclear and controversial (Salvaya et al.
, 2.
In Indonesia.
Vitex trifolia leaves have been demonstrated to possess anti-inflammatory properties, effective against both immunological and non-immunological inflammatory responses (Ikawati, 2.
Vitex trifolia extract could enhance cancer treatment efficacy by overcoming chemotherapy resistance (Lubis, 2.
Vitex sp as anti-COVID-19 plants in East Kalimantan (NoorAoan.
Legundi leaf essential oil has good potential to be explored further, especially to better understand the chemical activities of its compounds, since it shows toxic properties (Setianingsih et al.
, 2.
Therefore, it is necessary to carry out molecular identification to identify species that have potential as herbal medicines so that the public can avoid counterfeiting and errors in the use of herbal products (SuAoudi et al.
, 2.
Recently.
DNA barcoding has developed into an effective taxonomic classification technique for species identification.
It is considered a promising technique for accurate species identification by effectively using short regions of specific DNA sequences (Bhavana et al.
, 2.
DNA barcoding is a molecular technique used to identify species using DNA code-based similarities combined with morphological characteristics, which minimizes errors from conventional identification (Wardani et al.
DNA barcoding can be used for the characterization of new species, cryptic species, or unknown species at the species level (Letsiou et al.
, 2.
Chloroplast DNA .
pDNA) is a specific type of DNA barcode that is commonly used in plants (Sundari et al.
, 2.
Chloroplast DNA sequences that are likely to be used for DNA barcoding are the genes atpF-H, matK, ndhF, rpoC1, trnH-psbA, rbcL, rpl32-trnL, and rpoB (Paksoy et al.
, 2.
The matK and rbcL genes have been used as standard coding for plant DNA Therefore, in this study, these two genes were selected for DNA coding in Vitex trifolia plants.
The matK and rbcL genes can be used to distinguish different types of plant species and subspecies due to sequence variability, including acacia species (Suriani et al.
, 2.
, orchids (Worthy et , 2.
, bamboo (Yong et al.
, 2.
Cucurbitaceae family (Ho & Nguyen, 2.
, and Coelogyne genus (Pratiwi et al.
, 2.
The matK gene marker has a total sequence length of 1500 bp, and rbcL has about 1400 bp, which has high accuracy for DNA barcoding (Utama et al.
, 2.
The matK and rbcL genes are crucial in the authentication of medicinal plants, ensuring the right species is used in medicinal ingredients, which is important for conservation and treatment effectiveness (Cahyaningsih et al.
, 2.
In addition, the combination of matK and rbcL genes can discriminate about 90% of flowering plants (CBOL .
Hartanto et al.
Ae Development of primers amplifying DNA barcoding genes matK and rbcL of Legundi .
Edubiotik : Jurnal Pendidikan.
Biologi dan Terapan Vol.
No.
, pp.
398 Ae 411 However, more conserved genomic regions can be identified and used to detect adulteration of medicinal materials (Wattoo et al.
, 2.
The use of appropriate primers is one aspect that can affect the success of DNA barcoding.
Primers are short nucleotide or oligonucleotide polymers composed of DNA or RNA that play an important role in the PCR .
olymerase chain reactio.
process (Salsabila et al.
, 2.
The main cause of unsuccessful PCR is the selection of primers that have low quality and are not appropriate (Fulghum et al.
, 2.
Primers are an important component during the amplification process, which consists of forward and reverse primers, where the first step of successful DNA sequencing of a gene is the use of appropriate primer design.
Inaccuracy in primer design can cause the resulting PCR product to be non-specific and unable to amplify the entire DNA sequence (Indradewi, 2.
In some studies, it was found that universal primers failed to amplify various groups of species (Bahram et al.
, 2019.
Dahl et al.
, 2022.
Cryutlein et al.
In addition, both primers matK and rbcL failed to discriminate Calligonum species (Kipkiror et al.
In the study Corvalyn et al.
, the rbcL primer covering more than 99% of the sequence while the matK primer less than 85% despite optimization, thus emphasizing the limitations of universal primers and the need for the development of taxa-specific primers.
Several studies reported that no single universal primer set can successfully amplify all land plants, and matK primers, although informative, frequently fail to amplify consistently across taxa without optimization (Yu et al.
, 2.
