the construction and development of a building or an object. DNA .... Mutations in key nucleotides of a coding sequence may ..... cladogram not on the basis of high genetic similarity between them calculated ..... Answer. : A DNA polymorphism that can be easily detected by molecular or ...... white kernel,let aromatic variety.
Molecular Diagnostics of Crop Varieties
Molecular Diagnostics of Crop Varieties
First Edition: 2012
Copyright 2012 Orissa University of Agriculture & Technology, Bhubaneswar, Odisha
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the
Anuprita Ray, K.C.Samal, G.R.Rout
publisher.
Printed at: M.S. Imfotech,
DEPARTMENT OF AGRICULTURAL BIOTECHNOLOGY, COLLEGE OF AGRICULTURE ORISSA UNIVERSITY OF AGRICULTURE & TECHNOLOGY BHUBANESWAR- 751 003, ODISHA
2012
ABBREVIATIONS
CONTENTS Page Acknowledgement Abbreviation PREFACE Chapter-I Introduction Chapter-II Equipments Required For Molecular Diagnostics of Crop Plants Chapter-III DNA: Blueprint of Life
1 1 2 5 5
Chapter –IV Markers for Diagnostics
8
Chapter-V Different protocols
5
Chapter-VI Frequently asked questions on Molecular diagnostics techniques Chapter-VII Detection of adulteration in Basmati rice employing molecular diagnostics technique
5 5 5 6 4
Chapter-VIII Molecular diagnostic techniques in disease management
4
Chapter-IX Molecular diagnostic techniques in seed purity assessment
4
Chapter-X Molecular marker and transgenic crops
4
Chapter- XI Molecular markers and plant breeding
4
Chapter- XII Molecular markers and plant diversity
2
Note
1
% AFLP bp cDNA cm conc. CTAB DNA dNTPs DUS e.g. EDTA EDV EST EtBr etc g g/l GM GMO h ISSR kb m M mA MAS mg mg/l MI min ml mm mM mmol N NAA NaOAc
-
per cent amplified fragment length polymorphism base pair(s) complementary DNA centimeter concentration N-cetyl –N, N,N-trimethyl ammonium bromide deoxyribonucleic acid deoxyribonucleotide triphosphates distinctness, uniformity, and stability exemplia gratia (Latin : for example) ethylenediamine tetraacetate essentially derived varieties expressed sequence tag ethidium bromide et cetera (Latin: and so on) gram (s) gram(s) per liter genetically modified genetically modified organism hour (s) inter simple sequence repeat kilbase pairs meter molar milliampere marker aided selection milligram milligram/litre marker index minute milliliter millimeter millimolar millimole normality 1-naphthaleneacetic acid sodium acetate
ng oC PBR PCA PCR PGR pH
-
nanogram degree celsius plant breeders’ rights principal component analysis polymerase chain reaction plant genetic resources potentia hydrogine (negative logarithm of hydrogen ion concentration) PIC polymorphism information content PPV & FRA - Protection of Plant Varieties and Farmers’ Rights Authority PVP polyvinylpyrrolidone QTL quantitative trait locus RAPD random amplified polymorphic DNA RFLP restriction fragment length polymorphism RNA ribonucleic acid RNase ribonuclease rpm revolution per minute SDS sodium dodecyl sulphate sec seconds SNP single nucleotide polymorphism SSR simple sequence repeat SSR simple sequence repeat STMS sequence-tagged microsatellite sites STS sequence tagged site TAE tris acetate EDTA buffer Taq DNA Polymerase - Thermus aquaticus DNA polymerase TBE tris boric acid EDTA buffer TE tris ethylenediamine tetraacetate buffer UPOV Union for the Protection of New Varieties of Plants v/v volume by volume viz videlicet (Latin : namely) w/v weight by volume wk week wt weight ìg microgram ìl microlitre ìM micromolar
ACKNOWLEDGEMENT With great pleasure and deep gratitude we record our sense of indebtedness to Prof. D.P. Ray, Vice-chancellor, Orissa University of Agriculture and Technology, Bhubaneswar for his help, constant encouragement for bringing out this book. We are also thankful to the Registrar and Comptroller for their kind support. We wish to express our deep sense of gratitude to Prof. S. S. Nanda, Dean of Research, Prof. D. Naik, Dean, College of Agriculture and Prof. M. Kar, Director Planning
Monitoring & Evaluation for their support
and encouragement for bringing out this book. We also express our thanks to Dr. F.M. Dash, Asst. Seed Production Officer for his help. The financial support from Indian Council of Agricultural Research (ICAR), New Delhi and Rashtriya Krishi Vikash Yojana (RKVY), Ministry of Agriculture and Cooperation, New Delhi is duly acknowledged. G.R.Rout KC.Samal
PREFACE Agriculture in India is the means of livelihood of almost two thirds of the workforce in the country. It employs nearly 62% of the country’s total population and occupies 42% of its total geographical area. Agriculture and allied activities constitute one of the main contributors to the Gross Domestic Product of the Nation. The increase in agricultural production has been brought about by bringing additional area under cultivation, extension of irrigation facilities, the use of seed of improved high yielding varieties, better production technologies evolved through agricultural research, water management, and plant protection through judicious use of fertilizers, pesticides and cropping practices. The greatest threats to agriculture have always come from nature: freezing, temperatures, flooding, droughts, and pests and pathogens. New and emerging diseases are an ever-increasing reality for phytopathologists. The deliberate release of a crop pathogen is not a new threat. Deliberate releases of plant pathogens onto crops have been reported since biblical times. An accidental introduction of a regulated plant pathogen could easily cause losses to the economy in the tens of billions of dollars. The food production and distribution network is susceptible to contamination with human pathogens such as Escherichia coli and Salmonella species. E. coli increases in numbers very rapidly on small fruits and vegetables following infection with plant pathogens. If no plant pathogens are present, numbers of E. coli remain below thresholds needed for human infection. Also, plant pathogens themselves can pose real threats. Several plant pathogenic fungi and bacteria produce animal and human toxins in plants. Hybrid vegetable seed, which is mostly produced abroad by local contract growers, can serve a major avenue for accidental or deliberate introduction of seed borne pathogens. Presumptive diagnosis of plant diseases in plants showing symptoms can be relatively simple when typical, definitive symptoms are evident. However, symptoms are not always unique and can be confused with other diseases. Typical examples are halo blight of beans, caused by the regulated Pseudomonas syringae pv. phaseolicola, and brown spot of beans, caused by the unregulated organism P. syringae pv. syringae. The lesions can normally be differentiated on green leaves because of the yellow halo produced by P. syringae pv. phasaeolicola. However, no halos are visible on dried pods; both pathogens produce indistinguishable brown spots. Diagnosis of plant diseases can be even more difficult with infected seeds or asymptomatic infected propagative materials such as tree-grafting stocks or potato tubers. Traditional isolation and pathogenicity tests require 10 to 20 days or longer, enough time for bacteria and aerial fungi to spread dramatically, causing severe epidemics. Many fungal diseases, such as rusts, are amenable to rapid chemical controls once a positive identification has been made. However, with bacteria and viruses, rapid eradication and containment are the only available control measures. Successful disease eradication requires quick reaction such as occurred with the discovery of PCR. The PCR confirmation clearly contributed to the quick and successful containment of
this highly regulated. PCR-based assays have been performed on several genera of nematodes, although most have not focused on the development of diagnostic assays using such techniques. Many of these nematodes are not necessarily of federal quarantine significance nor are they likely candidates for intentional introduction because epidemics of nematodes develop slowly. Sampling nematodes requires extraction from soil or roots, which can be time consuming, though maceration of endoparasitic nematodes within roots may be considered. Fortunately, only a single juvenile or cyst is required for acceptable detection, although the resilient nature of the cuticle hinders rupturing of vermiform types. Many of the same challenges in developing PCR-based assays for fungi occur with plant-parasitic nematodes. Development of unique PCR primers and probe sequences, optimizing assay conditions, and testing against closely related species will be required. Species-specificity of primers to the target nematode must be achieved though this may be difficult for all populations of a species. Currently more and more diagnostic laboratories and inspection agencies are using molecular methods for detection and identification of pathogens. The development of more versatile robust and cost effective systems, allowing for greater sensitivity and specificity, elevated throughput and detection of multiple microbes will continue over the coming years. Pathogen detection is only the first step; quantification and isolate characterization are crucial elements in diagnostics. Diagnostic technology is moving from qualitative to quantitative and there is no doubt that most tests will be quantitative in the future. Microarray-based technology is the most suitable technique for multiple pathogen detection in a single assay. Currently, microarrays can be expensive for routine application. However, with reducing fabrication costs, the cost per sample will be significantly lower. The effort to add a quantitative aspect to microarrays must continue and more work is needed to address the challenges of working on environmental samples where contaminants (organic substances, heavy metals etc) may interfere with DNA hybridization and affect the performance of microarrays. Adding innovative molecular tools for differentiating viable from non-viable organisms should be given emphasis in developing diagnostic assays. This manual enumerated the major technical demands that must be met and how the existing and emerging technologies are developing to fulfill these demands and what limitations still exist. In developing a tool for pathogen detection, issues such as detection specificity and sensitivity are very important. In addition multiplexing, quantification and cost effectiveness are increasingly becoming important features of a diagnostic technology. There is also a growing need for a field deployable portable rapid detection system that provides the capability for pathogen testing and identification in the field. Early detection plant diseases will great help in crop productivity and susceptible agriculture.
G.R.Rout KC.Samal
abundantly. One of the most important advantages that molecular based detection has over conventional diagnostic detection methods is the high specificity. That is the ability to distinguish closely related organisms. The specificity of PCR is it conventional or real-time, depends upon the designing of proper PCR primers that are unique to the target organism. Highly conserved gene regions are often the target for designing primers. Closely related microbial species often differ in a single (single-nucleotide polymorphism (SNPs) to few bases in such genes. PCR allows detection of such SNPs. With the advancements in high throughput DNA sequencing more and more genomes of plant pathogens is sequenced and nucleotide sequence data will be available increasing the possibility for designing unique primers and probes for specific detection of pathogens. Nucleic acid based detection methods can be designed to detect either DNA or mRNA. Whereas DNA based detection method is often more straightforward than that of mRNA, the stability of DNA leads to the possibility that DNA based methods yield positive results from non-viable or dead pathogens. One of the main goals of pathogen detection system, besides determining the presence and absence of the pathogen, is the viability since in the event of positive result it is important to know whether the pathogen detected poses threat to crop production, public health or food safety. The lack of discriminating viable from dead cells is a pitfall common to the nucleic acid based detection systems including microarrays. The diagnostic PCR demonstrated that prolonged detection of non viable cells led to potential over estimation in the quantitative real time detection of Escherchia coli. In order to circumvent this problem many studies consider enrichment culturing (BIO PCR) instead of direct PCR. While the system allows the detection of only viable cells and helps in elimination of possible PCR inhibitors, it is not appropriate approach for quantitative assay. Therefore, the lack of ability to distinguish between viable and dead cells, and the lack of sample preparation methods that do not involve enrichment culturing are currently limiting the implementation of quantitative PCR for routine diagnostic use. Although new, rapid detection and identification technologies are becoming available for various pathogens, pathogen quantification remains to be one of the main challenges in the disease management of many crops. Quantification of a pathogen upon its detection and identification is an important aspect as it can be used to estimate its
3
potential risk regarding disease development, establishment and spread of inoculums and economic loss. In addition it provides information for well informed disease management decisions. PCR is ideal for detection of small amount of the target but one of its limitations has been quantification. Three PCR variants namely limiting dilution PCR, kinetic PCR and competitive PCR have been used for quantitative analysis of DNA. However, all are based on end point measurements of the amount of DNA produced which makes estimation of initial concentration of DNA and quantification rather problematic. Even the emerging microarray technology has limitations with respect to microbial quantification in complex environmental samples due to the fact that microarray hybridization signals could vary depending on target abundance and hybridization efficiency. In other words, a low abundance target with high genetic similarity to a microarray probe might produce a stronger hybridization signal compared with a higher abundance target that has low similarity to the same microarray probe. Due to the advancement of fluorogenic chemistry, a second generation PCR known as real time PCR has become an emerging technique for the detection and quantification of micro organisms in the environment. In PCR the target DNA sequence is amplified over a number of denaturation-annealing-extension cycles. In a conventional PCR, only the final concentration of the amplicons may be monitored using a DNA binding fluorescent dye. However, in the quantitative real time PCR, the concentration of the amplicons is monitored throughout the amplification cycles using a group of fluorescent reagents. The fluorescence intensity emitted during this process reflects the amplicons concentration in real time. The real time data will serve as useful basis for establishing inoculum threshold levels and detailed analysis of disease epidemics. The DNA microarray technology originally designed to study gene expression and generate single nucleotide polymorphism (SNP) profiles, is currently a new and emerging pathogen diagnostic technology offers a platform for unlimited multiplexing capability. The unlimited capability for simultaneous detection of pathogens makes microarrays to be an approach with a potential capacity of detecting all relevant pathogens of a specific crop.
4
Chapter-II
Bioreactor
:
Elisa reader
:
Electrophoresis
:
Gel documentation
:
HPLC
:
Equipments Required For Molecular Diagnostics of Crop Plants Name of the Equipment
:
Purpose
Centrifuge
:
Used for the separation of different biomolcules like pigments, carbohydrates, lipids, proteins, nucleic acids etc
Autoclave
:
Used for sterilization of empty glasswares, test tubes containing medium, surgical tools, scalpels, scissors, needles, cotton etc
Deep freeze
:
Storage of heat labile chemicals, reagents such as Taq DNA polymerase enzymes, primers, dNTPs, antigens, antibodies, homones, vitamins etc
Balance
:
For taking the weights of of deferent chemicals for preparation of various solutions/reagents
pH meter
:
For adjustment of pH of the solution to a desired level by adding either 0.1N NaOH or 0.1N HCl dropwise as per the requirement of the stock solution;
Fluorescence microscope
:
For viewing the detail structure, components, biomolecules in cells/ tissues/ organelles labeled with fluorescent dyes
BOD incubator
:
For incubation of microorganisms, cells to facilitate for better growth under controlled environmental condition by regulating temperature, light & humidity etc
Micro centrifuge :
For separation of different biomolcules like pigments, carbohydrates, lipids, proteins, nucleic acids etc. Also used for settle down of reagents adhered to the wall of micro-centrifuge tube prior to PCR reaction and other investigation
5
For mass production of microorganisms, plant or animal cells by growing these cells under proper physical and chemical condition. Also used for the study of growth kinetics. There is provision of adjusting pH, aeration, adding of fresh medium or chemicals and withdrawal of used medium or products during the growth process. For detection and quantification of coloured product formed by the conversion of the colourless substrate to the coloured product in the presence of secondary antibody coupled with the enzyme. Absorbance at a particular wavelength of each well of micro-title plate is recorded. Used to separate proteins or other biomolecules on the basis of their charge and/ or size. Also a mixed population of DNA and RNA fragments is separated on basis of their length to estimate the size of DNA and RNA fragments after PCR reaction or restriction endonuclease digestion. These bio-molecules are separated by applying an electric field to move the charged molecules through an matrix (agarose gel or polyacrylamide gel or SDS gel) Used for the imaging and documentation of nucleic acid and protein polyacrylamide or agarose gels typically stained with ethidium bromide or other fluorescent dye (SYBR Green). The banding pattern of DNA, RNA or protein in the stained gel is documented for further analysis. Used to separate a mixture of compounds or bio-molecules with the purpose of identifying and quantifying the individual components of the mixture.
6
HPTLC
Gradient PCR
Hybridization oven
Lypholizer
Environmental shaker
7
: It is needs to use the basic method of thin layer chromatography but with a number of enhancements to automate the different steps, to increase the resolution achieved and to allow more accurate quantitative measurements. It is primarily used to separate a mixture of compounds or biomolecules for the purpose identification, quantification and purification. : Used to amplify the segments of DNA via the polymerase chain reaction (PCR) process under in vitro condition. It has a gradient function, which allows different temperatures in different parts of the block. This is particularly useful when testing suitable annealing temperatures for primers. : Transfer of electrophoresis-separated DNA fragments to a filter membrane was done through electroblotting. The membrane is then baked in a Hybridization oven at 80 °C for 2 hours to permanently attach the transferred DNA to the membrane and subsequent fragment detection by probe hybridization. : Used to preserve a perishable material or microorganism or plant or animal cell by dehydrating it. It is used for freezing the material and then reducing the surrounding pressure to allow the frozen water in the material to sublimate directly from the solid phase to the gas phase. : Used for agitation or shaking of the culture medium inoculated with micro-organism or plant cell under controlled environmental condition i.e. light, humidity and temperature.
PCR
:
UV-Vis : spectrophotometer
Rocker
:
Water purifier
:
Laminar air flow
:
Used to amplify segments of DNA via the polymerase chain reaction (PCR) process under in vitro condition. A thermo cycler is used for this purpose Routinely used for the quantitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules by recoding the absorbance or transmittance of light passed through the test solution. For cyclic immersion and de-immersion of gel. For removing undesirable chemicals, biological contaminants, suspended solids and gases from contaminated water with a objective to produce water fit for a biochemical and molecular analysis. Used for providing an aseptic area for transfer of microorganism/ plant tissue or explant onto the sterilized medium under aseptic condition.
