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Crop breeding has remained one of the key operations of agricultural R&D organisations around the world. Breeding initiatives in the post-Green Revolution era aim to increase yield potential and sustain current yield levels. The transmission of improved varieties from breeding programs, when associated with favourable agronomic practices and increased market access, is found to promote agricultural production, profitability, and household nutrition, as well as system resilience against the detrimental effects of climate change. Traditionally, species and varieties are identified using distinctness, uniformity, and stability (DUS) approach, based on morphological descriptors. These morphological descriptors are less informative, time-consuming, and unreliable due to multigene nature and environmental interaction. Moreover, environmental fluctuations, poor sampling techniques and unknown genetic control make morphological characters less effective for variety identification. To address these challenges, DNA fingerprinting has appeared as a potent molecular tool for clear-cut varietal identification, genetic purity assessment, and germplasm conservation. DNA fingerprinting comprises the use of molecular markers to produce unique genetic contours of plant varieties, empowering breeders and researchers to distinguish between closely related cultivars. DNA fingerprinting has its roots in forensic science. Central work by Jeffreys in the 1980s ensued in the understanding that the revealing of variation in short sequences of DNA (minisatellites) could be used to identify a unique pattern for each individual (i.e., a DNA fingerprint). The traditional technology used restriction fragment length polymorphisms (RFLP) and was dependent on cutting DNA into various fragment lengths by restriction enzymes, splitting the fragments based on their size on an electrophoresis gel, then gestating the DNA with labelled microsatellite probes. The subsequent unique outline of bands on the gel was the DNA fingerprint. Progress in genotyping technology have impressively taken over this original methodology, giving way to lowcost, high throughput systems proficient in differentiating among narrowly related crop varieties. Presently genotyping methodology usually used in DNA fingerprinting studies include genotype by sequencing (GBS) and DArTSeq. GBS and DArTseq are reduced-representation methods, using restriction enzymes to cut the genome in repetitive regions, netting most of the functional chunks. The results from sequencing technique are greatly reproducible and

Introduction The identification of crop varieties is crucial in improving agricultural productivity, ensuring quality control, and supporting breeding programs. The correct identification helps improve food security, conserve biodiversity, and manage pests and diseases. Traditionally, farmers were relying on visual traits and phenotypic characteristics, but these methods had their own limitations. Modern molecular techniques, such as DNA fingerprinting and genotyping, have revolutionized variety identification, rendering it both more accurate and efficient. This has allowed for early identification of varieties, assisting in the selection of desired traits and enhancement of crop resilience, thus contributing to more sustainable and productive agricultural practices (Azizi et al., 2021; Khan et al., 2024). Importance of Crop Variety Identification in Agriculture Crop variety identification remains one of the most important components in agriculture since it can improve crop production, enhance food security, and improve adaptability of the crops to diverse environmental conditions (Hasan et al., 2021). The major aspects related to correct identification of crop varieties include: • Quality control and standardization: It is often to measure the quality of crops that could meet market demand, for optimum consumption, and for export. • Biodiversity conservation: This helps to conserve native crop varieties and regards the preservation of genetic diversity that is key for breeding and in future agricultural resilience. • Breeding program: It allows the identification of a variety for selecting traits such as disease resistance, drought tolerance, and yield potential for improving agricultural productivity through its effectiveness. • Pest and disease management: It contributes to pest and disease management too since different crop varieties may express varying levels of resistance which also reflects in pest control strategy. • Market preferences and economic value: This helps the farmers to supply some specific markets depending on the market demand of certain varieties so as to gain a profit (Hussain & Nisar, 2020; Sharma & Singh, 2021; Tazeb et al., 2018; Dheer et al., 2020; Jamali et al., 2019).

Introduction In recent years crop yields have increased significantly due the development of new varieties and cultivars. Certain characteristics, like disease resistance, plant height, yield potential, and maturity period, set one variety of a cultivated crop species apart from other variations of the same species. A key element of contemporary agriculture is the precise identification of crop types, which is essential for plant breeding, seed certification, and the defence of intellectual property rights. The increasing demand for improved crop varieties with increased yield, disease resistance, and environmental adaptability has demanded the development of accurate methods for differentiating cultivars (Hannah, 2024). Traditional techniques of variety identification rely on morphological, biochemical, and agronomic features, which can be altered by environmental conditions and are generally time-consuming and labour-intensive. Various molecular marker systems have been developed to differentiate plant varieties and species of agricultural and horticultural significance. For effective seed quality control programs and the preservation of breeders’ rights, it is essential to define the identity, genetic purity, and stability of plant varieties. According to Yuan et al., (2022), precise variety identification is essential for determining genetic purity, analyzing genetic diversity, improving germplasm for better agronomic qualities, and getting rid of duplicate accessions. For plant protection and production systems of Intellectual Property Rights (IPR) and Plant Biodiversity Register (PBR), accurate cultivar identification is crucial. According to Ghosh et al. (2001), molecular marker approaches are pertinent to PBR registration in the following ways. (1) In order to establish a set of comparison cultivars, genetic distance analysis between the candidate cultivar and the current pool of cultivars is performed. (2) The contribution of the comparison cultivar to PBR registration is then determined, and (3) the use of DNA markers to identify cultivars in cases where PBR infringement is alleged is investigated. The capacity of PCR-based markers to amplify particular DNA sequences has made them popular among molecular marker techniques, enabling accurate identification of genetic variations. Although conventional approaches are still employed for Plant Variety Protection (PVP), molecular techniques can be useful adjuncts that increase the effectiveness of accomplishing this goal. During variety certification, additional information from isozyme analysis, RFLP, SSR, SNP, or other genetic fingerprinting methods can be added to cultivar descriptions. Plant varieties can be legally protected on the basis of precise and unique molecular marker data. Crops with little morphological variation benefit greatly from molecular methods. To guarantee complete protection of cultivars, it is ideal to use both botanical and molecular evaluations (Yu and Chung, 2021). Amplified Fragment Length Polymorphism (AFLP), Random Amplified Polymorphic DNA (RAPD), Simple Sequence Repeat (SSR), and Inter Simple Sequence Repeat (ISSR) are the four main PCR-based markers used in crop variety identification that are the subject of this chapter. In plant genetics, each of these marker systems has special qualities, benefits, and uses. While AFLP markers give high resolution and genome-wide coverage, RAPD markers offer a rapid and economical method of polymorphism detection. Due to their high polymorphism and co-dominant inheritance, SSR markers—also referred to as microsatellites—are perfect for genetic mapping

