eChapters

Introduction
Molecular markers have revolutionized various fields of biological research by providing precise tools to elucidate genetic diversity, map genes, and facilitate marker-assisted selection (MAS) in breeding programs. These markers, ranging from simple sequence repeats (SSRs) to single nucleotide polymorphisms (SNPs), offer invaluable insights into the genetic architecture of organisms, enabling researchers to link specific DNA sequences to desired traits. By harnessing the power of molecular markers, scientists can expedite the development of stress-tolerant crops, enhance agricultural productivity, and address global challenges such as food security and climate change.
A. Definition of molecular markers: Molecular markers play a crucial role in identifying genetic variations or disparities among individuals, populations, or species. These markers are specific sequences of DNA (or RNA) that can be found in blood, other body fluids, or tissues, serving as indicators of normal or abnormal processes, conditions, or diseases. Additionally, they can be fragments of DNA associated with a genome, aiding in the identification of specific DNA sequences. Molecular markers are indispensable tools in the fields of genetics, molecular biology, and biotechnology, enabling the identification of specific DNA sequences and genetic variations within populations.

Molecular markers, including DNA based genetic markers have found their applications in almost all areas of biology, more so in evolutionary biology, genetics and breeding in order to identify a particular sequence of DNA in a pool of unknown DNA. For many applications, DNA markers are preferred over other molecular markers since DNA is stable within an organism over time and among stages of development whereas other molecules are dynamic. A molecular marker is a DNA sequence that is readily detected and whose inheritance can be easily monitored. The uses of molecular markers are based on the naturally occurring DNA polymorphism. Most commonly used molecular markers in biological science include:

1. Restriction Fragment Length Polymorphism (RFLP)

RFLP is the first molecular marker technique which was invented by Botstein et al. (1980). The technique is based on restriction fragment length polymorphism; a molecular marker based on the differential hybridization of cloned DNA to DNA fragments in a sample of restriction enzyme digested DNAs; the marker is specific to a single clone/restriction enzyme combination. It consists of the following steps:

Introduction

Millions of dollars are lost each year due to insect crop devastation, and the use of chemical pesticides has unwanted side effects such as resistance and unintentional impacts on species that are beneficial (Kumar et al. 2019). For farmers, authorities, and academics, finding additional target-selective pest management techniques is of utmost importance. Enhancing performance, cost-effectiveness, and increasing the markets for bio pesticides are all made possible by biotechnology. Molecular methods are used to detect and track the emergence and spread of particular types that are natural enemies (Tipvadee, 2002). It offers chances for the growth of beneficial insect natural enemies with features including pesticide resistance, cold tolerance, and sex ratio change.

The diversity of the haloarchaea found within a crystallizer pond of a solar saltern located in Siridao and Ribandar, Goa, India was examined. Both cultured-based and culture independent methodology were employed which included  surface spreading of samples on different media containing 25% crude salt/NaCl, DNA extraction from sediment and water samples, amplification of 16S rRNA gene and DGGE.

Abstract

Molecular modeling is a computational technique used to simulate the three-dimensional interactions between atoms and molecules. It allows for the visualization, manipulation, and prediction of molecular structures and chemical reactions. In this chapter, we discuss the different types of molecular models used in computational chemistry, including ball-and-stick models, space-filling models, wireframe models, and ribbon models. Ball- and-stick models depict atoms as spheres and bonds as sticks. Molecular modeling has numerous applications in drug discovery, materials science, and other fields of research, making it a valuable tool for computational chemists and molecular biologists. Here, this chapter focuses on different types of molecular models.

Abstract

Molecular phylogeny is an important method for understanding the evolutionary relationships between different organisms. Molecular phylogeny is based on the idea that organisms that share a more recent common ancestor will have more similarities in their DNA or protein sequences. The chapter seems to cover the basic concepts and terminology used in molecular phylogeny, including the difference between rooted and unrooted trees, as well as various methods used for constructing phylogenetic trees. Further, chapter also covers some common tools and software used for analyzing molecular data and constructing phylogenetic trees. Overall, this chapter provides a useful introduction to molecular phylogeny and the methods used for understanding the evolutionary relationships between different organisms. Understanding these concepts is important for many areas of biological research, including evolutionary biology, ecology, and biotechnology.