8
9
10
11
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Chapter-III DNA: Blueprint of Life Introduction A blueprint is a detailed drawing or map which identifies and directs the construction and development of a building or an object. DNA, or deoxyribonucleic acid, is the hereditary material in all living organisms. Nearly every cell in an organism has the same DNA. DNA is the blueprint that guides the construction and development of living organisms. In the nucleus of each cell, the DNA molecule is packaged into threadlike structures called chromosomes. Each chromosome is made up of DNA tightly coiled many times around proteins that support its structure. The order, or sequence, of these bases determines the information available for building and maintaining an organism. Deoxyribonucleic acid (DNA) DNA is a nucleic acid containing the genetic instructions used in the development and functioning of all known living organisms (with the exception of RNA viruses). It is one of the major macromolecules that are essential for all known forms of life. It consists of two long polymers of simple units called nucleotides, with backbones made of sugars and phosphate groups joined by ester bonds. These two strands run in opposite directions to each other and are therefore anti-parallel. Attached to each sugar is one of four types of molecules called nitrogenous bases (A,T.G.C). Everyone’s chemical structure of DNA is same (A,T,G,C) only difference is in the ordering of these four nitrogenous bases. It is the sequence of these four nitrogenous bases along the backbone that encodes information.. Within cells DNA is organized into long structures called chromosomes. During cell division these chromosomes are duplicated in the process of DNA replication, providing each cell its own complete set of chromosomes. Eukaryotic organisms (animals, plants, fungi, and protists) store most of their DNA inside the cell nucleus in pairs of chromosomes, each inherited from one of the parents and some of their DNA in organelles, such as mitochondria or chloroplasts. In contrast, prokaryotes (bacteria and archaea) store their DNA only in the cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA. These compact structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed. Gene Gene is a segment of DNA that encodes a polypeptide (protein) or an RNA molecule. It is associated with regulatory regions, transcribed regions, and or other functional sequence regions and considered as a unit of inheritance. The DNA segments, carrying this genetic information, are called genes. In case of eukaryote (animals, plants, fungi, and protists), each gene in an individual, therefore, has two copies, called alleles, one on
13
each chromosome of a pair. In eukaryotes, genes are scattered along chromosomes, separated by long, mainly repetitive, DNA sequences. Genes are formed by coding sequences (exons) separated by introns. The latter carry no protein coding information, but sometimes play a role in the regulation of gene expression. The instruction encoded by genes is put into action through two processes. The first is transcription (copy) of genetic information into another type of nucleic acid, RNA (ribonucleic acid). Both exons and introns are transcribed into a primary messenger RNA (mRNA) molecule. This molecule is then edited, a process which involves removing the introns, joining the exons together, and adding unique features to each end of the mRNA. A mature mRNA molecule is, thereby, created, which is then transported to structures known as ribosomes located in the cell cytoplasm. Ribosomes are made of ribosomal RNA (rRNA) and proteins, and provide sites for the second process – translation of the genetic information, previously copied to the mRNA, into a polypeptide (an entire protein or one of the chains of a protein complex). The mRNA molecule is read or translated three nucleotides (a codon) at a time. Complementarity between the mRNA codon and the anti-codon of a transfer RNA (tRNA) molecule which carries the corresponding amino acid to the ribosome ensures that the newly formed polypeptide contains the specific sequence of amino acids required. Not all genes are translated into proteins; some express their function as RNA molecules (such as the rRNA and tRNA involved in translation). Recently, new roles of RNA in the process of mRNA editing and in the regulation of gene expression have been discovered. Indeed, non-coding RNAs appear to be key players in various regulatory processes. Thus, three types of molecules are available for investigating genetic characteristics at cellular, tissue and whole organism levels: the DNA which contains the encoded instruction; the RNA which transfers the instructions to the cell “factory”; and the proteins which are built according to the instructions, and make functioning cells and organisms.
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Chapter-IV Markers for Diagnostics Marker in life sciences is defined as a trait or an allele or a DNA sequence or a cytogenetic segment or a chromosome fragment or a protein or an enzyme used as an experimental probe to keep track of an individual, a tissue, a cell, a nucleus, a chromosome or a gene. In general. Different types of markers are used in life sciences. Markers are broadly classified into morphological, biochemical and molecular markers. Morphological markers Morphological markers (or also called phenotypic markers) are those distinguishable traits that are evident to human naked eyes. These markers are selected based on the experience of the breeder to correlate a phenotypic trait with a trait of interest. Morphological markers differ among species, genus and varieties of plants and animals. It is the easiest and quickest way to identify or detect the presence of a morphological marker with that of a trait for improvement. Morphological markers are detected and well established in different plant species such as different growth, yield and disease resistance traits that are related to markers such as purple hull, phenol staining, glabrous leaf and hull, purple node, awned panicle, lazy growth habit, brown pericarp and others. Requires less investment at initial stage Limited number (difficult to differentiate closely related varieties/ duplicates). Most of them are quantitative or continuous characters Influenced by environment Not suitable for large scale testing and automation Biochemical markers Isozyme is a biochemical marker system based on the staining of proteins with identical function, but different electrophoretic mobilities. The weakness of isozyme markers is that each of the proteins that are being scored might not be expressed in the same tissue and at the same time in development. Therefore several samplings of the genetic population need to be made. Biochemical constituents (e.g. Secondary metabolites in plants) and macromolecules, viz. proteins. Analysis of secondary metabolites is restricted to those plants that produce a suitable range of metabolites which could be easily analyzed and which could be distinguished between varieties. These metabolites, which are being used as markers should be neutral to environmental effects or management
15
practices. These markers are useful, as they are based on the expressed loci of the genome; however, these markers are also less reliable because of cell/tissue/organ-specific expression of characters, as well as influence of the developmental stage and/or environment. Biochemical markers are limited in number- thin coverage of the genome hence reveals low polymorphism. Dimeric and multimeric enzymes/proteins add complexity Less reliable due to stage specific, tissue expression of characters, influence of environment. Influenced by environment. Not suitable for large scale testing and automation Requires less investment at initial stage Many of these complications of phenotype and biochemicalbased assay can be overcome through direct identification of genotypes with DNA-based diagnostic assay. For this reason, DNA-based genetic markers are being integrated into several plant systems and are expected to play an important role in the future. Molecular markers Molecular markers have wide application in different branches of life sciences. In genetics, molecular marker is defined as a fragment of DNA sequence that is associated to a part of the genome carrying genes responsible for a trait. It is usually defined as an allele or DNA sequence or chromosome fragment indicating the existence of a metabolism or chemical or physical process. Sometimes this category of markers is referred to as bio-markers or bio-signatures or molecular signatures. Molecular markers are also sub-divided into Biochemical markers (Eg: Isozymes, Allozymes), DNA based markers (Eg: RFLP, RAPD, SSRs), Physiological markers, Biomarkers and Protein markers (also called biochemical markers) depending on the biochemistry, physiology and origin of markers within the plant. Molecular markers are phenotypically neutral. This is a significant advantage compared to traditional phenotypic markers. DNA based markers Diversity among organisms is a result of variations in DNA sequences and of environmental effects. Genetic variation is substantial, and each individual of a species possesses a unique DNA sequence. DNA variations are mutations resulting from substitution of single nucleotides (single nucleotide polymorphisms – SNPs), insertion or deletion of DNA fragments of various lengths (from a single to several
15
thousand nucleotides), or duplication or inversion of DNA fragments. DNA variations are classified as “neutral” when they cause no change in metabolic or phenotypic traits, and hence are not subjected to positive, negative, or balancing selection; otherwise, they are referred to as “functional”. Mutations in key nucleotides of a coding sequence may change the amino acid composition of a protein, and lead to new functional variants. Such variants may have an increased or decreased metabolic efficiency compared to the original “wild type”, may lose their functionality completely, or even gain a novel function. Mutations in regulatory regions may affect levels and patterns of gene expression; for example, turning genes on/off or under/ over-expressing proteins in specific tissues at different development or physiological stages. Several DNA-based molecular markers are available They differ in principle, detection of polymorphism rate, application, cost and time requirement. Numerous, highly polymorphic Not influenced by environment (phenotype neutral) Expressed in all tissues Most of DNA markers are co-dominant and can differentiate heterozygote from homozygote whereas morphological and biochemical markers are usually dominant/ recessive Advantages of DNA based molecular markers DNA-based markers provide a valuable tool for genetic analysis and plant breeding. DNA-based molecular markers offer several advantages over traditional phenotypic markers and have found their own position in various fields. Unlike morphological and biochemical markers, DNA-based markers are phenotypically neutral as alternate alleles at molecular markers loci caused no obvious changes in the phenotype of the organisms. These markers can be tested and can give results after a single collection of vegetable material from the field. They are independent of developmental stage and environment. These methods are rapid, relatively cheap and eliminate the need to grow plants to maturity. They have also high discrimination power enabling distinction of even closely related genotypes. These markers have been used to identify trees with desirable plant and fruit characteristics within the gene pool and breeding population. By applying the specific molecular marker at the seedling stage, tree and fruit characteristics can be identified early without having a wait for a mature tree to grow. This early characterization of tree will save money and accelerate the process of breeding and selection of superior varieties.
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Molecular markers can also be used to confirm the parents of handpollinated crosses and to determine the paternity of seedlings derived from open-pollinated crosses. This approach should enable us to produce much greater number of crosses and that will accelerate the breeding program. Hence, amongst the molecular markers used, DNA markers are more suitable and ubiquitous to most of the living organisms. The following properties are desirable for ideal DNA markers: Codominant inheritance (determination of homozygous and heterozygous states of diploid organisms)
Easy access (availability) and easy exchange of data between laboratories
Easy and fast assay
Frequent occurrence in genome
High reproducibility and Highly polymorphic nature
Selective neutral behaviour (the DNA sequences of any organism are neutral to environmental conditions or management practices)
It is extremely difficult to find a molecular marker which would meet all the above criteria. Types of molecular marker Several different types of molecular markers are being developed continuously. Detection of polymorphism at the molecular level is usually based either on restriction patterns or differential amplification of DNA. Restriction fragment length polymorphism (RFLP) was the first molecular marker generated for genome analysis and mapping. However, the development of the polymerase chain reaction (PCR) technology has introduced a considerable number of useful molecular markers, e.g., RAPDs and SSRs. A combinational advantage of the use of restriction endonucleases and PCR-based amplification as explicit in AFLP has further strengthened molecular analysis. The following are the examples of DNA based molecular markers: • Restriction Fragment Length Polymorphisms (RFLPs) • Random Amplified Polymorphic DNAs (RAPDs) • Amplified Fragment Length Polymorphisms (AFLPs) • Simple Sequence Repeats (SSRs) • Sequence Tagged Microsatellite Sites (STMS) • Sequence Tagged Sites (STS)
18
• Single Nucleotide Polymorphism (SNP) The detailed description of the above markers are described below Restriction fragment length polymorphisms (RFLPs) Restriction fragment length polymorphisms (RFLPs) are identified using restriction enzymes that cleave the DNA only at precise “restriction sites” (e.g. EcoRI cleaves at the site defined by the palindrome sequence GAATTC). At present, the most frequent use of RFLPs is downstream of PCR (PCR–RFLP), to detect alleles that differ in sequence at a given restriction site. A gene fragment is first amplified using PCR, and then exposed to a specific restriction enzyme that cleaves only one of the allelic forms. The digested amplicons are generally resolved by electrophoresis. Advantages RFLPs are co-dominant and can differentiate heterozygote from homozygote. It is more sensitive and most reliable marker technique. It can able to identify a unique locus Disadvantage The technique is laborious, costly and involves several time consuming, tedious steps. The detection system uses radioisotope or complex biochemistry. It requires large amount of high quality DNA. It requires species specific primers/ probes. It is not suitable for high scale analysis of varieties/ genomes. Automation is not possible Random Amplified Polymorphic DNA marker (RAPD) RAPD is a PCR-based method which employs short synthetic oligo-nucleotides (10 – 12 bases long) of random sequences as primers to amplify DNA fragments from genomic template DNA under low annealing temperatures. Amplification products are generally separated on agarose gels and stained with ethidium bromide. The amplified DNA fragments have been visually scored and used for different analysis. Advantage
19
The RAPD technique is simple, cost effective and requires no radioactivity. The procedure requires very small amounts of DNA and don’t require cloning or prior knowledge of sequence of genome. Same primer can be used across the genome. Suitable for large scale analysis of genotypes Automation is possible Disadvantage RAPDs are commonly dominant markers. The heterozygote can’t be differentiated from homozygote. RAPD is less reliable. Microsatellites or SSR (Simple Sequence Repeats) marker Microsatellites or SSR (Simple Sequence Repeats) or STR (Simple Tandem Repeats) consist of a stretch of DNA a few nucleotides long – 2 to 6 base pairs (bp) – repeated several times in tandem (e.g. CACACACACACACACA). They are spread over a eukaryote genome. Microsatellites are of relatively small size, and can, therefore, be easily amplified using PCR from DNA extracted from a variety of sources including blood, hair, skin or even faeces. Polymorphisms can be visualized on a sequencing gel, and the availability of automatic DNA sequencers allows high-throughput analysis of a large number of samples. Microsatellites are hyper variable; they often show tens of alleles at a locus that differ from each other in the numbers of the repeats. They are still the markers of choice for diversity studies as well as for parentage analysis and Quantitative Trait Loci (QTL) mapping, although this might be challenged in the near future with the development of cheap methods for the assay of SNPs. Advantage The technique is simple It requires little DNA, faster and cost effective. Microsatellite markers are co-dominant. These markers are abundant, distributed evenly throughout the genome, show high level of polymorphism compared to other marker. It is useful especially for analyzing closely related genotypes. Suitable for large scale analysis of genotypes.
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Disadvantage It requires species specific primers The technique requires development of marker. The cost of microsatellite markers is high. Minisatellites Minisatellites share the same characteristics as microsatellites, but the repeats are ten to a few hundreds bp long. Micro and minisatellites are also known as VNTRs (Variable Number of Tandem Repeats) polymorphisms. Amplified fragment length polymorphisms (AFLPs) Amplified Fragment Length Polymorphism is a molecular marker generated by a combination of restriction digestion and PCR amplification. Advantage AFLPs are highly polymorphic, evenly distributed throughout the plant genome and hence serve as useful tool for various genetic studies. It is suitable for large scale analysis of genotypes. The technique can be used for DNA of any origin or complexity It combines the advantages of both RFLP and RAPD Disadvantage AFLPs are mostly dominant in nature and hence heterozygote can’t be differentiated from homozygote. Requires high quality DNA. Procedure is little bit complex and requires careful handing Sequence Tagged Site (STS) STS (Sequence Tagged Site) are DNA sequences that occur only once in a genome, in a known position. They needn’t be polymorphic and are used to build physical maps. Single Nucleotide Polymorphism (SNP) SNPs are variations at single nucleotides which do not change the overall length of the DNA sequence in the region. SNPs occur throughout the genome. They are highly abundant. Most SNPs are located in non-coding regions, and have no direct impact on the phenotype of an individual. However, some introduce mutations in expressed sequences or regions influencing gene expression (promoters, enhancers), and may induce changes in protein structure or regulation. These SNPs have the potential to detect functional genetic variation.
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Comparison of different molecular marker systems Feature
RFLP
RAPD
AFLP
SSR
SNP
10
0.02
0.5-1.0
0.05
0.05
DNA quality
High
High
Moderate
Moderate
High
Ease of use
Not easy
Easy
Easy
Easy
Easy
PCR based
No
Yes
Yes
Yes
Yes
Reproducibility
High
Unreliable
High
High
High
No. of polymorphic loci
1.0-3.0
1.5-50
20-100
1.0-3.0
1.0
Low
Moderate
Moderate
High
High
Development cost
Low
Low
Moderate
High
High
Cost per analysis
High
Low
Moderate
Low
Low
DNA required (μg)
Amenable to automation
Application of molecular markers Assessment of genetic variability and fingerprinting of genotypes Mapping of monogenic and qualitative trait loci (QTL) of economically important traits Estimation of genetic distance or degree of relatedness between population, inbreeds and breeding materials or among groups of accessions in germplasm. Identification of sequences for candidates genes and economic breeding traits Marker assisted selection for crop improvement Genetic purity testing of seeds and micro-propagated plantlets Characterization and evaluation of plant genetic resources and its conservation Screening transgenic plants for resistance genes using linked molecular markers Cloning and analysis of cDNA encoding a specific synthesis, thus understanding the nature of basic control mechanisms.
22
Chapter-V Different Protocol DNA isolation & Purification The young emerging healthy leaves were collected and immediately brought to the laboratory. The leaves were washed with distilled sterile water and cleaned with moist tissue paper. The leaf sample (2-3 g) was cut into small pieces and placed in a pre-cooled mortar, frozen by adding liquid nitrogen and crushed vigorously with pestle to a fine powder. The ground powder was transferred into a clean sterile 50 ml centrifuge tube and 10 ml of pre-warmed (60 oC) extraction buffer (details given below) was added to it. The content was shaken vigorously by inversion to form slurry. The tubes were incubated at 60 oC in circulating water bath for 1 h with intermittent shaking to form an emulsion. After that equal volume of chloroform: isoamyl alcohol (24:1) was added and mixed by gentle swirling for 15 to 20 min. After complete emulsion formation, centrifugation was done at 10,000 rpm for 10 min at 25 oC. The aqueous phase was transferred to a fresh centrifuge tube and then equal volume of chilled isopropanol was added. The content was mixed by gentle inversion. The tubes were kept for overnight at 4 oC. The DNA was spooled out carefully and kept in 1.5 ml eppendorf tubes, which were then spun at 10,000 rpm for 10 min. The aqueous part was decanted. The pellet was washed with 70% ethanol. The tubes were spin at 10,000 rpm for 10 min at 25 oC. The aqueous part was decanted and the pellet dried free of ethanol. The DNA pellet was dissolved in 100µl of TE (10:1) buffer (pH 8.0) and kept overnight for complete dissolution. All the dissolved samples were treated with RNase A. One microlitre solution of RNase enzyme (10 µg/ml) per 100 µl was added to the DNA solution and mixed gently. The content was incubated at 37 ºC for 1 h for the removal of RNA. Equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added and mixed properly for 5 min. Sample tubes were centrifuged at 10,000 rpm for five min and the supernatant was collected in fresh tubes. Equal volume of chloroform: isoamyl alcohol was added, mixed well and centrifuged for 10 min. The aqueous layer was removed and the process was repeated to produce a creamy solution. Then one-tenth volume of 3M sodium acetate and 2.5 volume of absolute chilled ethanol were added. The content was mixed gently to precipitate DNA and thereafter kept overnight at -20oC. The solution was then centrifuged at 8000 rpm for 5 min and the supernatant was decanted off. Extra salts were removed by two washing with 70% ethanol. DNA was dried under vacuum and dissolved in TE (10:1) buffer at room temperature and stored frozen at - 20oC.
