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10 From genes to greens: Approaches to molecular authentication

11 of medicinal plants
12 Abstract
13 In medicinal plant research, numerous applications have been established to harness the
14 phytochemicals derived from these plants having biological importance, particularly drug
15 development. However, proper identification of plants prior to research is fundamental for scientific
16 accuracy, safety, therapeutic efficacy, and importantly conservation. There are a myriad of plant
17 identification techniques, ranging from basic morphological methods to advanced molecular
18 approaches encompassing DNA barcoding, polymerase chain reaction (PCR), next-generation
19 sequencing (NGS), metabolic profiling, etc. This review examines the pros and cons of those
20 techniques extensively used in plant authentication. Here, we delve into the principles and applications
21 of key techniques, with special attention to their strengths and limitations, taking factors such as
22 accuracy, sensitivity, cost, and ease of implementation into account. Moreover, we highlight state-of-
23 the-art techniques and innovative approaches in molecular plant identification, encompassing the
24 integration of bioinformatics tools and the progression of multiplex assays.
25 Keywords: DNA microarray; LAMP; pPlant authentication; SNPs; Sspecies-specific molecular
26 markers

27 Introduction
28 India is home to a rich and diverse array of medicinal plants, with approximately 7,000 species
29 known for their use in various medicinal purposes (1). Ayurveda, one of the oldest whole-body healing
30 systems, has more than 90% plant-based formulations. Historically, the exploration of plants for
31 treating various disorders was initiated centuries ago. Subsequent investigations often relied on the
32 traditional knowledge of these folk medicines as a ground basis for further research. However, the
33 foundation of any research concerning plant-based drug discovery or any plant-based study lies in their
34 meticulous, unbiased identification. For example, if a phytochemical is found in a plant species listed
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35 as endangered, the exploitation of these plants for compound extraction is prohibited, necessitating the
36 synthesis of analogues instead. Crucially, aAccurate plant identification is paramount to determine if a
37 species is endangered, highlighting the importance of proper plant identification.
38 Plant adulteration, the deliberate or accidental replacement of plant materials with other
39 substances, poses significant challenges across various industries such as pharmaceuticals, food, and
40 herbal supplements. This deceptive practice can compromise product quality, efficacy, and safety,
41 leading to potential health risks for consumers and economic losses for producers. The complexity of
42 plant identification and the increasing globalization of supply chains exacerbate the problem, making it
43 difficult to detect and prevent adulteration. As a result, there is a growing need for robust techniques
44 for plant authentication to ensure the integrity and authenticity of plant-based products. Addressing
45 plant adulteration is crucial not only for consumer protection but also for maintaining trust in the
46 industry and protecting biodiversity and ecosystem health. A large numbers of plant identification
47 methods have been adopted over the years which include morphological identification, metabolites-
48 profiling, molecular approaches, etc. Morphological identification relies on the appearance of plant
49 structures such as leaves, stem, buds, fruits, etc. (2). However, relying solely on morphological
50 appearance for plant identification is not always sufficient. In such instances, a combination of
51 techniques, such as DNA barcoding, is necessary to reach a definitive conclusion (Table 1).
52 Conversely, advanced molecular techniques like next-generation sequencing (NGS) offer exceptional
53 accuracy in plant identification, yet they may not always be cost-effective. In summary, the application
54 of techniques for plant identification is not universal, as each method's suitability depends on the
55 specific circumstances at hand. With this premise, the current review focuses on comprehending the
56 merits and demerits of the techniques employed in plant authentication.
57 Evidence of adulteration in herbal medicines
