Traditional herbal and herbs-mineral drugs have been used since the dawn of civilization to maintain and alleviate human sufferings from diseases. According to an estimate of the World Health Organization (WHO) , about 80 per cent of the world population still uses herbs and other traditional medicines for their primary health care needs. Use of indigenous drugs from plant origin forms a major part of complementary and alternative medicine/traditional medicine (CAM/TM). The world market for herbal medicine, including herbal products and raw materials has been estimated to have an annual growth rate between five and 15 per cent.

The total global herbal drug market is estimated as US $62 billion and is expected to grow to US $5 trillion by the year 2050. India has a great wealth of traditional knowledge and wisdom. Ayurveda contributes Rs. 3500 crores (US $813 million) annually to the internal market. The Indian medicinal plants-based industry is growing at the rate of 7–15 per cent annually. The value of medicinal plants-related trade in India is estimated at Rs.5000 crores per annum.

From the very beginning, herb authentication has presented a great challenge for people using them for medical purposes. The authentication of medicinal plants is a critical issue for the protection of consumers as well. Usage of wrong herb may be ineffective or may worsen the condition and even cause death. Herbal drugs are normally processed parts of various plants, such as roots, stems, leaves, flowers, fruits, seeds etc. Therefore, authentication should be done from the harvesting of the plant material to the final product. Due to popularity of herbal drugs globally, their adulteration/ substantiation aspects are gaining importance at the commercial level.

Pharmaceutical companies are procuring materials from traders, who are getting these materials from untrained persons from rural and/or forest areas. This has given rise to wide-spread adulteration/ substitution, leading to poor quality of herbal formulations. Misidentification of herbs can be non-intentional (processed plant parts are inherently difficult to distinguish) or intentional (profit driven merchants sometimes substitute expensive herbs with less-expensive look-alike ones).

Conventional methods vs advanced techniques
Initially people authenticated herbs by their appearance, smell or taste. However, these methods were dependent on personal skills. With the advent of microscopy, herbs were authenticated by inspection under microscopes, where the shape and content of various plant cells were examined and analyzed. These methods, based on organoleptic markers or anatomical characters, are sometime imprecise. Further, analytical chromatography techniques, such as thin-layer chromatography, high-performance liquid chromatography, or liquid chromatography were used for herb authentication. Chemical profiling establishes a characteristic chemical pattern for a plant material, its fractions or extracts. Thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) are routinely used as valuable tools for qualitative determination of small amounts of impurities. In addition, many analytical techniques such as volumetric analysis, gravimetric determinations, gas chromatography, column chromatography, high performance liquid chromatography and spectrophotometric methods are also frequently used for quality control and standardization. However, during last two decades, molecular markers have rapidly complemented the classical strategies. Molecular markers are generally referred to as biochemical constituents, including primary and secondary metabolites in plants and macromolecules. Secondary metabolites as markers have been extensively used in quality control and standardization of herbal drugs, but these also suffer with few limitations. Recently, the focus has shifted to the development of markers based on genetic composition which are unique, stable, and ubiquitous to the plants. These DNA-based markers are not influenced by age, physiological condition as well as environmental factors. Figure 1 gives brief outline about techniques used in conventional and advanced methods.

Types of molecular markers used in plant genome analysis
Various types of DNA-based molecular techniques are utilized to evaluate DNA polymorphism. These are hybridization-based methods (Non PCR based), polymerase chain reaction (PCR)-based methods and sequencing-based methods (Figure 2).

Molecular markers differ in many qualities and must therefore be carefully chosen and analyzed differently with their differences in mind. To assist in choosing the appropriate marker technique, an overview of the main properties of the marker technologies is described in Table 1.

SCAR markers
SCAR (Sequence Characterized Amplified Region), was initially developed for downy mildew resistance genes in lettuce. A SCAR can be used to rapidly amplify a diagnostic nucleic acid from herbal materials using a pair of specific oligonucleotide primers designed from polymorphic RAPD or AFLP fragments. Polymorphic regions from RAPDs, AFLPs or ISSR are selected among amplified fingerprints. After cloning and sequencing for the selected polymorphic regions, pairs of internal primers are designed to amplify a unique and specific sequence designed as a SCAR marker (Figure 3). PCR results in a positive or negative amplification in target-containing and nontarget-containing samples correspondingly or amplification products of different sizes in the case of closely related samples.

Merits of this technique over other are: they are monolocus, less sensitive to reaction conditions, and they can be potentially converted into codominant markers, easy to use, high reproducibility. Prior sequence information (i.e., sequencing the polymorphic fragments) is required for designing the primers contiguous the polymorphic region.

Application of SCAR Markers
Authentication and identification of medicinal plants
The botanical sources of herbal supplements and medicines are identified at the species level by their Latin scientific names. Unambiguous identification and authentication of the plants used for production is consequently an elementary and critical step at the beginning of an extensive quality assurance process. Authentication is also of importance for the characterization of the genetic diversity, phylogeny and phylogeography, as well as the protection and management of endangered species.

SCAR markers have been developed for authentication of various medicinal plants including Artemisia, Panax, Atractylodes, Echinacea, Pueraria tuberose Roxb. ex. Wild, Angelica decursiva, Jatropha curcas, Curcuma alismatifolia and Lonicera japonica. SCAR marker has been used for identification of Phyllanthus emblica Linn., Litchi chinensis Sonn and Flammulina velutipes. SCAR marker is also developed for Zingiber officinale Roscoe from crude drugs and multicomponent formulations, and identification of different species of Phyllanthus viz. Phyllanthus amarus,

P. fraternus, P. debilis and P. urinaria.
Detection of adulteration/ substitution
Uncontrolled collection and sale of large quantities of plant material from forest can lead to adulteration of many medicinal plants with easily accessible plants on the basis of their significant morphological similarities. This adulteration not only decreases the therapeutic efficacy of indigenous drugs, but also hazardous effects on the body.

