DNA Barcoding in Plants and Animals: A Critical Review

Systematics plays the most crucial role in biodiversity conservation which is at stake due to anthropogenic activities and environmental degradations. The ever-increasing decline of classical taxonomic expertise drives the need to develop molecular marker-based tools for quick, efficient, and reliable detection of organisms, to assess their ecological impacts for deepening our understanding of systematic and evolutionary relationships between organisms which is central to biology. The pace of alpha taxonomy has quickened by its integration with an increasingly fashionable and novel concept called DNA barcoding which utilizes a short genetic marker or barcode to categorize species for enhanced biodiversity assessment. As a supplementary but not complete alternative of systematics research, DNA barcoding, however, not error-free, brings precision in identification by solving existing problems of classical taxonomy and phylogenetics, irrespective of the growth stage of organisms, particularly for known taxa rather than unknown ones. Mitochondrial gene Cytochrome C Oxidase 1 (COI) serves as a universal animal barcode but there is no such universal barcode for plants and developing a suitable one is more challenging. With the recent advancement of Next Generation Sequencing (NGS), DNA metabarcoding technology is advancing rapidly. Still, ambiguity and error prevail with the correct identification of species due to some problems. After extensive analysis of the existing DNA barcoding papers, this review discusses commonly used DNA barcodes in plants and animals, their roles, advantages, limitations to solve existing problems of conservation biology and add the author’s views and recommendations. The Bar-HRM method is feasible for high throughput


Introduction
Species identification is the fundamental step for measuring biodiversity and developing our primary understanding of the biological world. This critical attempt is somewhat hindered by the shortage of professional knowledge of classification and sometimes due to the limitation of morphology-based identification (Chase et al., 2005). The traditional morphometric taxonomic study is time-consuming as it is dependent on the growth stage of the organism. Such studies are also laborious that are dependent on pre-determined classifications and expertise (Costion et al., 2011;Huang et al., 2015). As taxonomic expertise is declining worldwide because of scientific reductionism (Crisci et al., 2020), an integrative approach becomes highly necessary for the survival of this magnificent, crucial and quintessential branch of science.
The genetic barcoding approach has been implemented in different groups of organisms including vertebrates, nonvertebrates, bacteria, bryophytes, gymnosperms, algae, fungi, angiosperms, and so on to solve their problematic taxa and improve their phylogeny. DNA barcoding serves a wider range of purposes including the safety of plant and animal-based natural products used in traditional medicine, protection of endangered species from illegal trading and poaching, revealing cryptic diversity, assessing ecological impact in the community level study, forensic analysis, phylogenetic analysis, food safety, supporting ownership or intellectual property rights (IPR), etc. (Penton et al., 2004;Nithaniyal et al., 2021). The recent rapid advancements of bioinformatics and molecular biology make molecular data readily available from different databases, which improve the practical and analytical scope of DNA barcoding. With the impressive progress of high throughput next-generation sequencing (NGS) technology over the years, DNA barcoding is transforming into DNA metabarcoding to eliminate the limitations of single species sequencing by enabling multi-species sequencing simultaneously in a single PCR run to overcome the previous timeconsuming specimen sorting and single species dependency. Metabarcoding enables multiplexing of hundreds of taxa from large diverse groups to study species composition and estimate population size in a complex mixed community which significantly increases surveillance measures for constructive, affordable, and dynamic management responses (Piper et al., 2019;Gao et al., 2019).
Although molecular barcoding can help to solve the problems of phenotypic plasticity, it never can be an alternative way of classical taxonomy as it relies heavily on some predetermined threshold value of bioinformatic analysis which is not the proper way to identify a new species (Waugh, 2007). The heavy dependence on the reference database with existing sequences is another challenge of this technique. Despite these challenges, the number of DNA barcoding studies is on the rise for its benefit to detect unknown samples quickly and efficiently ( Figure 1). This review aims to shed light on the role of DNA barcoding in different sectors, commonly used barcodes, recent advancements of DNA barcoding technology in different areas, and future prospects of this technology through a problem-based discussion.