Antimicrobial resistance (AMR) is a mounting global health emergency that threatens the effectiveness of modern medicine and veterinary care. In India, the challenge is compounded by high antibiotic consumption, unregulated access to antimicrobials, and widespread use in agriculture and animal husbandry. The World Health Organization has identified the Eskape pathogens— Enterococcus faecium, Staphylococcus aureus, Klebsiellapneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp, as priority organisms due to their ability to “escape” the effects of antibiotics and cause life-threatening infections. Simultaneously, zoonotic transmission of resistant bacteria from animals to humans is emerging as a critical concern, particularly in regions with close human-animal interfaces like Jammu & Kashmir. The importance of Eskape pathogens to the establishment and promotion of antimicrobial resistance in hospitalized patients was first recognized in a 2008 by Rice. The morbidity and mortality associated with Gram-negative Eskape pathogens is particularly concerning, as new antimicrobial agentsthose with activity against multidrug-resistant and pan-resistant Gram-negative strainshave not emerged as quickly as needed. Consequently, nosocomial infections continue to pose a serious threat to patient health, especially among critically ill inpatients and those undergoing invasive procedures or requiring the placement of medical devices. Eskape pathogens are well known for their intricate resistance mechanisms, often leaving clinicians with limited therapeutic options. Due to their significant role in driving antimicrobial resistance within hospital settings, consistent and systematic monitoring of their resistance profiles is crucial for guiding effective infection control and therapeutic strategies.This also serves as a critical indicator of regional resistance trends among hospitalized populations. In animal sector, the wastewater of livestock slaughterhouses, animal products (raw or processed) have been considered a source of multidrugresistant bacteria with clinical implications as well as their dissemination into the environment and entry into human food chain.Bacteria from the livestock can act as opportunistic pathogens and carry resistance genes that are important for both veterinary and human health. Since many antibiotics are used in both human and animal health fields, the resistant bacteria enter the environmentespecially through wastewater treatment plants affected by slaughterhouse wasteposing risks to public health. Among the various sources, the water used during poultry slaughtering often contains multidrug-resistant bacteria and can expose slaughterhouse workers to infection. To reduce these risks, it’s important to develop strategies that limit bacterial release into the water systems and prevent their spread to people and the environment. The development of AMR has been attributed to several factors comprising of microbial mutations rendering the antibiotics ineffective, human interference such as over use and over-prescription of antimicrobials, agricultural and commercial application of antimicrobials in the animal sector, and human behavioural factors (Vijayalaxmi et al., 2021). An expected annual rise of antimicrobial resistance (AMR) by 5–10 % annually has been predicted by the Indian Council of Medical Research (ICMR) Annual Report 2021 due to wide spread abusive use of broad-spectrum antibiotics (ICMR Annual Report 2021). Twelve families of priority drug-resistant bacteria (critical, high and medium) posing great threat to human health in terms of resistance to selected antimicrobials have been identified in priority pathogen list (PPL) of Global Action Plan, World Health Organisation (WHO) to stimulate the research towards the development of new antimicrobials against a particular drug-resistance (WHO,2017). From the Indian perspective, AMR is a grave concern as the Indian population is the highest consumer of antibiotics in the world (10.7 units per person). The poor public health systems, hospital infections, high rate of infectious diseases, easy availability of inexpensive

Introduction

The term ‘lectin’ was coined by William C. Boyd in 1954 for their property of selectivity (Latin ‘legere’, to pick up or choose). In 1888 H. Stillmark of Russia gave the first description of what we now know as lectin. While investigating the toxic effects on blood of extracts of the castor bean (Ricinus communis), Stillmark observed that the red blood cells were being agglutinated by a protein and gave it the name ‘ricin’. Shortly afterward ‘arbin’ was extracted from Arbus precatorius. Because they were first isolated from plants, lectins came to be known as phytohaemaglutinin. Now it has become clear that lectins are present not only in plants but also in some bacteria, vertebrates and invertebrates. However, lectins are most widely distributed in plants, reported in almost 1000 plant species.

Plant lectins are a very heterogeneous group of proteins; as far as their occurrence within the plant kingdom and distribution over tissues are concerned. They show high degree of heterogeneity with respect to biochemical and physiological roles, because of their widely different carbohydrate binding specificities (Etzler 1986; Rüdiger 1988; Goldstein and Hayes, 1978). Besides the fact that they all exhibit carbohydrate binding activity, most of the plant lectins behave as typical storage proteins (Peumans and Van Damme, 1995 b).

The harmony between human beings and plants is one of the evergreen reasons that maintains and supports life on earth. This fact clearly explains why any hazard to the life of plants prominently affects man. One important feature distinguishing plants from other complex multicellular organisms is that plants are sessile and thus have to endure environmental challenges (Zhu, 2001).

Crop productivity is limited by stress factors. The stresses which pose major threats to survival of plants belong to two categories- abiotic and biotic stress. Drought, salinity, high or low temperatures, light, deficient or excess nutrients, heavy metals, pollutants etc., either individually or in combination causes abiotic stresses. Biotic stress refers to any stress provided by living organisms, be it unicellular or multicellular. Plants facing a variety of biotic and abiotic stresses are able to protect themselves by means of tolerance mechanisms which result from changes at the molecular level. They are often confronted by a multitude of stresses in their natural environment. To overcome this, plants have evolved various defence strategies, which involve comprehensive reprogramming of the cellular metabolism.

Introduction

Genetic resistance to diseases is a great resource to control and prevent diseases, and to improve the productivity in livestock and poultry. The emergence of virulent and drug resistant pathogens, restriction on use of antimicrobials and appearance of certain diseases associated with selection for production traits emphasize the possible exploration for genetic resistance to diseases (Pinard et al., 1992). Genetic resistance to diseases is a composite trait and difficult to measure directly. The disease resistance measurement was first started as difference in mortality among populations. Later on, several other criteria related to expression of infection such as antibody response, tumour production count or expression in quantitative scale such as survival time and density of pathogen in target organs were used as a measure of disease resistance.

ABSTRACT

Frankia is a gram-positive, microaerophilic, actinomycete that enters into symbiotic associations with a number of woody dicotyledonous plants called actinorhizal plants. Frankia can fix atmospheric nitrogen through such associations. The actinorhizal-Frankia association is beneficial for the environment, since it increases the fertility of soils. Extensive studies on the biochemical and molecular aspects helped in the classification of Frankia. PCR-RFLP studies of the 16S rRNA and the nif regions of Frankia revealed considerable information regarding the diversity of Frankia strains, isolated from different parts of the world. The completion of three whole genomes of Frankia has bolstered the study of the molecular organization of the genome. Comparative genomics study of the three complete genomes revealed that, the strains reflected the biogeographical history of the host plants they infected. Striking similarity in the codon usage patterns highlights the fact that they may have evolved as a unit. In the present review we highlighted the taxonomy of Frankia and the host plants, the Frankia-actinorhizal association, regulation of infectivity, biochemical and molecular characterization, phylogeny and genomics of Frankia and stressed the need for metabolomic study of the Frankia-actinorhizal association.