Introduction Crop improvement is critical for tackling global food security challenges caused by population growth, resource scarcity, and climate change (Singha and Singha, 2024). Traditional breeding techniques, such as Sanger sequencing, have been around for a long, but their accuracy, speed, and usefulness are typically limited. Sanger sequencing is useful for small-scale inquiries, but its laborintensive procedures and limited throughput make it unsuitable for large-scale genome analysis. Molecular marker-based breeding strategies have proven to be effective tools for addressing these difficulties by increasing plant growth and overall quality (Bohar et al., 2020). In this respect, molecular markers like as single nucleotide polymorphisms (SNPs), insertions/deletions (indels), simple sequence repeats (SSRs), and structural variations (SVs) are expected to be useful for future breeding operations (Panahi et al., 2024). The discovery and characterization of these molecular markers is strongly reliant on advances in sequencing technology, namely next generation sequencing (NGS). Furthermore, NGS technologies have shown to be crucial for functional genomics, allowing for large-scale transcriptome and whole genome sequencing across plant taxa (Singh et al., 2024). These vast databases give a comprehensive picture of gene expression and genetic variation, making it easier to identify functional markers linked with certain features (Figure 1). The integration of NGS and bioinformatics technologies has simplified the study of these datasets, enabling more efficient discovery of markers and improving the use of marker-assisted selection (MAS) in breeding programs (Panahi et al., 2024). The development of high-throughput genotyping technology has enabled substantial SNP-based research, which has improved our understanding of genetic diversity, population structure, and disease association (Yirgu et al., 2023). With the progress of genotyping by sequencing (GBS), SNPs have emerged as the most extensively utilized DNA markers in the twenty-first century (Thomson, 2014). SNP markers have significantly advanced plant breeding across a variety of crops, including tomato (Peterson et al., 2012), maize (Junta et al., 2020), rice (Razak et al., 2020), wheat (Wei et al., 2011), soybean (Kim et al., 2010), oil palm (Maryanto et al., 2020), cassava (Karim et al., 2020), and taro (Helmkampf et al., 2018). Overall, SNP markers have emerged as critical tools for genetic improvement and precision breeding across a wide range of crop species. SNPs initially trailed behind SSRs in DNA fingerprinting because they have fewer polymorphisms

Introduction The distinctions that differentiate one genotype from another are encoded in their hereditary material i.e., DNA (deoxyribonucleic acid). DNA is packaged into chromosomal pairs, with one strand of genetic material derived from each parent. The genes that govern a plant’s traits are situated on distinct portions of each chromosome (King and Stansfield, 1990). Depending on the context, a genetic marker may be defined as: (a) a chromosomal landmark that facilitates the tracing of a specific DNA region; (b) a distinct DNA segment with a known genomic position; or (c) a gene whose phenotypic expression is easily observable, and used to identify an individual and used as a probe to spot a nucleus, chromosomes, or locus. Molecular markers are distinguishable DNA sequences located at specific loci within the genome, inherited according to the established principles of inheritance from one generation to the subsequent generation. The presence of several molecular approaches, along with their distinct concepts and procedures, necessitates meticulous evaluation when selecting one or more marker types or loci (King and Stansfield, 1990). Various molecular markers have been described, based on mode of transmission, mode of gene action and method of analysis. Single nucleotide polymorphisms (SNPs) are variations in DNA sequences characterized by a single nucleotide (A, T, C, or G) that differs among individuals, serving as biological markers for the identification of genes linked to phenotypes. The genome sequences in public domain from various organisms have facilitated the examination of sequence variations among individuals, among cultivars, and among subspecies. These investigations demonstrated that single nucleotide polymorphisms (SNPs) are prevalent and dispersed across the genome across numerous species, including plants (Drenkard et al., 2000; Nasu et al., 2002).The prevalence of SNPs polymorphisms in plant genomes renders the SNP marker system a valuable marker technology for mapping, marker-assisted breeding, and map-based cloning (Rafalski, 2002a; Batley et al., 2003).A single nucleotide polymorphic (SNP) marker is a single base alteration in a DNA sequence, with a typical alternative of two possible nucleotides at a specific position. In recent years, numerous SNP genotyping methodologies have been established, utilizing diverse techniques for allelic discrimination and detection platforms (Rafalski, 2002b; Vignal et al., 2002; Sobrino et al., 2005; Tost and Gut, 2005).At now, SNPs are preferred for DNA fingerprinting, investigations of genetic diversity and association studies, phylogenetic linkages, studies of evolutionary history of species and population, genome-wide association mapping, and genomic selection. Single nucleotide polymorphisms (SNPs) are the most prevalent and reliable markers, suitable for automated high-throughput genotyping, highly reproducible, and capable of identifying variants, therefore superseding older markers due to their high throughput, efficiency, and cost-effectiveness (Alkan et al, 2011; Amdadul et al, 2016; Close et al., 2009; Muñoz-Amatriaín et al, 2014; Li et al., 2020; Qiuetal., 2012). The swift advancement and use of NGS technology have led to the creation and application of diverse reduced-representation genome sequencing methods for SNP genotyping. The methodologies encompass GBS (Elshire et al., 2011),

