eChapters

SCARs are DNA fragments amplified by the Polymerase Chain Reaction (PCR) using specific 15-30 bp primers, designed from nucleotide sequences established in cloned RAPD (Random Amplified Polymorphic DNA) fragments linked to a trait of interest. By using longer PCR primers, SCARs do not face the problem of low reproducibility generally encountered with RAPDs. Obtaining a codominant marker may be an additional advantage of converting RAPDs into SCARs. However, SCARs may exhibit dominance when one or both primers partially overlap the site of sequence variation. SCARs are locus specific and have been widely applied in gene mapping studies and marker assisted selection (Paran & Michelmore, 1993).

Synonym : PCR-RFLP (PCR- Restriction Fragment Length Polymorphism)

The Cleaved Amplified Polymorphic Sequence or CAPS method is a technique in molecular biology for the analysis of genetic markers. It is an extension to the Restriction Fragment Length Polymorphism (RFLP) method, using polymerase chain reaction (PCR) to more quickly analyse the results.

Like RFLP, CAPS works on the principle that genetic differences between individuals can create or abolish restriction endonuclease restriction sites, and that these differences can be detected in the resulting DNA fragment length after digestion.

In the CAPS method, PCR amplification is directed across the altered restriction site, and the products digested with the restriction enzyme. When fractionated by agarose or acrylamide gel electrophoresis, the digested PCR products will give readily distinguishable patterns of bands. Alternatively, the amplified segment can by analyzed by Allele specific oligonucleotide (ASO) probes, a process that can often be done by a simple Dot blot.

The ultimate success of commercial propagation depends on the capacity to transfer plants out of culture on a huge range, at low cost and with high survival rates. The plants multiplied in vitro are exposed to a unique set of growth conditions (high levels of inorganic and organic nutrients, growth regulators, sucrose as carbon source, high humidity, low light, poor gaseous exchange) which may support rapid growth and multiplication but also induce structural and physiological abnormalities in the plants, rendering them unfit for survival under in vivo conditions. Poor control of water loss and heterotrophic mode of nutrition are main cause of deficient growth of in vitro plants.  Hence, gradual acclimatization is necessary for these plants to survive transition from culture to the greenhouse or field. During acclimatization the in vitro formed leaves do not recover but the plant develops normal leaves and functional roots. Once roots are well formed the plants are ready to be transferred into soil.The procedure of acclimatization of regenerated plantlets are: 

1. ICAR-National Research Centre for Banana, Trichi

2. ICAR-National Research Centre for Grapes, Pune

3. ICAR-National Research Centre for Litchi, Muzaffarpur

1. ICAR-National Bureau of Plant Genetics Resources, New Delhi

2. ICAR-National Bureau of Agriculturally Important Micro-organisms, Mau, Uttar Pradesh

Directorates/Project Directorates

1. ICAR-Directorate of Groundnut Research, Junagarh, Gujarat

2. ICAR-Directorate of Soybean Research, Indore, Madhya Pradesh

Agricultural Research Centers in World

1. Consultative Group on International Agricultural Research (CGIAR Centers)- Montpellier, France (1971)

2. International Center For Tropical Agriculture (CIAT)-Colombia (1969)

Phenotype is the product of genotype and environment. In presence of genotype x environment interaction (differential performance of genotypes over the environments), phenotype is the product of genotype, environment and genotype x environment interaction. A stable genotype is one which interacts less with the environment or shows a minimum of g x e interaction. When g x e interaction is significant, one must investigate the specific types of g x e interactions such as genotype x location, genotype x year and genotype x location x year interactions by growing selected genotypes over multiple locations and years. Normally, the genotypes are evaluated for three years. A typical analysis of variance for g x e interaction is given in Table 7.1 (Allard, 1960).

A variety produced by crossing inter se a number of genotypes selected for good general combining ability in all hybrid combinations with subsequent maintenance of the variety by open-pollination. According to Hayes and Garber (1919), the synthetic production of an improved variety by inbreeding and cross-breeding seems a reasonable plan. Synthetic cultivar is a commercial entity and a combination of number of inbred lines, sibbed lines, clones or other populations of cross-pollinated crops. The features of the components are as follows:

1. They will have been tested for their combining ability.
2. They will be preserved for future syntheses of the synthetic variety.
3. The component lines will be combined in a manner so as to allow random mating.