23
Quantification of isolated DNA The concentration of DNA was estimated by the measurement of the UV irradiation absorbed by nucleic acid bases. First the spectrophotometer was calibrated using 2000 µl of TE in a quartz cuvette at 260 nm and 280 nm. Then five µl of DNA sample was added to 1995 ml of TE, mixed well and absorbance (OD) was taken at 260 nm and 280 nm. The concentration of the DNA in the sample was estimated as follows Concentration of DNA (µg/ml ) = OD at 260 x Dilution factor x 50 The ratio between readings at 260 nm and 280 nm (OD260/ OD280) provided an estimate for the purity of nucleic acid. Any sample showing the ratio below 1.8 or above 2.0 was further subjected to purification. Quality of isolated DNA Further in order to know intactness of genomic DNA, presence of proteins and/or RNA contaminants, an aliquot (2 µl) of each sample was subjected to agarose gel (0.8 % w/v) electrophoresis for about 2 h along with 500 ng of molecular weight marker ( Lambda / EcoRI digest). The gel was stained with ethidium bromide (0.5 µg/ml), viewed under UV Transilluminator and photographed immediately for further interpretation using a Gel-Doc system. By comparing the fluorescent intensity of the bands with the standard, DNA concentration was also estimated following the method described by Sambrork et al. (1989). Part of stock DNA samples were diluted with appropriate amount of TE buffer to yield a working concentration of 10 ng/µl and stored at 4 oC. Polymerase Chain Reaction (PCR) The PCR mixture consisted of Taq DNA polymerase, PCR buffer, dNTPs, MgCl 2 , oligonucleotide primers and genomic DNA. Optimization of concentration of PCR components was carried out for MgCl2, Taq DNA polymerase, and genomic DNA concentration. To determine optimal amplification reaction conditions, a factorial experiment was carried out at three concentrations of MgCl2 (2.0 mM, 2.5 mM and 3.0 mM), three concentrations of Taq DNA polymerase (0.5 U, 1.0 U, 1.5 U),
24
three concentrations of template DNA (10 ng, 25 ng, 50 ng) and 10 pmole primer in a volume of 25 µl. PCR was carried out using Thermal Cycler (Bio-Rad, USA), PCR conditions that gave better amplified DNA profile .were determined and presented below.
PCR constituents optimized for RAPD and ISSR analysis Component RAPD analysis PCR Buffer with MgCl2 (15mM)
Stock
Quantity in µl
Final concentration in the reaction mixture
10 x
2.5
1x
dNTPs mix Taq DNA polymerase
10 mM I U/µl
2.0 1.0
200 µM each 1 unit
RAPD Primers
250 pM
1.0
10 pmoles
Sterile DNase, RNase free water
17.5
Total
24.0
Template DNA
1.0
25 ng
10 x
2.5
1x
dNTPs mix
10 mM
2.0
200 µM each
Taq DNA polymerase ISSR Primers
I U/µl 250 pM
1.0 1.0
1 unit 10 pmoles
ISSR analysis PCR Buffer with MgCl2 (15mM)
Sterile DNase, RNase free water
17.5
Total
24.0
Template DNA
1.0
25 ng
For RAPD analysis, all PCR were carried out in a final volume of 25 ml reaction mixture containing 25 ng template DNA, 200 µM each dNTPs, 2 mM MgCl2, 10 pmoles primer, 1X Taq polymerase buffer and 1 unit of Taq DNA polymerase. The Thermal Cycler was programmed to include a pre-denaturation step at 94 oC for 3 min, followed by 44 cycles of denaturation at 94 oC for 1 min annealing at 37 oC for 1 min and extension at 72 oC for 2 min. The final extension was made for 7 min at 72 oC. For ISSR analysis, all PCR reactions were carried out in a final volume of 25 µl reaction mixture containing 25 ng template DNA, 200 µM
25
each dNTPs, 2 mM MgCl2, 10 pmoles primer, 1X Taq polymerase buffer and 1 unit of Taq DNA polymerase. Initially, the amplification was performed in programmable gradient Thermal Cycler with following programme: a predenaturation at 94 oC for 3 min followed by 44 cycles of denaturation at 94 o C for 1 min, annealing at gradient temperature for 1 min and extension at 72 oC for 2 min. The final extension was made for 7 min at 72 oC. The annealing temperature tested was 5 oC above and below the melting temperature (Tm) of the particular primer. PCR amplification at a particular annealing temperature that gave better resolution, that temperature was selected for optimum annealing temperature for that ISSR primer for final PCR amplification. Agarose gel electrophoresis Agarose gel (1.5%) was prepared by mixing 4.5 g of agarose in 300 ml of 1X TBE buffer. The content was boiled in microwave oven till it completely dissolved. During warming intermittent shaking was made (4-5 times) to prevent formation of clumps of agarose. The molten agarose was kept for cooling up to 50-60 oC and then ethidium bromide (1 µg/ml) was added. The molten agarose was poured into the clean, leveled casting plate containing 30 well combs and was kept for solidification. The gel was transferred to the electrophoresis unit containing 1X TBE buffer. The PCR reaction products were first mixed with 2.0 µl of loading dye (details given below) and spun for a while before loading into the wells of the gels. The medium range ruler (Bangalore Genei) was also loaded in first and/or last well of the gel to serve as standard molecular weight marker for determining the size of the amplified DNA fragments. The gel was run at 80 volts for 4 h. The run was stopped when bromophenol blue dye had travelled 2/3rd length of the gel. Gel documentation and photography After electrophoresis, the stained DNA gel was visualized under UV light in a Gel-Doc system and photographed. Fragment size of all amplification products were estimated from the gel by comparison with standard molecular weight marker (Medium Range Ruler). DNA bands were scored as discrete variable using ‘1’ to indicate presence and ‘0’ to indicate the absence of a band. Analysis of molecular data It is imperative to understand the different ways that the data generated by molecular techniques can be analyzed before considering their application to diversity studies. Two main types of analysis are frequently done Analysis of genetic relationships among samples, Calculation of population genetics parameters, in particular diversity and its partitioning at different levels.
26
The analysis of genetic relationships among samples starts with the construction of a matrix specifying the character-state of each marker for each sample. A sample will usually be DNA from an individual. Marker states may be binary, as in the presence or absence of RAPD bands or restriction sites (as revealed by RFLPs and related techniques). This sample x marker matrix of character-states is then commonly used to construct a sample x sample matrix of pair-wise genetic distances (or similarities). There are several different ways of calculating the genetic distance (or similarity) between two samples on the basis of the differences between them in the states of a set of genetic markers, but a commonly used index is Nei’s genetic distance (D) or Jaccard’s similarity coefficient (S). There are two main ways of analyzing the resulting distance (or similarity) matrix and displaying the results. One is to use Principal Coordinate Analysis (PCO) to produce a 2- or 3- dimensional scatter plot of the samples such that the geometrical distances among samples in the plot reflect the genetic distances among them with a minimum of distortion. Aggregations of samples in such a plot will reveal sets of genetically similar material. Another approach is to produce a dendrogram (or tree-diagram) linking together in clusters samples that are more genetically similar to each other than to samples in other clusters. Clusters are linked to each other at progressively lower levels of similarity until all the samples being analyzed are included in a single cluster. Such Cluster Analysis may proceed according to a range of different algorithms, but some of the more widely used ones include Unweighted Pair Group Method with Arithmetic Averages (UPGMA), Neighbour-Joining Method and Ward’s Method. Both PCO and cluster analysis are so-called ‘phenetic’ methods in that they are based on measures of overall distance or similarity among samples. However, there is another, philosophically quite distinct approach to the analysis of genetic relationships, referred to as ‘cladistics’. Cladistic analysis also begins with the sample x marker character-state matrix, and also results in dendrograms, though these are sometimes called cladograms to distinguish them from the phenograms of cluster analysis. The difference is that two samples are placed together in the same cluster (or clade) of a cladogram not on the basis of high genetic similarity between them calculated from all markers taken together, but because they share a particular state of a given marker (or markers). The two approaches are also sometimes distinguished as ‘distance’ and ‘character-state’ respectively. Because it is possible to generate many cladograms from a single dataset, due to conflicts among characters, so-called parsimony approaches are used to choose among them. A most-parsimonious cladogram is one that requires the least number of character-state changes. There is a wide range of parsimony algorithms, each with its own data requirements and assumptions. Some
27
require that the polarity of character changes be known, i.e. which character states are ancestral and which derived. Cladograms are reconstructions of phylogenies. RAPD data, because of uncertainty over the identity of bands, is not usually thought suitable for this kind of analysis. Turning now to the measurement of genetic diversity and genetic structure (among and within populations), the F-statistics are commonly employed. Estimates of these statistics are based on allele frequencies, and the most appropriate molecular data for such statistical analyses are clearly those in which allele frequencies can be determined directly, such as RFLPs, STMS and sequence haplotypes. Of these, sequences and restriction site data are unique among molecular markers in providing both frequency and phylogenetic information. Nevertheless, suitable statistical treatments are also available for dominant markers such as RAPDs. Solutions and reagents used DNA extraction, PCR réaction and electrophoresis DNA extraction 1. Liquid nitrogen 2. Tris HCl buffer (pH 8.0, 1 M) Tris salt (12.11 g) was dissolved in sterile de-ionised water and pH was adjusted to 8.0 with conc. HCl. Volume was made upto 100 ml with de-ionised water. The solution was autoclaved prior to use 3. Ethylenediaminetetraacetic acid (0.5 M EDTA, pH 8.0 ) EDTA (18.61 g) was dissolved in 70 ml of de-ionised water and adjust pH to 8.0 with 5 N NaOH, and volume was made up to 100 ml with de-ionised water and autoclaved prior to use. 4. Sodium chloride (5.0 M) Sodium Chloride (29.2 g) was dissolved in de-ionized water and volume was made up to 100 ml with water. The solution was autoclaved prior to use. 5. Cetyl Trimethyl Ammonium Bromide (CTAB) (10%) One hundred gram CTAB was dissolved in sterile distilled water and volume was made up to 1000 ml with the same after complete dissolution of the solute. The solution was then autoclaved. 6. 2-Mercaptoethanol (2 %) 2 % solution provided by manufacturer was stored in an amber coloured bottle and used directly 7. DNA extraction buffer DNA extraction buffer was prepared using following volumes of stock solutions of the individual components. Extraction buffer composition
28
Component
CTAB (freshly prepared) NaCl Tris HCl (pH 8.0) EDTA ß mercapto ethanol PVP Water (de-ionized water)
Conc. of Stock solution 10 % 5M 1M 0.5 M 2% -
Conc. of Working solution 2% 1.4 M 100 mM 20 mM 0.4% 2.0% -
Volume taken to prepare 100 ml buffer 20 ml 35 ml 10 ml 4 ml 2 ml 2gm 34ml
Components was dissolved with de-ionized water and autoclaved before use 8. Chloroform: Iso-amyl alcohol Mixture (24:1) 96 ml of chloroform was mixed with 4 ml of isoamyl alcohol and was stored in an amber coloured bottle 9. 70 % Ethanol 70 ml of absolute ethanol was mixed well with 30 ml sterile water and stored in a stopper bottle till use. 10. Iso-propanol (cold) Filter sterilized and store at 4 °C DNA purification 1. RNase (1 ml) solution Component 1M Tris-HCl (pH 8.0) 5 M NaCl RNase
Volume taken to prepare 100 ml buffer 100 µl 300 µl 10 mg
Final Conc. in solution 10mM 10mM
Sterile water was added to make the volume to 1 ml. The solution was heated to 100 °C for 15 minutes to inactivate any DNase present and then stored in aliquots at -20 °C 2. Sodium acetate solution (3 M, pH 6.8 ) Sodium Acetate (30.75 g) was dissolved in sterile distilled water, pH was adjusted to 6.8 with glacial acetic acid, and made up to 100 ml with de-ionised water and autoclaved. 3. Phenol : Chloroform : Isoamyl (25 : 24 :1) 100 ml Tris saturated phenol was added to a mixture of 96 ml chloroform and 4 ml Isoamyl alcohol. The mixture was vortexed
29
well prior to use and stored in amber coloured bottle. The mixture was prepared afresh as and when necessary. Solvent for DNA 1. TE (10: 1) 1 M Tris- HCl (pH 8.0) = 1 ml 0.25 M EDTA (pH 8.0) = 0.4 ml Sterile water was added to make the volume to 100 ml. The solution was autoclaved before use Gel electrophoresis 1. Agarose gel (1.5 %) 4.5 g Agarose was dissolved in 300 ml 1x TBE buffer. The contents were mixed thoroughly and boiled till completely dissolved. The molten gel was cooled down to 45- 50 0C and was casted in a gel tray with a comb to produce well. 2. 10X TBE (pH 8.0)
Trizma base Boric acid 0.5 M EDTA (pH 8.0)
108g 55g 40 ml
Sterile water was added to make the volume to 1000 ml. The solution was autoclaved before use. 3.
Loading Buffer (6x solution) Bromophenol Blue = 50 mg Glycerol = 30 mg 0.25M EDTA = 4 ml Volume was adjusted to 100 ml with de-ionized water and stored at 40c. PCR reaction master mix 1. Taq DNA polymerase A stock solution of 1 unit/µl was provided by the Banglore Genei company which was stored at -20 0C. One unit /reaction mix was used. 2. 10x Assay buffer 10x PCR Assay buffer for Taq DNA polymerase containing 15 mM MgCl2 supplied by the Bangalore Genei was used. The buffer was stored at -20 °C
30
3. Deoxyribonucleotide triphosphate (dNTPs)
10.
After completion, the filter paper stack and the Whatman papers are removed and position of wells are marked with soft pencil.
11.
Peel off the membrane from the gel with blunt ended forceps, air
dNTP mix (10 mM) containing nucleotide (2.5 mM each) supplied by Bangalore Genei was used.
dry at room temperature and store in vacuum dessicator until further use.
4. Primers Primer was provided by the manufacture in a lyophilized form. Based on molecular weight of a given primer, a working solution was prepared by adding the required amount of sterile distilled water and stored at -20°C.
Radioactive Probing a) Random labelling 1.
Mix 200 ng of insert DNA (5 μl ), 19 µl sterile water and 10 ìl of random primer in a microfuge tube and incubate in a 95-100o C water bath for 5 min.
2.
Spin briefly and set up the reaction as follows:
Southern hybridization Blotting of DNA on nylon membrane (common to both probing methods) Treat the agarose gel after electrophoresis with 0.25 N HCl for 15
Above mix
34 μl
min at room temperature with gentle shaking.
DNA polymerase buffer (10X)
5 μl
2.
Wash the gel with sterile distilled water.
100 mM dNTP mix
5 μl
3.
Again treat it with 0.4 M NaOH for 30 min at room temperature with
d CTP á-32P labelled (3000µCi / m mole) 5 μl
1.
gentle shaking. 4.
Total
face the plate. 5.
Cut Hybond N+ membrane exactly the same size of the gel, soak in
3.
6.
Place exactly the same size of Whatman 3 mm filter papers, soaked in 0.4 M NaOH on the membrane.
7.
Place a stack of blotting paper (5-6 cm thick) of relatively small size on the Whatman filter papers.
8.
Put another glass plate on the stack along with 400-500 g weight.
9.
Allow the transfer to continue for 16-18 h.
31
4.
=
50 μl
Incubate at 37oC for 5 min and terminate the reaction by adding 2
0.4 M NaOH and carefully place on the gel without any trapped air bubble between the plate and the membrane.
1 μl
T7 DNA Polymerase (2 U / µl )
Place the gel upside down on a clean glass plate so that the wells
μl of 0.5M EDTA.
Labeled DNA fragments may be purified through Sephadex G-50 column.
b) Pre-hybridization and hybridization 1. Add 100 µl / cm2 hybridization solution into the hybridization tube or bag containing the membrane. 2. Seal the bag or tighten the tube carefully preventing any leakage. 3. Incubate at 65oC in water bath (for bags) with gentle shaking for 8-10 h.
32
4. Denature the radiolabelled probe at 100 oC for 10 min and immediately chill on ice. 5. Take out the bag, cut at one end, and drain out half of the hybridization solution.
Salmon sperm DNA 20X SSC NaCl
175.3 g
Sodium Citrate
88.2 g
Total volume in water
1 lit
100X Denhardt’ Solution BSA (bovine serum albumin)
2% w/v
Ficoll
2% w/v
7. Gently massage the bag and incubate at 65oC in a water bath (specially for bags) with gentle shaking overnight.
PVP (polyvinyl pyrrolidone)
2% w/v
8. W ash the blots as described below:
c)
Autoradiography
1.
Place the membrane on a used X-ray sheet and cover with Saran Wrap.