Introduction DNA barcoding is a molecular taxonomic method used to identify species by analyzing variations in conserved DNA sequences that differ among species. The concept of “barcoding” refers to pooling multiple samples and sequencing and analysing them collectively. This technique has been widely applied to identify organisms ranging from simple to complex forms. Unlike other genetic markers, such as short tandem repeats (STR), which are typically used to identify individual organisms, DNA barcoding enables broader species identification. However, identifying species-specific DNA sequences across different taxa can be complex and time-consuming. For accurate species differentiation, the variation in DNA sequences must be sufficiently high to clearly distinguish between closely related species (Antil et al., 2023). DNA barcode sequences are stored in databases like the Barcode of Life Data Systems (BOLD), which is an online repository containing reference DNA sequences for comparison with unknown samples. These databases store speciesspecific genetic sequences, specimen images, and related data, and also track updates to barcode libraries (Jin et al., 2020). DNA barcoding has progressed alongside genomics research, with large online libraries serving as reference databases for identifying samples from various environments like gardens, forests, and even commercial products (Seethapathy et al., 2019). As research advances, the number of conserved genomic regions used for DNA barcoding continues to grow. For phylogenetic analysis, the rbcL gene locus is commonly used, along with other regions such as the trnL-F intergenic spacer, matK, ndhF, and atpB (Shaw et al., 2005). The CoxI gene has been wellestablished for barcoding animals and plant species. The trnH, psbA region of the plastid has been proposed as a universal barcode for terrestrial plants (Ward et al., 2005). Several other conserved genomic regions have been established by a “plant working group” for molecular taxonomy (Singh et al., 2012). Despite these advancements, further critical evaluations of the authenticity of these DNA barcoding markers in assessing biological diversity are still needed. Over the last few decades, DNA barcoding has become a widely used tool for confirming species identification, as evidenced by the growing body of research publications on the topic (Rob et al., 2019).Publications on DNA barcoding have seen a notable increase, as shown by a search using specific keywords in the CabDirect database (http://www.cabdirect.org/). This trend highlights the growing importance of DNA barcoding in taxonomic research

Introduction Pathogenic organisms that cause diseases in plants are generally referred to as plant pathogens. Fungi, bacteria, viruses, viroids, algae and protozoa are the important phytopathogenic organisms responsible for plant diseases and they cause immense economic losses to growers. Recent estimates indicate that crop losses due to diseases range from 17-30%, primarily caused by fungal, oomycete, and nematode pathogens (Agrios, 2005). Beyond biotic factors, crop losses can also be attributed to abiotic causes, such as poor agricultural practices, nutrient deficiencies, changes in temperature and rainfall patterns, inadequate/overirrigation and weather extremes, all of which exert selection pressure on plant pathogens, potentially leading to new host infections. Hence, for effective disease management strategy, accurate identification and diagnosis of plant pathogens are of paramount importance before the pathogen induces any epidemic leading to immense loss of crop plants which is certain. Often, identification can be accomplished either visually through microscopic observation of disease-causing micro-organisms or molecular diagnosis based on nucleic acid detection methods. Microscopic observations alone are inadequate for species-level identification of plant pathogens and can often lead to taxonomic errors due to the potential for misidentification of organisms. Nucleic acid-based diagnostic tools allow for the sensitive detection, precise quantification, and specific differentiation of pathogens.Although numerous nucleic acid-based methods are available for plant pathogen detection, PCR-based diagnostics, especially those employing DNA barcodes, are still considered highly reliable and accurate. Scientists, especially plant pathologists, can now easily identify and diagnose most plant diseases as many standardized phytopathogen barcodes facilitate rapid, early, and accurate identification of plant diseases.

Introduction DNA fingerprinting is a powerful molecular technique used to identify individuals based on their unique genetic makeup. First developed by Sir Alec Jeffreys in 1984, this method has revolutionized fields such as forensic science, paternity testing, genetic research, and biodiversity conservation. We present here the account of variations in DNA sequences, particularly of hypervariable regions like minisatellites and microsatellites in reference to the DNA fingerprinting. Advances in PCR, hybridization-based markers, and next-generation sequencing (NGS) have further refined its accuracy and efficiency enabling precise identification of genetic relationships. Additionally, genetic distance analysis and clustering methods help in understanding evolutionary relationships, population structure, and genetic diversity among species. These methods serve as tools to quantify the magnitude and order of DNA sequence polymorphisms which exist at population level. DNA Fingerprinting History of DNA Fingerprinting: It was 10th date of September 1984, when geneticist Sir Alec John Jeffreys (the father of DNA fingerprinting) wrote three words - “33 autorad off” - in his red desk diary. The implicit phrase suggested the completion of an experiment by him pertaining to the study about inherited illnesses descended through families. He was one of the first to discover inherited variation in human DNA that can be utilized to resolve issues of disputed parentage and forensic investigations.