58 Primarily, aAdulteration involves substituting the ingredients of herbal medicines with inferior
59 or ineffective compounds to gain financial benefits (3). Due to the high demand for affordable
60 medicines, counterfeit medications have garnered global attention and pose an increasingly
61 challenging problem for pharmaceutical industries. The adulteration of herbal medicines is not limited
62 to developing countries; it's also prevalent in developed nations. Reports indicate that around 10% of
63 herbal medicines are adulterated and sold in retail pharmacies, while a staggering 50% are distributed
64 through online channels (4). Based on their intent, there are two types of adulterations: intentional and
65 unintentional. Intentional adulteration involves purposefully substituting components with the specific
66 objective of gaining benefits, while unintentional adulteration occurs by chance, often due to the lack
67 of knowledge or carelessness of producers.
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68 A systematic review aimed at identifying the prevalence of ginger fraud in the Chinese-
69 European supply chain revealed that adulteration is a significant concern in ginger products,
70 particularly ground ginger, which is the most vulnerable species (5). SimilarlyAdditionally, the study
71 noted that the ginger supply chain from China to Europe, consisting of nine stages, exhibits a medium
72 vulnerability to fraudulent activities. SimilarlyLikewise, Tulsi (Ocimum tenuiflorum), often dubbed the
73 "queen of herbs," is also threatened by adulteration. This plant contains plentiful biologically
74 important molecules and plays a crucial role in maintaining good human health. However, in herbal
75 medicine, Tulsi can be surrogated with other Ocimum species, which may pose significant health risks
76 to humans (6). Furthermore, the role of Ganoderma, also known as the "king of herbs," in various
77 biological applications cannot be overstated. It is renowned for boosting immunity, fighting infections,
78 providing hepatoprotection and liver detoxification, balancing blood sugar, and combating cancer, etc.
79 (7). Unfortunately, some unethical practices involving adulteration of products derived from the plant
80 such as Gonoderma lucidum spore oil (GLSO) with low-cost vegetable practices have also been
81 reported (8). Strikingly, adulteration in herbal products is not limited to the aforementioned plant
82 species; a multitude of plants are affected by this widespread phenomenon. Here, only particular plants
83 are highlighted because they are among some of the most highly recognized for their medicinal
84 purposes. On this ground, the focus of the present review was centred on assessing the suitability of
85 various molecular approaches for plant authentication.
86 Isoenzyme markers as a tool for plant identification
87 Isoenzymes, otherwise known as isozymes, are different molecular forms of the same enzyme
88 that catalyse the same chemical reaction but differ in their amino acid sequences and often in their
89 kinetic properties, regulatory mechanisms, and physical properties such as isoelectric point and
90 electrophoretic mobility. Isoenzymes provide a means of fine-tuning metabolic processes and can be
91 used as markers in plant identification and genetic studies due to their distinct and inheritable patterns
92 (9). To exemplify its their application in plant identification, Krulíčková and others exploited
93 isoenzymes such as acid phosphatase and esterase for the identification of Flax and Linseed cultivars
94 which that were ‘morphologically distinct’ (10). Similarly, in the family Lamiaceae, isoenzyme
95 markers have been used to measure genetic variability within and among plant populations,
96 highlighting the widespread application of isoenzymes in plant identification (11). Conversely, this
97 technique also has several demerits, including: 1) the discriminatory power of isoenzyme markers in
98 distinguishing the samebetween species or varieties is much lower due to their limited polymorphism,
99 2) the expression of isoenzymes can be influenced by environmental factors, leading to non-genetic
100 variability, and 3) the expression of isoenzymes may not be uniform throughout a plant, which
101 challenging their use for whole-plant identification (12).
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102 Restriction Fragment Length Polymorphism (RFLP)

