eChapters

DNA Sequencing

DNA sequencing is the determination of the precise sequence of nucleotides in a sample of DNA. Sequencing can be done on a short fragment of DNA to cover the entire genome. The maps thus constructed using the nucleotide sequences in the correct order is called as sequence map.

In 1977, twenty-four years after the discovery of the structure of DNA, two separate methods for sequencing DNA were developed: the chain termination method (Sanger et al., 1977) and the chemical degradation method (Maxam and Gilbert, 1977). Both methods were equally popular to begin with, but, for many reasons, the chain termination method is the method more commonly used today. In chemical degradation method, chemicals specific to each base remove the last base, which has been labelled. The method uses toxic chemicals which required high care while handling.

In chain termination method, DNA polymerase extends a DNA strand in which dideoxynucleotide becomes incorporated terminating the chain extension (phosphodiester formation) at the 3’OH site making an array of fragments differing in length. This method is based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another using polyacrylamide gel electrophoresis.

DNA can be sequenced in both double stranded and single stranded templates by cloning in plasmid or phage vectors. There are two sequencing methods.

Principle

Determination of the precise sequence of nucleotides in a sample of DNA is called “DNA Sequencing”. The most popular method for doing this is called the “Dideoxy method or Sanger Method” (named after Frederick Sanger, who was awarded the 1980 Nobel Prize in chemistry for inventing it). DNA is synthesized from four deoxynucleotide triphosphates. The top most figure is a normal nucleotide: deoxythymidine triphosphate (dTTP). During polymerization each new nucleotide is added to the 3' -OH group of the last nucleotide added.

The dideoxy method has been coined after the synthetic nucleotides that lack the –OH at the 3’carbon atom of the sugar and it plays an important role in this process atom (red arrow). In the figure below dideoxythymidine triphosphate — ddTTP, a dideoxynucleotide has been shown that can be added to the growing DNA strand. But the chain elongation ceases when the next nucleotide tries to get attached to the former as there is no 3' -OH for the next nucleotide to be attached to. Hence, the dideoxy method is also called the chain termination method. 

Identification of DNA as the Genetic Material

Experiments with pneumococcus suggested that DNA is the genetic material

Hershey and Chase provided evidence that the genetic material injected into the bacterial cytoplasm is T2 phage DNA

RNA functions as the genetic material in some viruses Work Before Watson and Crick

Nucleotides are the building blocks of nucleic acids

Nucleotides are linked together to form a strand

In 1869, Miescher first isolated DNA and named it “nuclein.”

In 1902, Archibald Garrod linked heredity to enzyme (protein) defects. In 1909 he published a book entitled “Inborn Errors of Metabolism.”

In 1928, Griffith identified a “transforming factor” that transmitted infectiousness in bacteria.

In 1944, Avery, MacLeod and McCarty showed that the transforming factor was DNA)

In 1950, Hershey and Chase, using their famous “blender” experiment, confirmed that DNA, not protein, is the genetic material.

The work of chemist Phoebus Levene showed that DNA includes deoxyribose, nitrogenous bases, and phosphates, in equal proportions.

Chargaff s data showed that the number of purines equals the number of pyrimidines.

Using Rosalind Franklin’s X-ray diffraction patterns, Watson and Crick deduced that DNA is a double helix

In 1868, Johann Friedrich Miescher, a young Swish medical student got excited with an acidic substance that he isolated from pus cells obtained from bandages used to dress human wounds. To obtain large quantities of pus for his experiment, Miescher began purchasing used bandages from surgical clinics in Tuebingen, Germany. He separated pus cells from bandages and treated the cells with pepsin, a proteolytic enzyme that he isolated from stomach of pigs. After pepsin treatment, he recovered an acidic substance that was named as “nuclein” by him. This nuclein was unusual in the sense that it was rich in nitrogen and phosphorous, two elements known at that time to coexist only in certain types of fat. Miescher’s paper on this investigation was published in 1871. At that time, importance of Miescher’s nuclein was not fully appreciated. Even the existence of polynucleotide chains, the key component of the acidic material in Miescher’s nuclein was not documented until 1940s. The role of nucleic acid in storing and transmitting genetic information remained un-established until 1944 and the double-helix structure of DNA was not discovered until 1953 by James Watson and Francis Crick. Even in 1953, many geneticists remained skeptical to accept the role of nucleic acid as the genetic information carrier rather than proteins as nucleic acids exhibited less structural variability than the proteins (Snustad et al., 1997).