Therefore, this study aims to design and evaluate species-specific primers based on the matK and rbcL genes using an in silico DNA barcoding approach, and to assess their effectiveness for accurate species-level molecular identification of Vitex trifolia.
RESEARCH METHODS
The research was conducted from August to November 2024 at the Biology Laboratory Research Department of Biology Education.
Faculty of Teacher Training and Education.
Muhammadiyah University of Surakarta.
Indonesia.
Data were obtained through DNA barcoding by including several stages such as DNA extraction.
PCR.
DNA visualization and qualification, and DNA sequencing.
Then, the data were processed using bioinformatics analysis, which can be seen in Figure 1.
Plant material in the form of leaves from the Vitex trifolia tree was collected from Makassar.
Indonesia.
The plants are maintained in a controlled greenhouse environment with natural light.
The full sequences of Vitex trifolia were obtained through the database from NCBI (National Center of Biotechnology Informatio.
In the genomic DNA extraction and DNA concentration stage, the genomic DNA was extracted from 150 g of Vitex trifolia leaves.
This consideration is carried out based on the criteria of the plants needed in the study, which are the leaves of the Vitex trifolia species with the morphology of three-fingered compound leaves, slightly ovoid, and grayish-green colour (Gentallan et al.
Samples were sterilized with 70% alkohol and grinded it with nitrogen liquid using a pestle and mortar, and extraction was done using a GeneAid plant genomic DNA mini kit.
The extracted DNA was quantified using the BioDrop spectrophotometer, which was used for measuring the extracted DNA concentrations in the samples.
DNA was visualized using gel electrophoresis on a 1% agarose gel to verify the quality and quantity of the extracted DNA.
The DNA was stored at Ae20AC for subsequent amplification experiments.
Hartanto et al.
Ae Development of primers amplifying DNA barcoding genes matK and rbcL of Legundi .
Edubiotik : Jurnal Pendidikan.
Biologi dan Terapan Vol.
No.
, pp.
398 Ae 411 Figure 1.
Workflow of DNA Barcoding and Species-Specific Primer Development in Vitex trifolia The primer design stage.
The primer design development was carried out by selecting the Vitex trifolia matK and rbcL gene sequences from the NCBI database .
ccession number NC_062602.
downloaded in FASTA format, and aligned using MEGA version 11 to assess nucleotide similarity and sequence identity (Patel et al.
, 2.
Forward primers .
Ae30 b.
were manually designed in the 5AIe3A direction, while reverse primers were generated from the terminal region and reverse-complemented using bioinformatics tools .
ttps://w.
org/sms/rev_comp.
Primer specificity was evaluated in silico by analyzing hairpin formation, melting temperature (T.
, self-annealing, and GC content (Sen et al.
, 2.
and optimized using the PCR primer stats tool .
ttps://w.
org/sms2/pcr_primer_stats.
to meet optimal criteria, including primer length of 18Ae30 bp (Batubara et al.
, 2.
GC content of 40Ae60% (Messe et al.
, 2.
, and a maximum Tm difference of 5AC between primer pairs (Praja & Rosalina, 2.
Polymerase chain reaction (PCR) and DNA visualization stage.
PCR was used to amplify the isolated DNA through repeated cycles of denaturation, annealing, and extension (Zhang et al.
, 2019.
Shahzad et al.
, 2.
, and amplification success was evaluated by gel electrophoresis (Dzikrina et al.
Each reaction was prepared in a total volume of 50 AAL containing 25 AAL MyTaq Red Mix, 22 AAL ddHCCO, 1 AAL of each primer, and 1 AAL DNA template.
The PCR program for matK was as follows: initial denaturation at 94AC for 300 s.
35 denaturing cycles at 94AC for 30 s, annealing at 55AC for 45 s, extension at 72AC for 60 s.
and final extension step at 72AC for 300 s.
The DNA was visualized by gel electrophoresis using 1% agarose gel.
Furthermore, 4 AAL of the sample was inserted into the well.
After all the samples were put into the wells, 4 AAL of DNA ladder was run in electrophoresis at 100 volts for 30 In the final step, the DNA bands that appeared on the agarose gel 1% under UV light using BluPAD Dual LED Blue/White Light Transilluminator (Bio-Heli.
The DNA sequencing stage.
DNA fragments obtained after electrophoresis were sequenced at PT Genetics Science Indonesia using the Sanger method.