103 This technique employs restriction enzymes, also known as 'molecular scissors,' which are so -
104 called because they can cleave DNA at specific sites determined by the nucleotide sequence. Each
105 restriction enzyme is unique in terms of its restriction site, and most are co-dominant and locus-
106 specific. When DNA containing numerous restriction sites is allowed to react with restriction enzymes,
107 it cleaves the DNA into small pieces of varying lengths. Upon separating these fragments by gel
108 electrophoresis, unique banding patterns, otherwise known as DNA fingerprints, are generated for each
109 genetically distinct DNA sample (13). Sometimes, gel electrophoresis is coupled with Southern
110 blotting, where the DNA bands from agarose gels are transferred to a membrane. This step may beis
111 often followed by hybridization with labeled DNA probes that bind to specific nucleotide sequences,
112 enhancing the detection of polymorphisms. The resulting DNA fingerprints can then be compared
113 across different plant samples. Each plant species or genotype has a unique pattern, allowing for
114 identification by comparing it with reference DNA fingerprints. There are a few someevidence of
115 RFLP being used as a molecular tool to discriminate between the plant species belonging to the same
116 genus (14-17). Notably, while this technique is still in use, the RFLP was extensively used in the
117 1990s. However, it is now often regarded as an obsolete method in plant identification (18).
118 Random Amplified Polymorphic DNA (RAPD)
119 Unlike RFLP, the RAPD is a polymerase chain reaction (PCR)-based technique where a single
120 ‘arbitrary’ primer is used to target multiple sites within a single genomic DNA (13). To identify a plant
121 species, genomic DNA isolated from a plant sample is subjected to PCR using an arbitrary primer.
122 This primer initiates DNA synthesis at multiple sites in the target DNA, yielding a mixture of DNA
123 fragments. Gel electrophoresis then separates these fragments based on their molecular size, producing
124 a unique DNA fingerprint for each plant. The number and length of each fragment are determined by
125 the distribution of DNA synthesis sites, which are detected by the arbitrary primers. Earlier, this
126 technique has been employed to identify adulterated Cuscuta reflexa which carries commendable
127 medicinal properties (19). There are also proof that RAPD is often applied to authenticate the plant
128 species to prevent adulteration (20-22). Similarly, another study used RAPD markers to assess the
129 genetic diversity among the Cardiospermum halicacabum Linn. Populations were collected from five
130 different regions in Kerala (23). One of the notable advantages of the RAPD technique is that it doesn't
131 require any prior information about the targeted DNA, particularly about nucleotide sequence. Further,
132 this technique can target both coding and non-coding regions of the DNA. On the other hand, most of
133 the primers used in RAPD are were dominant, and designing arbitrary primers can be technically
134 challenging, representing significant drawbacks. Moreover, this technique relies heavily on PCR,
135 where maintaining precise cyclic conditions is crucial. As a result, reproducibility is challenging
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136 because RAPD uses short primers (10-12 nucleotides long), which may lead to non-specific
137 amplifications.
138 Amplified Fragments Length Polymorphism (AFLP)
139 The AFLP technique is generated by amalgamatingcombines elements of the two previously
140 discussed techniques, namely RFLP and RAPD. Here, the plant DNA is initially digested with two
141 restriction enzymes, typically a frequent cutter and a rare cutter to create a large numbers of fragments
142 of varying length. This will be accompanied by an adaptor ligation step wherein a short double -
143 stranded adaptor will be added to the sticky end of each DNA fragment produced by restriction
144 enzymes (Fig. 1 and; figFig. 2). Next, a first round of PCR will be employed to using the primers that
145 are complimentary to adaptors plus one additional selective nucleotide to amplify a subset of the
146 restriction fragments. Further, inIn the second round of PCR, further selectivity is achieved using more
147 selective PCR is conducted using primers with additional selective nucleotides (typically three). This
148 step further narrows down the subset of fragments amplified, resulting in a manageable number of
149 fragments for analysis (24). Even though AFLP takes advantage of integrating two promising
150 molecular markers (RFLP and RAPD), it fails to fully address the shortcomings of individual
151 techniques.
152 Simple Sequence Repeats (SSR)
153 The SSR, also known as a microsatellite, is a tract of repetitive DNA in which certain DNA
154 motifs (ranging from 1 to 6 base pairs in length) are repeated in tandem, typically 5-50 times. These
155 sequences are distributed throughout the genome and are highly polymorphic, making them useful
156 markers for genetic studies (25). Unlike the aforementioned techniques, SSR entails prior information
157 about the plant genome, particularly the identification of microsatellite regions. Using this sequence
158 information, primers are designed to flank the microsatellite regions, which will then be amplified by
159 PCR (Fig. 3). This process creates copies of SSR regions that are specific to each plant type. The
160 resulting differential banding pattern can be visualized using gel electrophoresis, followed by staining