Synthetic varieties are normally used in cross-pollinated crops in those situations where hybrid development and organized seed production are in the preliminary stages. Inbred lines are common components of synthetics because they are easy to maintain and can be used again and again as per requirement of reconstituting the synthetics. When selfing is not possible, sibbing may be used. In case of perennials, clones are used as components. Combining ability of the component lines is determined in different ways. General combining ability is desired more than the specific combining ability because there are several components to a synthetic variety and each is required to combine well with the rest other lines. Random crossing of components of a synthetic variety is assumed and each line should contribute the same amount of seed to the next generation. Because a synthetic variety is a mixture of many genotypes, it might change under selection pressure during multiplication.

Abstract

Bioinformatics is an important interdisciplinary field that brings together several areas of science, including molecular biology, biostatistics, and computer science (vis-a-vis information technology). With the rapid increase in the sequencing of DNA/RNA/protein molecules, as well as the growing understanding of the interconnections between different areas of biological science, the importance of bioinformatics is only increasing. This chapter seems to provide a thorough introduction to the definition, concept, and importance of bioinformatics. It also appears to highlight the relevance and potential scope of the subject for students interested in pursuing a career in the field. As bioinformatics continues to evolve and expand, it is likely to become an increasingly important area of research and development in the life sciences.

This book is designed to introduce to the students some of the most widely used experimental procedures in biotechnology, including DNA/RNA isolation, manipulation, cloning, protein purification and characterization. The students will also get familiarized with some of the equipments frequently used in biochemistry and molecular biology.

The biotechnology laboratory course is an exploration of procedures. Hence, in order to get full benefit from the course, the students need to read the manual, which is self explanatory. The more effort they put into the course work, the more they will learn. Before the start of actual lab works, one needs to spend some time reading the Laboratory Manual. This reading will provide background information and an outline of the procedures to be performed.

The biotechnology laboratory is conducted as a “directed” research project. This means that although the general procedures are well established, the overall goal of each experiment is the acquisition of new information. Because of the nature of scientific research, predicting the outcome of experiments that have not previously been performed is difficult. It may therefore be necessary to design new experiments based on the results of previous ones, or to repeat experiments that yielded uninterruptable or ambiguous results.

Abstract

This chapter provides a brief overview of the key milestones in the development of bioinformatics that would help to understand the historical context and evolution of a subject. As an interdisciplinary field, bioinformatics has grown gradually from molecular biology, with the development of various algorithms, databases, and other important tools that have helped to advance computational biology. Some important events in the history of bioinformatics might include the development of the first algorithms for sequence alignment and comparison, the creation of early databases such as GenBank and Swiss- Prot, and the development of software tools for molecular modeling and drug design. Other key milestones might include the sequencing of the first genomes, the development of high-throughput sequencing technologies, and the emergence of new areas of research such as systems biology and synthetic biology. By understanding the historical context of bioinformatics, students and researchers can gain a deeper appreciation for the current state of the field and the ongoing efforts to advance our understanding of the molecular mechanisms that underlie life.

In molecular biology and biochemistry, the ability to accurately and reproducibly measure and transfer small volumes of liquids is critical for obtaining useful results. For volumes less than 1 ml, the most common method for measuring liquid volumes involves the use of a device known as a pipetman or micro-pipette.

The pipetmen can be both fixed and variable volumes. Commonly used pipetmen are: P1000, P200, P20, P10 etc. P1000 are useful for volumes from 200 to 1000 µl. P200 are useful for volumes from 20 to 200 µl. P20 are useful for volumes from 0.5 to 20 µl. For beginners fixed volume pipetmen are more useful. It is important to ascertain whether the correct pipetman is chosen for measuring the volume one needs.