Introduction The identification of agricultural crops with accuracy determines how precision farming works and affects biodiversity protection and food safety standards in addition to pest control practices. In modern agricultural practice, crop species identification relies on the combination of three traditional methods: morphological characteristics and biochemical markers, and remote-sensing technologies. These methods face a large variety of constraints such as environmental differences, high costs, and time-consuming operations. Genome-editing tools, especially CRISPR, a modern molecular identification tool for crops that increases efficiency and accuracy, have largely solved the problems traditional methods faced. Identifying agricultural crops correctly is an essential operation for resource management to be used efficiently in the hope that businesses meet regulatory standards and do their share in stopping trade-related fraudulent activities with incorrect labeling. The early and exact identification process helps pest and disease control because different crop species need different levels of resistance management (Rahayu et al., 2025). A reliable identification methodology remains important because sustainable agricultural practices and climate-ready crop varieties reach higher popularity in current times. The identification method based on morphological classification takes too much time and gets misdirected because of environmental conditions (Mascellino et al., 2024). High-resolution satellite data together with advanced computational tools needed for remote sensing-based identification cannot easily be accessed by small-scale farmers (Sharma et al., 2024). The high specific identification of crop species becomes achievable through CRISPR-based technology which utilizes genetic markers as its primary feature. CRISPR enables fast and exact and affordable crop classification through its ability to detect and analyze specific genetic patterns. The accuracy of crop identification improves while real-time evaluation of plant diversity alongside disease. Alert traits becomes achievable through this method (Kanapiya et al., 2024). CRISPR-based biosensors have experienced recent advancements that enabled portable field-deployable identification tools which eliminate the need for complex laboratory setups (Lagener et al., 2025). Crop monitoring and breeding programs will undergo revolution through CRISPR technology integration in agricultural systems which will support the creation of climate-adaptive highyield crop varieties. The evolution of CRISPR application research demonstrates its expanding function in farming efficiency together with food security establishment. The chapter explores CRISPR-based methods’ identification potential for crops using an evaluation of their superiority over conventional identification techniques and their expected contributions to future agricultural developments

Introduction Bioinformatics software plays an important role in the identification of plant varieties through employing genetic and genomic data to distinguish various kinds from each other. The software uses such approaches to DNA sequencing, molecular markers, and computational algorithms to increase precision in crop identification. Techniques such as SNP genotyping, DNA barcoding, and machine learning models are helpful in easily classifying and validating crop varieties. Farmers and breeders, for instance, will benefit from bioinformatics because it ensures genetic purity, improves genetic selection in crops, and provides more yields. It also incorporates bioinformatics tools that are revolutionizing plant breeding and conservation and allow the accurate identification of diverse highquality crop varieties for sustainable agriculture and food security (Derreper et al., 2011; Heo et al., 2024). Crop variety identification has prime importance as per agriculture, food security, and conservation of biodiversity it maintains genetic purity for crops resulting in more yields with disease resistance and adaptability to climate. Correct identification will help farmer’s select superior seeds and maximize resources for increased productivity; further crop variety identification would participate in breeding programs ensuring superior crops varieties with desirable characteristics like drought tolerance, pest resistance, and better nutritional content. Accurate crop variety identification, in terms of food security, creates a steady supply of food through supporting continuous cultures of high-yielding and hardy varieties for the farm. Welfare in crop variety identification would help prevent fraud and misrepresentation in the food because they would be able to ensure that what customers receive is the type of crop they were looking for both in planned nutrition and costs. Facilitate seed certification schemes for protection of farmers from counterfeit seeds that would result in low yields. Maintenance of genetic resources through proper identification of crop varieties ensures conservation of biodiversity. Crop variety identification and registration is a huge stroke towards conservation of wild and traditional crop species, which are very important for breeding and climate resilience. Such an event becomes important in climate change where genetic diversity ensures the abilities of crops to adapt to changing environmental conditions (Kim et al., 2021; Nguyen et al., 2025; Pavan et al., 2020).

Introduction Information on genetics is also crucial in agriculture research-related advancements in crop and livestock improvement, sustainability, and food security. Genetic information is analyzed by scientists so that important traits such as disease resistance, drought tolerance, and improved yield can be bred into plants and animals with many attributes. Selective breeding programs and subsequent genetic manipulation to improve agricultural production while reducing dependence on chemical inputs like pesticides and fertilizers are then developed (Bohra et al., 2020; Li & Yan, 2020). Precision agriculture also boasts genetic data for informed decisions on the genetic composition of crops and livestock. Genetic data are thus used to optimize resource use as global demand for food resources continues to grow with increasing population needs against the backdrop of climate change resilience of crops and nutritional quality of agricultural products. Genetic studies also serve purposes of biodiversity conservation through ex-situ preservation of native and rare species presumed to have important genetic assets worthy for future breeding programs (Chaudhary et al., 2023; Varshney et al., 2021; Thudi et al., 2021). Overview of Challenges in Analyzing Genetic Data It may prove difficult for agricultural research to analyze genetic data because of its wide and varied capabilities: Data Complexity and Volume Advances in genomic sequencing technologies produce huge amounts of data that necessitate specialized computational machinery for processing. The most complex aspect of large-scale data is reliable storage, retrieval, and analysis. Data Integration and Standardization Genetic data comes from different sources and different sequencing techniques and institutes of research. Variations in format, methods, and terminologies complicate comparison of findings across studies. There are standardized protocols for collection and analysis of genetic data that ensure its consistency and reliability (Lassoued et al., 2021; Mulder et al., 2017). Computational and Bioinformatics Challenges Genetic data processing needs very high-performance computational resources and sophisticated bioinformatics tools. Most agricultural research institutions, particularly in developing countries, are poorly equipped with such technologies,