2.
Place the wrapped membrane in an exposure cassette.
3.
Take the cassette to the dark room, place an X-ray film on the membrane and close the cassette.
4.
Cover the cassette with black cloth and keep at -70oC for 1h to 3 days depending upon the counts.
5.
Carry out the remaining operations in dark.
6.
Develop the film in Kodak developer (5 min) and rinse with cold distilled water.
7.
Transfer the developed film to the Kodak fixer (5 min).
8.
Wash the film thoroughly in running tap water and air dry.
9.
Analyse.
o
2nd washing with 0.5X SSC and 0.1% SDS at 65 C for 15 min 3rd washing with 0.1X SSC and 0.1% SDS at 65oC for 15 min Wrap the blot with Saran Wrap, check the count and perform auto radiography.
Estimation of protein by Lowry Method
Hybridization buffer SSC
5X
Denhardt’ Solution
2.5X
Sodium phosphate buffer(pH 7.0)
0.05 M
33
μ g / ml
6. Add probe carefully to the buffer in the bag and seal.
1st washing with 1X SSC and 0.1% SDS at 65oC for 15 min
9.
100
Protein can be estimated by different methods as described by Lowry and also by estimating the total nitrogen content .No method is 100% sensitive . Hydrolysing the protein and estimating the amino acid alone will give the exact quantification .The method developed by Lowry et el is sensitive enough to give a moderately constant value and hence
34
largely followed . Protein content of enzyme extracts is usually determined by this method .
2.
Pipette out 0.1ml and 0.2ml of the sample extract into other two test tube.
Principle:
3.
Make up the volume to 1ml in all the vtest tube .A tube with 1ml of water serves as the blank
4.
Add 5ml of reagent C each tube including the blank .Mix well and allow to stand for 10 min.
5.
Then add 0.5 ml of reagent D mix well and incubate at room temp in the dark for 30 min .Blue colour is developed.
Material:
6.
Take the readings at 660nm.
1. 2.
7.
Draw a standard graph and calculate the amount of protein in the sample.
The blue colour developed by the reduction of the phosphotungstic components in the Folin –ciocalteau reagent by the amino acid tyrosine and tryptophan present in the protein plus the colour developed by the biuret reaction of the protein with the alkaline cupric tartrate are measured in the Lowry method.
3. 4.
2%sodium carbonate in 0.1 N sodium Hydroxide (Reagent A) 0.5%cupper sulphate (CuSO 4 5H 2O) in 1%potassium sodium tatarate (Reagent B) Alkaline copper solution : Mix 50ml of A and 1ml of B prior to use (Reagent C) Foline –ciocalteau reagent (Reagent D) :
Reflux gently for 10 hr a mixture consisting of 100g sodium tungstate (Na2 WoO4 2H2O), 25 g sodium molybate (Na2MoO4 2H2O), 700, l water, 50ml of 85%phosporic acid and 100ml of concentrated hydrochloric acid in a 1.5 L flask . Add 150g lithium sulphate , 50ml water and a few drop of bromine water. Boil mixture for 15 min without condenser to remove excess bromine cool dilute to 1 L and filter. The reagent should have no greenish tint. (Determine the acid concentration of the reagent by titration with 1 N NaOH to a phenolphthalein end point)
NOTE 1.
For complete enzyme extraction sometimes the chemicals like ethylenediamine teraacetic acid (EDTA) magnesium salt and mercaptoethanol are included . This method of protein estimation should not be followed if the extratant contains K+ Mg++, Tris , EDTA and thiol (mercaptoethanol) compound as they interfere with this procedure. When these chemicals are presented in the extract precipitate the protein by the adding 10% TCA centrifuge and dissolve the precipitate in 2 N NaOH and proceed for protein estimation .
2.
If the protein concentration of the sample is high (above 500 µg/ ml) measure the colour intensity.
Weigh accurately 50mg of bovine serum albumin (Fraction) and dissolved in distilled water and make up to 50ml in a standard flask .
3.
Rapid mixing as the folin reagent is add is important for reproducibility.
Working standard
4.
A set of standards is needed with each group of estimation preferably in duplicate . Duplicate or triplicate unknown are also recommended.
Dilute 10ml of the stock soluation to 50 ml with distilled water in a flask one ml of this solution contains 200µg protein.
5.
Folin –ciocalteau reagent can be purchased commercially. Store refrigerator in amber bottle. A good quality reagent is straw yellow in colour.
6.
If the protein estimation is desired in a sample with high phenolic or pigment content extract should be prepared with a reducing agent preferably cystine and NaCl. Precipitate the protein with TCA separate the protein and dissolved in 2N NaOH and proceed.
5. Protein solution (Stock standard)
Procedure: Extraction of protien 1.
35
Pipette out 0.2, 0.4, 0.6, 0.8, and 1ml of the working standard into a series of test tube.
36
Protein estimation – Bradford method
NOTE
The protein in solution can be measured quantitatively by the different methods .The methods described by the Bardford use a different concept –the proteins capacity to bind a dye quantitatively .The method is simple rapid and inexpensive .
1.
It important to use a protein as similar in its properties to your sample as possible. If your sample is unknown, use antibody protein as reference. BSA usually gives a 2-fold higher value in this assay and therefore, cannot be as a general standard .
Principle:
2.
As in the case of Lowry protein assay procudre detergents such as SDS Nonidet p40 Triton x100 etc. Interfere with this protocol too.
3.
Several blue G dye is another dye used in place of coomassie blue G 250 with similar properties.
4.
The dyes exist as to forms (blue and orange) in acid solution. The protein bind the similar properties. Preferentially.
5.
Check the absorption of working dye solution at 550nm is 1.18 if necessary adjust either with the powder or water are required.
The assay is based on the ability of proteins to bind coomassie brilliant blue G 250 and from a complex whose extinction cofficent is much grater than that of the free dye . Material: 1.
Dye (concentration) Dissolve 100gm of coomassie brilliant blue G 250 in 50 ml of 95%ethanol.
Add 100 mg of conc. ortho phosphoric acid. Add distilled water to final volume of 200ml of store refrigerated in umber bottles. The solution is stable for at least six months Mix 1 volume of concentrated dye solution with 4 volume of distilled water for use 2.
Phosphate – buffered saline (PBS)
Procedure: 1.
Prepare a series of protein sample in test tube in the concentration. This preferably prepared in PBS.
2.
Prepare the experimental sample (a few dilutions) in 100 µl of PBS
3.
Add 5ml of diluted dye binding solution each tube.
4.
Mix well and allow the colour to develop for at lest 5 min but no longer than 30 min . The red dye turn blue when it binds protein .
5.
Read the absorbance at 595nm.
6.
Plot a standard curve using the standard protein absorbance Vs concentration. Calculate the protein in the experimental sample using the standard curve.
37
SDS -PAGE Gel electrophoresis for protein analysis Electrophoresis is widely used to separate and characterize protein by applying electric current . Electrophoresis producer are rapid and relatively sensitive requiring only micro-weight of proteins .Electrophoresis in the polyacralimide gel is more convenient than any other medium such as paper and starch gel. Electrophoresis of proteins in polyacrylamide gel is carried out in buffer gels (non- denaturing) as well as in sodium dodecyl sulphate (SDS) containing (denaturing)gels. Separation in buffer gel relied on both the charge and size of the protein whereas it depends only upon the size in the SDS-gel. Analysis and comparison of protein in a large number of sample is easily made on polyacrilamide gel slabs. Polyacralimide gels are formed by polymersing acrylamide with a cross linking agent (bisacrylamide) in the presence of a catalysts (persulphate ion) and chain initiator (TEMED N, N, N, N,- tetramethylethylene dopamine). Solutions are normally degassed by evacuation prior to polymerization since oxygen inhibits polymerization.The porosity of the gel is determined by the relative proportion of acryl amide monomer to bisacrylamide. Gel is usually referred to in terms of the total percentage of acrylamide and bisacrylamide present and most protein separation are
38
preformed using gel in the range 7-15%. A low percentage gel (with large per size) is used to separate high molecular weight protein and vice-versa .At high concentration of the persulphate and TEMED the rate of polymerization is also high. Among a number of method commonly used the sodium dodecyl sulphate-polyacrylamide gel electrophorsis (SDS-PAGE) in slab (facilitating characterization of polypeptides and determination of their molecular weight by co-electrophoresis) is described bellow. Principle: SDS is an anionic detergent which binds strongly to and denatures proteins. The number of SDS molecules bounds to a polypeptide chain is approximately half the number of amino acid residues in that chain. The proteins –SDS complex carries net negative charges hence move towards the anode and the separation is based on the size of the preparation. Materials: a.
b. c. d.
Stock acrylamide soluation Acrylamide- 30% 30g Bisacrylamide -0.8% 0.8g Water to 100ml Separating gel buffer 1.875M (pH 8.8) 22.7g -Tris-HCL Water to 100ml Stacking gel buffer 0.6M Tris-Hcl (pH 6.8) 7.26g. Water to 100ml Ammonium persulphate 5% - 0.5 g in 10ml. Prepare freshly before use
e. f.
TEMED - 20 l Fresh from the refrigerator Electrode buffer (Used for three times) 0.05M Tris 12g 0.192 M Glycine 28.8g pH 8.2-8.4 (No adjustment required) 0.1%SDS 2g Water to 2000 ml
g.
Sample Buffer (5x concentration) Tris-HCl buffer pH 6.8 5ml SDS 0.5g Sucrose 5g Mercaptoethanol 0.25ml
39
Bromophenol Blue (0.5% (W/V) solution is water) Water to
1ml 10ml
Store frozen in small aliquots. Dilute to 1x concentration and use. h.Sodium dodecyl sulphate 10% solution at room temperature. i. Standard marker proteins
protein ά- Lactalbumin Trypsin inhibitior soyabean Trypsinogen Carbonic anhydrase Glyceraldehyde-3-phospate dehydrogenase Albumin, egg Albumin bovine
MW Daltons 14, 200 20, 100 24, 000 29, 000 36, 000 45, 000 66, 000
Dissolved the above proteins in single strength sample buffer at the conceration each of 1mg per ml . Load the well 25-50 l j. Protein staining solution Coomassie brilliant blue R-250 0.1g Methanol 40ml. Acetic acid 10ml Water 50ml First dissolve the dye in methanol and use fresh preparation every time k. Destaining Solution : All above solution except cormassie brulliant blue R-250 Procedure 1. Thoroughly clean and dry the glass plates and spacer. Assemble them properly hold the assembly together with bulldog clips clamp in an up right position white petroleum jelly or 2%agar (melted in a boiling water bath )is then applied around the edges of the scarper to hold them in place and seal the chamber between the glass plates
40
electrophoresis set can be kept cool using suitable facility so that beat generated during the run is dissipated and does not affect the gel and resolution.
2. Prepare a sufficient volume of separating gel mixture (30 ml for a chamber of about (18x 9x 0.01cm ) by mixing the following. Stock
15% gel
Stock Acrylamide solution Trish- HCl (pH8.8) Water Degas on water pump for 3-5 min and then add Ammonium persulphate 10% SDS TEMED
10%gel
20ml 8ml 11.4ml
13.3ml 8ml 18.1ml
0.2ml 0.4ml 20 μ l
0.2ml 0.4ml 20 μ l
3.
Mix gently and carefully pour the gel solution in the chamber between the glass plates. Layer distilled water on top of the gel and leave to set for30-60 min
4.
Prepare staking gel (4%) by mixing the following solution (total volume 10ml)
6.
Prepare sample for electrophoresis following suitable extraction procedure. Adjust the protein concentration in each sample using the 5 strength sample buffer and water in such a way that the same amount of protein is presented per unit volume. Again the concentration should be such as to give a sufficient amount of protein (50-200 μ g) in a volume (25-50 μ l)not greater than the size of the sample well. As general practice heat sample solution in boiling water for 2-3 min to ensure complete interaction between protein and SDS.
7.
Cool the sample solution and take up the required volume in a micro syringe and carefully injects it into a sample well through the electrode buffer marking the position of well as the glass plate with a marker pen and the presence of bromophenol blue in the sample buffer facilitate easy loading of the sample. Similarly load a well with standard marker proteins in the sample buffer.
8.
Turn on the current to 10-15 mA for initial 10-15 min until the sample travel through the stacking gel.Then continue the run at 30mA until the bromophenol blue reach the bottom of the gel (about 3h) However the gel may be run at high current (60-70mA) for short period (1 h) with proper cooling.
9.
After the run is complete carefully remove the gel from between the plate and immerse in staining solution for at least 3hr or over night with uniform shaking .The proteins absorb the coomassie brilliant blue.
10.
Transfer the gel to a suitable container with atleast 200-300ml destining solution and shake gently and continuously. Dye that is not bound the protein is thus remove. Change the destainer frequently particularly during initial periods until the background of the gel is colourless. The protein fractionated into band are seen colored blue as the protein of minute quantities are stained finely. Destining process should be stopped as appropriate stage
Stock acrylamide soluation Trish –HCl(pH-6.8) Water Degas as above and then add Ammonium persulphate soluation (5%) 10% SDS TEMED
= 1.35ml = 1ml = 7.5ml = 50 μ l = 0.1ml = 10 μ l
Remove the water from the top of the gel and wash with a little stacking gel solution pure the stacking gel mixture place the comb in the stacking gel and allow the gel to set (30-60)min 5.
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After the stacking gel has polymerized remove the comb without distorting the shape of the well. Carefully install the gel after removing the clips agar etc in the electrophoresis apparatus fill with eletrode buffer and remove any trapped air babuls at the bottom of the gel. Connect the top and turn on the DC power briefly to check the electrical circuit. The electrode buffer and
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to visualized as many band as possible .The gel can now be photographed or stored in polythene bag or dried in vacuum for permanent record. Note 1.
2. 3.
4. 5. 6. 7.
All chemicals and distilled water should be high quality. The solution prepared should be filtered before use .The solution can be store refrigerated-1-2 weeks. Aged solution result in poor resolution of proteins. Acrylamide as a monomer, is highly neurotoxin handle with extreme care Prefer to use the gel immediately following polymerization although the separation gel after setting can be store over night by waiting four fold diluted separation gel buffer or with stacking gel and comb placed over it to avid drying. Degassing of gel mixing should be adequate f or easy polymerization. During polymerization of the gel heat is evolved. The water layered over the separation gel should be completely removed for quick polymerization of the stacking gel. Some troubles and remedies are follows.
Trouble a)Failure or slow polymerization of the gel
b)poor sample well c) Long duration of the run d)Staining is poor
The stain is patchy The stain bands are decolorized
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Cause I) Presence of oxygen ii) Absence of catalysts iii)Stock solution aged iv) Glass plate Stacking gel and comb Air bubbles interference The dye absorption is not efficient Solid dye The dye is removed excessively Insufficient electrophoresis Separation gel Excess persulphate
Remedy i) Degas the solution sufficiently ii)Check if all solution mixed iii)Use fresh solution iv) Degrease the plates with ethanol. Fit remove the comb carefully Flush air bubbles The dye may be hold hence use a strong solution of dye or change to a more sensitive stain Dissolved the dye completely or filter
e)Protien bands are inadequately resolved f)Protien bands are wavy
Proteins remain arranged denatured or insoluble
Band have become starker
Insufficient cooling
Gels partly insulated by air bubbles
Sample density g)Protien dye migration is not even h)The protein band lane broadens at the bottom of separation gel Sample diffuse while loding the wells
Low density of sample
Restrain the gel and stop destining appropriateluy. Run for longer time Change the percentage of the gel Use optimum concentration of persulphate Use fresh sample buffer or extract SDS or centrifuge the sample extract sufficiently Remove air bubbles before electrophoresis Improve the cooling run at a low current Load equal volume of sample in each well equal strength sample buffer leaves no empty wells in the middle Increase the concentration of sucrose or glycerol in the sample buffer.
8. 9.
Handle the polyacrylamide gel carefully to avoid any breakage The slab gel along the glass plate placed vertically in the electrophoresis tank and it is there fore called vertical slab gel electrophoresis. 10. In10%polyacyrlamide gels the molecular weight (10,000daltons) polypeptides will migrate diffused for fine resolution of this polypeptides use gel of higher (15%) acrylamide concentration. 11. Any band of 0.1 μ g protein is visualized by coomassie brilliant blue staining in SDS-PAGE form visualizing protein of lower concentration below 0.1 μ g high sensitive method is preferred. Western Blotting It is used for confirmation of transgene expression in genetically modified (GM) or transgenic plant. Principle: In this test, the protein (trans-protein) of the introgressed gene separated in the SDS-PAGE gel is transferred to a solid support (nitrocellulose membrane) through electroblotting. The first antibody (raised against the trans-protein) is allowed to bind to the antigen (trans-protein) on the nitrocellulose membrane. Then anti-FC antibody coupled with an enzyme is allowed to bind 1st primary antibody. The enzyme produces a colour product
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on addition of the substrate and this is only possible when the test antibody binds to the solid support. The development of colour indicates the antigen –antibody reaction and the test is positive, Reagents and materials 1. Transfer buffer Trisbase (15.6 mM) 1.93 g Glycine (120 mM) 9.00 g Adjust the pH to 8.1 to 8.4 and make volume upto 1000 ml with double distilled water and autoclave 2. TBS (50 mM Tris – HCl, pH – 8.0, 150.0 mM NaCl) 3. 3% BSA in TBS 4. 0.5% bovine serum albumin (BSA) 5. Alkaline phosphatase buffer Tris-HCl (pF-9.5) - 0.1 M NaCl - 0.1 M MgCl2 - 5 mM 6. BCIP (5-Bromo 4-Chloro Inolylphosphate ) solution BCIP in 100% DMF - 50 mg/ml 7. Nitro Blue Tetrazolium p-NBT solution p-NBT in 70% DMF - 50 mg/ml 8. Developing reagent NBT solution 66 µl Alkaline phosphatase buffer 10 ml BCIP solution 33 µl 9. Primary antibody (1st Ab) 10. Conjugated secondary antibody (2nd Ab) 11. Extra-thick blotting pads 12. Paper cutter 13. Micropipettes, tips, etc. 14. Electro-blotting apparatus 15. Blot development/ staining tray Procedure: (a) Transfer of protein from gel to nitrocellulose paper 1. Cut the nitrocellulose membrane and whatman 3 µm filter paper of the size of the gel. 2. Mark the gel and nitrocellulose membrane for proper orientation. 3. Soak the nitrocellulose membrane in transfer buffer for 15-20 minutes.