161 the agarose gel with an appropriate stain. Rather than mere plant identification, SSR is widely used in
162 investigating the genetic diversity among the same plant species (26). The major pitfalls of this
163 technique include: 1) it cannot be applied to plants lacking genomic data, 2) it is expensive, labor-
164 intensive, and time-consuming, 3) SSR markers developed for one species may not be applicableapply
165 to other, even closely related species, limiting their use across different species, 4) SSRs can have a
166 high mutation rate, complicating the interpretation of results, especially in evolutionary studies, 5)
167 during PCR amplification, stutter bands (artifacts) can occur, which are shorter than the actual SSR
168 amplicons and can make scoring of alleles difficult, and 6) since SSR markers are co-dominant, one of
169 the alleles in a heterozygous individual may sometimes not amplify well, leading to incorrect
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170 genotyping (allelic dropout). Despite these challenges, SSR markers remain a valuable tool in genetic
171 research due to their high polymorphism, co-dominant inheritance, and wide distribution across the
172 genome.
173 Inter Simple Sequence Repeats (ISSR)
174 Inter-Simple Sequence Repeats (ISSRs) are DNA regions located between microsatellite loci
175 that are used as molecular markers. This technique involves amplifying these regions using primers
176 anchored in the microsatellite repeats, resulting in highly polymorphic and reproducible DNA
177 fingerprints (Fig. 4.) useful for studying genetic diversity and phylogenetics studies (Fig. 4.). This
178 technique is comparatively advanced and attested to be a reliable method especially to identify the
179 different cultivars in many plant species (27). For instance, cultivars among the plant species Rheum
180 officinale, R. palmatum, and R. tanguticum were identified using ISSR primers, while the same
181 technique was applied to differentiate Cissampelos pareira from its adulterants Cyclea peltata and
182 Stephania japonica (28-29). The greatest advantage of this technique is its versatility, as it can be
183 applied to a wide range of plant species even without genomic information. Furthermore, this
184 technique offers exceptional coverage, with ISSR primers targeting multiple loci across the plant
185 genome. Conversely, despite its versatility, the primers may not be 100% successful in all cases.
186 Overall, ISSR markers are a valuable tool for genetic analysis, particularly when prior genomic
187 information is lacking, but their dominant nature and sensitivity to PCR conditions require careful
188 consideration and optimization.
189 Sequence Characterization of Amplified Regions (SCAR)
190 SCAR markers, in fact, are designed by drawing inspiration from molecular markers like
191 RAPD or RFLP. These markers are developed by sequencing the ends of a polymorphic DNA
192 fragment and designing specific primers that amplify this fragment in a PCR reaction (Fig. 5). Hence it
193 is a potential tool for authenticating the plant species (30). The first step in this method involves the
194 identification of polymorphic regions using preferably by RAPD or other similar techniques. Then, the
195 polymorphic regions are sequenced in order to determine the end sequence, based on which SCAR
196 primers are designed. These primers are typically 18-24 nucleotides long and are highly specific to the
197 target regions, thereby minimizing the potential for non-specific PCR amplification. The SCAR
198 markers have extensively been used in genetic linkage mapping to locate genes associated with
199 specific traits (31). Further, these markers are promising for the authentication of a variety of plant
200 species (32). However, considering the fact that SCAR markers alone cannot detect polymorphism but
201 rather depend on RAPD, and given the technical challenges and expenses involved in developing these
202 markers, their application may have certain restrictions.
203 Allelic-Specific Diagnostic PCR
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204 It is a technique used to identify specific alleles (variants of a gene) within a plant genome. The
205 principle involves using primers that are designed to match specific nucleotide sequences unique to
206 particular alleles. These primers will only amplify the DNA if the target allele is present, allowing for
207 precise identification of genetic variants. Apart from the variety authentication of plants, this technique
208 has also been used in screening of the pathogen resistance to diseases of pests (33). The major
209 limitation is that this technique is restricted to known variants, and novel or unknown variants cannot
210 be detected. Additionally, it is primarily useful for small-scale studies. For large-scale genotyping
211 projects, a refined technique known as Kompetitive Allele-Specific PCR (KASP) offers a high-
212 throughput and automated approach with fluorescence-based detection, making it particularly useful
213 (34).
214 Amplification Refractory Mutation System (ARMS)
215 It is again a PCR-based technique that detects specific mutations or variations by exploiting the
216 sensitivity of the Taq polymerase enzyme to mismatches between the primer and template DNA at the
217 3' end of the primer. This method is particularly effective for distinguishing between alleles that differ
218 by a single nucleotide polymorphism (SNP) (35). The protocol involves designing two sets of primers,
219 one for the wild-type allele and one for the mutant allele. During PCR, if there is a mismatch at the 3'