Introduction As the seed market has become more globalized, seed trade has seen a rise in competitiveness as well as stricter quality requirements. Throughout the entire seed manufacturing process, thorough management is necessary to ensure high seed quality, from initial planning to final distribution. Among the various seed quality parameters, genetic purity is particularly critical in determining the authenticity and planting value of a seed lot. Genetic purity refers to the degree of purity within a seed lot, the genetic integrity of an F1 hybrid in a hybrid seed lot, or the difference between a submitted seed sample and the stated variety. During manufacturing, achieving complete (100%) varietal purity is challenging, even when recommended protocols are strictly followed. Outcrossing, partial off-type removal, and physical mixing during harvest, storage, or seed handling are a few examples of these factors can result in unintended varietal impurities (Bradford, 2006). Seed firms, producers, and suppliers conduct laboratory-based evaluations for internal quality control or post-control grow-out testing on seed lots to guarantee the necessary levels of purity. Assessments for genetically modified trait purity and the accidental inclusion of genetically modified (GM) seeds in non- GM seed lots are now necessary due to the introduction of transgenic varieties into the worldwide market, which has further broadened the scope of seed purity testing. Thus, seed purity testing is a cornerstone of agricultural production, ensuring that the seeds sown in the fields conform to their designated genetic identity. This process is essential for maintaining varietal integrity, meeting regulatory requirements, and ensuring high agricultural productivity (Sandra et al., 2023). The quality and genetic purity of seeds all directly impact crop productivity, pest and disease resistance, and overall farm profitability. Traditional seed purity assessment methods rely heavily on morphological, physiological, and biochemical traits. These methods include visual examination of seed characteristics, seedling grow-out tests, electrophoresis-based protein profiling, and chemical marker-based identification. However, these approaches often suffer from several limitations, such as the influence of environmental conditions on phenotypic traits, subjective assessments, and the need for significant time and labour. The accuracy and reproducibility of such conventional techniques can be further affected by variations in experimental conditions and the expertise of personnel conducting the tests.The advent of molecular biology has brought a paradigm shift in seed purity testing. DNA-based technologies provide precise, reliable, and high-throughput solutions that overcome the limitations of traditional methods. These molecular approaches allow for direct examination of genetic material, offering greater accuracy in varietal identification, hybrid seed purity assessment, and detection of genetic contamination. DNA markers can distinguish even closely related varieties, enabling seed companies, regulatory bodies, and farmers to ensure the authenticity of seed lots with high confidence. This chapter provides an overview of various DNA-based techniques employed in seed purity testing, including polymerase chain reaction (PCR)-based markers, single nucleotide polymorphism (SNP) markers, and high-throughput sequencing (HTS) technologies.

Introduction Rice is a key crop that directly feeds about half of the global population, with most of it consumed in developing countries (Wang et al., 2018). It is cultivated in many different types of environments and is always being enhanced by the creation of new varieties and the assessment and conservation of germplasm collections. To improve rice diversity and increase its genetic range, numerous researchers and organizations have been attempting to bring long-forgotten types back into production.This variation among varieties is essential for breeders as it provides genetic traits that can be incorporated into commercial varieties, such as resistance to diseases and pests, tolerance to abiotic stresses and improvements in grain nutritional quality, among others. In some cases, specific new traits areintroduced through crosses made with distantly-related genotypes. As a result of these efforts, despite being one of the first domesticated crops, cultivated rice continues to tap into a vast gene pool (Vieira et al., 2022). There are over 500,000 rice accessions stored in gene banks around the world (Genesys 2021). To further explore the molecular diversity of this germplasm International Rice Research Institute (IRRI), Chinese Academy of Agricultural Sciences and BGI-Shenzheninitiated the 3K Rice Genome Project. This project focused on the most important rice species, Oryza sativa, and involved resequencing 3,000 rice genomes selected from the available genetic diversity, creating an invaluable resource for large-scale genetic variation discovery in this species (Vieira et al., 2022). Varietal identification and classification in breeding programs are typically assessed using the distinctness, uniformity, and stability (DUS) approach, which relies on morphological descriptors (Tiwari et al., 2013). However, using morphological data/markers to determine genetic variation has limitations due to the labor-intensive, time-consuming nature of the process, as well as theagronomical traits and season-specific that are heavily influenced by ecologicalaspects. The development of genetically and morphologically similar varieties has made the task of clearly distinguishing between them increasingly complex. These environmental challenges necessitate the use of new techniques that are fast, reliable and environmentally independent.Biochemical and chemical tests fail to accurately estimate genetic distances among closely related genotypes. PCRbased molecular markers, however, are fast, reliable, and effective for determining the genetic purity of crop varieties (Rana, 2006). Hypervariable microsatellites (Teymnykhet al., 2011) offer a valuable source of polymorphic DNA markers, connecting genetic maps to genomic sequences and, ultimately, to phenotypic variation. SSR-based “DNA barcodes” are unique patterns of SSR polymorphism that assist in the precise identification of varieties, providing stability and resistance to environmental influences, epistatic interactions, and pleiotropic effects.