A laboratory is essential for research work in science and development. Most of the research institutes have dedicated laboratories for researchers and students to conduct experiments, perform research work, as well as to provide hands-on learning/training to students. There are certain rules and regulations laid down by the laboratory in-charge which need to be followed strictly for safety purposes. Some of the laboratories keep ‘safety manual’ for researchers to read before entering into the laboratory to conduct experiments. Whenever safety is compromised in a laboratory, there is increase in risk of unwanted incidents in the laboratory. Storing food/beverages in the same refrigerator where laboratory chemicals are kept may lead to disastrous effects on health. This chapter aims to provide instructions in brief on the things to be performed (Do’s) before and after entering onto a laboratory as well as the things never to be done (Don’ts) in a laboratory for the safety reasons.

Good Laboratory Practices (Do’s)

• P Wear apron/labcoat while working in the laboratory.
• P Use gloves/respiratory mask/fumehood while handling toxic/volatile/inflammable chemicals/reagents.
• P Use pippet or pippetor for safety in transfer of solutions.
• P Protect eyes, face and naked parts of the body from UV radiations. Use safety goggles or face mask/apron.
• P Keep your eyes away from Liquid Nitrogen and use cryogloves while handling it.
• P Arrange all the necessary materials/solutions before starting the experiment.
• P Periodically fumigate/ozonize culture and inoculation rooms.
• P Autoclave all contaminated materials before discarding.
• P Pre-treat gel or solution containing Ethidium Bromide (EtBr) before discarding into laboratory waste-disposal bin, and use gloves while handling them.
• P Burn all the transgenic plant materials in an incinerator after taking necessary observations.
• P Make entry in the designated log-book and clean the instrument after using it properly.
• P Switch-off the instrument after use or when not in use.
• P Wash hands with soap and remove the labcoat before leaving/going out of the laboratory7.
• P Ensure that switches for fans/lights, and water taps are off while leaving the laboratory.
• P Follow the biosafety guidelines suggested for handling and disposal of the hazardous chemical.

INTRODUCTION

Ethno-entomophagy is the term used to describe the practice of consumption of insects as food by native human populations. Insects are an important source of protein for humans, either through direct consumption or as food supplements for livestock (DeFoliart, 1989). They are nutritious in terms of protein (40–75 g/100g dry weight) and minerals. Their protein is of high quality and has high digestibility (77–98%) with good concentration of essential amino acids (46–96% of the nutritional profile) (Verkerk et al., 2007). The biochemical composition and chemical analysis have revealed that they are comparable with other foods enriched with high proteins, fats, lipids, vitamins and mineral salts (Prasad, 2007). Insects generally have higher food conversion efficiency than other animals. They show a food conversion efficiency of about twice as efficient as pig and boiler chick and more than five times as efficient as beef cattle (Durst and Shono, 2010). Some of the important edible insects include grasshoppers, caterpillars, beetle grubs, adult winged termites, bees, wasps, winged ants, cicadas and a variety of aquatic insects.

1. Introduction

Phytochemists and pharmacologists from all over the world obtain several patents for herbal drugs developed on the basis of the indigenous knowledge systems of different ethnic groups. Many countries rich in traditional medicinal knowledge have been conducting ethnobotanical survey of medicinal plants (Patil and Bhaskar, 2006). In India the native people have exploited a variety of herbal medicines for effective curing of various diseases. The plants used, preparation and administration of drug varies from area to area (Rajaram, 2006). The ethnobotanical research still plays its evident scientific role in stimulating further phytochemical and pharmacological studies (Leporatti and Corradi, 2001).

The objective of this study is to interact with Kani traditional healers and document their traditional knowledge. The Kani-tribals are one of the most primitive hill tribes of Western Ghats. They occupy the slopes of the Agasthiarmalai in Tirunelveli, Tamil Nadu, India. Some ethnobotanical studies have been done in this area (Anonymous, 1948-76, Chopara et al., 1956, Satyavati, 1976, Jain, 1981, Nagendra Prasad et al., 1996). The impact of deforestation, urbanization and modernization makes the tribals shifting from their natural habitats to urban areas. Documenting the traditional knowledge through ethnobotanical studies is important for the conservation of medicinal plants and traditional medicines.