220 end, the extension step will not proceed efficiently because DNA polymerase can only extend primers
221 that perfectly match the template at the 3' end. Therefore, ARMS can be used to authenticate the plants
222 which that uniquely carry specific alleles. For instance, Panax ginseng, used in herbal medicine for
223 regulating diabetes, cholesterol, and inflammation, are often adulterated with materials from other
224 Panax species. However, Yang and his team employed the ARMS technique to authenticate the plant
225 accurately (36). Specifically, they designed a tetra-primer ARMS-PCR assay to detect the SNPs within
226 trnL-trnF locus. Similarly, another study with a parallel objective of preventing fraudulent labelling of
227 plants, designed a pair of diagnostic primers for ARMS (37). On one hand, the technique is praised for
228 its specificity, high sensitivity, and cost-effectiveness; on the other hand, it faces major drawbacks
229 such as the complexity of primer design and its limitation to known variations.
230 Single Nucleotide Polymorphisms (SNPs)
231 SNPs claim a pivotal role in plant authentication by providing a reliable approach for
232 identifying and differentiating plant varieties through SNP profile comparison (38). It They also serves
233 as a potential marker for the identification of individual plants when SNP profiles are matched with a
234 reference database of known species. Further, SNP analysis in association with other PCR-based
235 markers can be implemented to detect adulteration in plant products by identifying the presence of
236 foreign or unintended plant species. This is particularly important in the food and herbal medicine
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237 industries, where as product purity and authenticity are critical essentail for consumer safety and
238 regulatory compliance (39).
239 In fact, SNPs can occur in plant genomes owing to a variety of biological and environmental
240 reasons such as error in DNA replication, DNA damage and repair, mutations, natural selection,
241 population mixing, genetic drift, etc (40). Even though these variations can occur in both coding and
242 non-coding regions, their frequency is higher in non-coding regions. Similarly, while restriction sites
243 are distributed throughout the genome, they tend to be more abundant in non-coding regions. This is
244 because mutations that create or eliminate restriction sites in coding regions can alter protein
245 sequences, potentially impacting protein function and organism fitness. If SNPs occur at the restriction
246 sites of specific restriction enzymes, they serve as Cleaved Amplified Polymorphic Sequence (CAPS)
247 markers (41). CAPS markers exploit SNPs that introduce or remove restriction enzyme recognition
248 sites in DNA sequences (Fig. 6). In this method, genomic regions containing the SNPs are PCR-
249 amplified using specific primers. To infer the SNP genotypes, these amplicons are subjected to
250 restriction enzyme digestion, and the presence or absence of restriction sites is determined by
251 analyzing the band patterns on gel electrophoresis. Owing to its specificity and precision, this
252 technique is widely used in plant authentication. For example, the authentication of Tetrastigma
253 hemsleyanum has been successfully achieved using CAPS markers along with other molecular markers
254 (42).
255 Loop Mediated Isothermal Amplification (LAMP)
256 LAMP is a technique used to amplify DNA with the help of a temperature, primers, and
257 enzymes, similar to what PCR does. However, interestingly, LAMP is not a PCR-based method and is
258 distinctly different from the principles and workflow of PCRs (Fig. 7). The key differences between
259 them include the following: PCR requires three different temperatures for denaturation, annealing, and
260 extension of primers, whereas LAMP operates at a constant (isothermal) temperature typically around
261 60°C-65°C. Additionally, unlike PCR, which uses only one forward primer and one reverse primer,
262 LAMP requires two inner primers and two outer primers to target six distinct regions on the target
263 DNA, increasing the specificity and precision (43). Notably, the isothermal nature and rapid
264 amplification ability of LAMP make it ideal for point-of-care diagnostics and field applications.
265 In the field of plant authentication, LAMP has gained momentum over PCR owing to its rapid
266 amplification abilities and limited instrument requirements. To distinguish Panax ginseng from its
267 adulterant P. japonica, LAMP was employed to target the 18S ribosomal RNA conserved in P.
268 ginseng. This approach yielded a positive response only for P. ginseng DNA (44). Similarly, ITS2
269 region conserved in Crocus sativus was targeted to differentiate from the adulterants using LAMP
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270 (45). Noteworthy, by leveraging advantages, LAMP provides a robust and efficient method for plant
271 authentication, ensuring the integrity and quality of plant-based products in various industries.
272 Array Based Markers (Hybridization)
273 So far, we discussed a variety of techniques that have wide applications in plant authentication.
274 Strikingly, all the aforementioned techniques can target only one or a few loci in the plant genome.
275 However, a microarray marker has the ability to analyse thousands of genes simultaneously. In fact,
276 mMicroarray system works on the ground nucleic acid (DNA/RNA) hybridization to complimentary
277 complementary sequences attached on a solid surface (46). In this method, specific DNA sequences