The resulting sequences were assembled into contigs with DNA Baser Assembler and then analyzed using BLAST on the NCBI website .
to determine sequence similarity with reference data in the database.
The data analysis, primer candidates that have been obtained in the previous stage, are tested for primer Hartanto et al.
Ae Development of primers amplifying DNA barcoding genes matK and rbcL of Legundi .
Edubiotik : Jurnal Pendidikan.
Biologi dan Terapan Vol.
No.
, pp.
398 Ae 411 specifications in-silico using the PCR primer stats tool from the Bioinformatics website .
ttps://w.
org/sms2/pcr_primer_stats.
to determine the secondary structure of each of the candidate primers that exist.
The DNA sequencing products are qontique using DNA baser In the contiq process, the ambiguous sequence was cut.
The results of the nucleotide sequence were aligned with MEGA version 11, used for the alignment of nucleotides with reference to Vitex trifolia sequences that used matK and rbcL gene from NCBI (Budiman et al.
, 2.
Then the alignment results were analyzed for the highest sequence similarity using the BLAST (Basic Local Alignment Search Too.
tools on the NCBI website .
ttps://blast.
gov/Blast.
can find regions that have similarities with other sequences in existing data centers, so it can be known that there is a genetic relationship between sequences with other species or just a coincidence (Budiarsa et al.
, 2.
In addition, to validate BLAST
BOLD
ttps://v3.
org/index.
php/IDS_BlastReques.
BOLD can identify by matching sequences with the most closely related individuals available in the database by generating inter and intraspecific genetic distance graphs, barcode gaps, reconstruction trees, and haplotype distributions (Imtiaz et al.
FINDING AND DISCUSSION
Based on the results of a DNA sequence search using the NCBI database, one of the Vitex trifolia trnK gene sequences was obtained and then downloaded in FASTA format.
The length of the V.
trnk gene sequence obtained is around 2,580 bp .
ase pai.
, then converted into FASTA format as a template to facilitate the design of primer design manually, where this format is a text-based format to show nucleotide sequences without numbering (Sihotang et al.
, 2.
In the trnK gene sequence, there is a matK gene that overlaps the intron part of the trnK gene with a size of around 1500 bp.
The matK gene is a gene located within the intron of the trnK gene, where it overlaps with the intron of the trnK gene (Figure .
MatK is a chloroplast coding gene that is nested between exons 5' and 3' of the trnK .
RNA-lysin.
gene (Udensi et al.
, 2.
To obtain the full-length sequence of the matK gene, a portion of the trnK gene sequence can be used so that the entire matK sequence can be amplified.
The length of the trnK gene region overlapping with matK is in the order of 2214 to 3744 bp .
530 b.
Primer design to get the desired primer candidates previously needed to be done manually.
Based on the DNA consensus sequence obtained, the primer candidate was obtained with the Forward and reverse primers (Figure .
Furthermore, the obtained primer candidates can be tested in silico using PCR primer stats tools from the Bioinformatics website .
ttps://w.
org/sms2/pcr_primer_stats.
to determine the secondary structure of each candidate primer.
The secondary structures observed include melting temperature (T.
GC count (%).
GC clamp, self-annealing, and hairpin formation (Table .
From these tests, it will be known whether the primer candidates meet the requirements of a good primer so that the resulting primer is specific.
Each candidate is given a special naming to make it easier to recognize each primer, namely the forward primer with the letter symbol AuFAy and the reverse primer with the letter symbol AuRAy.
The results of the test show that the primer candidate that meets the criteria of a good primer is the matK primer candidate .
orward and revers.
, where a good primer meets the criteria of the prime parameter (Aulia et al.
, 2.
Good primer parameters include primer lengths ranging from 18-30 bp, g 40-60% guanine (G) and cytosine (C) bases, and the melting temperature (T.
between the forward and reverse primers is not too far apart (Nuryady et al.
, 2.
Meanwhile, the rbcL primer candidate obtained low GC content results, namely 35% and 25%, where the amount is <40 - 60% as the ideal limit of the Hartanto et al.
Ae Development of primers amplifying DNA barcoding genes matK and rbcL of Legundi .
Edubiotik : Jurnal Pendidikan.
Biologi dan Terapan Vol.
No.
, pp.
398 Ae 411 amount of guanine (G) and cytosine (C) contained in a primer to ensure the stability of the bond between the primer and the template (Suwarny et al.