A. Answer the following questions

1.    Why protein requirement for maintenance in cat is high?

Ans: It is due to the inability of the nitrogen catabolic enzymes in the cat’s liver to adapt to changes in dietary protein intake.

1.    Why arginine is dietary essential in cat?

Ans: Because, cat is unable to synthesize ornithine which is an arginine precursorwithin theurea cycle. Aminoacid glutamic acid and proline is precursor of ornithine in the intestinal mucosa of all mammals.However, an enzyme thatis required in the pathway, proline-5-carboxylate synthatase is absent in cat’s intestinal mucosa. In the absence of dietary arginine, a high catabolism of protein generates a load of ammonia that cannot be detoxified by its incorporation to urea and hyper ammonia develops.

2.    Why taurine is dietary essential in cat?

The full name of an organism technically has eight terms. For the dog it is: Eukarya, Animalia, Chordata, Mammalia, Carnivora, Canidae, Canis, and lupus. Notice that each name is capitalized except for species, and the genus and species names are italicized. Scientists generally refer to an organism only by its genus and species, which is its two-word scientific name, or binomial nomenclature. Therefore, the scientific name of the dog is Canis lupus. The name at each level is also a taxonomy Carnivora is the name of the taxon at the order level; Canidae is the taxon at the family level, and so forth. Organisms also have a common name that people typically use, in this case, dog. Note that the dog is additionally a subspecies: the “familiaris” in Canis lupus familiaris. Subspecies are members of the same species that are capable of mating and reproducing viable offspring, but they are separate subspecies due to geographic or behavioural isolation or other factors. Dogs, which are under the species Canis lupus familiaris, are known to have a total of 78 chromosomes (2n). The diploid genes can be classified into 38 pairs with two sex chromosomes.

Dolichos bean or Hyacinth bean, Lablab purpureus (L).Sweet. (Syn. Dolichos lablab Roxb.) under the family Fabaceae (Leguminosae) and having Chromosome No.: 2n = 2x = 22 is one of the most ancient among the cultivated plants. Young, immature pods are cooked and eaten like green beans. It is grown throughout the tropical regions of Asia, Africa and America. In India, it is grown extensively in the cool season in Tamil Nadu, Andhra Pradesh, Karnataka, Madhya Pradesh, West Bengal and Maharashtra. The pods are one of the rich sources of protein and it also provides fair amount of carbohydrates, calcium, phosphorus sodium, magnesium and iron, b-carotene, riboflavin, thiamine and nicotinic acid.
Origin, domestication and adaptation in India
Dolichos bean or Hyacinth bean has been originated in India as wild forms of this bean are found in this country. From India, it was introduced to China, Western Asia, Egypt and other tropical countries of South and South-east Asia and Africa. Vegetable and pulse type dolichos bean belong to two distinct botanical varieties under Lablab purpureus viz., Lablab purpureus var. typicus and Lablab purpureus var. lignosus, respectively. Various forms and cultivars of both the cultivated types are available in India.

Dolichos bean is known as Lablab bean, hyacinth bean or sem. Grown as field crop in MP, Maharashtra, AP and TN. Genus Dolichus is renamed as Lablab (Syn. D. lablab, D. purpureus, Lablab niger, L. bvulgaris) and Lablab purpureus name is widely accepted. Dolichos lablab L. (synonyms Lablab purpureus (L.) Sweet.) is leguminous pod vegetable grown in tropics and few temperate countries. Lablab niger var. lablab is synonym to Dolichos lablab var. typicus where long axis of seed is parallel to suture and is vegetable type. Lablab niger var. lignosus synonym to Dolichos lablab var. lignosus where long axis of seed is perpendicular to suture and is pulse type.

