Genetic markers can be used to track genes and chromosomes during genetic analysis. There are four main types of genetic markers: morphological, biochemical, cytological, and DNA markers. DNA markers are now widely used as they are not influenced by the environment and show high levels of polymorphism. Common types of DNA markers include restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPDs), microsatellites, and single nucleotide polymorphisms (SNPs). DNA markers have advantages such as being easy to detect, exhibiting simple inheritance patterns, and showing minimal environmental influences. They have become powerful tools for applications like genetic mapping, diversity analysis, and gene tagging.
Genomics and its application in crop improvementKhemlata20
meaning ,definition of genome ,genomics ,tools of genomics ,what is genome sequencing ,methods of genome sequencingand genome mapping ,advantage of genomics over traditional breeding program, examples of some crops whose genome has been sequenced, important points about genomics, work in the field of genomics ,applications of genomics .classification of genomics .different Omics in genomics like Proteomics ,Transcriptomics ,Metabolomics ,Need of genome sequencing
Genomics and its application in crop improvementKhemlata20
meaning ,definition of genome ,genomics ,tools of genomics ,what is genome sequencing ,methods of genome sequencingand genome mapping ,advantage of genomics over traditional breeding program, examples of some crops whose genome has been sequenced, important points about genomics, work in the field of genomics ,applications of genomics .classification of genomics .different Omics in genomics like Proteomics ,Transcriptomics ,Metabolomics ,Need of genome sequencing
Introduction
History
Genetic mapping
DNA Markers
Physical mapping
Importance
Drawback
Conclusion
References
uses genetic techniques to construct maps showing the positions of genes and other sequence features on a genome.
Genetic techniques include cross-breeding experiments or, in the case of humans, the examination of family histories (pedigrees).
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
Introduction
Transcriptome analysis
Goal of functional genomics
Why we need functional genomics
Technique
1. At DNA level
2.At RNA level
3. At protein level
4. loss of function
5. functional genomic and bioinformatics
Application
Latest research and reviews
Websites of functional genomics
Conclusions
Reference
A new era of genomics for plant science research has opened due the complete genome sequencing projects of Arabidopsis thaliana and rice. The sequence information available in public database has highlighted the need to develop genome scale reverse genetic strategies for functional analysis (Till et al., 2003). As most of the phenotypes are obscure, the forward genetics can hardly meet the demand of a high throughput and large-scale survey of gene functions. Targeting Induced Local Lesions in Genome TILLING is a general reverse genetic technique that combines chemical mutagenesis with PCR based screening to identity point mutations in regions of interest (McCallum et al., 2000). This strategy works with a mismatch-specific endonuclease to detect induced or natural DNA polymorphisms in genes of interest. A newly developed general reverse genetic strategy helps to locate an allelic series of induced point mutations in genes of interest. It allows the rapid and inexpensive detection of induced point mutations in populations of physically or chemically mutagenized individuals. To create an induced population with the use of physical/chemical mutagens is the first prerequisite for TILLING approach. Most of the plant species are compatible with this technique due to their self-fertilized nature and the seeds produced by these plants can be stored for long periods of time (Borevitz et al., 2003). The seeds are treated with mutagens and raised to harvest M1 plants, which are consequently, self-fertilized to raise the M2 population. DNA extracted from M2 plants is used in mutational screening (Colbert et al., 2001). To avoid mixing of the same mutation only one M2 plant from each M1 is used for DNA extraction (Till et al., 2007). The M3 seeds produce by selfing the M2 progeny can be well preserved for long term storage. Ethyl methane sulfonate (EMS) has been extensively used as a chemical mutagen in TILLING studies in plants to generate mutant populations, although other mutagens can be effective. EMS produces transitional mutations (G/C, A/T) by alkylating G residues which pairs with T instead of the conservative base pairing with C (Nagy et al., 2003). It is a constructive approach for users to attempt a range of chemical mutagens to assess the lethality and sterility on germinal tissue before creating large mutant populations.
Vector mediated gene transfer methods for transgenesis in Plants.Akshay More
Presentation include Vector mediated gene transfer methods for trans-genesis in Plants. Only Vector-based methods are covered. Vectors includes Bacteria, Viruses, transposable genetic elements. Other possible vectors for transgenesis are also covered.
Molecular Marker and It's ApplicationsSuresh Antre
Molecular (DNA) markers are segments of DNA that can be detected through specific laboratory techniques. With the advent of marker-assisted selection (MAS), a new breeding tool is now available to make more accurate and useful selections in breeding populations.
The different types of external stresses that influence the plant growth and development.
These stresses are grouped based on their characters
Biotic
Abiotic
Almost all the stresses, either directly or indirectly, lead to the production of reactive oxygen species (ROS) that create oxidative stress in plants.
This damages the cellular constituents of plants which are associated with a reduction in plant yield.
Molecular markers for measuring genetic diversity Zohaib HUSSAIN
Molecular markers for measuring genetic diversity
Introduction:
The molecular basis of the essential biological phenomena in plants is crucial for the effective conservation, management, and efficient utilization of plant genetic resources (PGR).