278 complementary to the target genes are synthesized and coated on a solid surface, preferably a glass
279 slide or silicon chip. The nucleic acid samples from the plant tissue may be extracted and labelled with
280 fluorescent dyes, followed by fragmentation to ensure the efficient hybridization. While scanning the
281 array using a laser, the fluorescent labels, hybridized with the probes may shift to an excited state,
282 emitting fluorescence that can be detected and quantified. Intensity The intensity of fluorescence is
283 directly proportional to the amount of DNA hybridization. Therefore, this data can be analyzed to
284 determine gene expression levels or the presence of specific genetic variations. By leveraging
285 multiplex detection of genes, microarray provides a high-throughput and a benchmark method for
286 varietal identification by designing probes targeting the cultivar-specific loci (47). Similarly, by
287 targeting the conserved regions of plants, species identification can easily be achieved by microarray.
288 One of the commendable advantages of this technique is its ability to provide comprehensive analysis,
289 capable of providing broad genetic information of plants from a single experiment. Additionally, it
290 offers much higher speed compared to conventional Sanger sequencing. On the contrary, microarray
291 technology is reliedrelies on prior knowledge of the genome, which limits its ability to detect novel
292 sequences or unexpected mutations. Additionally, microarrays can suffer from cross-hybridization,
293 where non-specific binding occurs, leading to potential false positives or inaccuracies in data
294 interpretation. Though a sequence-independent microarray system, known as sequence-independent
295 single primer amplification (SISPA) microarray, has been introduced, its applicability is questioned
296 due to low specificity and data complexity (48).
297 DNA barcoding
298 Much like libraries employ barcodes to catalogue books, DNA barcoding identifies organisms,
299 including plants, by analyzing specific regions of their DNA. This technique functions on the ground
300 that DNA segment varies enough between species to be distinctive but is ‘conserved’ enough within a
301 species to be reliable. In plants, the conserved region preferably used for this purpose includes the part
302 of chloroplast DNA (49). Numerous studies illustrated the efficacy of DNA barcoding in the
303 authentication of plant species. For instance, the Internal Transcribed Spacer of nuclear ribosomal
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304 DNA (nrDNA-ITS) and chloroplast spacer sequences rpoB and rpoC1 which code for DNA directed
305 RNA polymerase beta and gamma chains, respectively have been used to differentiate the Ruta
306 graveolens from its adulterant Euphorbia dracunculoides (50). Similarly, PsbA-TrnH (Photosystem-II
307 protein D1-structral RNA His tRNA) intergenic spacer was used for the identification of different
308 Aconitum species (51). Despite its widespread application across various fields, a significant limitation
309 of DNA barcoding is that the plastid genome, which is maternally inherited in almost all flowering
310 plants, can lead to inaccurate results in hybrid plants during DNA barcoding. This issue similarly
311 affects polyploid plants.
312 Next-Generation Sequencing (NGS)
313 NGS is considered as the gold standard in plant authentication methods, facilitating
314 comprehensive analysis through whole-genome sequencing of an organism, encompassing all plant
315 species. NGS allows for the sequencing of entire genomes or specific genomic regions at
316 unprecedented speed and accuracy. This capability is particularly advantageous for plant
317 authentication, as it provides comprehensive genetic information that can distinguish between closely
318 related species and even different varieties within a species. By targeting multiple genetic loci
319 simultaneously, NGS enhances the resolution of plant identification far beyond what is achievable with
320 traditional methods. For instance, DNA barcoding, which uses short genetic sequences from a
321 standardized region of the genome, can be exponentially more powerful when combined with NGS
322 (52). This combination allows for the identification of plants at the species level with high confidence,
323 even in cases where morphological distinctions are subtle or non-existent. Interestingly, those
324 molecular markers, tailored for plant authentication which requires sequence information, are
325 developed largely based on the NGS-generated sequence data. Apart from exploiting existing
326 molecular markers, NGS also facilitates the discovery of novel markers, effectively addressing the
327 shortcomings of current plant authentication techniques. One of the primary advantages of NGS is its
328 high-throughput nature, enabling the simultaneous analysis of numerous samples. This is particularly
329 beneficial for large-scale studies and commercial applications where the rapid and accurate
330 authentication of many plant samples is required. For example, in the agriculture industry, NGS can be
331 employed to verify the authenticity of crop varieties, ensuring that farmers receive the correct seeds
332 and protecting against the adulteration of agricultural products. Till date, tThere are a myriad of
333 sequencing platforms available in the market encompassing Illumina, PacBio, Oxford Nanopore, etc.
334 However, choosing the right platform will depend on the specific needs of the plant authentication
335 project, and in some cases, a combination of platforms may be used to achieve the best results.
336