Introduction Maize (Zea mays L.) is one of the most widely cultivated cereal crops globally and holds significant economic and food security value across diverse agroclimatic regions. In India, its multifaceted utility as food, feed, and industrial raw material underscores its relevance in national agriculture. With the expansion of hybrid cultivation, climate-resilient breeding, and commercial seed markets, the need for precise and dependable varietal identification has become increasingly critical. Traditional methods of cultivar identification, which relied primarily on morphological characteristics evaluated under the Distinctness, Uniformity, and Stability (DUS) framework, have shown considerable limitations due to their environmental dependence, subjectivity, and limited resolution among closely related genotypes (Verma et al., 2023; Jamil et al., 2021). Morphological traits, although historically pivotal in classification, are often confounded by genotype-environment interactions, multigenic inheritance, and developmental plasticity. Consequently, accurate varietal authentication using such traits becomes impractical, particularly in crops like maize where morphological similarity among hybrids is frequent (Poets et al., 2020; Verma et al., 2023). This challenge has driven the development and adoption of molecular techniques such as DNA fingerprinting, which offers a robust, reproducible, and environmentindependent alternative for cultivar and germplasm identification (Jamil et al., 2021). DNA fingerprinting involves the use of specific DNA markers to distinguish and characterize plant genotypes based on their genetic makeup. The concept, introduced by Paul Hebert in 2003, was originally proposed as a supplement to morphological taxonomy and has since evolved into a cornerstone of modern plant breeding and germplasm management. This technique has been instrumental in multiple domains of crop improvement, including genetic diversity analysis, heterotic grouping, marker-assisted selection, and varietal purity testing. Moreover, in the context of intellectual property rights and seed certification, DNA-based identification plays a vital role in enforcing breeder rights and ensuring seed quality in both public and private breeding sectors (Poets et al., 2020). Its utility is further underscored by its ability to detect essentially derived varieties (EDVs), resolve disputes in seed authenticity, and support biodiversity conservation efforts through accurate germplasm cataloging (Jamil et al., 2021; Verma et al., 2023). Given these wide-ranging applications and advantages, the present chapter aims to comprehensively assess the evolution and current status of DNA fingerprinting technologies, with specific emphasis on their application in maize variety and germplasm identification.

Introduction Legumes that produce dry seed suitable for human consumption are known as pulses, and they are agronomically valuable plants in the field and in the food chain. In the past ten years, grain legumes—particularly pulses—have become more popular as a means of addressing global agricultural concerns. (Bohra et al. 2015, Varshney et al. 2015).The best plant-based sources of dietary protein, as well as other minerals including iron, zinc, magnesium, and dietary fiber, are pulses. (Bohra et al.2014, Kouris-Blazos and Belski 2016, Maphosa and Jideani 2017). According to Simpson and Campbell (2015), a plant-based agricultural diet high in fruit or legume fiber helps to improve microbial diversity and has a beneficial effect on short-chain fatty acid levels, both of which are critical for preserving gut health. Because of their unique ability to fix nitrogen symbiotically with Rhizobium spp. in their root nodules, legumes not only improve nutritional health but also increase soil fertility (Graham and Vance 2003, Stagnariet al. 2017). The Leguminosae, often known as the Fabaceae, are the third largest family of flowering plants, behind the orchid (Orchidaceae) and sunflower (Asteraceae) families (Walters 1960), with 750 genera and 20,000 species (Polhill 1981).With a productivity of 1016 kg ha-1, 93.23 million tons (m t) of pulses are harvested worldwide from 91.77 million (m ha) of land (http://faostat.fao.org/). With a cumulative average productivity of 1030 kg ha-1, the major pulse crops—dry bean (primarily common bean), chickpea, dry pea (pea), cowpea, pigeonpea, lentil, and faba bean—share 92.58% (84.05 million ha) of the world’s total pulse production, or 92.82 percent (86.53 m t) (http://faostat.fao.org/). According to their ability to adapt to tropical and temperate agroclimatic conditions, pulse crops can be broadly divided into two groups: 1) warm season crops, such as common bean, pigeonpea, and cowpea, and 2) cool season crops, such as pea, chickpea, lentil, and faba bean (Cannon et al. 2009, Young et al. 2003, Zhu et al. 2005). Chickpea, pea, and lentil are regarded as one of the founding grain crops and helped start modern agriculture because of its early domestication (around 11,000 years ago) (Zohary and Hopf 2000).Over the past 60 years, the global area under pulses has increased from 64 to 91.77 million hectares (http://faostat.fao.org/), and numerous high-yielding varieties have been developed and released as a result of extensive research on conventional breeding for pulse improvement due to their high agricultural value (Singh 2005, Saxena 2008, Pérez de la Vega et al. 2011, Torres et al. 2011, Gaur et al. 2012).Pulses are less productive than other crops, particularly cereals, due to factors such growing in risky settings, unpredictable rainfall, extended dry spells, and susceptibility to a range of pests and diseases (Borlaug 1973, Varshney et al. 2013a).