SCAR marker has been developed for detection of adulterants Cistus incanus L., Rubus caesius L. and Rhus coriaria L. in Mediterranean oregano, adulteration in Lycium barbarum (wolfberry), detection of papaya seed adulteration in traded black pepper powder, also for the identification of Oleae uropaea L. adulterant in commercial Mediterranean oregano, Ipomoea mauritiana Jacq.

Marker-assisted selection of desirable chemotype for plant breeding
Molecular Marker-assisted selection (MAS) is an approach that has been developed to avoid problems associated with conventional plant breeding. It provides new solutions for selecting and maintaining desirable genotype. MAS is being used to breed plants which have resistance to pathogens, insects and nematodes, tolerance to abiotic stresses, improve yield, and varieties with more desirable characters.

SCARs have been developed for downy mildew resistance genes in lettuce and nematode resistance gene in tomato. SCAR was developed for producing various disease/pest resistant plants e.g. against gall midge in Orseolia oryzae, sugarcane mosaic virus in Maize, rhizomania in Sugar beet, powdery mildew in Tomato, powdery mildew in Apple. SCAR was also developed for improvement of quality e.g. in tomato for total soluble solids (sugars and acids) and fruit elongation; in wheat (Triticum aestivum) to improve dough strength. RAPD derived SCAR marker was developed for marker-assisted selection of seedless grapevine cultivars.

Sex determination
SCAR marker provides immediate and consistent identification of sex types in plantlets growing in field. During the cultivation of dioecious plant sex determination is more useful and profitable. E.g. papaya species have three sex types (male, female and hermaphrodite) determined by a multiallelic locus. Increase cultivation of female plant increases production of fruit and papain production making plantation more profitable. SCAR allows the identification of sex type of papaya plant at the plantlet level.

SCAR marker was developed for sex identification in Pistacia chinensis Bunge, Hakea salicifolia and H. tibetana, Cannabis sativa L., Mercurialis annua L., Carica papaya L., Eucommia ulmoides Oliv.

Germplasm characterisation
Crop genetic diversity must be conserved to fight new pests and diseases, and to produce better adapted varieties for the changing environments. Seed conservation has vital role in preservation of genetic variability as it is simple to handle, economical and capable of maintaining genetic stability over long time periods. Characterization of germplasm is essential to provide information on the traits of accessions assuring the maximum utilization of the germplasm collection to the final users. SCAR marker is useful for adequate characterization for agronomic and morphological traits, necessary to facilitate utilization of germplasm by breeders.

SCAR markers were developed for germplasm characterisation of Olea europea L., Jatropha curcas L. and strawberry.

Research at PERD
At PERD Centre, research is being conducted to develop molecular markers for various applications. SCAR markers have been developed for some of the plant species under study.
a)    Differentiate male and female plant in Jojoba at early stages of development to reduce the economic burden.
b)    Identify high and low yield of specific biological markers in Bacopa monnieri, Andrographis paniculata and Adhatoda vasica.
c)    Identify adulteration and substitution in genuine raw material of medicinal plants like Terminalia arjuna, Saraca asoca, Phyllanthus amarus, Sida cordifolia.

Future prospects
Along with authentication of species identity, prediction of the concentration of active phytochemicals may be required for quality control in the use of plant materials for pharmaceutical purposes. Development of SCAR markers that can correlate DNA fingerprinting data with quantity of selected phytochemical markers associated with that specific medicinal herb would have extensive applications in quality control of raw materials. These designed markers would act as a qualitative/quantitative diagnostic tool for identification of genuine medicinal herb, from harvesting to finished product. Plant breeders can further use SCAR marker developed for a specific trait or superior varieties for medicinal crop improvement and conservation.

Conclusion
The confirmation of herbal drugs and discrimination of adulterants from genuine medicinal herbs are essential for both pharmaceutical company as well as public health. There are various types of molecular markers available in the literature for the identification of herbal drugs. Limitations of chemical and morphological, microscopical methods for authentication have generated a need for newer methods in quality control of herbal formulations and drugs. SCAR, a PCR-based marker, represents single, genetically defined locus identified by amplification of genomic DNA with a pair of specific oligonucleotide primers. SCAR markers may contain high-copy number and dispersed genomic sequences within the amplified region. Thus, they are valuable in large-scale and locus-specific applications such as marker-assisted screening and map-based gene cloning. A SCAR marker, generated from polymorphic regions that differ in size between species, permits sample authentication based on SCAR size shifts. Different DNA-based markers viz. AFLP, SSR, ISSR and RAPD can be used to generate these markers. However, high reliability of SCAR markers could lead to the displacement of AFLP and other DNA-based markers which are costly, time consuming and tedious and using some radioactive materials in detection step. Also high detection sensitivity and avoidable electrophoresis make it an economical molecular tool. Thus, so far SCAR marker seems to be the best suitable technology for authentication of traditional medicinal herbs. Molecular characterization by SCAR markers allows effective and reliable authentication and discrimination of herbs from their adulterants and identification of superior verities. The plant species, which are morphologically similar, can be differentiated using these molecular markers. Once a SCAR marker is developed for a particular species, it can be used to detect the same in homogeneous and heterogeneous formulations used in traditional system of medicines for the authentication of traditional herbal drugs.