Dolichos bean, [Lablab purpureus (L.) Sweet] var. typicus (2n=22) also known as Indian bean or sem is one of the vital leguminous vegetables grown in India for its tender green pods. Both pole types and bush types are commercially grown. It is predominantly cultivated in Maharashtra, Tamil Nadu, Andhra Pradesh, Uttar Pradesh and North-East India. Protein, minerals, vitamins and f ibers are abundant in Dolichos bean. Bush-type vegetable dolichos unlike the pole-type dolichos (which are generally grown during the winter season as they require short day) are thermosensitive and can be grown year-round.

Dolichos is a versatile and valuable leguminous crop, offering multiple benefits. It boasts a high protein content of 3.6% and fiber content of 1.8%. The dry seed is composed of a protein content ranging from 23.0% to 28.0%. The pods are abundant in phenols, with levels ranging from 1.7 to 9.67 mg/100 g, making them a valuable source of antioxidants. Furthermore, dolichos serves as an excellent source of amino acids, particularly lysine, along with vitamins such as A, C, and riboflavin, and essential minerals (Ca, Fe, Mg, S, Na and P). The seeds of dolichos contain a flavonoid, kievitone, recognized for its potential in combating breast cancer. Additionally, the presence of tyrosinase in the seeds indicates promising possibilities for treating hypertension in humans. Dolichos beans are utilized for various medicinal purposes, acting as stomachic, anthelmintic, diuretic, aphrodisiac, antispasmodic, digestive, febrifuge, and carminative agents. Furthermore, they are rich in bioactive compounds that offer significant benefits in treating specific ailments such as liver problems, diabetes and tumors.

Physical ergonomics is concerned with human anatomical, anthropometric, physiological and biomechanical characteristics as they relate to physical activity. This is the ergonomics domain we are most concerned with in the workplace, and most of the content on this site is very much focused on workplace ergonomics.Physical ergonomic principles have been widely used in the design of both consumer and industrial products for optimizing performance and to preventing / treating work-related disorders by reducing the mechanisms behind mechanically induced acute and chronic musculoskeletal injuries / disorders.

There are about 6.5 million coconut farmers in the country. Coconut production, processing, marketing and other connected activities support the livelihood of over 12 million people. Coconut crop provides natural health drink, nutritious edible products and oil for edible and non edible uses, fibre of commercial value, shell and wood for fuel and industrial uses, and a variety of miscellaneous products of utility. Coconut is a symbol of national integration and prosperity. The crop is considered reverential cutting across linguistic and religious barriers promoting national integration. Contribution of coconut to the GDP of the country is around Rs. 100,000 million.

Introduction
Domestication is the process of adapting wild plant species for human cultivation and consumption and it has played a pivotal role in shaping modern agriculture. Since the dawn of civilization, humans have selectively bred plants to enhance desirable traits, such as increased yield, improved taste, and resistance to pests and environmental stresses. Through domestication, early agriculturalists inadvertently altered the genetic makeup of wild progenitors, favouring traits that aligned with their needs and preferences. This process involved selecting and propagating plants with desired characteristics over successive generations gradually transforming once-unruly wild species into more docile, productive and palatable varieties. The domestication of crop plants was a gradual and ongoing process, often spanning thousands of years. It required keen observation experimentation, and an inherent understanding of plant biology and genetics even before these concepts were formally understood. This patient and persistent effort by our ancestors paved the way for the development of diverse and nutritious food sources, enabling the establishment of sedentary communities and the rise of complex civilizations. While modern breeding techniques have advanced significantly, the fundamental principles of domestication remain a cornerstone of crop improvement efforts. By understanding the historical significance and underlying mechanisms of domestication, we can better appreciate the invaluable contributions of our ancestors and continue to build upon their legacy, ensuring a sustainable and diverse food supply for generations to come.
The current state of food systems is being challenged due to the heavy reliance on a limited number of resource-intensive staple crops. The emphasis on maximizing yield and the consequent reduction in diversity over the course of domestication history has led to the development of contemporary crops and agricultural practices that are environmentally unsustainable, susceptible to the impacts of climate change, lacking in essential nutrients, and contributing to social inequalities. For many years, researchers have advocated for diversification as a viable solution to tackle the issues surrounding global food security. In this context, we present a vision for a new phase of crop domestication, which aims to expand the range of crop diversity. This approach involves active engagement and benefits for three key components of domestication: a) the cultivated crops themselves, b) the surrounding ecosystems, and c) the human populations depending on these crops. We delve into the potential of current tools and technologies to initiate a revival of diversity within existing crops, enhance the value of underutilized crop varieties, and introduce new crops into cultivation. This multipronged strategy aims to reinforce genetic diversity, promote resilient agroecosystems, and establish a more varied and robust food system. The successful execution of this renewed phase of domestication hinges on a bold commitment from researchers, funders, and policymakers to invest in both fundamental and practical research efforts. As we navigate the challenges of the anthropocene epoch, characterized by significant human impact on the environment, the process of domestication offers a pathway to construct more diverse and adaptable food systems that can better serve humanity’s needs.