DNA barcoding: Commonly Followed Procedures
To extract DNA, the technique begins with collecting samples, including fresh samples, mixed samples, dry samples, and processed products, as per the requirement of the experiment. Then DNA is extracted from the collected samples using one or many of the available methods such as CTAB (Doyle & Doyle, 1987), SDS method, PVP method, Phenol -Chloroform method, etc. After extracting, DNA samples are amplified using appropriate markers via PCR following the three stages-denaturation, annealing, and extension. The PCR products are then purified and band samples are used for DNA sequencing. Different types of sequencing technologies are available, such as Sanger sequencing, nextgeneration sequencing, third-generation sequencing technologies, etc.
After getting sequenced DNA data of the samples, similarity search begins using bioinformatic techniques and relevant databases and tools such as NCBI, BLAST, etc. Proper data analysis at that stage is considered crucial for generating a new barcode as well as reporting a new record for an existing barcode . If the similarity search becomes statistically authentic, the sequence is submitted to GenBank (Clark et al., 2016) to get the accession number. Data from NCBI subsequently gets mined into the Barcode of Life Database (Ratnasingham & Hebert, 2007) to identify species.
There are different software used for multiple sequence alignment, molecular data analysis and the most commonly used ones are MEGA 10 (Tamura et al., 2021), ABGD (Puillandre et al., 2012), Taxon DNA (Meier et al., 2006), Geneious vR6.1.6, MAFFT v7.017 (Katoh et al., 2002). Kimura 2 parameter (K2P) for genetic distance correction is used profusely to measure sequence divergences among organisms (Hebert et al., 2003). Gene efficiency is tested by comparing intra-and interspecific genetic distances, focusing on the existence or lack of a barcoding gap. Species identification depends on the use of thresholds, set to differentiate between interspecific variation and intraspecific divergence. Intraspecific average genetic distances should be at least ten folds smaller than interspecific average genetic distances (Meyer & Paulay, 2005).

Major Databases holding DNA Barcode Data
Continuous improvement of DNA sequencing technology has expanded the availability of DNA barcode data in public databases such as GenBank (Clark et al., 2016), EMBL (Kanz et al., 2005), DDBJ (Mashima et al., 2017). It is unequivocal that the progress of DNA barcoding depends largely on the improvement of public databases. The   Figure 3).