Introduction Plant genetic resources (PGRS) encompasses everything that is on the earth related to the plants which are beneficial to mankind (Appazov,2008). So, it is the basic raw material for crop improvement (Somsenet al., 2002). In the present regime of transgenic crops, you can have ‘designer crops’- crops of your willingness (Wisner et al., 2001). From the heritage of mankind to the sovereign rights of the states this is the path that PGRs have traversed, earlier before CBD (Conventional Biological Diversity)(Chakraborty et al., 2018) PGRs were considered as a heritage of mankind freely available to everybody but after signing that they became the sovereign rights of the states with sovereign rights of the nation (Brahmiet al., 2015). In such a scenario where states have their rights on those plants’ genetic wealth, protection becomes very important (Clevelandet al., 1997). DNA fingerprinting is one of the technologies which can facilitate that protection (Wanget al., 2019).Rapeseed-mustard (Brassica spp.) is a vital oilseed crop cultivated extensively in India and other parts of the world (Kumaret al., 2008). It plays a crucial role in ensuring edible oil security and contributes significantly to the agricultural economy(Pahariyaet al., 2007). The genetic diversity within rapeseed-mustard species is a key factor in improving yield potential, disease resistance, and oil quality through breeding programs (Chandet al., 2021). However, accurately identifying and characterizing varieties is a challenge due to phenotypic similarities, environmental influences, and potential genetic erosion(Chhikaraet al., 2024). To address these challenges, DNA fingerprinting has emerged as a powerful molecular tool for precise varietal identification, genetic purity assessment, and germplasm conservation (Hosseinpouret al., 2024).DNA fingerprinting involves the use of molecular markers to generate unique genetic profiles of plant varieties, enabling breeders and researchers to differentiate between closely related cultivars (Singhet al., 2022; Singhet al., 2014). This technology is instrumental in breeding programs, ensuring the registration and protection of plant varieties under legal frameworks such as the Protection of Plant Varieties and Farmers’ Rights Act (PPV&FRA) in India (Williamet al., 2024). Additionally, DNA-based markers provide valuable insights into genetic relationships, aiding in the efficient selection of parent lines for hybridization and improving crop resilience to biotic and abiotic stresses (Nadeemet al., 2018; Haqueetal., 2021).Over the past two decades, several DNA fingerprinting techniques, including Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Repeats (SSR), and Single Nucleotide Polymorphisms (SNPs), have been widely applied to characterize rapeseed-mustard varieties (Nybomet al., 2014; Panjabietal.,2019) Advances in high-throughput genotyping and nextgeneration sequencing (NGS) technologies have further enhanced the resolution and accuracy of genetic profiling, paving the way for marker-assisted selection (MAS) and genomic-assisted breeding strategies (Sinhaet al., 2023).

Introduction DNA fingerprinting envisages a very significant molecular methodology employed for identification and characterization of wheat varieties and germplasm. It is of utmost importance in varietal authentication, rights protection of breeders, and analysis of genetic diversity. Currently employed molecular markers like SSRs, SNPs, and DArT are applied extensively for wheat fingerprinting. Such markers precisely differentiate closely related varieties and aid in breeding programs (Babu et al., 2020; Kiss et al., 2021). The current standing of wheat DNA fingerprinting shows a trend toward high-throughput genotyping and genomic selection strategies to accelerate crop improvement. Future prospects would entail the integration of advanced sequencing technologies such as Next-Generation Sequencing (NGS) and machine learning for genetic analysis. These technologies will ease wheat breeding and dramatically contribute to food security and climate-resilient agriculture. Expanding the global DNA fingerprinting database would further enhance global wheat germplasm conservation and utilization (Sandhu et al., 2021; Ahmadi et al., 2020). Wheat (Triticum spp.) is one of the major staple crops of the world, supporting the sustenance of over one-third of the total world population. It is the main source of carbohydrates, proteins, fibers, and other important micronutrients, making it very important for human nutrition. Wheat is cultivated mainly in all types of agro-climatic zones, with major producers being China, India, Russia, United States, and the European Union. Wheat is highly valuable not only in terms of food security, but also economically with millions of farmers involved in its production and trading worldwide. Wheat is an important raw material for many food products, such as bread, pasta, and baked goods. Wheat production faces challenges from climate change, pests, and emerging pathogens. Therefore, continuous research on wheat, genetic improvement for desirable traits, and sustainable agricultural practices are needed to enhance yield, resilience, and in turn global food security. Genetic diversity in wheat is pertinent for fulfilling food security at the global level and for the success of breeding programs, apart from giving diverse traits for resistance against pests, diseases, and environmental stresses like droughts

Introduction In the realm of modern agriculture and plant breeding, the integration of advanced biotechnological tools has revolutionized the traditional methods of crop improvement. Traditional approaches to genetically improving horticultural crops that are primarily propagated asexually encounter multiple challenges. These include an extended juvenile phase, the absence of seeds, a high level of intraand interspecific incompatibility, frequent heterozygosity, seed sterility, and, most importantly, the restriction of certain valuable traits to wild species (Mehlenbacher, 1995). Conventional or classical breeding techniques rely on natural processes and traditional tools to develop new plant varieties or cultivars. However, these methods are often time-consuming, labor-intensive, and less efficient. The lengthy process, coupled with the challenges of maintaining desirable traits, makes it difficult to achieve rapid genetic improvements. As a result, there is a growing need for more advanced and precise breeding methods to enhance crop development (Jain and Kharkwal, 2004). A molecular marker is a specific DNA segment that signifies genetic variations at the genome level, aiding in genetic analysis and breeding (Dhutmal et al., 2018). These are composed of nucleic acids as well as proteins and used on basis of naturally occurring polymorphism (Thottappilly et al., 2000; Ahmad et al., 2020). These markers are closely related with the target gene and they act as sign or flags (Nadeem et al., 2018), aiding in the identification of specific regions associated with advantageous characteristics such as disease resistance, yield potential, nutritional content, and environmental adaptability (Meena et al., 2023).The advancement of high-throughput molecular markers and genetic maps has enabled the precise identification of genes responsible for agronomically important traits, potentially enhancing breeding through markerassisted selection (MAS). Compared to traditional breeding methods, MAS overcomes the challenges of phenotypic selection and significantly improves selection efficiency. This approach accelerates crop improvement by ensuring the effective transfer of desirable traits with greater accuracy and reliability (Wani et al., 2022). The incorporation of next-generation sequencing (NGS) technologies and high-throughput genotyping platforms has also facilitated genome-wide association studies (GWAS) and the discovery of single nucleotide polymorphisms (SNPs) linked to important traits, further enhancing the resolution of genetic analysis. In recent years, molecular markers have been extensively used to characterize and enhance genotypic and phenotypic traits in temperate fruit and nut crops. Studies have focused on assessing genetic diversity in almonds, identifying genes responsible for red skin color in apples, developing sharka disease-resistant apricots, and sequencing the almond genome. These markers have been successfully used for genetic studies in many horticultural crops including vegetables (Gulsen et al., 2007; Kumar et al., 2008 a and b; Schafleitner et al., 2013; Yadav et al., 2015; Sharma et al., 2016; Kumar et al., 2017 and Kumar et al., 2019) and flowering crops (Kumar et al., 2019; Kumar et al., 2016 and Kumar et al., 2018a and b). These advancements highlight the crucial role of molecular markers in improving breeding strategies, accelerating genetic improvements