337 Conclusions
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338 Plant adulteration, the intentional or accidental substitution of biologically important plant materials
339 with unclaimed substances, is not uncommon, especially in industries like pharmaceuticals, food, and
340 herbal supplements. In this regard, techniques for plant authentication play a pivotal role in
341 successfully addressing plant adulteration. Molecular markers are essential tools for plant
342 authentication, providing the precision and reliability needed to distinguish plant species accurately.
343 Techniques such as DNA barcoding, using regions like rbcL and matK, offer a standardized approach
344 for species identification. Microsatellites (SSRs) and single nucleotide polymorphisms (SNPs), on the
345 other hand, enable fine-scale discrimination, particularly useful for identifying closely related species
346 or different cultivars. Advances in next-generation sequencing (NGS) have further enhanced marker
347 discovery and validation, allowing for high-throughput analysis and the development of novel markers.
348 Each type of molecular marker has its strengths, making them collectively invaluable for various
349 applications in agriculture, pharmaceuticals, and conservation. However, choosing the right technique
350 depends entirely on the specific needs of the study, and often, a combination of two or more
351 techniques is required to draw a definitive conclusion.

352 Did you use generative AI to write this manuscript?

353 We didn’t use any AI support while drafting this manuscript.
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739 Figure Captions

740 Fig. 1.Schematic Representation of AFLP process. Frequent cutter restriction enzyme MseI and
741 Rare cutter EcoRI are shown here. A). Since the adapter sequence and the sticky ends of the
742 template DNA are compatible, the adaptor binds to the sticky ends of the DNA. B).Primers are
743 designed using the sequence of adaptor. In Pre-amplification primer is supplemented with
744 additional base. C) In selective Amplification step primer is supplemented with additional two
745 bases.
746 Fig. 2. Mismatch amplification using four selective nucleotides. Here D can be (G, C, A).
747 Fig. 3. Schematic representation of steps of SSR technique.
748 Fig. 4. Schematic representation of steps of ISSR technique.
749 Fig. 5. Schematic representation of SCAR analysis
750 Fig. 6. CAPS technique : Schematic representation
751 Fig. 7. Diagramatic Representation of LAMP Amplification Products
752
753
754
755
756
757
758
759
760
761
762
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763
764
765
766
767
768
769
770
771 Table.1. Application of various molecular techniques for authentication of herbals

SL No Molecular technique Medicinal plant Reference

1 DNA barcoding Piper nigrum L. (53)

2 DNA-based methods (genome Cinnamomum burmannii, (54)

skimming, PCR with mini C. cassia, and C. verum
markers)

3 PCR–RFLP Orthosiphon stamineus (55)

4 PCR- RFLP Ocimum tenuiflorum (56)

5 qPCR Cinnamomum verum (57)

6 DNA barcoding Phyllanthus species (58)

7 Duplex-PCR Moringa oleifera (59)

8 DNA barcoding through Tinospora cordifolia (60)

chloroplast genome sequencing

9 ISSR Mentha species (61)

10 DNA Barcoding Trillium govanianum Wall. ex D. Don (62)

11 ISSR and SCOT Dendrobium officinale (63)

12 ISSR Matricaria recutita L. (64)

13 SCAR Marker-based PCR Bacopa monnieri (65)

14 (PCR-RFLP) Portulaca oleracea (66)