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4. 5.
6. 7. 8.
(b) 1.
2. 3. 4. 5. 6. 7.
8. (c.) 1. 2.
3. 4.
Soak the gel in transfer buffer for 5 minutes. Arrange the sandwich in blotting apparatus in the following order from bottom to top, one filter pad (extra thick), nitrocellulose membrane, gel, one filter pad (extra-thick). Remove trapped air bubbles by rolling a clean glass pipette over the sandwich. Place the upper electrode (cathode) gently on the top of the sandwich and close the transfer cassette. Fill the tank with transfer buffer Attach to electrodes and switch on the assembly by 100 volt for 1 hour or 30 volt for over-night. The resolved bands were transferred from the gel to the nitrocellulose membrane during this period. Detection of proteins by antibodies Dissemble the apparatus and transfer the membrane to a small container or petriplates. Mark on the top right corner of nitrocellulose paper for identification. Add at least 8 ml 3% BSA/ TBS Rock gently for 30 min to 1 hour For washing, pour off BSA solution and rinse briefly with TBS three times for three minutes each. Add primary (1st) antibody (Ab) at appropriate dilution in 0.5% BSA and incubate the membrane by rocking it gently for 2 hours at 25oC For washing, pour of 1st antibody solution and wash twice for 10 min with TBS Add secondary antibody (Ab) at appropriate dilution in 0.5% BSA and incubate the membrane by rocking it gently for 2 hours at 25oC. Wash the secondary antibody (Ab) solution and rinse for 30 min with TBS for three times. Developing nitrocellulose membrane Add developing reagent to the nitrocellulose membrane and incubate the membrane at 37 oC for 30 minutes. The conjugated enzyme covalently linked to secondary Ab react with a BCIP-NBT (5-Bromo 4-Chloro Inolylphosphate- Nitro Blue Tetrazolium) substrate to give a dark purple precipitate. Wash nitrocellulose membrane for 5 minutes in alkaline phosphatase buffer (APB). Stop reaction by rinsing the membrane with 20 mM EDTA in TBS. Dry the blot in filter paper and label the bands.
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pattern of a gene that has not yet been identified, but whose approximate location is known.
Chapter-VI Frequently asked questions on Molecular diagnostics techniques Question
:
What is DNA?
Answer
:
DNA (Deoxyribonucleic acid), the chemical basis of life that complexes with proteins to form the chromosomes. Structurally, DNA is a double helix-two thread like long strands of genetic material spiraled around each other. DNA is a polymer of deoxyribonucleotides composed of base [Adenine (A), Thymine (T), Guanine (G), Cytosine (C)], Sugar and a Phosphate. An A on one strand always pairs with a T on the other through two hydrogen bonds, while a C always pairs with a G through three hydrogen bonds. The two strands are, therefore, complementary to each other. The sequential arrangement of the individual nucleotides is responsible for giving uniqueness to any individual living form be it humans, animals, plants, or microbes.
Question
:
What is Complementary DNA (cDNA)?
Answer
:
DNA sequences generated from the reverse transcription of mRNA sequences. This type of DNA includes exons and untranslated regions at the 5’ and 3’ ends of genes, but does not include intron DNA.
Question
:
What is RNA?
Answer
:
Ribonucleic acid is a single stranded nucleic acid consisting of three of the four bases present in DNA (A, C and G). T is, however, replaced by uracil (U).
Question
:
What is Genetic marker?
Answer
:
A DNA polymorphism that can be easily detected by molecular or biochemical analysis. The marker can be within a gene or in DNA with no known function. Because DNA segments that lie near each other on a chromosome tend to be inherited together, Markers are often used as indirect ways of tracking the inheritance
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Question
:
What is Microarray technology?
Answer
:
A new way of studying how large numbers of genes interact with each other and how a cell’s regulatory networks control number of genes simultaneously. The method uses a robot to precisely apply tiny droplets containing functional DNA to glass slides. Researchers then attach fluorescent labels to mRNA or cDNA from the cell they are studying. The labelled probes are allowed to bind to cDNA strands on the slides. The slides are put into a scanning microscope that can measure the brightness of each fluorescent dot; brightness reveals how much of a specific mRNA is present, an indicator of how active the gene producing that mRNA is.
Question
:
What is Primer?
Answer
:
A short (single strand) oligonucleotide sequence used in a polymerase chain reaction (PCR)
Question
:
What is PCR?
Answer
:
The development of the polymerase chain reaction (PCR) was a technological breakthrough in genome analysis since it enabled the amplification of specific fragments from the total genomic DNA. The principle of PCR is very simple. It is based on the function of a copying enzyme, Taq DNA polymerase, which is able to synthesise a duplicate molecule of DNA from a DNA template which is bracketed by the primer. The product of duplication of the original template DNA becomes a second template for another round of duplication. Repeated duplications thus lead to an exponential increase in DNA product accumulation. Even when starting from a single DNA molecule, detectable amounts of target DNA are generated by PCR in a few hours.
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Question
:
What is DNA fingerprinting?
Answer
:
It is a technique, by which an individual can be identified at molecular level. With the advancement of science and technology, STR analysis has become very popular in forensic laboratories. Scientists have chosen repeating sequences in the DNA, which are present in all individuals on different chromosomes, and are known to vary from individual to individual. These are used as genetic markers to identify the individual.
Question
:
What is the use of DNA fingerprinting technology?
Answer
:
DNA fingerprinting technology has made it possible to identify the source of biological samples/ identification of plant species or cultivar, detection seed purity, adulteration in food and seed and other planting material. This will resolve disputes of maternity / paternity, identification of cultivars or breeding material, forensic wildlife, protection of farmers rights and biodiversity. This remarkable technology provides positive identification with virtually 100% precision.
Question
:
Is there any chance of DNA profile being the same among the individuals related or non-related?
Answer
:
DNA profile of an individual is unique. It can never be identical even in biologically related individuals except for the identical (monozygotic) twins.
Question
:
What are the samples requ ired for DNA fingerprinting examination?
Answer
:
Any biological material such leaf, seed, plant parts in case of plant and a drop of blood, saliva, semen, and any body part such as bones, tissue, skull, teeth, hair with root in case of animal and human beings.
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Chapter-VII Detection of adulteration in Basmati rice employing molecular diagnostics technique Rice is the staple food for more than half of the world’s population. In the evolution of rice and its genetic differentiation into distinct varietal groups, consumer quality preferences have played a significant role besides agro-ecological factors. One such varietal group comprising of aromatic rice of Indian sub-continent is known as ‘Basmati’. It is the highly priced rice in the domestic as well as international markets. Originated in the foothills of the Himalayas Basmati rice is characterized by extra long slender grain, pleasant and distinct aroma and soft and fluffy texture of cooked rice. The grain also possesses a unique shape, which on cooking elongates to almost double its length whilst its width remains the same These unique features of Basmati rice said to be the culmination of centuries of selection and cultivation by farmers, are well preserved and maintained in their purest form in the traditional Basmati (TB) varieties. The historical and archeological findings infer that the varieties with such unique morphological and quality attributes are not present in traditional rice growing areas anywhere in the world. Traditional Basmati rice is not only in great demand in the domestic markets, but is also seen in the menu of connoisseurs worldwide. Basmati rice qualifies as a Geographical Indication (GI Detection and quantification of adulteration of Basmati rice Basmati rice, in addition to the desired quality traits, harbours many undesirable traits that include tall stature, low yield and photosensitivity. Attempts since 1970s to develop high yielding Basmati rice varieties by cross breeding, though, resulted in many high yielding Evolved Basmati (EB) varieties; they fell short of the quality standards of TB varieties. The evolved and non-Basmati rice looking similar to TB varieties in appearance have suited the interests of unscrupulous traders to use them as adulterants in Basmati trade. The practice of Basmati adulteration has would seriously jeopardize our export trade of over Rs.2000 crores. Hence, identifying genuine Basmati variety from other look-alike long grain non-Basmati varieties and Evolved Basmati
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(EB) varieties is important from the viewpoint of sustainable trade and to protect the interests of consumers and rice-trading community in general. Traditionally employed morphological and biochemical assays for detecting adulteration in Basmati rice have not been found to be discriminative enough warranting more precise high-throughput techniques. Traditionally used morphological and chemical parameters have not been found to be discriminative enough to differentiate traditional Basmati and evolved Basmati and non-Basmati rice varieties, warranting more precise techniques. Several molecular techniques are available for detecting genetic differences within and among cultivars. Among these, simple sequence repeat (SSR) markers are efficient and cost-effective and detect a significantly higher degree of polymorphism in rice. They are ideal for assessing genetic purity study.
Protocol
The Basmati Verifiler™ Kit; the world’s first product for establishing the authenticity of Basmati rice samples was developed by a group of scientists from the Centre for DNA Fingerprinting and Diagnostics (CDFD) in Hyderabad, India. The kit is now manufactured and marketed by Labindia on behalf of Centre for DNA Fingerprinting and Diagnostics. The kit uses a PCR amplification technique based on Simple Sequence Repeats (SSR) that provides the single most discriminating assay for Basmati genotyping. The high resolution of the fluorescence-based microsatellite assay provides highly reproducible data with as low as 5 ng of DNA per PCR reaction. The Basmati Verifiler Kit simultaneously amplifies 8 SSR loci in a single, robust amplification reaction. The use of SSR by the Basmati Verifiler Kit is efficient enough to detect genetic diversity within and among various Basmati rice cultivars. This makes the kit highly efficient and cost-effective in detecting high levels of polymorphism in rice. The kit exploits wellcharacterized Basmati rice specific molecular markers which are used as molecular tags for the identification of true Basmati varieties. The Basmati Verifiler Kit employs a robust, eight-locus multiplex PCR, which significantly reduces the preparation time and effort required for amplification and analysis.
PCR amplification: Set up a 10µl PCR reaction comprising of DNA template, PCR mixture (AmpliTaq Gold, dNTPs, MgCl2) along with labeled primers. After an 2 initial denaturation step, the PCR mix is subjected to a fixed number of amplification cycles followed by the extension step.
Each Basmati Verifiler Kit contains a pre-formulated PCR reaction mix, blended primer set along with pure Basmati DNA as a control. The kit provides sufficient reagents for 100 reactions. The PCR reaction components, primer sequences and amplification protocols have all been developed to provide specific, robust amplification. The procedure involves the following basic steps: DNA extraction: Isolate DNA from rice grains using any commercial column-based DNA extraction kit. Each rice sample should have at least 100g of grain powder, from which a minimum of three sub-samples of 1g each should be randomly drawn and bulked for DNA extraction. Quantify the DNA and adjust to an optimum level.
Genotyping: This can be performed on any of the AB Genetic Analyzers using specific running modules. The PCR product is mixed with size standard GeneScan™ ® 500 LIZ before being injected on the instrument. Subsequently, allele sizes are called using the ® GeneMapper software from AB. Kit Components.
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Chapter-VIII Molecular diagnostic techniques in disease management Introduction Accurate identification and early detection of pathogens is a crucial step in disease management and environmental monitoring. The failure to adequately identify and detect plant pathogens using conventional, culture based morphological techniques has led to the development of nucleic acid based molecular approaches. Molecular diagnostic began to develop a real momentum after the introduction of polymerase chain reaction (PCR) in the mid 1980s. To date an increasing number of agricultural research centre is adapting molecular methods for routine detection of pathogens. With the advances in molecular biology and biosystematics, the techniques available have evolved significantly in the last decade, and besides conventional PCR other technologically advanced methodologies such as the real time PCR and microarrays which allows unlimited multiplexing capability have the potential to bring pathogen detection to a new and improved level of efficiency and reliability. Detection specificity and sensitivity Sensiti v ity and speci f icit y are num eric measures of effectiveness of a detection system. Diagnostic specificity is defined as a measure of the degree to which the method is affected by non target components present in a sample, which may result in false positive responses. Diagnostic sensitivity is defined as a measure of the degree to detect the target pathogen in the sample, which may result in false negative responses. Too low sensitivity often leads to false negatives. Thus, a high degree of diagnostic accuracy is characterized by the ability to detect, true and precisely the target micro organism from a sample without interference from non target components. The high degree of sensitivity of molecular methods made pre-symptomatic detection and quantification of pathogens possible. One of the most important advantages that molecular based detection has over conventional diagnostic detection methods is the high specificity. That is the ability to distinguish closely related organisms. The specificity of PCR, be it conventional or real-time,
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depends upon the designing of proper PCR primers that are unique to the target organism. Highly conserved gene regions are often the target for designing primers. Closely related microbial species often differ in a single (single-nucleotide polymorphism (SNPs) to few bases in such genes. PCR allows detection of such with the advancements in high throughput DNA sequencing. More and more genomes of plant pathogens is sequenced and nucleotide sequence data will be available increasing the possibility for designing unique primers and probes for specific detection of pathogens. PCR is a highly sensitive technology. However, its sensitivity is greatly affected by the presence of inhibitors which prevent or reduce amplification. A wide range of inhibitors are reported. Although their mode of action is not clear, these inhibitors are believed to interfere with the polymerase activity for amplification of the target DNA. On the other hand, it is worth mentioning that the high sensitivity of PCR also causes one of the limitations of PCR, that is detection sensitivity exceeding threshold levels or clinical significance and false positive results from slight DNA contamination. Hence, stringent conditions are necessary in conducting the assay and proper negative controls must be included in the test. It is also recommended to have separate dedicated areas for pre- and post PCR handling. Determination of viability Nucleic acid based detection methods currently applied in pathogen detection are based on nucleic acid hybridization or PCR. These methods can be designed to detect either DNA or mRNA. Whereas, DNA based detection method is often more straightforward than that of mRNA, the stability of DNA leads to the possibility that DNA based methods yield positive results from non-viable or dead pathogens. One of the main goals of pathogen detection system, besides determining the presence and absence of the pathogen, is the viability since in the event of positive result it is important to know whether the pathogen detected poses threat to crop production, public health or food safety. The lack of discriminating viable from dead cells is a pitfall common to the nucleic acid based detection systems including microarrays and diagnostic PCR demonstrated that prolonged detection of non viable cells led to potential overestimation in the quantitative real time detection of Escherchia coli. In order to circumvent this
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problem many studies consider enrichment culturing (BIO PCR) instead of direct PCR. While the system allows the detection of only viable cells and helps in elimination of possible PCR inhibitors, it is not appropriate approach for quantitative assay. Therefore, the lack of ability to distinguish between viable and dead cells, and the lack of sample preparation methods that do not involve enrichment culturing are currently limiting the implementation of quantitative PCR for routine diagnostic use. Molecular methods for inferring pathogen viability focus on detecting mRNA in a sample as mRNA species are believed to be labile with a very short half life (seconds to minutes) after cell death. However, although it is in theory a more accurate indicator of viable micro organisms there has been report of poor correlation between the two variables. More work is still needed to verify the question. Pathogen quantification Although new, rapid detection and identification technologies are becoming available for various pathogens, pathogen quantification remains to be one of the main challenges in the disease management of many crops. Quantification of a pathogen upon its detection and identification is an important aspect as it can be used to estimate its potential risk regarding disease development, establishment and spread of inoculum and economic loss. In addition it provides information for well informed disease management decisions. PCR is ideal for detection of small amount of the target but one of its limitations has been quantification. Three PCR variants namely limiting dilution PCR, kinetic PCR and competitive PCR have been used for quantitative analysis of DNA. However, all are based on end point measurements of the amount of DNA produced which makes estimation of initial concentration of DNA and quantification rather problematic. Even the emerging microarray technology has limitations with respect to microbial quantification in complex environmental samples due to the fact that microarray hybridization signals could vary depending on target abundance and hybridization efficiency. In other words, a low abundance target with high genetic similarity to a microarray probe might produce a stronger hybridization signal compared with a higher abundance target that has low similarity to the same microarray probe. Slide to slide variations from a particular probe, same hybridization condition and amount of DNA was also reported leading to a speculation that variation
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from the printing of probes from slide to slide may contribute to such differences. On the other hand, efforts are underway towards adding a quantitative aspect in the array technology. Due to the advancement of fluorogenic chemistry, a second generation PCR known as real time PCR has become an emerging technique for the detection and quantification of micro organisms in the environment. In PCR the target DNA sequence is amplified over a number of denaturation-annealing-extension cycles. In a conventional PCR, only the final concentration of the amplicons may be monitored using a DNA binding fluorescent dye. However, in the quantitative real time PCR, the concentration of the amplicons is monitored throughout the amplification cycles using a group of fluorescent reagents. The fluorescence intensity emitted during this process reflects the amplicons concentration in real time. Undoubtedly most of the future tests will be quantitative in nature and the real time detection system will be a method of choice. The real time data will serve as useful basis for establishing inoculum threshold levels and detailed analysis of disease epidemics. Multiplexing Crops can be infected by numerous pathogens and they may be present in plants in complexes. Therefore, it is desirable to develop technology that can detect multiple pathogens simultaneously. The methodological limitations however, are in many cases the reasons for developing simplex or assays only including few targets. Multiplex PCR, a PCR variant which is designed to amplify multiple targets by using multiple primer sets in the same reaction, has been applied in many tests. Multiplex PCR assays can be tedious and time consuming to establish requiring lengthy optimization processes. Among the drawbacks of such variant PCR assays are that the sensitivity is decreased enormously and the number of different targets to be amplified in one assay is limited. Moreover, the dynamic range of the target present in the sample to be tested is not always reflected in the outcome of the test. That is targets that are present in very low amounts will most of the time not amplified in contrast to those that are present abundantly. The real-time PCR offer better multiplexing possibilities, however, multiplexing is still limited by the availability of dyes emitting
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fluorescence at different wavelengths. Thus, detection of more than few pathogens is currently not possible using these systems. The DNA microarray technology originally designed to study gene expression and generate single nucleotide polymorphism (SNP) profiles, is currently a new and emerging pathogen diagnostic technology which in theory, offers a platform for unlimited multiplexing capability. The principle of microarray is the hybridization of fluorescently labelled sequences or targets to their complementary sequences spotted on solid surface, such as glass slides, serving as probes. Tens of thousands of such DNA probes can be spotted in a defined and addressable configuration on the glass slide forming the chip. The unlimited capability for simultaneous detection of pathogens makes microarrays to be an approach with a potential capacity of detecting all relevant pathogens of a specific crop. Development of microarrays for diagnostic applications is a recent history. In plant pathology the method was applied for identifying oomycete, nematode bacterial and fungal DNA from pure cultures. However, for application in practice, pathogens should be detected from environmental samples (plants, soil, etc.). Recently the possibilities of parallel detection of pathogens from such environments were shown. In contrast with studies using pure cultures, microarray-mediated analysis from environmental samples presents several challenges that must be addressed.