DNA barcoding in Animals
DNA barcoding technique relies on three important principles such as standardization, minimalism, and scalability. Considering these principles, selecting suitable barcoding depends on selecting single or multiple standard loci for routine sequencing with the highest reliability in a very comprehensive and diverse set of samples to make data sets easily comparable to distinguish one species from another. The 5'end of Cytochrome C Oxidase subunit 1 (COXI, COI, or COI-5P) having 600 to 1000 base pairs DNA sequences, in general, fits the purpose for interspecific variability and is considered as universal species level barcode for animals (Kress & Erickson, 2012). It is a haploid, maternally inherited, single locus with protein-coding region having high copy number per cell assuring sequence recovery from poorly preserved samples (Hebert et al., 2003;Fazekas et al., 2009;Hollingsworth et al., 2011). COI is prioritized over other mitochondrial genes due to the reason that primers of COI are highly specific, robust, and show the highest degree of accuracy to retrieve 5' end of target DNA (Folmer et al., 1994). Since the rate of mutation in DNA shows an inverse relationship with the size of the genome, mitochondrial DNA undergoes relatively high mutation for their smaller size compared to the nuclear DNA and that makes mitochondrial COI more capable as a universal animal barcode than nuclear rbcL, matK or other nuclear barcodes (Drake et al., 1998;Waugh, 2007). Intraspecific variation of COI is generally less than 10 % than interspecific variation, where deletions and insertions are rare (Blaxter, 2004).
Degree of morphological variation, poor phylogenetic info due to lack of knowledge on anatomical features, the occurrence of cryptic species drives the need for a morpho-molecular approach to bring precision in identification and solve existing problems of the animal world . Understanding and recording cryptic biodiversity (morphologically similar species but genetically distinct) of species are critically important to achieving effective species conservation by unmasking phenotypic similarity and predicting biodiversity responses to environmental changes. Since frogs are considered as a target group for cryptic species investigation, a modern molecular systematics study of amphibian Limnonectes Kuhlii using mtDNA data has revealed 22 distinct evolutionary lineages, 16  DNA barcoding approach significantly emphasizes the need for taxonomic revision of different animal genera due to the advent of cryptic species and newly discovered species. An underestimated genus Triplophysa has been studied in Qinghai-Tibet Plateau, a biodiversity hotspot, to find out 24 native species with 2 cryptic species namely T. robusta and T. minxianensis by DNA barcoding of 1630 specimens . Besides, the application of high throughput technologies for sensitive identification purposes is on the rise. Traditional trap-based surveillance strategies to control the arrival of invasive insects are less fruitful solely than when accompanied with DNA metabarcoding as the latter helps in the simultaneous, multi-species identification of mixed populations (Piper et al., 2019). Insects are expected to become temperature sensitive because of their short life cycle, for example, significant poleward shifting of non-migratory butterflies in Europe, which is heavily dominated by temperatures (Parmesan et al., 1999;Bale et al., 2002).
Changes in the insect community are critical to understanding modifications in ecological parameters including decomposition pattern, nutrient cycling, primary productivity as well as biodiversity assessment. Combining community datasets with high throughput DNA barcoding technologies has potential aspects to minimize various logistical, financial, and systematic impediments for large-scale observation. Arctic arthropod community has been explored focusing on Arachnida, Collembola and Insecta to develop 18, 096 (75%) barcodes using mitochondrial COI to be assigned to BINs, having GenBank accession number ranging from MN665381 to MN683476, of 24,198 specimens collected during study (Pentinsaari et al., 2020).
In a primate study, to suggest appropriate marker for species identification, the efficiency of 12 mitochondrial proteincoding genes is tested to explore that NADH dehydrogenase subunit 5 (ND5) and cytochrome c oxidase subunit II (COII) produce the largest barcode gaps within genus and family than between species rather than COI, which is suggestive of more conservative nature of the barcodes in the species level (Jackson & Nijman, 2020). 3 species of Canidae, one of the most interesting families of Mammalia, which is threatened due to illegal poaching, poisoning, and habitat loss, have been explored by DNA barcoding approach to validate COI barcode and detect genetic divergence. The mean sequence divergence among and within species was 12.32% and 0.61% respectively indicating relatively higher genetic diversity than previously reported studies (Aksöyek et al., 2017). The consistency between DNA barcoding and morphological identification for a particular species depends on the presence or absence of sibling species that are morphologically and genetically similar. Lack of conspecific barcodes, sharing the same DNA barcode may create ambiguity to identify lower operational taxonomic units (OTUs). In addition to that, misidentification and mislabeling of samples results in potential error and hesitation in later research ( Pleijel et al., 2008;Dixon, 2012).
In an approach to correctly identify Spanish Blackfly as a preventive measure of the outbreak in Spain, to generate important information about their species distribution, vector control strategy, disease dissemination, Ruiz-Arrondo et al., (2018) used the COI gene as a potential barcode and explored 6 new records out of 22 species from 239 specimens with the average intraspecific and interspecific genetic divergence of 1.47% and 12.25%, respectively. All the sequences were submitted to the GenBank database with accession numbers ranging from MG894170 to MG894340 and five new species were registered in the BOLD database for the first time with complete DNA barcodes (Ruiz-Arrondo et al., 2018). Barcodes showing high intraspecific divergence between randomly selected sibling species than morphospecies may indicate the presence of hidden diversity (Ruiz-Arrondo et al., 2018). However, no uniform threshold has been reported yet for species delimitation. Maximum conspecific and minimum congeneric differences have been reported as more effective to define barcode gap than average intraspecific and interspecific sequence divergence (Meier et al., 2008). Although it is a matter of dispute, distance-based technique remains as the most followed method in DNA barcoding (Reid et al., 2011;Srivathsan & Meier, 2012).
Gastropods, as the most abundant group of mollusks, are different to identify morphologically for their different morphological characters at various growth stages and they form an important part of marine biodiversity with 80,000 species existing worldwide, face threats due to overexploitation as they are economically significant (Bieler, 1992;Schmidt et al., 2002). Accurate species detection plays a pivotal role in the conservation of flora and fauna. To make an efficient fisheries resource survey and natural resource management using COI gene as potential mitochondrial DNA barcode, Ran et. al. (2020), developed a barcode reference library with 306 barcode sequences, GenBank accession number ranging from MN388943 to MN389209, obtained from 120 species containing 3 new records in China, belonging to 35 families and 7 orders, to validate the efficiency of COI gene as barcode sequence. The K2P average intraspecific and interspecific sequence divergences were 0.9% and 14.7%, respectively and a slight increase within the higher taxonomic levels of families and orders was reported but the rate of increase in higher taxa was lower due to substitutional saturation (Iyiola et al., 2018; Ran et al., 2020).
Ornithologists without the help of molecular tools, sometimes face challenges to provide the right identity of birds. DNA barcode reference libraries, as a useful tool, can help them to expand geographical sampling for COI sequences. A total of 26 COI sequences from bird blood samples were obtained from 18 species belonging to 10 families. A boreal migrant bird was re-identified as a different species after molecular study (Pulgarín-R et al., 2021).
Overfishing of sharks is a growing problem in Brazil as the country imports most of the shark meats in the world, the majority in Asia.