15 DNA barcoding Viola philippica Cav. (67)

16 DNA barcoding and HPLC Labisia pumila (68)

17 DNA barcoding Wikstroemia indica (69)

18 SCAR Viscum coloratum (70)

 25

19 DNA barcode marker ITS2 Gnaphalium affine (71)

20 DNA barcoding Withania somnifera (72)

21 DNA Barcoding Species-Specific Loranthus tanakae Franch. & Sav (73)

Primers based on matK Sequences

22 K - mer based approach combined Lupinus species (74 )

with metagenomic sequencing

23 DNA barcoding Ligusticum sinense (75)

24 SSR and SNP markers Glycyrrhiza glabra L. (76)

25 ISSR markers and FTIR analysis Cassia tora L. (77)

26 DNA barcoding Physalis (78)

27 DNA barcoding Smithia conforta Sm. (79)

28 DNA Barcoding Lasianthus Jack (80)

(Lasiantheae: Rubiaceae)

29 ISSR Centella asiatica (81)

30 DNA metabarcoding Veronica officinali (82)

31 SCAR Adenophorae radix (83)

32 NGS Valeriana officinalis (84)

33 ISSR Tetrastigma hemsleyanum (42)

34 DNA Barcoding dendrobium species (85)

35 DNA barcoding Sida cordifolia (86)

36 DNA barcoding Swertia chirayita (87)

37 PCR- SRAP molecular markers Hedera species (88)

38 Barcoding with High-Resolution Acanthaceae Species (89)

Melting Analysis

39 DNA barcoding Peucedanum praeruptorum (90)

40 RAPD and ISSR Ganoderma species (91)

41 SCAR Aconitum heterophyllum (92)

42 SCAR Crocus sativus L. (93)

43 ISSR Cissampelos pareira L. var. hirsuta (29)

(Buch.-Ham. ex DC.)
 26

44 DNA barcoding Asparagus racemosus willd (94)

45 DNA barcoding Rhododendron species (95)

46 DNA barcoding Boerhavia diffusa (96)

47 RAPD Ipomoea mauritiana Jacq. (97)

48 DNA barcodes family Araliaceae (98)

49 DNA barcoding followed by the Ruta graveolens (50)

development of SCAR markers

50 AFLP Zingibera officinale (99)

51 RAPD profiling Senna angustifolia (21)

52 DNA barcoding Sabia parviflora Wall. ex Roxb. (100)

53 DNA barcoding Taxillus chinensis (101)

54 DNA barcode marker Medicinal Pteridophytes (102)

55 RAPD Cuscuta reflexa (19)

56 DNA barcoding Fabaceae (103)

57 DNA Barcoding Lonicera japonica (104)

58 RAPD Chlorophytum species (105)

59 DNA barcoding Valeriana jatamansi (106)

60 RAPD Desmodium species (107)

61 SCAR Cynanchum wilfordii (108)

62 RAPD Glycyrrhiza glabra Linn. (109)

63 RAPD Myristicaceae family (110)

64 PCR-amplified ITS2 with specific Aristolochia manshuriensis (111)

primers

65 Tinospora cordifolia (Willd.) miers ex (112)

RAPD hook f. & thomas

66 ISSR Asparagus acutifolius L. (113)

67 DNA microarray Datura inoxia (47)

68 RAPD Curccuma species (114)

69 RAPD Astragali radix (115)

 27

70 species-specific PCR Camellia sinensis (116)

71 RAPD-PCR analysis Cimicifuga species (117)

72 RAPD Scutellaria galericulata (118)

73 AP-PCR Ginseng roots (119)

74 RFLP Duboisia (14)

772
773 Fig. 7. Schematic Representation of AFLP process. Frequent cutter restriction enzyme
774 MseI and Rare cutter EcoRI are shown here. A). Since the adapter sequence and the
775 sticky ends of the template DNA are compatible, the adaptor binds to the sticky ends of
776 the DNA. B) .Primers are designed using the sequence of adaptor. In pPre-amplification
777 primer is supplemented with additional base. C) In selective aAmplification step primer is
778 supplemented with additional two bases.
 28

779
780
781
782
783
784
785
786
787
788

789
791 Fig. 8. Mismatch amplification using four selective nucleotides. Here D can be (G, C, A).
 29

792

793 Fig. 9. Schematic representation of steps of SSR technique.

794
795
796
 30

797
798 Fig. 10. Schematic representation of steps of ISSR technique.
 31

799
800 Fig. 11. Schematic representation of SCAR analysis.

801

802
 32

803
804 Fig. 12. CAPS technique : Schematic representation.
 33

805

806 Fig. 7. Diagramatic rRepresentation of LAMP aAmplification pProducts.

807
808
809
810
811
812
813
814
815
816
817
818
819