, 2.
A low amount of GC content can reduce primer specificity because it leads to a low optimal annealing temperature (So et al.
, 2.
In addition, unspecific and too low primer annealing temperatures can lead to amplification of unwanted DNA segments (Mubarak et al.
, 2.
Figure 2.
The Developed Primers of matK and rbcL of Vitex trifolia based on Full-Length Mitochondrial Sequences Table 1.
The Output of Primer-Stats for Sequence Accession No Primers Name NC_062602.
NC_062602.
NC_062602.
NC_062602.
Length
Melting Temperature (Tm EE) Content
(%)
Clamp Pass Pass Pass Pass SelfAnnealing Pass Pass Pass Pass Hairpin Formation Pass Pass Pass Pass Visualization of electrophoresis results from Vitex trifolia DNA extraction using 1% agarose gel with staining using Florovue gel stain that allows DNA to fluoresce when illuminated with UV light.
DNA bands were compared with a DNA ladder (Figure .
and showed fragment sizes of approximately 10,000 bp.
DNA concentration was measured with a NanoDrop spectrophotometer, giving a value of 100 AAg/mL for the Makassar sample, a method widely recognized for accurately assessing DNA concentration and purity (Garcya-Alegrya et al.
, 2.
Three main factors affect the success of the DNA extraction process, namely the quality of the plant tissue used, the quantity of plant tissue, and the technique of destroying plant tissue (Nugroho et al.
, 2.
Good DNA quality obtained from extraction results is a basic requirement that must be met in molecular studies (Jayanti & Mushlih, 2.
Good DNA quantity can affect the success of subsequent DNA barcoding steps such as PCR.
DNA sequencing, and electrophoresis (Sittenthaler et al.
, 2.
Vitex trifolia DNA sequences were tested for concentration and purity by PCR using both candidate primers, namely matK and rbcL, obtained DNA amplification results that have been visualized by electrophoresis using 1% agarose gel (Figure .
proving that the DNA was successfully amplified by looking at the presence of fluorescent DNA bands and measuring more than 1500 bp (Anissa et al.
, 2.
The size of the DNA band formed can determine the high and low concentration of the DNA sample, the thicker the DNA band produced, the higher the concentration and vice versa (Setiati et al.
, 2.
addition, good quality DNA looks thick, solid, and has no smears (Ramlan et al.
, 2.
In the amplification Hartanto et al.
Ae Development of primers amplifying DNA barcoding genes matK and rbcL of Legundi .
Edubiotik : Jurnal Pendidikan.
Biologi dan Terapan Vol.
No.
, pp.
398 Ae 411 results of the two primer candidates, it is known that both have high DNA concentrations and sizes ranging from 1500 to 2000 bp, but the quality of the DNA is low, marked by smears.
Smears that appear at the electrophoresis stage can be caused by degradation or contamination of the DNA sample (Kusuma.
Figure 3.
The Result of gDNA Extraction Figure 4.
The Amplification Result of Vitex trifolia matK and rbcL Genes Considering the quality of the matK primer against the in-silico test and the amplification results showing thick DNA bands and thinner smears than the rbcL primer, the matK gene will be used as the candidate primer tested in the next stage.
Vitex trifolia matK gene sequences were tested using BLAST tools on the NCBI website .
ttps://blast.
gov/Blast.
with various parameters such as species name, max score, total score, etc (Table .
To determine the similarity/compatibility of a DNA sequence with the available database, the percent identity value is used.
the higher the percent identity, the more significant the match, so that it can estimate biological similarity (Samal et al.
, 2.
In the table, the highest percent identity is 100.
00% in the species Vitex rotundifolia.
Vitex trifolia, and Vitex So from these results, it is known that the matK primer successfully amplified the Vitex trifolia Hartanto et al.
Ae Development of primers amplifying DNA barcoding genes matK and rbcL of Legundi .
Edubiotik : Jurnal Pendidikan.
Biologi dan Terapan Vol.
No.
, pp.
398 Ae 411 sequence with a percent identity of 100.
The BOLD result details of the Vitex trifolia matK sequence can be seen in Table 3.
Table 2.
NCBI Blast Result Detail of Vitex trifolia matK Sequence Max Query Species Score Total Score Cover value Vitex rotundifolia Vitex trifolia Vitex trifolia Vitex bicolor Vitex rotundifolia Vitex negudo var Vitex negudo Vitex negudo Vitex tripinnata Vitex negudo var Table 3.