challenges of working on environmental samples where contaminants (humic matter, organic substances, heavy metals etc) may interfere with DNA hybridization and affect the performance of microarrays. Adding innovative molecular tools for differentiating viable from nonviable organisms should be given emphasis in developing diagnostic assays. Conclusion However, while the specificity and sensitivity of detection of pathogens are greatly improved and pathogen detection is becoming simpler and faster, there are still major challenges, technical and economic nature, which need to be addressed to ensure the emergence of reliable detection system for routine applications. In developing a tool for pathogen detection, issues such as detection specificity and sensitivity are very important. In addition multiplexing, quantification and cost effectiveness are increasingly becoming important features of a diagnostic technology. There is also a growing need for a field deployable portable rapid detection system that provides the capability for pathogen testing and identification in the field. These major required features are summarized below.
Prospects Currently more and more research centre and laboratories are using molecular methods for detection and identification of pathogens. The development of more versatile robust and cost effective systems, allowing for greater sensitivity and specificity, elevated throughput and detection of multiple microbes will continue over the coming years. Pathogen detection is only the first step; quantification and isolate characterization are crucial elements in diagnostics. Diagnostic technology is moving from qualitative to quantitative and there is no doubt that most tests will be quantitative in the future. Microarray-based technology is the most suitable technique for multiple pathogen detection in a single assay. Currently microarrays can be expensive for routine application. However, with reducing fabrication costs, the cost per sample will be significantly lower. The effort to add a quantitative aspect to microarrays must continue and more work is needed to address the
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Chapter-IX Molecular diagnostic techniques in seed purity assessment
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Traditionall y, morphological comparisons of seeds and grown plants have formed the basis for genetic purity evaluations and certification. ‘Grow Out Test’ (GOT), based on morphological traits, is carried out for assessment of purity of seeds. A set of morphological descriptors are currently used for varietal identification, description and seed purity assessment. ‘Grow Out Test’ involves growing plants to maturity and assessing several morphological and floral characteristics that distinguish the cultivars/ genotypes. It is time consuming (takes one full growing season for completion), space demanding and often does not allow the unequivocal identification of genotypes. In India, while hybrid rice seed production is generally taken up in the rabi season (January-April), and the hybrid crop is raised in the kharif season (June to October). Hence, there is a need for an assay to assess the genetic purity of hybrid seeds that is both accurate and faster, so that the seed produced in the rabi season can be released for commercial cultivation in the ensuring kharif season, without waiting for the traditional and time consuming, subjective GOT. Though widely adopted and practiced, purity assessments based on morphology is often affected by environment, beside the on time and resources. Morphology cannot provide information on the purity of specific genetic attributes that relate to grain quality or to pest or herbicide resistance bred into varieties. Furthermore, many of the modern high yielding varieties and hybrids are phenotypically less distinct making morphological evaluation more difficult. Newer DNA-based technologies such as restriction fragment length polymorphisms and more recently developed methods that use the polymerase chain reaction can allow even more discriminative and faster identification of varieties. Molecular Characterization Molecular marker has been widely used to assess the genetic purity of seed and its certification. Assessment of purity of seeds and maintenance of genetic purity of parental lines & hybrids is crucial for the successful adoption modern agro-technology. Molecular markers particularly microsatellites have the potential in assessing the genetic purity of seeds. DNA fingerprinting techniques were first reported by Jeffrey et al., (1985). Polymorphism between individuals can be generally detected more efficiently based on DNA markers than morphological or protein based markers.
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Microsatellite markers have been used for genetic characterization of cultivars in wheat, maize, sunflower and tomato. The Biochemical and Molecular Techniques Group of the International Union for the Protection of New Varieties of Plants (UPOV) is evaluating different DNA marker parameters prior to its routine use in establishing distinctness, uniformity and stability (DUS) of plant varieties. W ith increasing number of public as well as private bred rice hybrids under commercial cultivation, quality control in terms of monitoring seed genetic purity at both parental and hybrid seed production stages is vital for the success of hybrid rice technology. Considering the innate disadvantages of GOT for seed purity analysis, marker based seed purity assay which could be an alternative, is receiving the attention. Replacement of GOT with a marker-based assay demands characterization of the parental lines with a large set of hyper polymorphic markers to identify ‘informative’ markers. The utility of these markers in detection of impurities is also clearly demonstrated through cost saving strategies of grow out matrix based bulked sample analysis and multiplex PCR. With respect to hybrid seed production in rice and other selfpollinated crops, monitoring of seed genetic purity is thus imperative in the context of detection of pollen contamination from wild relatives. In a self-pollinated crop like rice, one of the challenges is the production and supply of adequate quantities of pure hybrid seed to the farmers. For breeding material maintenance of high level of genetic purity of hybrid is essential to exploit the moderate level of heterosis observed in this crop. The success of improved variety / hybrid in the farmers field depends upon the availability of seed with high genetic purity, which decides the effect of all other inputs in increasing the productivity. The genetically pure hybrids have recorded a yield advantage of 15 to 20 per cent over semi dwarf high-yielding varieties (HYVs) in farmers fields. DNA Finger Printing For accurate detection of genetic impurities in seed lots, it is essential to identify a set of informative SSR markers which can clearly distinguish the parental lines and amplify specific or unique allele combinations in the hybrids, not present in any other rice line. The fingerprinting of rice hybrids and identification of their genetic relationships are very important for plant improvement, variety registration system, DUS testing, seed genetic purity testing and the Protection of Plant Variety and Breeders Rights. Accordingly, clear-cut identification of elite crop varieties and hybrids it is essential for protection and prevention of unauthorized commercial use. On the other hand, purity of hybrid seeds
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supplied to farmers must surpass 95 per cent. During last ten years, techniques based on DNA markers along with morphological traits have been used to detect variation at DNA level to distinguish closely related genotypes. DNA marker is a new approach based on DNA polymorphism among tested genotypes, and thus applicable to biological research. It offers many advantages over other categories of markers such as morphological, cytological or biochemical markers. DNA marker can cover the whole genome and, therefore, is much larger in quantity. There is more polymorphism in DNA markers, which are able to reveal the variation and allelism. Many DNA markers are co-dominant and can differentiate between the homozygous and heterozygous genotypes. Furthermore, DNA markers are “neutral” and they have no epistatic effect, and are not influenced by environmental conditions and developmental stages. Therefore, DNA marker is simple, quick, less environmentally conditioned, and also experimentally reproducible. It has been applied widely in the identification, registration of plant variety, and in monitoring of the seed purity and the authenticity with high accuracy, high reliability and low cost. DNA fingerprinting approaches based on polymerase chain reaction have become methods of choice for germplasm characterization, diversity studies and seed purity assays. A variety of DNA markers are now available for fingerprinting of cultivars and for marker assisted selection. Of these, SSRs are the preferred ones for rice due to their abundance, co-dominant nature and their distribution throughout the genome and user-friendly nature Fast track DNA finger printing protocol for genetic purity assessment As per Indian Minimum Seed Certification Standard (1988), for assessment of genetic purity, 800 plants (Foundation Seed) and 400 plants (Certified Seed) are observed for the entire crop duration to assess the morphological characters. Of late, DNA finger printing approaches based on PCR (Polymerase Chain Reaction) is being recommended by Government of India for seed genetic purity analysis in the Seed Testing Laboratory. However, assessment of genetic purity through DNA finger printing on individual seed basis is very difficult for 400 or 800 seeds. Therefore a simpler, accurate method involving recommended sample size has to be developed. To address this problem, a Fast track DNA finger printing protocol for rice crop has been designed. It is a simple technique of bulking single leaf bits from 49 seedlings was proposed for testing the genetic purity of certified seed lot of crop varieties. Presence of extra alleles other than the allele ‘specific’ and ‘unique’ to the test variety, will confirm that at least one of the 49 seeds tested is impure. This means that genetic contamination is at the level of > 2 per cent level. As per Indian Minimum
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Seed Certification Standard (1988), this lot can be rejected. Since the Indian Minimum Seed Certification Standard (1988), recommends 400 plants for testing genetic purity, 8 batches of 49 seeds can be tested. The inference will be that “Even if one batch reveals an extra allele the lot can be rejected” since the genetic purity level exceeds the permissible limit of 2 per cent. Similarly, for foundation seeds the lots containing greater than 1 per cent contamination should be rejected, to estimate this 800 seeds have to be tested as per Indian Minimum Seed Certification Standard (1988). Therefore, bulking of single leaf bits from 99 seedlings were analyzed through multiplex PCR involving two markers, in 8 batches. Even if one batch reveals heterozygous amplification, it can be interpreted that the lot contains >1 per cent genetic contamination which can be rejected. By bulking 49 seedlings (8 batches) for certified stage and 99 seedlings (8 batches) for foundation stage is sufficient to identify whether the presence of genetic contamination >1 per cent (foundation stage) and >2 per cent (certified stage) which is the threshold level as per Indian Minimum Seed Certification Standards (1988). Therefore, individual seedling analysis of all 400 seedlings is not required and the PCRs can be restricted to just 8 reactions. Thus bulked method of sample analysis will be of immense help for the seed industry to assess the genetic contamination of the seed lots, through DNA finger printing. The advantages are restriction of PCR samples to 8 numbers which is much user friendly quicker and cheaper than analysis 400 /800 individual seed sample. Future research should concentrate on identifying one or more primers that are capable of discriminating as many varieties as possible in all economically important crops. This technology has been filed for receiving a patent, so that further commercialization could be achieved in the seed certification process. Through use of the DNA based molecular technique, genetic purity in crop varieties must be achieved and maintained for agronomic performance as well as to encourage investment and innovation in plant breeding and to ensure that the improvements in productivity and quality imparted by breeders are delivered to the farmer and, ultimately, to the consumer Conclusion: However, none of the DNA methods have replaced biochemical and morphological methods for seed purity assays, other than in a relatively select group of crops with very high seed value, due to their high data point cost. It will require further miniaturization, automation and enhanced capabilities to process numerous samples simultaneously before newly developed methods supersede biochemical and morphological methods for routine usage in purity testing.
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Chapter-X Molecular marker and transgenic crops Molecular marker technologies and the development of transgenic plants are the two major areas of today’s molecular breeding strategies. When used as a strategy on its own, MAS relies on the primary and secondary gene pool and meiotic chromosome recombination. All genes reside in their natural chromosomal location, thus minimizing the possibility of gene silencing or other epigenetic interactions. In addition, MAS traits are truly allelic. In contrast to MAS, with genetic transformation genes and regulatory elements are removed from their genetic environment and mostly derive from totally unrelated species. The genetic elements can be manipulated and altered before they are newly combined and transferred into a new context to produce a so called genetically modified organism (GMO) or transgenic organism. The introduction of genes from unrelated species is a characteristic attribute of gene transformation and cannot be achieved with MAS. However, only one or a few genes can be transferred by genetic transformation, and neither the number of copies nor the position of the transgenes introduced into the recipient genome can be regulated. Also, there are no different alleles of GMO traits. Marker technology is an indispensable tool when producing GMOs as markers are needed to identify and locate the transgenes within the GMOs. Also, MAS and GM approaches can be combined by using MAS to identify recombinants with transgenic traits. Often, these are disease or pest resistances. However, some of the main agricultural breeding goals like yield, stress resistance or long-term disease protection are quantitative traits that require the combination of several to many genes. The targeted recombination of a multitude of genes can be done efficiently with marker assisted selection. MAS can help in the early detection of desirable recombinants which would go undetected based on the phenotype alone. Thus MAS can accomplish what would be very difficult to achieve using a transgenic approach. Some goals that have been reached through a transgenic approach can also be achieved through MAS. An example is the so called ‘Golden Rice’, a genetically modified rice variety containing high â-carotene levels as an approach to address dietary vitamin A deficiency in the developing world. Through association mapping identified favorable alleles in maize that lead to an increased â-carotene content in maize grains. These alleles were introgressed via MAS into tropical maize germplasm adapted to developing countries. Other examples are the non-GM maize variety ‘Sunrise’ which is – similar to some of only recently authorized GM maize lines – resistant to the coleopteran pest i.e. corn root worm (Diabrotica virgifera). Similarly a non-GM potato variety developed through MAS that
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produces nearly pure amylopectin like the GM potato ‘Amflora’. Thus, to increase the â-carotene content in cereal grains, to alter the starch composition in potato or to confer pest resistance in maize transgenic approaches are no longer mandatory. Same achievement can be achieved through marker assisted breeding. GMOs are facing several legislative constraints due to biosafety concerns and ethic considerations. The introduction of varieties obtained through MAS is not subject to such restrictions. As MAS does not necessarily include genetic engineering the thought that it will not be subject to public distrust as are GMOs. Organizations that criticize genetic engineering seem to accept MAS to a large extent. In several publications the hope is expressed that by means of MAS products of modern biotechnology can be introduced into the market without experiencing the skepticism transgenic crops are facing worldwide. Dot-immuno Binding Assay for detection of transgene Dot blot immune binding assay is an efficient technique for detection of expressed protein from transgene in genetically modified (GM) plant in a short period. The technique is also semi-quantitative. It can be even carried out in the field condition. Principle: In this test, the protein (trans-protein) of the introgressed gene is transferred to a solid support (nitrocellulose membrane). The first antibody (raised against the trans-protein) is allowed to bind to the antigen (transprotein). Then anti-FC antibody coupled with an enzyme is allowed to bind 1st primary antibody. The enzyme produces a colour product on addition of the substrate and this is only possible when the test antibody binds to the solid support. The development of colour indicates the antigen –antibody reaction and the test is positive, Requirements: 1. Phosphate buffer saline (0.15M pH 7.5) NaCl 8.0 g KCl‘ 0.2 g KH2PO4 0.2 g Na2HPO4 2H2O 1.42 g Adjust the pH to 7.5 and make volume upto 1000 ml with double distilled water and autoclave 2. Blocking solution:- PBS + 0.25% BSA 3. Substrate solution:- BCIP/ NBT
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Procedure: 1. Wash the nitrocellulose membrane in sterile distilled water and make the imprint by dot blot apparatus. 2. Aliquots (2 µl) of sample containing required 1 µg of antigen (crude protein extract of genetically modified (GM) or transgenic plants) was spotted on the glossy/ dull surface of nitrocellulose membrane and allow it for drying for 15 min. 3. Treat the nitrocellulose with 2.0% blocking solution for 2 hours. 4. Wash the nitrocellulose blot thrice with PBS and 0.25% BSA for 5 min 5. Incubate the membrane with required dilution of 1st antibody (1:500) at room temperature for 2 hours. 6. Wash the nitrocellulose blot thrice (5 min each) in the PBS + 0.25%. BSA 7. Again incubate the membrane with alkaline phosphatase conjugated secondary antibody (diluted to 1: 1000 in antibody dilution buffer) at room temperature for two hours. 8. Wash the membrane thrice (5 min each) with PBS + 0.25%. BSA 9. Add 2 ml alkaline phosphatase colour development solution BCIP/ NBT (5-Bromo 4-Chloro Inolylphosphate- Nitro Blue Tetrazolium) and incubate for 10 min. 10. Stop the reaction by adding distilled water and by removing the substrate. 11. Record the development of colour. Development of colour indicates the test is positive i.e. the genetically modified (GM) or transgenic plant has successfully synthesized the protein from the transgene. Immunodipstick test Immunodipstick test is a ready to use kit for detection of transgene expression in genetically modified plants in shortest period even under field condition. Principle: The dipstick test was developed for the detection of the protein expressed by the introgressed gene in transgenic plants, These dipsticks were screened of reactions of various expressed proteins for various agronomic traits. In dipstick test the anti-FC antibody is tagged by an enzyme and the antigen is coated on a solid support. The first antibody (raised against desired trans-protein) reacts with expressed protein extracted from the transgenic plant (test plant) and then antigen- antibody complex subsequently
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bind with secondaary antibody coupled with enzyme. The enzyme produces a colour product on addition of the substrate and this is only possible when the test antibody binds to the solid support. The development of colour indicates the antigen –antibody reaction and the test is positive, Requirements: Bt- express kit Procedure 1. Pick up a cotton seed or a piece of leaf tissue 2. Break the seed and collect the white coloured internal embryo matter and transfer it into the vials provided. 3. Add 0.5 ml extraction buffer (provided in the BT-Expressed kit) into the vial containing the broken seed embryo or leaf tissue. 4. Crush the embryo / leaf disk using the pestle provided with the kit. 5. Dip the ‘Cry1 Ac Bt instant check strip’ into the vial. Take care that only the end of the strip marked as ‘sample’ is dipped into the sample. Wait until the sample solution (purple coloured) travels till the top end of the strip and the filter pad at top end is almost completely wet. This should take about 15-30 minutes. 6. If two bands develop, one band develops half way through the strip and another band at the top of the strip, indicate the presence of Cry 1 Ac protein in the sample. It infers that it is a positive Bt cotton sample. 7. If only one band develops at the top of the strip, it indicates that the sample is negative for Cry 1 Ac protein. It infers that it is a non-Bt cotton sample. Precautions 1. Clean the pestle thoroughly. Any minor contamination of the sample with a positive sample can lead to false positive. 2. Keep the strips in airtight pack in refrigerated condition to ensure sustained activity. 3. If unused the strips may loose activity in prolonged storage of 6 months. 4. Do not keep the strip outside the seal pack. In a humid environment the strips will absorb the moisture and have a slow flow rate. 5. Remove the strips from the sample after 20-25 minutes to avoid a possible reverse flow and appearance of false positive. 6. Leaves of Bt plant older than 90 days have less Cry 1 Ac protein, hence the sample line intensity is usually very light. It is recommended that the younger leaves may be used for the test.
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Chapter- XI Molecular markers and plant breeding To improve plant varieties, plant breeders relied on assessment of external and internal phenotypic traits such as plant habits, disease resistances, yield, or quality traits. New, improved varieties were developed by solely selecting plants with desirable phenotypes. Plant breeding techniques became very sophisticated over the years but time demanding too. Developing a new, improved plant variety by means of phenotypic selection can easily exceed 10 years. Only with the advent of molecular markers in the late 1970s, it became possible to select desirable traits more directly. Easily detectable DNA markers can now be used in plant breeding. Marker-assisted selection (MAS) has turned into a tool for the development of improved varieties, allowing for a breeding approach based on the genotype of plants rather than assessing the phenotype only. DNA markers are sections of the genome of the organisms in question which are used for recognition. They can be understood as naturally occurring tags attached to specific segments of a chromosome, which in turn are associated with specific phenotypes. A marker can either be located within the gene of interest or be linked to a gene determining a trait of interest, which is the most common case. Thus MAS can be defined as selection for a trait based on genotype using associated markers rather than the phenotype of the plant. Many agriculturally important traits such as yield, quality and some forms of disease resistance are controlled by many genes and are known as quantitative traits. The regions within genomes that contain genes associated with a particular quantitative trait are known as quantitative trait loci (QTLs). The identification of QTLs based only on conventional phenotypic evaluation is not possible. A major breakthrough in the characterization of quantitative traits that created opportunities to select for QTLs was initiated by the development of DNA (or molecular) markers in the 1980s. One of the main uses of DNA markers in agricultural research has been in the construction of linkage maps for diverse crop species. Linkage maps have been utilised for identifying chromosomal regions that contain genes controlling simple traits (controlled by a single gene) and quantitative traits using QTL. The process of constructing linkage maps and conducting QTL analysis–to identify genomic regions associated with traits–is known as QTL
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mapping (also ‘genetic,’ ‘gene’ or ‘genome’ mapping). DNA markers that are tightly linked to agronomically important genes (called gene ‘tagging’) may be used as molecular tools for marker-assisted selection (MAS) in plant breeding. MAS involves identification of the desired plant using the presence/absence of a marker as a substitute for or to assist in phenotypic selection, in a way which may make it more efficient, effective, reliable and cost-effective compared to the more conventional plant breeding methodology. The use of DNA markers in plant (and animal) breeding has opened a new realm in agriculture called ‘molecular breeding’. DNA markers are widely accepted as potentially valuable tools for crop improvement and play a vital role in enhancing global food production by improving the efficiency of conventional plant breeding programs. Essentially, DNA markers may reveal genetic differences that can be visualised by using a technique called gel electrophoresis and staining with chemicals (ethidium bromide or silver) or detection with radioactive or colourimetric probes. DNA markers are particularly useful if they reveal differences between individuals of the same or different species. These markers are called polymorphic markers, whereas markers that do not discriminate between genotypes are called monomorphic markers. Polymorphic markers may also be described as codominant or dominant. This description is based on whether markers can discriminate between homozygotes and heterozygotes. Codominant markers indicate differences in size whereas dominant markers are either present or absent. Strictly speaking, the different forms of a DNA marker (e.g. different sized bands on gels) are called marker ‘alleles’. Codominant markers may have many different alleles whereas a dominant marker only has two alleles. Construction of linkage maps A linkage map may be thought of as a ‘road map’ of the chromosomes derived from two different parents. Linkage maps indicate the position and relative genetic distances between markers along chromosomes, which is analogous to signs or landmarks along a highway. The most important use for linkage maps is to identify chromosomal locations containing genes and QTLs associated with traits of interest; such maps may then be referred to as ‘QTL’ (or ‘genetic’) maps. ‘QTL mapping’ is based on the principle that genes and markers segregate via chromosome recombination (called crossing-over) during
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meiosis (i.e. sexual reproduction), thus allowing their analysis in the progeny. Molecular marker and marker-assisted selection: Marker-assisted selection (MAS) is a method whereby a phenotype is selected on the basis of presence or absence of a maker on the genotype of the test plant. Selecting plants in a segregating progeny that contain appropriate combinations of genes is a critical component of plant breeding. Moreover, plant breeders typically work with hundreds or even thousands of populations. Marker-assisted selection increases the efficiency and effectiveness in the selection process of the desired plant type in plant breeding as compared to conventional breeding methods. The breeders use specific DNA marker alleles as a diagnostic tool to identify plants carrying the genes or QTLs. The advantages of MAS include: time saving from the substitution of complex field trials (that need to be conducted at particular times of year or at specific locations, or are technically complicated) elimination of unreliable phenotypic evaluation associated with field trials due to environmental effects; selection of genotypes at seedling stage; gene ‘pyramiding’ or combining multiple genes simultaneously; avoid the transfer of undesirable or deleterious genes (‘linkage drag’; this is of particular relevance when the introgression of genes from wild species is involved); selecting for traits with low heritability; testing for specific traits where phenotypic evaluation is not feasible (e.g. quarantine restrictions may prevent exotic pathogens to be used for screening). All markers that are tightly linked to QTLs could be used for MAS. However, due to the cost of utilizing several QTLs, only markers that are tightly linked to no more than three QTLs are typically used. Even selecting for a single QTL via MAS can be beneficial in plant breeding; such a QTL should account for the largest proportion of phenotypic variance for the trait. As phenotypic evaluation may be timeconsuming and/or difficult and therefore using markers may be cheaper and preferable
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Breeding strategies in marker-assisted breeding The breeding strategies for which MAS is used most frequently, are selection of simple traits or QTLs from breeding lines/populations, introgression of genes from breeding lines or wild relatives, MABC, marker-assisted recurrent selection (MARS), and pyramiding of genes. (a) Selection from breeding lines/populations Markers are used for selecting qualitative as well as quantitative traits. MAS can aid selecting for all target alleles that are difficult to assay phenotypically. Especially in early generations, where breeders usually restrict their selection activities to highly heritable traits because a visual selection for complex traits like yield is not possible with only few plants per plot being available, MAS is said to be effective, cost and time-saving. To improve early generation selection, markers should decrease the number of plants retained due to their early generation performance, and at the same time they should ensure a high probability of retaining superior lines. An important prerequisite for successful earlygeneration selection with MAS are large populations and low heritability of the selected traits, as under individual selection, the relative efficiency of MAS is greatest for characters with low heritability. Markers are also frequently used to select parents with desirable genes and gene combinations, and MARS schemes involve several successive generations of crossing individuals based on their genotypes. The achievable genetic gain through MARS is probably higher than that achievable through MABC. (b) Marker-assisted backcrossing (MABC) Backcrossing is used in plant breeding to transfer (introgress) favorable traits from a donor plant into an elite genotype (recurrent parent). In repeated crossings the original cross is backcrossed with the recurrent parent until most of the genes stemming from the donor except the target desirable gene are eliminated. However, the donor segments attached to the target allele can remain relatively large, even after many backcrossing generations. It typically takes 6–8 backcrosses to fully recover the recurrent parent genome. Recurrent Generation parent genome is 75% (for the entire BC1 population), BC2 (87.5%), BC3 (93.8%), BC4 (96.9%), BC5 (98.4%) BC6 (99.2%) so on.
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In order to minimize this linkage drag, marker assays can be of advantage. Markers can be used in the context of MABC to either control the target gene (foreground selection) or to accelerate the reconstruction of the recurrent parent genotype (background selection). In traditional backcross breeding the reconstruction of the recurrent parent genotype requires more than six generations, while this may be reduced to only three generations in MABC. At the moment MABC also is and will probably remain the preferred means of backcrossing transgenes into elite inbred lines, which is also considerably contributing to its popularity. MABC is especially efficient if a single allele is to be transferred into a different genetic background, for example, in order to improve an existing variety for a specific trait. However, if the performance of a plant is determined by a complex genotype it is unlikely that this ideal genotype will be attained through MABC only. To overcome the limitation of only being able to improve existing elite genotypes, other approaches like marker-assisted recurrent selection (MARS) have to be considered. Although the initial cost of marker-assisted backcrossing would be more expensive compared to conventional breeding in the short term, the time savings could lead to economic benefits. This is an important consideration for plant breeders because the accelerated release of an improved variety may trans-late into more rapid profits by the release of new cultivars (c) Marker-assisted recurrent selection (MARS) The improvement of complex traits via phenotypic recurrent selection is generally possible, but the long selection cycles impose restrictions on the practicability of this breeding method. With the use of
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markers, recurrent selection can be accelerated considerably. In continuous nursery programs pre-flowering genotypic information is used for marker assisted selection and controlled pollination. Thus, several selection-cycles are possible within one year, accumulating favorable QTL alleles in the breeding population. Additionally, it is possible today to define an ideal genotype as a pattern of QTLs, all QTLs carrying favorable alleles from various parents. If individuals are crossed based on their molecular marker genotypes, it might be possible to get close to the ideal genotype after several successive generations of crossings. (d) Pyramiding Using MAS, several genes can be combined into a single genotype. Pyramiding is also possible through conventional breeding but phenotypically testing individual plants for all traits can be time-consuming and sometimes very difficult. The most frequent strategy of pyramiding is combining multiple resistance genes. Different resistance genes can be combined in order to develop broad-spectrum resistance to, e.g., diseases and insects. Either qualitative resistance genes can be combined or quantitative resistances controlled by QTLs. In order to pyramid disease or pest resistance genes that have similar phenotypic effects, and for which the matching races are often not available, MAS might even be the only practical method – especially where one gene masks the presence of other genes As the disease is caused by various strains, pyramiding resistance genes seems an intelligent strategy. Thus, MAS offers promising opportunities. What has to be taken into account when applying such strategies in practical breeding is the fact that the pyramiding has to be repeated after each crossing, because the pyramided resistance genes are segregating in the progeny. Marker and essentially derived varieties (EDV) Registered plant varieties are protected against plagiarism. However, protected germplasm is available for the development of new varieties, which is fixed in the concept of “breeder’s exemption” in the convention of the Union for the Protection of New Varieties of Plants (UPOV). Using modern molecular breeding methods such as genetic engineering or marker-assisted backcrossing the breeder’s exemption can be misused by adding only a few new genes to an existing variety or by selecting for lines which are very similar to one of their parents. If plant variety protection is claimed for such a “new” variety, the breeder of the original variety is not compensated for his or her investments. In cultivar registration, molecular markers could offer assistance in the evaluation of crop cultivars for distinctness, uniformity, and stability (DUS). Conclusion An understanding of the basic concepts and methodology of DNA marker development and MAS, including some of the terminology used by
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molecular biologists, will enable plant breeders and researchers working in other relevant disciplines to work together towards a common goal – increasing the efficiency of global food production. MAS will probably never replace phenotypic selection entirely. Especially for disease resistances a final testing of breeding lines is always required, regardless how tight a marker is linked to a gene or QTL. Working with improvement of drought adaptation, it is no doubt that the collection and use of very high quality phenotypic data are critical for the application of MAS. It is “risky to carry out selection solely on the basis of marker effects, without confirming the estimated effects by phenotypic evaluation”.
Wheat (Triticum aestivum L.) Variety
: Sagarika
Parentage
: NP 798 X Kalyansona
Year of release
: 1983
Duration
: 95-110 days
Yield (Average)
: 28 q/ha
Yield (Potential)
: 57 q/ha
Salient characters
: Height- 75-90 cm, stiff straw, semi-compact ear head, white bold grains
Area of adaptability : Suitable for normal as well as late sown condition in costal and interior districts of Odisha under irrigated condition Special features
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: Tolerant to temperature stress and moderately resistant to blight disease
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Variety Parentage Year of release Duration Yield (Average) Yield (Potential) Salient characters
Area of adaptability Special features
: Utkalika : HD 1742 X FA0 106/68R : Proposed in1983 : 95-110 days : 27 q/ha : 55 q/ha : Height- 80-100 cm, (single dwarf) compact tillers, stiff straw, compact ear head with high spikelet density, medium bold white grains : Suitable for normal sowing condition in costal and interior districts of Odisha under irrigated condition : Tolerant to temperature stress and moderately resistant to blight disease
Variety Parentage Year of release Duration Yield (Average) Yield (Potential) Salient characters
: : : : : : :
SARALA (B-12-4) A MUTANT SELECTION FROM T. 9 VARIETY 1980 SUMMER 70-75; KHARIF 80-90 DAYS. 8.5 q/ha 12.0q/ha Short plant height, photo-insensitive, compact in habit with large number of branches borne on shot inter nodes. Leaf lanceolate and dark green seeds, light black in colour, protein content-24% Area of adaptability : Suitable for entire Odisha during kharif and summer season. Special features : Resistant to yellow mosaic virus and powdery mildew.
BLACK GRAM (URD BEAN) (Vigna mungo (L.) Hepper) Variety
: Ujala (OBG-17)
Parentage
: Mutant of B 3-8-8
Year of release
: 2006
Duration
: Early (65-70 days)
Yield (Average)
: 7.65 q/ha
Yield (Potential)
: 9.00 q/ha
Salient characters : Light purple stem base, Non-hairy oblong black pod, Medium size attractive Brownish black colour seed with prominent white hilum and Early maturity Area of adaptability : Kharif, Pre-rabi and rabi seasons in Coastal Orissa & Kharif and Pre-rabi in interior districts of the State Special features
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: Resistant to Yellow mosaic virus (YMV) and CLS, Moderately resistant to, LC & LCV and powdery mildew. Moderately resistant to Pod borer, White fly and Jassids.
Variety Parentage Duration Yield (Average) Yield (Potential) Salient characters
: : : : : :
T-9 Pure line selection SUMMER 75-80; KHARIF 85-95 DAYS. 8 q/ha 10 q/ha Erect, dwarf, stem purple, 35 to 40 cm in height hastate leaves. Flower characters Yellow, Pod characters, Black with nil to light pubescence 4.5 cm long, turns black on ripening. Seed are black colour with prominent hilum with green tinge. Area of adaptability : Suitable for entire Odisha during kharif and summer season Special features : Can be grown both during kharif and summer season
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SESAMUM (Sesamum indicum)
Variety
: Vinayak
Parentage
: A pure line selection from local collection
Year of release
: 1972
Duration
: 85-90 days(Kharif) 80-85(Rabi/Summer)
Yield (Average)
: 5.0q/ha
Yield (Potential)
: 9.0q/ha
Salient characters : Less compact branching, capsules glabrous, seed colour light brown, oil content 48.5% Area of adaptability
: Suitable for entire Odisha during kharif/ summer season.
Special features
: Moderately resistant to major disease and pests.
Variety
: Kanak
Parentage
: Vinayak XT4
Year of release
: 1979
Duration
: 85-90 days(Kharif) 80-85(Rabi/Summer)
Yield (Average)
: 6.0q/ha
Yield (Potential)
: 10.0q/ha
Salient characters : Less compact branching, capsules pubescent, seed coat colour grey, bold seeds, oil content 47%
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Area of adaptability
: Suitable for entire Odisha during kharif and summer season.
Special features
: Moderately resistant to cercospora leaf spot.
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Variety
: Kalika
Parentage Year of release Duration
: Mutant of Vinayak : 1980 : 90-95 days(Kharif) 80-85(Rabi/Summer) : 7.0q/ha : 12.0q/ha
Yield (Average) Yield (Potential) Salient characters
: Compact branching, capsules glabrous, seed coat colour brown, oil content 48.7% Area of adaptability : Suitable for entire Odisha during kharif and summer season. Special features
: Less susceptible to cercospora leaf spot, stem rot and root rot
Variety
: Uma (KA-5)
Parentage Year of release Duration
: Mutant of Kanak : 1980 : 75-80 days(Kharif) 65-70 (Rabi/Summer) Yield (Average) : 8.0q/ha Yield (Potential) : 14.0q/ha Salient characters : Medium height, moderately branched capsules bold, short, hairy, compact, on-shattering, synchronous in maturity, seeds small with thin seed coat, seed colour pale whitish brown, oil content 53.1% Area of adaptability : Suitable for entire Odisha during kharif and rabi/summer sowing, performs well in late sowing condition in all the zones of Odisha. Special features : Resistant to leaf curl, phyllody and moderately resistant to blight, stem rot, powdery mildew, cercospora leaf spot. Variety was notified by CVRC for zone-II (U.P. Bihar, Assam, Tripura, M.P. West Bengal, Odisha and N.E. States)
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Variety Parentage Year of release Duration
: Usha (KA-7) : Mutant of Kanak : 1990 : 85-90 days(Kharif) 75-80(Rabi/Summer) Yield (Average) : 10.0q/ha Yield (Potential) : 15.0q/ha Salient characters : Medium height, moderately branched capsules compactly arranged, bold and hairy, seeds bold, shining biscuit coloured, oil content 49% Area of : Suitable for entire Odisha during kharif/ summer season adaptability Special features : Resistant to cercospora leaf spot and blight, moderately resistant to leaf curl, phyllody, powdery mildew. Toria (Brassica campestris L.) Variety Parentage
: :
Year of release Duration Oil content (%) Yield (Average) Yield (Potential) Salient characters
: :
Area of adaptability
:
Special features
:
: : :
Parbati (ORT-2-4 ) BT4 selection from 100KGR (GAMMA RAY) 2001 Early maturing type (70-75 DAYS) 42 13.8 q/ha 16 q/ha Uniform plant height; no. of primary branches exceeds the secondary ones; siliquae is medium-long, slender and round bilocular; no. of seeds is twenty per siliquae. Seeds are medium bold, flat and brownish red in colour; main stem height is 50 cm; average no. of siliquae in main stem is forty seven. Leaf colour is dark green. White bold grains This variety can be grown in upland and medium land situation of coastal Orissa, sowing dates being last week of September to 15th October. Tolerant to temperature stress
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Variety Parentage Year of release Duration Oil content (%) Yield (Average) Yield (Potential) Salient characters
: : : :
Area of adaptability
:
87
: : :
Anuradha (ORT-6-2) TS- 29, selection from 80 KR (GAMMA R AY) 2002 Early maturing type (70-75 DAYS) 44 14.60 q/ha 16.5 q/ha plant height:67- 85cm; uniform plant height; no. of second ary branches exceeds; primary branches; siliqua is moderately thicker and long flat and round and biocular; no. of seeds are 15-18 siliqua; seeds are medium bold and smooth slightly flat and browinish red in colour; main stem height is 45-48 cm. av. no. of siliqua in main stem is 45.; basal leaves are stalk ed broad, less hairy round terminal lobe entire, white mid rib and light green in colour; flower colour is deep yellow with bigger petals. Suitable for normal sowing condition in costal and interior districts of Odisha under irrigated condition. This variety can be grown profitably by last week of September to 15th October.
88
TOMATO (Lycopersicon esculentum) Variety Parentage Year of release Duration Yield (Average) Yield (Potential) Salient characters
BRINJAL (Solanum melongena) Utkal Tarini : Parentage : PUSA Kranti X Gopa local Year of release : 1992 Duration : 90-95 days(Up to first harvest) Yield (Average) : 340.0q/ha Yield (Potential) : 809.0q/ha Salient characters : Resistant to bacterial wilt, plant stature 90-100 cm, fruit weight 120-150 gm, deep purple, solitary fruiting habit but sometimes two fruits per cluster, less seeded. Area of : It can be grown in the entire Odisha state in all types adaptability soil prefarabely sandy loam. It is suitable for kharif and rabi season. Any other relevant : It performs well in rabi season, popular in states of information Odisha.
: Utkal Dipti : Punjab chhuhara X AC-142 XBWRS-1 : 1992 : 85-90 days (Up to first harvest) : 412.0q/ha : 704.0q/ha : Resistant to bacterial and nematode wilt, less susceptible to fruit borer,determinate growth habit, slightly curved leaves, plant height 56-60 cm, fruit size is medium small, round shaped fruit with a nipple at basal end, average fruit weight 35-45 gm, less seeded, deep red, pulpy with good keeping quality. Area of : Suitable for kharif and late rabi season. It performs adaptability well in all types of soil preferably in sandy loam soil, can be grown in saline soil. Any other relevant : It is a very popular variety in the states of Odisha information and Andhra Pradesh. It requires more irrigation in comparison other varieties for better performance.
Variety
89
Variety Parentage Year of release Duration Yield (Average) Yield (Potential) Salient characters
: Utkal Pallavi : Punjab chhuhara X AC-142 XBWRS-1 : 1992 : 90-95 days(Up to first harvest) : 370.0 to 490.0q/ha : 611.0q/ha : Resistant to bacterial and nematode wilt, determinate growth habit, slightly curved leaves, plant height 60-65 cm, fruit size is medium to small, average fruit weight 40-50 gm, less seeded, deep red, pulpy with good keeping quality. Area of adaptability : Suitable for kharif and late rabi season. It performs well in all types of soil prefabely in sandy loam soil, can be grown in saline soil. Any other relevant : It is a very popular variety in the states of Odisha and other states information of country where bacterial wilt is a probleam. It requires more irrigation in comparison other varieties for better performance.
90
GINGER (Zingiber officinale) : Suprabha : A selection from kunduli local : 1988 : 230 : 165.0q/ha : 230.0q/ha : Rhizome plumpy with flattened fingers, whitish yellow flesh, finger nodes covered with light brown scales leaves bright glazy skin, • Oleoresin - 8.9 %, • Essential Oil - 1.9 %, • Dry Recovery 20.5 % • Crude fibre - 4.4 % Area of : Suitable for rainfed and irrigated areas of Koraput, Kalahandi, adaptability Phulbani, Ganjam and Dhenkanal districts of Odisha. Any other relevant : Less susceptible to soft rot, leaf spot diseases and scale information insect. Variety Parentage Year of release Duration Yield (Average) Yield (Potential) Salient characters
Variety Parentage Year of release Duration Yield (Average) Yield (Potential) Salient characters
: Suravi : Rudrapur local mutant. : 1992 : 225 : 180.0q/ha : 265.0q/ha : Tall pseudo shoots with deep green foliase plumpy rhizomes of dark glazy skin and dark yellow flesh, finger nodes covered with deep brown scales, • Oleoresin - 10.2 % • Essential Oil - 2.1 %, • Dry Recovery - 23 %, Crude fibre - 4 % Area of : Suitable for rainfed and irrigated areas of Koraput, Kalahandi, adaptability Phulbani, Keonjhar and Mayurbhanja districts of Odisha. Any other relevant : Less susceptible to soft rot, leaf spot diseases and scale information insect.
91
92
Variety Parentage Year of release Duration Yield (Average) Yield (Potential) Salient characters
: Suruchi : A selection from kunduli local : 1991 : 218 : 136.0q/ha : 230.0q/ha : Profuse tillering, rhizome, slender cylindrical and noddy finger with round tip, greenish yellow flesh finger nodes prominent and covered with reddish brown scale leaves, reddish bright skin • Oleoresin - 10.0 % • Essential Oil - 2.0 %, • Dry Recovery - 23.5 %, • Crude fibre - 3.8 % Area of : Suitable for rainfed and irrigated condition, and perform adaptability well in late sown condition. Any other relevant : Less susceptible to soft rot, leaf spot . information
93
CHILLI (Capsicum annuum) Variety : Utkal Rashmi Parentage : Pusa Jwala X BCG-4 Year of release : 1993 Duration : 125-135 days(Up to first harvest) Yield (Average) : 21.83q/ha (as dry chilli) Yield (Potential) : 26.48q/ha (as dry chilli) Salient characters : Plant type is medium, plant height 75-80 cm, downward fruiting, fruit lenth 8-10 cm, highly pungent, medium seediness, deep red colour at ripening, tolerant to fruit borer and less susceptible to thrips and mites suitable as dry chilli. Area of adaptability : Suitable for late kharif and rabi season. It performs well in sandy loam soil, can be grown in all zones in Odisha. Any other relevant : It retains it’s red colour and pungency after drying. information
94
Cashew (Anacardium occidentale) Variety
: Bhubaneswar-1
Nut wt (g)
: 4.6 g
Nut Yield (Average)
: 1.58 q/ha
Shelling (%)
: 30
Special features
It is a selection of WBDC-5 (Vengurla 36/3),plant height 2.6 m, trunk girth 30.76, canopy spreading 2.33 in East - West and 3.33 in North – South direction, Grown in coastal area.
Variety
: Jagannath
Nut wt (g)
: 8.6 g
Yield (Average)
: 3.30 q/ha
Shelling (%)
: 35
Special features
It is a hybrid from Bhubaneswar cluster -2 x VTH 711/4, plant height 2.42, trunk girth 31.84, canopy spreading 3.49 in East - West and 3.65 in North – South direction, Grown in coastal area.
Variety
: Balabhadra
Nut wt (g)
: 7.4 g
Yield (Average)
: 3.69 q/ha
Shelling (%)
: 33.0
Special features
It is a hybrid from Bhubaneswar cluster -1 x H 2/16, plant height 2.50, trunk girth 31.13, canopy spreading 3.24 in East - West and 3.23 in North – South direction, Grown in coastal area.
95
96
weighting 15-18 kg each with uniform long finger throughout he bunch. It has about 6-7 hands with bold, stout fruits, turning golden yellow on ripening. Plants take approximately 13-15 months to complete their life cycle. Hard lumps and fruit cracking are the major physiological disorders.
Banana (Musa sp)
Champa
: It is a cultivar in Odisha. It is distinguished from other cultivar by its pink pigmentation on the ventral side of the midrib of leaf when young. It bears bunches weighing 16-20 kg each with closely packed short and stout fruits having a conspicuous beak. The fruit or bunch varies from 150 to 300. Though the fruits are slightly acidic and crop duration is 16-17 months, its ease of cultivation and hardiness make Champa a popular cultivar. However, it is severely affected by banana streak virus.
Morphological characteristics of OUAT Released Rice Varieties Used for SSR Primers.
Important characteristics of Musa species Robusta
Grand Naine
Amrithapani
97
: Semi-tall variety, sport of dwarf Cavendish and is an important cultivar Odisha. The plant bears bunches weighting 25-30 kg each with good sized slightly curved fruits. Plants take approximately e year to complete their life cycle. It is highly susceptible to Sigatoka leaf spot disease limiting its cultivation in humid areas. But it is resistant to Panama wilt. : Tall variety, It is a mutant of dwarf Cavendish. It is a popular cultivar in Odisha. The plant bears bunches weighting 25-30 kg each with uniform long finger throughout he bunch. Plants take approximately a year to complete their life cycle. It is resistant to Panama wilt. : Plant is medium statured. Consumers prefer this variety because the fruit is tasty, crisp, good sour sweet blended taste and pleasant flavour. The plant bears bunches
Sl. No
Variety
Parentage
1
Keshari ( OR 63-252 )
Kumar / Jagannath
2
Meher (ORS 26-20008-4) Birupa (OR 253-2) Mahalaxmi (OR 621-6) Manik ( OR 624-46 )
6
SR-26-B
7
Jajati (OR 47-2)
8
Badami (OR 164-5) Samanta (OR 487-30-3)
3 4 5
9
10 11
Santep Heap ( OR 142 -99 ) IR-64
Maturity Duration (days) 90-95
Yield Q/ha.
Important Features
32
OBS677 / IR2071// Vikram/ w1263 ADT 27/ IRB// Annapurna Pankaj / Mahsuri
140
45
135
42
150
45
CR 210-1010 /Obs 677
155-160
45
Selection from kalambanka Rajeswari / T141
150
20
Semi-dwarf, photo –insensitive, deep green short narrow erect leaves, profuse tillering ability. Medium bold, Multiple resistance. Medium bold, Multiple resistance. High grain Number, suitable for shallow low land Resistant to sheath blight, moderately resistant to blast, sheath rot, BLB and BPH. Long bold , saline tolerant.
135
40
Suphala / Annapurna T90/IR8/ Vikram/Sanja29/ Mahsuri Pankaj / Sigadis
100
35
140
44
150-155
45
Introduction
120
40
Medium slender, intermediate height, good grain quality matures 10 days earlier in summer, adopts well under low and high fertility condition. Medium bold, resistant to BLB and Blast, Red kernel. Medium bold, Multiple resistance. Resistant to BLB, sheath rot, sheath blight and gall midge. Medium slender, Multiple resistance.
98
12
CR-1017
13
Pratikshya ( ORS 201-5 )
14 15 16 17
Prachi (OR-888-430) Khandgiri(OR 8112) Rajeswari ( OR 10-193 ) Lalitgiri
Swarna / IR 64
155
48
142
48
IR-9764-45-22/CR149-3244-198 Parijat / IR1342994 T 90/ IRS
153
42.7
95
35
130-135
40
Badami / IR-19661364 Pankaj/ W 1263
95
32
150
35
40
18
Rambha ( OR 143-7 )
19
Lalat (OR 26-2014-4)
OBSM 677/ IR2071/ Vikram/W1263`
125
20
Kanchan (OR 609-15)
Jajati / Mahsuri
155
40
21
Nilgiri (OR 163-104 ) Urbashi (OR 645-18 ) Surendra ( OR -447-20-P )
Suphala / DZ 192
95
35
Kalinga -3 ( CR 237- -1 ) Mandakini
22 23
24 25 26 27
Gouri (OR 148-1-4 ) Annada (CR 222 MW 10 )
Rajeshwari / Jajati
145
40
OR 158-5/ Rasi
130-137
47.1
AC540 / Ratna
75
30
Ghanteswari / IR 27069 Rajeshwari / Vikram MTU 15/Yai kyaku kantoku
100-105
45.80
135
43
110
35
135
45
28
Uphar
29
Indrabati ( OR-1128- 7- 51 )
IR-56 / OR-142-99
152
40.7
30
Ghanteswari (OR 377-85-6)
IR 2061-628/ M22
95
35
31
Jogesh (OR-1519-2)
CR 544-1-3-4 / NDR 1008
89-90
30-40
99
Medium bold, suitable for shallow low land. Plant height semi-dwarf, long panicles, low tillering, stount stem, high grain number, MS grains, golden coloured hull with white kernel. Medium bold grains, Brown spotR, BPH,WBPH-MR. Medium slender, Suitable for gall midge and BPH endemic areas. Semi –dwarf, photo-insensitive, profuse tillering. Resistance of BLB, BLS, GM, BPH. Medium bold, suitable for shallow and semi deep low lands. Medium slender, Multiple resistance.
Medium slender, tall, suitable for shallow and semi deep low lands. Medium bold , Moderately drought tolerant. Medium bold, Tall, suitable for shallow low lands. Medium bold grains, suitable for rainfed a irrigated medium lands, Resistant to blast , sh. rot, GM, BPH WBPH. Long slender, Tall suitable for drought prone area. Adaptable to upland, Blast, Sheath blight. Medium slender, multiple resistance and good grain. Short bold, Moderately drought tolerant, suitable for rainfed upland and medium land. Medium bold, Multiple resistance. Medium slender grains, leaf blast-R, Brown spot-R, sh.rot-R, BPH,WBPH-MR. Medium bold, Resistant to BLB ,Blast, Gall midge and moderately tolerant for drought, Red kernel. Rainfed , irrigated uplands , resistant to BS , neck blast, mod. rest. to blast and Sh.B .
32
Manaswini
Swarna/ Lalat
140
49-75
33
P arijat ( OR 34-16 )
TN 1/ TKM6
90-95
35
34
B asumati -7
35
P ratap ( OR 131-3-1 )
Kumar / CR57-49
135-140
40
36
K alajeera
37
P athara (OR 85-23)
38
150-160
95
35
S idhant ( OR 102-4 )
95
35-45
39
MTV 1010
110
35-45
40
Mahanandi (OR-1301-13 ) B hubana (OR 447-20 ) U adaygiri (OR 752-38-1)
IR -19661-131-1-31-3/ Savitri OR 158-5/ Rasi
150
44.21
135
42
IR AT138/IR 1354366
90-95
35
43
R anidhan ( C R 260-30 )
CR 151-179/ CR 1014
180
40
44
IR 36/ / Hema / Vikram IR -13429-196-1120
140
40
130
44
46
B hanja ( OR 443 -80-4 ) G ajapati (OR 136-3) P umpudibasa-55
47
N eelabati -51
48
S uphala (OR 45-61-23/ IET 3274)
41 42
45
Co 18/ Hems.
20-25 20-28 T 141/ T (N)
90-95
35
R esistant to brown spot, blast, S heath blight, W BPH and BPH. Medium slender grains, semi dwarf, photo-insensitive, deep green leaves. Grain wt. 10.3%, white grain with white kernel,let aromatic variety. S emi dwarf leaves, medium bold grains with translucent white kernel. W ell adopted rice variety in orrisa, let variety, black colour grain with husk, fragrance like jeera. Medium bold , Moderately drought tolerant, suitable for upland and low land. Intermediate height, golden hull, tolerant of RTV and leaf folder. A dopted to rainfed and i rrigated uplands. E arly variety, grains medium slender, husk straw coloured, moderately resistant to stem borer, BPH . Medium bold grains, Resistant to blast, brown spot, sh.rot. Medium bold, Multiple resistance. S uitable for renfed and i rrigated uplands of Moderate tolerant to drought Resistant to leaf blash brown spot, stem borer. Medium slender ,Tall , suitable for semi deep low land, tolerant flood. Medium bold, Multiple resistance. R esistance of BLB