Animal based Traditional Medicine and DNA barcoding
Traditional medicines derived from animal sources play a significant role in zootherapy, the availability of potential compounds for drug discovery and therapeutic practices including Traditional Chinese Medicine (TCM), Kampo medicine, Ayurvedic medicine, American folk medicine (Alves & Alves, 2011;Liu et al., 2016). Numerous important medicinal animal species are threatened due to illegal hunting, poaching, and several other anthropogenic activities resulting in the increased demand for animal products to be used for medicinal purposes. To detect processed animal products rapidly for the authenticity of traditional medicines from adulterants as well as to protect threatened species, DNA barcoding can be revolutionary. (Yang et al., 2018). One of the biggest challenges to reaching a common conclusion regarding the selection of a universal plant barcode was the paucity of comparative data enclosing all candidate markers and broad range taxonomic sampling. The Consortium for the Barcode of Life (CBOL) Plant Working Group, after testing the efficacy, proposed a two-locusbased approach using rbcL and matK as the core barcode and trnH-psbA intergenic spacer as a supplementary barcode. Instead of using a single gene locus, this multi-locus approach is taken because of the intra and interspecific variation and divergence between families and genera that shows no genetic gap in all the cases to be found (Janarthanan et al., 2020). Despite lower discrimination success in plant species compared to mitochondrial COI in animals, the combination of rbcL with matK is recommended for some reasons. As both of them are plastid coding regions, it is possible to rectify sequence orientation by checking assembly errors during translation through in-silico analysis as well as to detect the presence of pseudogenes (Hollingsworth et al., 2011).
ITS has been reported as an additional candidate plant DNA barcode by China Plant Barcode of Life (China Plant BOL Group). In a comparative study seven markers namely trnH-psbA, matK, ycf5, rbcL, rpoC1, ITS, ITS2 from medicinal plant species were tested and ITS2 was found as the best potential marker with a successful species identification rate of 92.7% . ITS is considered as a specific reliable marker because it utilizes welldefined barcode gap to discriminate species, showing comparatively higher interspecific variation than intraspecific variation (Schoch et al., 2012). However, for some environmental DNA barcoding processes, ITS creates problems due to primer mismatching. To solve that problem, a primer combination is suggested for alternative ITS regions, in parallel along with standardizing changes in melting point (Bellemain et al., 2010). The presence of nuclear sequences of mitochondrial origin (NUMTs) creates difficulty in the barcoding approach. These are fragments of mitochondrial DNA that have been translocated into the nuclear genome (Williams & Knowlton, 2001) ranging from none or few in Anopheles, Caenorhabditis, and Plasmodium to more than 500 in human, rice, and Arabidopsis sp. (Vohra & Khera, 2013).