A spectrophotometer is an analytical instrument that measures the absorbance of a test sample (solution). The term “spectroscopy” comes from the word “spectrum”. “Spectroscopy” therefore implies the use of multiple wavelengths of light. Spectrophotometers have the ability to specifically measure absorbance at specific wavelengths. The most commonly used method to allow this involves a “monochromator”, a device (either a prism, or more commonly, a diffraction grating) that splits the incident light into its component wavelengths, and allows only light of the desired wavelength to reach the sample. The ability to measure absorbance at different wavelengths is very useful, because the extinction coefficient of a compound varies with wavelength. In addition, the absorbance spectrum of a compound can vary depending on the chemical composition of the compound, and depending on the nature of the solvent around the compound.

Absorbance is a useful quantity and follows the Beer-Lambert law. The extinction coefficient of a molecule at a given wavelength can be calculated using the Beer-Lambert equation from absorbance measurements for solutions of known concentration, which states that:

Abstract

Biological databases that are composed of a large collection of digital nucleic acid sequences, protein sequences, or other polymer sequences stored on a computer, which is made available to the research community worldwide using web servers. The vast amount of information generated in molecular biology is analyzed, curated, tabulated, and arranged in databases. There are different types of biological databases, based on their contents. Besides, the database management system, a program to manage the databases, can be of two types: distributed DBMS and relational DBMS. This chapter briefly narrates the importance and purpose of biological databases.

The crop production is affected by the plant type, climatic condition, soil type and management factors. The production of the plant might be increased by changing plant type and increasing the grain filling period under certain climatic condition where management and soil factors are not limited. There is a direct relationship between the plant type and crop yield because the orientation and number of leaves play an important role in carbon fixation (photosynthesis). Again any variety/cultivar will contribute 21-25 per cent to the total yield of any crop in general. In recent years due to development of photoinsensitive varieties and hybrids which are more fertilizer responsive and high yielding varieties lead to increase overall production in the country.

Plant ideotypes

Refers to plant type in which morphological and physiological characteristics are ideally suited to achieve high production potential and yield reliability. The concept of ideotype was given by Donald in 1968 and he illustrated that there should be minimum competition between the crops and crop must be competent to compete with weeds. Ideotype is defined as a biological model which is expected to perform or behave in a predictable manner within a defined environment. On the basis of environment, Donald and Hamblin (1976) identified two forms of ideotypes that is isolation ideotypes and competition ideotypes. Isolation ideotypes are suitable for mono/single cropping and competition ideotypes are suitable for mixed cultivation.

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.

Autoclave Operating Procedures

Place all material to be autoclaved in an autoclavable tray. All items should have indicator tape. Separate liquids and solids should be autoclaved separately. Ensure that the lids on all bottles are loose. Do not put a large number of items in tray, to ensure that all items reach the appropriate temperature. Also allow sufficient air/steam circulation.

Steps in autoclave

Pressure should read ‘O’ when you open the pressure chamber of the autoclave.

Place items to be autoclaved in the autoclave and close the door. Some autoclaves require that you also lock the door after it’s closed.

Set time - typically 20 minutes.

Temperature should be set at 121°C already, but double-check and change if necessary.

Set cycle: If liquid, set “liquid cycle” or “slow exhaust”. If dry, set “dry cycle” or “fast exhaust” + dry time.

Start the cycle. On some autoclaves, the cycle starts automatically at step 5. On others, turn to “sterilize”

An area with similar soil, climatic conditions and resource availability resulting in similar type of crops and cropping systems. Ex. Rice based cropping system, Ragi based cropping system, Wheat based cropping system, Cotton based cropping system etc.

Efficient cropping zone: An analysis of productivity efficiency of crop was made by Kanwar (1972) using relative yield index (RYI) and relative spread index (RSI).

A zone where both yield and spread indices are maximum that is above 90 per cent is called most efficient and in case of indices below 90 is the least efficient zone of a crop.

Abstract

PCR primer design is a crucial skill for molecular biologists, as it enables the study of gene expression, gene amplification, and molecular markers. Primer3 is an online tool widely used for designing optimal PCR primers, which are pairs of short oligonucleotides that initiate DNA synthesis in vitro using a thermocycler. This tool uses a range of parameters to generate high- quality primers, including target sequence length, melting temperature, GC content, specificity, and product size. Understanding and effectively utilizing these parameters is essential for achieving accurate and reliable PCR results. In this chapter, the practical aspect of primer designing using the online tool Primer3 has been discussed. The parameters and the purpose of their usage have been given in a tabulated manner.