Introduction to Molecular Markers in Vegetable Breeding As global demand for food continues to rise, the agricultural sector faces mounting challenges from climate change and resource limitations. These pressures necessitate an urgent transformation in farming practices, making molecular markers vital for enhancing crop improvement strategies (Tester and Langridge, 2010). Traditional breeding techniques, which often rely on phenotypic selection and protracted breeding cycles, find it increasingly difficult to produce resilient, high-yield cultivars (Bernardo, 2016). Molecular markers, including Single Nucleotide Polymorphisms (SNPs), Simple Sequence Repeats (SSRs), and Amplified Fragment Length Polymorphisms (AFLPs), provide direct access to plant genomes, allowing for the precise identification and manipulation of desirable traits regardless of environmental variability (Varshney et al., 2005). This approach is essential for expediting breeding programs and enhancing genetic gains, ultimately leading to crops better suited for diverse and evolving climates (Jannink et al., 2010).Additionally, the integration of genome-wide association studies (GWAS) and genomic selection (GS) harnesses high-density marker data to deepen our understanding of complex trait genetics, fostering sustainable agricultural practices and securing future food supplies (Hickey et al., 2019).

Introduction The accurate identification of crop varieties is crucial for agriculture, plant breeding, and intellectual property protection. DNA-based techniques have emerged as powerful tools for distinguishing between crop varieties due to their precision and reliability. However, several challenges persist in the adoption and implementation of these techniques. Conventionally, morphological descriptors are routinely used for establishing the identity of varieties. But these morphological descriptors suffer from many drawbacks such as influence of environment on trait expression, epistatic interactions, pleiotrophic effects etc. Furthermore, the paucity of sufficient number of these descriptors for unequivocal identification of increasing number of reference collection of varieties enforces to look for alternatives. Electrophoresis of seed proteins and isozyme analysis have overcome these limitations to some extent but now many powerful DNA based techniques are available. Since all genetic differences between individuals are laid down in the primary sequence of their genomic DNA, the straightforward method of identification of crop varieties and parental lines would be to determine this sequence for the genome under comparison. Sequencing requires huge monitory investment and it is practically impossible to sequence the whole genomes of all varieties. But with the automation in sequencing now possible, short stretches of amplified DNA can be sequenced. However, a more practical strategy would be to limit the comparisons to specific regions of the genome, which frequently differ between individuals.

Introduction Identification of crop varieties is one of the major tasks required in modern agriculture, plant breeding, and seed certification systems. The correctness of identification ensures that the varietal purity is sustained, advocates the rights of plant breeders, and guarantees quality seeds that will perform expectedly under specified agro-climatic conditions to farmers (Yu and Chung, 2021). The growing number of crop varieties due to the intensive breeding programmes all over the world makes it equally important to be able to differentiate between cultivars. Variety identification serves several purposes, such as: • Seed Quality Assurance: Used to prevent the sale of spurious or mislabeled seeds. • Intellectual Property Rights (IPR): Legal protection of new plant varieties by acts like the Plant Variety Protection (PVP). • Biodiversity Conservation: Tracking and documenting existing genetic resources. • Regulatory Compliance: Aids in Distinctness, Uniformity and Stability (DUS) testing protocols. • Traceability in Food Supply Chains: Ensures safety for consumers and compliance with labeling standards. Thus, crop variety identification forms the basis of integrity in agricultural systems and is the coordination mechanism for protecting innovations in plant breeding with regulatory frameworks both at national and international levels (Jones et al., 2013; Kage et al., 2016; Cockram et al., 2012).

Introduction With advancements in molecular biology, DNA-based technologies have shown great potential in enhancing the efficiency of crop breeding programs, preserving germplasm resources, improving the quality and yield of agricultural products, and protecting the environment. As a result, their role in modern agriculture is becoming increasingly important. DNA is the main genetic materials of all cellular organisms; preserving DNA itself is one way of preserving germplasm resources (Geptset al.,2006). The advancement of biotechnology and molecular biology has made it possible to regulate or even control plant traits using DNA sequence information, including its structure, function, and mechanisms.DNA technologies based on DNA molecular markers, transgenic technology and gene expression have been widely used in agricultural production which have showed great potential in improving agricultural yields and quality, reducing the loss that various biotic and abiotic stress caused, promoting the utilization of germplasm resource, improving breeding efficiency and strengthening the regulation of plant growth. (Garciaet al.,2005) These modern DNA technologies, with their high feasibility and necessity, play a crucial role in ensuring the sustainable development of agriculture. Although agriculture encompasses both plant and animal production, DNA technologies in these fields share similar technical purposes and types. Therefore, this study focuses on reviewing the agricultural applications of DNA technologies by exploring their utilization in plant production. Numerous reports have documented the application of various DNA technologies in agricultural production. However, as these technologies continue to evolve and advance, it becomes challenging to cover every DNA technique in detail..DNA technology holds immense promise for the future of crop identification, enabling precise and efficient breeding, accelerating the development of improved varieties, and contributing to sustainable food systems. Here’s a breakdown of the key future prospects: