eChapters

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:

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.

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.

Most of the DNA of an living organism is packaged and organized in a chromosome as well as in mitochondria and cell organelles (Feulgen and Rossenbeck 1924). DNA is not found on its own but rather is structured in long strands wrapped around protein complex called nucleosome consisted of a protein called histone. During most of the duration of cell cycle a chromosome consists of long double helix DNA molecule (with associated proteins)During S phase the chromosome gets replicated resulting in X –shaped structure (chromatids) and still attached to a single centromere. The basic role of the chromosome is to provide a frame work which allows each linear segment of the genome to replicate and segregate efficiently. Failure in either of processes cause chromosome imbalance in daughter cells. Three specific cis-acting sites are required for stable chromosome maintenance, i.e., an origin of replication, a centromere and telomeres.

Classical studies of the morphologyof individual chromosomesof the plant species begun by S. Nawaschin 1910-16 and continued by Taylor (1925), Sharp (1929) and Mc Clintock (1929-30). And eventually it was possible to set up working models which account for the internal structure and chemistry of the chromosome and the genetic material as well as genetical facts. The use of aceto-carmine with iron (Belling, 1921) and application of heat greatly improved the staining differentiation of chromosomes. Smears have practically replaced the paraffin techniques of studying chromosome. The gross morphology of chromosomes is generally worked out either at prophase stages of meiosis or metaphase in somatic tissues. The chromosomes may differ in thickness, total length, position of centromere or spindle fiber attachment region, chromomere pattern and number and position of secondary constriction. The total length of chromosomes at metaphase also differs in different tissues of a species and also in certain tissues chromosome present may be very different in appearance. Figure 1 shows various morphological features of a eukaryotic chromosome

In cell biology, a mitochondrion is a membrane-enclosed organelle found in most eukaryotic cells These organelles range from 0.5–10 micrometers (µm) in diameter. Mitochondria are sometimes described as “cellular power plants” because they generate most of the cell’s supply energy in form of adenosine triphosphate (ATP), used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases, including mitochondrial disorders and cardiac dysfunction, and may play a role in the aging process.

Several characteristics make mitochondria unique. The number of mitochondria in a cell varies widely by organism and tissue type. Many cells have only a single mitochondrion, whereas others can contain several thousand mitochondria. The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. In humans, 615 distinct types of proteins have been identified from cardiac mitochondria; whereas in Murinae (rats), 940 proteins encoded by distinct genes have been reported. The number of proteins encoded in plant mitochondrial DNA is probably not much higher than the number encoded in mammalian mitochondria.Experiments have shown that most of the protein synthesis in isolated maize mitochondria can be accounted for by some 18-20 polypeptidesThe mitochondrial proteome is thought to be dynamically regulated. Although most of a cell’s DNA is contained in the cell nucleus, the mitochondrion has its own independent genome. Further, its DNA shows substantial similarity to bacterial genomes.

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.

To isolate a streptomycin resistant mutant of E.coli by Direct selection using Gradient Plate Method.

Introduction

Zylbalskii devised this technique.

A mutation is any change in the nucleotide sequence of DNA. These changes are heritable. Although most mutations are harmful to the organism, a small percentage is beneficial under certain environmental conditions. Mutation can occur during chromosome duplication and they may be spontaneous of they may be induced by mutagenic agent. In this experiment, we select for a mutation causing streptomycin resistance in E. coli. Resistant mutants will be detected directly using thy gradient plate method.

Principle

Microbiologist developed a highly efficient method for isolation of antibiotic resistant mutant. The gradient is said as gradient of antibiotic formed on an agar plate. A gene imparting resistant toward an antibiotic is present in plasmid or chromosome. The occurrence of mutant with different level of streptomycin resistant involves mutation of gene with unequal potential. This means that under certain cells degree of mutation is more while few shows less mutation. Some cells show intermediate resistant while others show completes resistant. The resistant in cell against streptomycin of this type, while in case of penicillin resistant increases step arise & so resistant mutant of high concentration of penicillin cannot be isolated/ obtained. While it is possible with streptomycin obtaining high resistance to penicillin stepwise & so this stepwise increase in resistance involves mutation of gene with equal potential. This theory of mutation through multi step procedure. The gene direct the organism to produce an enzyme, which inactivates an antibiotic & thus resistance to antibiotic, is observed in case of cells. Resistance of antibiotic is a plasmid-mediated resistance.

To isolate a streptomycin resistant mutant of E.coli by Indirect selection using Replica Plate Method.

Introduction

J. Lederberg & his wife E. M. Lederberg devised this technique in 1952.

U.V. light coming from the sun emits strong radiation to extent the antibacterial effect in the microbial population. However it has a very short penetration power, UV light is used as a mutagenic agent & was used by Beedle & Tatum to obtain biochemical mutation. The mutation rate is only once in 10^-9 cells per population. Then hence using mutagens like UV light by 10 to 1000 folds will increase this mutation rate.

In isolation of the mutant using UV light the dose of UV light is measured by its units/nm² organisms. The time selected for the mutation is calculated on the basis of 0.1 to 1% survival & a suitable biochemical character is used to evaluate the mutation.

A detailed treatment on the concepts of mutagenic effectiveness and efficiency was given by Konzak et al. (1965). They proposed the term “effectiveness” as a measure of gene mutations in relation to the dose and “efficiency” as an estimate of the mutation rate in relation to other biological effects induced, such as lethality, injury or sterility. To obtain high efficiency, the mutagenic effect must greatly surpass other effects in the cell, such as chromosomal aberrations, and toxic effects which generally lead to damage

Mutations are sudden heritable changes in characteristic of the organism, that is other than due to recombination (Swaminathan, 1983). Mutations occur naturally as spontaneous mutations and induced as spontaneous mutations and induced artificially by treating the biological materials with physical and chemical agents (mutagens). Experimental mutagenesis is effective method to enlarge genetically controlled variation, considerably within a short time.

Darwin’s theory of evolution explains that “variation is the mainspring of evolution”. The extent of genetic variability available in the breeding population and the selection techniques determine the efficiency of any breeding programme. In classical plant breeding programme, variation is generated by hybridization and selections are made from the resulting segregating generations. Induced mutagenesis can supplement hybridization or replace as a source of variability. Thus, mutation provides the raw material for crop evolution (Mahadeva and Randerson, 1982) and it provides the fundamental variability required for crop improvement by breeding (Konzak, 1984).

The discovery of mutagenic effects of X-rays on the fruit fly (Drosophila) by H. Muller in 1927 and by L. J. Stadler in barley and maize in 1928 paved the way for researchers to experiment with its effects on various organisms. In 1928, H. Stubbe showed the use of mutagenesis in producing mutants in crop plants like tomato, soybean and others. The first commercial mutant was produced in tobacco in 1934. Reports by B. Sigurbjornsson and A. Micke mentioned 77 cultivars that were developed through mutagenesis prior to 1995. In 1995, this number was 484. This number has since been significantly increased covering a large number of crops, namely, corn, wheat, pea, chrysanthemum, poinsettia, dahlia, citrus, apple, peach and the modified traits include plant maturity, winter hardiness, lodging resistance, protein and lysine content and numerous ornamental mutants. By the end of 20th century, the number of mutant varieties rose to 2252 covering a range of crops like cereals, oilseeds, pulses, vegetables, fruits, fibres, and ornamentals. Majority of these mutants were released as direct mutants and the rest as a result of cross breeding with the mutants. Most of the mutant varieties have come as a result of using physical mutagens (X-rays, gamma rays, thermal and fast neutrons) with gamma rays alone accounting for more than 60% of the mutant varieties. By 2017, nearly 3250 mutant varieties belonging to 175 plant species have been developed and released where China, Japan, India, Russia, the Netherlands, Germany and USA are the leading countries. India with 345 commercialized mutants stands third after China (810 mutants) and Japan (481 mutants). Thus mutation breeding is a significant contributor to the development and release of commercial cultivars in several crops and for several traits.

Mutagenesis is the process whereby sudden heritable changes occur in the genetic information of an organism not caused by genetic segregation or genetic recombination, but induced by chemical, physical or biological agents.

Mutations were first identified by Hugo de Vries in the late nineteenth century, while experimenting on the ‘rediscovery’ of Mendel’s laws of inheritance. He coined the term ‘mutation’. Radiation-induced mutations as a tool for generating novel genetic variability in plants, advanced as a field after the discovery of the mutagenic action of X-rays demonstrated in maize, barley and wheat by Stadler. The discovery of X-rays by Rontgen in 1895 led to the application of X-rays for inducing mutations in fruit fly by Muller (1927). Mutation is of two types

Mutation is defined as a sudden heritable change in gene or in chromosome structure. The process of hereditary change is termed mutation and the individuals affected by the change are called mutants. Besides recombinations, mutations are the cause of genetic variation from which every breeding activity proceeds. By means of mutations, the multiplicity of the types of a population is not only increased by one genotype, but also by large number of further new genotypes due to possibility of subsequent recombinations of mutant with the existing genotypes.

Since genetic variability is essential for any crop improvement programme, the creation and management of genetic variability becomes central to crop breeding. Experimentally induced mutations provide an important source of variability. An advantage of such created variability is that the starting point can be an agronomically accepted cultivar rather than a genetic stock of no direct commercial value with many undesirable attributes. It should, however, be realized that none of the imposed treatments in a plant system has so far been able to direct a specific change. As a result, following mutagenic treatments, a mixed bag of induced variants is found and many of these may not be of any value. Also, many events take place concurrently in a treated cell to produce variation at several loci within a genome. Because of these reasons it will be unrealistic to expect miracles out of a mutation breeding programme. The desired results can be achieved only when the objectives are clearly defined and the experiments are specifically designed to achieve those objectives. When a mutant of the required type is obtained, we only reach the step of identification of a desired recombinant in a conventional breeding programme and all the succeeding steps of evaluation of the mutant are in no way different from those in the conventional breeding procedures.

A. Define/Explain the followings

1. Mutation: Sudden heritable change in the phenotype of an individual.

2. Macro-mutations: Mutations with distinct morphological changes in the phenotype are called macro-mutations. Such mutations are found in qualitative character and, therefore, are also called oligogenic mutations.

Abstract

The potent greenhouse gases N₂O and CO₂ are relased by aerobic/anaerobic respiring bacteria, archaea, fungi and protists in increasing amounts from converting virgin rain forest and permafrost soils, over fertilized agricultural fields and grasslands including animal husbandry, compost/waste water treatment plants, oceans, savannahs, forests of the temperate climate zones in annual amounts of 0.5 to 4.2 terragrams (Tg) N₂O-N into the atmosphere after autotrophic and hetreotrophic NH₄⁺ to NO₃- oxidation. With estimated 0.25% industries contribute to the annual atmospheric N₂O-N concentration increase.

Introduction

Fungicides are biocidal chemical compounds or biological organisms used to kill or inhibit fungi or fungal spores. An insecticide is a pesticide used against insects. Pyrethroids are widely used insecticides in agriculture as they control pests and diseases effectively in plants. Because of their low biodegradability and high persistence, they are extensively found in environmental samples such as air, water, soil, sediments, food and biological tissues [1-4]. Moreover, these chemicals induce cancer and act as endocrine disrupters in several organisms due to their high degree of toxicity. Although the use of most pyrethroids has been prohibited or limited in developed countries, they are used in many developing and under developed countries for controlling of pests and insecticides [5-8].