In recent years several plant pathological laboratories have been using molecular techniques for identification of the fungal plant pathogens isolated from the diseased plants. These are specifically used for the confirmation of the pathogen identified based on their cultural and microscopic characteristics. With the help of fungal genomic studies, not only the genus but also the species of the pathogen is identified based on the genomic sequences available with NCBI, and provide the accurate identification of the fungal isolate. This identification method involves the following steps:a

1. Introduction
Molecular techniques are laboratory methods used to study and manipulate DNA, RNA, and proteins. These techniques are crucial for analyzing and understanding the molecular basis of biological processes and have applications in various fields like medicine, agriculture, and forensics.
2. Molecular Marker Analysis
Molecular markers can be expressed as a DNA sequence or gene expression product that represents differences in genomic level in relation to a gene or a property. Molecular markers are markers that can be used to monitor differences at the DNA level and for a gene that is being investigated. DNA markers are also DNA regions in which polymorphism in individuals within a species can be determined.
Molecular markers are nontissue-specific DNA regions that are reliable, repeatable, standardizable, capable of identifying multiple regions in the genome, capable of identifying more than one region in the genome, independent of environmental conditions, dominant and codominant.
Molecular markers are classified as dominant and codominant markers. Heterozygous individuals cannot be distinguished from homozygous dominant individuals, since dominant markers are not suitable for identification of heterozygous individuals when related to dominance between alleles is dominated by dominant markers. Thus, three different individuals (AA, AA and AA) can be distinguished for any marker at any point.
The use of molecular marker systems based on this meta-analysis has become more prevalent in genetic studies conducted by the discovery of the polymerase chain reaction (PCR). The rapid development of technology and the accompanying needs, the facilities of the laboratories where the applications will occur, the biological properties of the species and the abundance of markers in the genome have contributed to the development of DNA markers [5]. Restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA markers (RAPD), amplified fragment length polymorphism (AFLP), sequence labeled sequences (STS), microsatellites (SSR), cleaved polymorphic sequence (CAPS), single strand such as complementary polymorphism (SSCP), amplicon length polymorphism (ALP), interspecific sequence repeat polymorphism (ISSR),

Introduction

In the ever-evolving realm of fisheries and aquaculture, the infusion of molecular technology has become a beacon of hope, transforming the way we diagnose diseases and identify pathogens. Imagine it as a kind of genetic detective work that not only helps us understand the health of aquatic life but also empowers us to protect it. It all begins with the delicate dance of DNA extraction, a bit like teasing out the secrets locked within the cells of aquatic organisms. This biological treasure map, once unveiled, guides us through the labyrinth of diseases. Enter the PCR protocol for Carp Edema Virus (CEV) detection—a sort of molecular magnifying glass that allows us to zoom in on specific genetic footprints of trouble. It’s like deciphering a genetic code, helping us pinpoint potential issues swiftly.

Use of morphological markers has continued till today for characterization of several crops including horticultural crops with the Mendel’s principles of inheritance by following visible traits in the progenies of sexual crosses. Genetic markers play an essential role  in the study of variability and diversity, in the construction of linkage maps, and in the diagnosis of individuals or lines carrying certain linked genes. Within this context, the limitations of morphological markers became quickly apparent. They tend to be restricted to relatively a few traits, display a low degree of polymorphism, are often environmentally variable in their manifestation, and can depend on the expression of several unlinked genes. Furthermore, some may affect plant viability or seed set, distorting gene frequencies in the progeny. Morphological markers were largely supplemented by biochemical markers, particularly isoenzymes that could be easily scored by electrophoresis (Ganapathy and Scandalios 1973; Tanksley 1983). The limitations of isoenzmyes as markers, in particular both the limited number of polymorphic enzymes that can be conveniently stained and the environmental effects on expression pattern, were apparent already twenty years ago (Tanksley 1983).

Introduction

Indian agriculture accounts for nearly 65% of the country’s employment, 23% of the total GDP and nearly 20% of total export earning and supplier of raw material to major industries. Agriculture is not only the backbone of Indian economy and food security but also a way of life, a tradition and anchor of overall livelihood opportunity for about 700 million of our one billion populations. Agriculture, therefore, is and will continue to be central to all strategies for planned socio-economic development of the country.