Algal Identification through DNA Barcode
DNA barcoding can play a great role to overcome insufficient algal genetic data to contribute by resolving numerous questions of algal systematics, addressing queries related to biogeography as well as establishing better costeffective biodiversity monitoring programs emanating from UN conventions and EU directives (Bartolo et al., 2020).
Red algae are economically important for carrageenan, a morpho-molecular study which using four different gene sequences namely COX1, COX2, COX2-3, and rbcL, proved the use of COX2 as a potential barcode to differentiate various commercially important carrageenophytes (Tan et al., 2012). Pyropia, a commercially important difficult genus, has been studied up to species level using COI-5P barcode, to reveal cryptic species. The diverse sample set suggests the probability of more biodiversity in the pristine habitat which has not been explored yet (Koh & Kim, 2018). Despite comprehensive monographs, identifying algal species can be difficult as seen in the brown alga Alaria. With the help of COX1, rubisco operon spacer (rbcSP), and ITS, a study reported species discrimination between closely related species of Alaria with the prediction of a probable collapse in the species barrier (Lane et al., 2007).
Green microalgae identification is always challenging. To reveal the cryptic diversity of Scenedesmus, barcode-based approaches were used using rbcL, tufA, ITS, and 16S where 5 cryptic species were revealed out of 11 recovered species, suggesting a combination of 3 genes is much better to attain high species resolution than single-locus approach (Zou et al., 2016).
Algae play a significant role in forensic analysis. Diatom has been identified from the internal extract of the victim using 16S, 18S, 23S, 28S, COI, rbcL, and ITS region through meta-barcoding aided with NGS. As most of the diatom sequence data in GenBank has been reported as mislabeled, developing standard barcodes for correctly identified voucher specimens is a great challenge (Liu et al., 2020). However, a two-step approach has been recommended by CBOL Protists Working Group, referring to the use of a universal barcode at the first step and a group-specific second barcode at the second step to increase species resolution for correct identification (Pawlowski et al., 2012).

Fungal Identification through DNA barcode
It is extremely difficult to build up a uniform, working species identification system that will work across all fungal species because fungi are extremely diverse ranging from microscopic to macroscopic life forms with a habit to colonize in every habitable niche on Earth (Xu, 2016). Proper identification of fungal species is highly significant to stop disease transmission, to monitor the spatial and temporal distribution and migration. Mitochondrial COI is not applicable for fungi as a universal barcode, though very few studies have reported its suitability for very few fungal genera for e.g. Penicillium sp.  , in a DNA barcoding approach, studied 49 moss species and 9 liverwort species and recommended rbcL, rpoC1, rps4, trnH-psbA, and trnL-trnF as the potential DNA barcode for mosses, while rbcL and rpoC1 were claimed as the most efficient single loci . Data deficiency is the prime cause of the establishment of a universal barcode for bryophytes. In a study on Fissidens, to build up a molecular phylogeny using rbcL and rps4, it was found that adaptation along with diversification in the tropical region is the prime causes behind their evolutionary build-up (Suzuki et al., 2018). More study is needed worldwide to build a separate reference DNA barcode database for bryophytes to understand their phylogeography and role in the environment through effective identification.

DNA Barcoding in Pteridophytes
Pteridophytes are the seedless, vascular, nonflowering, spore-bearing plants having distinct free-living gametophyte (n) and sporophyte (2n) generations. They are evidence of vascular system evolution, connecting non-vascular cryptogams with vascular phanerogams (Malati & Rao, 2020). Therefore, proper identification of pteridophytes with a morpho-molecular approach plays a significant role in our current understanding of biology.
Authentication of medicinal pteridophytes is significant to record their status of conservation as well as to control adulteration or mixing between species which may cause serious health problems. To substantiate this, psbA-trnH, rbcL, rpoB, rpoC1, matK were tested on herbal pteridophytes to find that psbA-trnH intergenic region as the best candidate among all with the highest amplification accuracy ( To reveal close similarity, Psilotum sp. was studied using rbcLa, trnA, trnV, matK, ITS, ycF3, and rpoB barcodes where rbcLa and trnA showed the highest species discrimination power with successful sequencing (Khan et al.,  2019).
A potential new ecotype of Pteris vittata L. has been explored in India detecting subtle changes in the size of mature sporophyte where the new ecotype was Pteris vittata L. ecotype nano, using rbcL as a sole locus which justifies the use of a single-locus approach for varietal identification of pteridophytes (Morajkar & Hegde, 2021).
Species complexity and polyploidization are often seen among the pteridophyte communities (Sigel, 2016). Asplenium normale D. Don shows species complexity in chromosome number and organization. An integrative morpho-cytomolecular approach was taken to clarify and revise its taxonomic state using plastid marker trnL-trnF, trnG-trnR, rps4-trnS and nuclear marker gapCp, pgiC, LFY to differentiate diploid and polyploid taxa distinctly (Chang et