Domestication of Livestock

Domestication (from Latin domesticus) is the process whereby a  population  of  animals  or plants is changed at the genetic level through a process of selection, in order to accentuate traits that benefit humans. It differs from taming in that a change in the phenotypical expression and genotype of the animal occurs, whereas taming is simply the process by which animals become accustomed to human presence. In the Convention on Biological Diversity, a domesticated species is defined as a “species in which the evolutionary process has been influenced by humans to meet their needs.” Therefore, a defining characteristic of domestication is artificial selection by humans. Humans have brought these populations under their control and care for a wide range of reasons: to produce food or valuable commodities (such as wool, cotton, or silk), for types of work (such as transportation, protection, and warfare), scientific research, or simply to enjoy as companions or ornaments. Plants domesticated primarily for aesthetic enjoyment in and around the home are usually called house plants or ornamentals, while those domesticated for large-scale food production are generally called crops. A distinction can be made between those domesticated plants that have been deliberately altered or selected for special desirable characteristics and those plants that are used for human benefit, but are essentially no different from the wild populations of the species. Animals domesticated for home companionship are usually called pets while those domesticated for food or work are called livestock or farm animals.

In literal sense a species is called underutilized whose potential has not been properly realized. These underutilized crops (referred to also by other terms such as minor, orphan, neglected, underutilized, underexploited, underdeveloped, lost, new, novel, promising, alternative, local, traditional, niche crops) have been included in world-wide plans of action after having successfully raised the interest of decision makers. Wealth of plant genetic diversity currently available holds vast potential. We use only small number of crop plants for our basic requirements. Many thousands of wild plants have great economic and cultural importance and tremendous market potential for vast numbers of people throughout the world. But the majority are still unknown to science. Plant genetic resources have gained global attention in  recent past by agricultural and environmental institutions. Conservation and use of genetic resources attracted  considerable attention. Value of biological diversity in economic terms is very important for human society.

Abstract

Pomegranate, Punica granatum L., is an ancient fruit tree native to the Middle East. All plant parts are rich source of the various secondary metabolites that accounts for its antioxidant and health-promoting activity. A strong genetic control on anthocyanin production is responsible for variability in the color of peel and arils of pomegranate. Characterization, cloning and expression of the genes related with anthocyanin and tannin production provides routes for engineering of these complex pathways. Recent studies on pomegranate genome sequencing have elucidated several biochemical pathways and candidate genes that could assist to increase production of potent bioactive compounds in near future. This will further facilitate our understanding to some exclusive biological processes that plays a significant role in pomegranate breeding.

Donkeys can also be termed as ass (Equus africanus asinus) and is a domesticated member of the horse family, Equidae with chromosome number 62. The Wild ass of Africa, E. africanus is the wild ancestor of the donkey. For approximately 5000-7000 years, the donkey has been used as a working animal.

An adult male donkey is a jack or jackass, an adult female is a jenny or jennet, and an immature donkey of either sex is a foal. Jacks are often mated with female horses (mares) to produce mules, the less common hybrid of a male horse (stallion) and jenny is called hinny. NBAGR-ICAR has registered three breeds of donkey till date.

1. Kachchhi

• These donkeys are found in Kachchh district of Gujarat.

• Coat colour is mainly grey (dorsal surface grey and ventral surface white) followed by white, brown and black.

• Forehead is convex. Nasal bone is straight.

• Height at wither ranges from 77 to 115 cm.

• Docile in temperament.

• Only donkeys are used for agricultural purposes like inter cultivation for weed removing.

• Also utilized for transportation as donkey cart, as pack animal during pastoralist migration, etc.