Losses due to insect pests represent one of the single largest constraints to crop productivity estimated at 14% of the total agricultural production. Besides, insects also serve as vectors of plant pathogens. The annual global cost of pest management through insecticides is more than US$10 billion per annum. However, over-reliance of insecticides creates agricultural, environmental and pest management problems, such as frequent evolution of insect resistance, destabilization of multitrophic interactions and environmental contamination. Broad spectrum insecticides often compound these problems. Insecticides often interfere with biological control, causing secondary pest outbreaks or catastrophic resurgence of insecticide resistant herbivores. The vicious cycle of pesticide treadmill effect is observed when more pesticides lead to insects developing resistance, which in turn necessitates use of more and newer pesticides. Therefore, now it is imperative to develop a holistic system of tackling pests to make it more eco-friendly, economically viable and socially acceptable for the farmers.

Two types of molecular weight (MW) standards are routinely used. The Lambda/HindIII and PhiX174/HaeIII MW standards provide a useful reference for calculating molecular weights of large and small DNA fragments, respectively, after electrophoretic separation; the “internal MW standards” provide a means for normalizing fragment migration distances within each lane to facilitate comparisons between lanes on the same or different luminographs in fingerprinting studies.

Recent years have seen a rapid increase in the application of the various techniques of molecular, cytogenetical and serological approaches to problems in plant Nematology. The application of these approaches has been considered to problems of genetic variation and classical taxonomy. Two principal approaches are available. Firstly the use of “random” techniques which aim to sample the entire genome of the animaIs or populations of interest and which are mostly used in studies of genetic variation. Secondly “non-random “ techniques which target defined regions of the genome such as particular genes or gene products and are most frequently used in identification to species or sub-species level. The defined region ofthe genome may be the ribosomal genes, the mitochondrial genome or a conserved nuc1ear gene. Whilst analysis of these regions may allow information about taxonomic relationships to be gathered, focusing on one region of the genome imposes certain limitations on the experimenter.

Vegetables have always been looked upon as an essential part of diet and are necessary for human health and well-being. Vegetables supply many kinds of nutrients, e.g., carbohydrates, proteins, minerals, vitamins, fibers, phytochemicals, etc. and also add flavour, colour and crispness to foodstuffs. For developing modern day vegetables, breeders have utilized an understanding of genetic principles and applied them for the improvement of these crops. They often make crosses (hybridization) between a crop plant and a related or wild species (a wide cross) enabling valuable genes to be used for genetic improvement of the crops. Over the last few decades, molecular breeding is supplementing plant breeder’s efforts for accelerated genetic enhancement bringing about a revolution in this area. This approach demands efficient procedures for the routine introduction of foreign DNA into plant genomes and utilizing them for the production of novel types of vegetable. In this chapter, attempts have been made to review most of the available molecular breeding techniques, which can routinely be employed in various aspects of vegetable improvement.

Cultivars are developed by selecting for desired traits or by removing undesirable traits that are controlled by genes (the blueprint for all characteristics in living organisms) from existing varieties or breeding lines. The plant species, varieties and clones are identified or selected by their physical features since olden times. However, these traits would change with respect to environmental and climatic conditions. The biochemical parameters like enzymes, proteins, alkaloids, etc. are also likely to change due to the stages of development of plant tissue. Successful selection strategies are dependent on the breeder’s ability to distinguish genetic effects from environmental effects on a visible or measurable trait. Therefore, the scientists resorted to use ‘genetic marker’, i.e., any stable and inheritable variation that can be measured or detected by a suitable method and can be used subsequently to detect the presence of a specific genotype or phenotype, which otherwise is very difficult to detect. Presently, the breeders and scientists all over the world are pursuing mostly DNA or molecular markers.

Introduction

Commercially laying of poultry birds carried out for a laying year or cycle after that as egg production decreases then they are usually culled when birds starts shedding of feathers. If we still keep these birds after allowing shedding and renewing of feathers birds take sufficient rest to builds up its body reserve of nutrients for next laying cycles. Their production regains 80-83% of the first laying cycle therefore recycling of laying can be carried out by shedding and renewing of feathers which is nothing but the “molting.”