DNA Barcoding in Gymnosperms
Gymnosperms are seed-bearing plants predominantly woody, used commonly to enhance the aesthetic beauty of parks and gardens. Very few studies have been reported to date for their molecular authentication and as these are economically and pharmacologically significant, the more molecular study is required to reveal their true identity and properties.
Primer assessment is critical in DNA barcoding strategy, as they play a key role in the amplification process. Centering to this objective, 1 rbcL and 9 matK potential primers were assessed to evaluate their universality in 57 species of Gymnosperms belonging to 40 genera and 11 families. The study proposed 1F/724R and Gym_F1A/Gym_R1A as the best universal primer for rbcL and matK respectively (Li et al., 2011).
Herbal dietary supplements of Ginkgo biloba L. helps to improve cognitive function via increasing blood perfusion (Kellermann & Kloft, 2011). To authenticate its herbal preparation matK mini barcode assay has been accomplished in the USA where 83.8% of samples were containing Ginkgo biloba L. DNA (Little & Gulick, 2014). Sass et al. (2007) performed a comprehensive study in some cycads, one of the world's most threatened group of gymnosperms, using matK, ndhJ, rpoC1, rpoB, trnH-psbA, accD, ycf5, ITS to test the potentiality of these markers to discriminate species where ITS and trnH-psbA showed the highest level of accuracy (Sass et al., 2007). Numerous species of Cycads were identified using rbcLa, matK as the core barcodes with the supplement of ITS and trnH-psbA from the traditional medicine market of South Africa during illegal trading from where near threatened, endangered, vulnerable, least concerned species were identified (Williamson et al., 2016).

DNA Barcoding in Angiosperms
Angiosperms are a large group of flowering plants, having seeds enclosed in carpel and they account for 295,383 species belonging to 13,164 genera under 416 families (Christenhusz & Byng, 2016). Therefore, the DNA barcoding approach may play a highly promising role for their effective identification, ecological assessment, and conservation in nature. Overcollection and habitat destruction pose a serious threat to Orchidaceae. DNA barcoding reveals a combination of atpF-atpH, psbK-psbI, trnH-psbA represents the best option for molecular identification of Korean orchids (Kim et al., 2014). Ornamental plants are economically important for the horticultural industry and is an approach to confirm their correct identity, a combination of rbcL and matK were used in Egypt where the success rate varied at 83.4% and 40% for genus and species level, respectively which emphasizes the use of ITS or trnH-psbA with rbcL+matK for much better result (Elansary et al., 2017).
Due to having no clear cut morphological differences, identification of the family Lauraceae becomes difficult, hence a molecular approach using matK, rbcL, trnH-psbA, ITS barcodes were taken in China for species identification, correcting the previous misidentification and reconstructing the phylogeny of the members and ITS was reported to show the highest accuracy (57.5%) to confirm species identity (Liu et al., 2017).
ITS 1 is recommended to be used as a potential barcode in highly diverse genera as reported in Passiflora where ITS 1 becomes highly accurate than other nuclear and plastid markers (Giudicelli et al., 2015). 12 medicinal plants were identified by DNA barcoding in Malaysia using ITS2, rpoC1, and trnH-psbA, where trnH-psbA showed the highest efficacy in the identification (Aziz et al., 2015). The molecular barcode can solve cryptic lineages of many angiosperms as reported in Dumasia, a taxonomically problematic genus of Fabaceae family, where matK, rbcL, trnH-psbA, trnL-trnF, psbB-psbF, ITS were tested to solve the lineage problem. The study revealed ITS alone or ITS in combination with any chloroplast marker can be used as potential barcodes for Dumasia (Jiang et al., 2020).