The BOLD Result details of the Vitex trifolia matK Sequence Phylum Class Order Family Genus Tracheophyta Magnoliopsida Lamiales Lamiaceae Vitex Tracheophyta Magnoliopsida Lamiales Lamiaceae Vitex Tracheophyta Magnoliopsida Lamiales Lamiaceae Vitex Tracheophyta Magnoliopsida Lamiales Lamiaceae Vitex Tracheophyta Magnoliopsida Lamiales Lamiaceae Vitex Tracheophyta Magnoliopsida Lamiales Lamiaceae Vitex Tracheophyta Magnoliopsida Lamiales Lamiaceae Vitex Tracheophyta Magnoliopsida Lamiales Lamiaceae Vitex Tracheophyta Magnoliopsida Lamiales Lamiaceae Vitex Tracheophyta Magnoliopsida Lamiales Lamiaceae Vitex Per.
Ident Acc.
Len Accession OQ942922.
ON711030.
NC_062602.
NC_065871.
NC_05091.
OP581016.
PP584504.
NC_057235.
MT473785.
PQ541021.
Species Agnus-castus Agnus-castus Score Similarity Furthermore, the same sequences and primer candidates were tested again using a different website known as BOLD .
ttps://v3.
org/index.
php/IDS_BlastReques.
In the test, there are parameters that can be observed, including phylum, class, order, family, genus, species, score, and similarity (Table .
When compared to the BLAST Bioinformatics tool, the BOLD system is easier to analyze for sequence similarity with the database and is more specific in taxonomy.
The similarity percentage for identifying the Vitex trifolia matK gene sequence showed a similarity of around 99.
with the Vitex trifolia species.
Thus, it is known that the matK primer candidate successfully amplified the Vitex trifolia sequence.
The similarity percentage of the Vitex trifolia matK gene sequence from both BLAST Bioinformatics and BOLD websites was successful in identifying up to the species level with a percentage of 99.
92-100%, so the matK primer candidate was specific in amplifying the Vitex trifolia This finding is consistent with previous studies, where Wei et al.
reported that amplification with the designed matK primer was more successful.
Korotkova et al.
observed successful matK amplification in Cactaceae, while Sahin et al.
noted moderate performance for matK and lower reliability for rbcL in species-level resolution.
Mishra et al.
indicated that the matK gene exhibits higher sequence variability than the rbcL region.
Overall, the findings demonstrate that the matK primers developed in this study exhibit higher reliability and specificity than the rbcL primers for molecular identification of Vitex trifolia, thereby supporting the use of matK as an efficient DNA barcode Hartanto et al.
Ae Development of primers amplifying DNA barcoding genes matK and rbcL of Legundi .
Edubiotik : Jurnal Pendidikan.
Biologi dan Terapan Vol.
No.
, pp.
398 Ae 411 for genetic exploration and accurate species-level identification of ethnopharmacologically important CONCLUSION The selection of specific primers is a success factor for DNA barcoding, especially in the PCR .
olymerase chain reactio.
The specific primer must meet the criteria as a good primer so that it is optimal in amplifying the target DNA sequence.
In this study, it is known that primer candidates that qualify as good primers are matK primer candidates consisting of forward primers .
AoTGTcGAGGTATCTATTCTTAC 3A.
and reverse primers .
AoCATTGCACACGGCtcTA 3A.
The results demonstrate that the designed matK primers show higher reliability and specificity than the rbcL primers for molecular identification of this species.
Successful amplification and high sequence similarity .
92-100%) confirmed the effectiveness of the matK marker in achieving species-level These findings imply that the matK primer can serve as a reliable molecular tool for accurate identification and genetic exploration of Vitex trifolia, supporting future studies in ethnopharmacology, biodiversity assessment, and conservation of medicinal plants.
ACKNOWLEDGMENT
The researcher would like to thank the Kolaborasi Riset Strategis (KATALIS) of Kementerian Pendidikan Tinggi.
Sains.
Dan Teknologi.
Indonesia, under contract number 003/LL6/P.
Katalis/AL.
04/2024 and 279.
07/A.
3-i/LRI/Vi/ for facilitating and financially supporting this research.
REFERENCES