Bar-HRM Technology
High-Resolution Melting Technology or HRM has been developed in recent years to enable genotyping of plants. In this technique, the melting curve of PCR amplicons is monitored in real-time by adding specific saturated dyes as the melting curve depends on the DNA base sequences. From different shapes of the melting curve, genotype or class of different test populations is detected (Sun et al., 2016;Mezzasalma et al., 2017). DNA barcoding technique has been amalgamated with HRM in the name of "Bar-HRM Technology'' which enables resolution accuracy to differentiate change in a single base, thus enhancing identification of medicinal plants greatly. Sequence-specific probes and sequencing are not required in this technique, thus rbcL, matK, trnH-psbA, rpoC, ITS, etc. can be employed in this technique to facilitate species identification (Yu et al., 2021). The Bar-HRM method is feasible for high throughput assay (Buddhachat et al., 2015), and by applying this market samples can be identified rapidly, reliably, and accurately.

DNA Mini-barcoding
DNA mini-barcoding is useful to overcome the limitations of DNA barcoding as mini-barcodes use a smaller length of DNA usually equal to or less than 200bp which can be amplified more rapidly than regular barcodes (Meusnier et al., 2008;Srirama et al., 2014). As the amplicon length is shorter, the PCR success rate is higher for DNA minibarcodes (Särkinen et al., 2012). The main purpose of developing a mini-barcode is to identify individual target species of herbal plants to stop adulteration of natural herbal products rather than achieving universality for most species (Gao et al., 2019). It becomes difficult to identify species present in natural herbal products using mini-barcode if the number of species is more than 10 in the herbal mixture. As DNA sequences may contain unstable mutation sites, developing a mini-barcode is critical considering the position and length to discriminate between multiple species (Hajibabaei et al., 2006).

DNA Metabarcoding with NGS
Along

Discussion and Future Prospects
DNA barcoding is incomplete without the integrity of the classical taxonomic approach to confirm species identity and it should never be replaced with the classical mainstream taxonomic study. As more DNA barcode data is generated nowadays, it's important to share equitable global access to reference databases and knowledge to ensure uniform infrastructure across all the country otherwise underdeveloped or developing countries will face difficulty to keep pace with the modern advancements. The rapid progress in NGS has been considered as a threat for DNA barcoding research in some studies as whole genome sequencing may produce better species resolution than an only barcode. But sequencing the whole genome is appropriate for studying genomic complexity, diversity, and function rather than species identification and biomonitoring (Grant et al., 2021). Thus in the future, DNA barcoding will provide affordable, efficient, and high throughput solutions. International Barcode of Life Consortium (iBOL) is supervising BIOSCAN project with 180 million dollars funding, assumed to be completed in 2027. In the next phase, iBOL has planned to initiate a "Planetary Biodiversity Mission" with an estimated cost of 500 million dollars and this project will be activated in January of 2026, aiming to complete the census of all multicellular species, to establish a global biosurveillance program and to construct a library of life by preserving DNA extracts from all species.
Emphasize should be given on which specific method is applicable for which species instead of better discrimination rate between species, as whole plastid genome sequencing technology is progressing rapidly . Rapid development and application of machine learning to check quality and disorientation of sequence data in the reference database will help to reduce errors due to mislabeling of barcodes with voucher specimens (Krachunov et al., 2019). More funding should be allocated in the herbaria for proper maintenance of voucher specimens as DNA barcodes obtained from well-curated specimens increase confidence and provide quality data stored in the reference database (Grant et al., 2021). Through biomonitoring using more available barcodes, it will be possible to detect more endangered species and invasive species in the future (Kuzmina et al., 2018). In the future, with the availability of exclusive bioinformatic tools, many DNA barcoding problems will be solved as biological data science is progressing rapidly.

Conclusions
DNA barcoding, significant development of molecular systematics, is a springboard to estimate diverse groups where the morphological study is painstakingly difficult and/or where the total number of species is beyond the grasp of classical taxonomists to investigate. With the advent of metagenomics and next-generation high throughput sequencing technology, DNA barcoding is progressing very fast. Integration of the taxonomic knowledge with the DNA barcoding approach is more satisfactory to bring a high level of accuracy. Beyond biodiversity monitoring, DNA barcoding knowledge can significantly reduce threats to global biodiversity by improving natural resource management, linking ecological assessments, and increasing awareness among the common mass.

Disclaimer
The author is solely responsible for the writing of this review paper.

Conflict of Interest Statement
The author has declared that there is no conflict of interest.