Determining genetic diversity can be based on morphological, biochemical, and molecular types of information. However, molecular markers have advantages over other kinds, where they show genetic differences on a more detailed level without interferences from environmental factors, and where they involve techniques that provide fast results detailing genetic diversity
Comparison of different methods
Morphological characterization does not require expensive technology but large tracts of land are often required for these experiments, making it possibly more expensive than molecular assessment. These traits are often susceptible to phenotypic plasticity; conversely, this allows assessment of diversity in the presence of environmental variation.
Biochemical analysis is based on the separation of proteins into specific banding patterns. It is a fast method which requires only small amounts of biological material. However, only a limited number of enzymes are available and thus, the resolution of diversity is limited.
Molecular analyses comprise a large variety of DNA molecular markers, which can be employed for analysis of variation. Different markers have different genetic qualities (they can be dominant or co-dominant, can amplify anonymous or characterized loci, can contain expressed or non-expressed sequences, etc.).
Genetic marker
The concept of genetic markers is not a new one; in the nineteenth century, Gregor Mendel employed phenotype-based genetic markers in his experiments. Later, phenotype-based genetic markers for Drosophila melanogaster led to the founding of the theory of genetic linkage. A genetic marker is an easily identifiable piece of genetic material, usually DNA that can be used in the laboratory to tell apart cells, individuals, populations, or species. The use of genetic markers begins with extracting proteins or chemicals (for biochemical markers) or DNA (for molecular markers) from tissues of the plant (for example, seeds, foliage, pollen, sometimes woody tissues).
Molecular markers In genetics, a molecular marker (identified as genetic marker) is a fragment of DNA that is associated with a certain location within the genome. Molecular markers which detect variation at the DNA level such as nucleotide changes: deletion, duplication, inversion and/or insertion. Markers can exhibit two modes of inheritance, i.e. dominant/recessive or co-dominant. If the genetic pattern of homozygotes can be distinguished from that of heterozygotes, then a marker is said to be co-dominant. Generally co-dominant markers are more informative than the
Introduction
History
Genetic mapping
DNA Markers
Physical mapping
Importance
Drawback
Conclusion
References
uses genetic techniques to construct maps showing the positions of genes and other sequence features on a genome.
Genetic techniques include cross-breeding experiments or, in the case of humans, the examination of family histories (pedigrees).
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
Introduction
Transcriptome analysis
Goal of functional genomics
Why we need functional genomics
Technique
1. At DNA level
2.At RNA level
3. At protein level
4. loss of function
5. functional genomic and bioinformatics
Application
Latest research and reviews
Websites of functional genomics
Conclusions
Reference
A new era of genomics for plant science research has opened due the complete genome sequencing projects of Arabidopsis thaliana and rice. The sequence information available in public database has highlighted the need to develop genome scale reverse genetic strategies for functional analysis (Till et al., 2003). As most of the phenotypes are obscure, the forward genetics can hardly meet the demand of a high throughput and large-scale survey of gene functions. Targeting Induced Local Lesions in Genome TILLING is a general reverse genetic technique that combines chemical mutagenesis with PCR based screening to identity point mutations in regions of interest (McCallum et al., 2000). This strategy works with a mismatch-specific endonuclease to detect induced or natural DNA polymorphisms in genes of interest. A newly developed general reverse genetic strategy helps to locate an allelic series of induced point mutations in genes of interest. It allows the rapid and inexpensive detection of induced point mutations in populations of physically or chemically mutagenized individuals. To create an induced population with the use of physical/chemical mutagens is the first prerequisite for TILLING approach. Most of the plant species are compatible with this technique due to their self-fertilized nature and the seeds produced by these plants can be stored for long periods of time (Borevitz et al., 2003). The seeds are treated with mutagens and raised to harvest M1 plants, which are consequently, self-fertilized to raise the M2 population. DNA extracted from M2 plants is used in mutational screening (Colbert et al., 2001). To avoid mixing of the same mutation only one M2 plant from each M1 is used for DNA extraction (Till et al., 2007). The M3 seeds produce by selfing the M2 progeny can be well preserved for long term storage. Ethyl methane sulfonate (EMS) has been extensively used as a chemical mutagen in TILLING studies in plants to generate mutant populations, although other mutagens can be effective. EMS produces transitional mutations (G/C, A/T) by alkylating G residues which pairs with T instead of the conservative base pairing with C (Nagy et al., 2003). It is a constructive approach for users to attempt a range of chemical mutagens to assess the lethality and sterility on germinal tissue before creating large mutant populations.
Vector mediated gene transfer methods for transgenesis in Plants.Akshay More
Presentation include Vector mediated gene transfer methods for trans-genesis in Plants. Only Vector-based methods are covered. Vectors includes Bacteria, Viruses, transposable genetic elements. Other possible vectors for transgenesis are also covered.
Molecular Marker and It's ApplicationsSuresh Antre
Molecular (DNA) markers are segments of DNA that can be detected through specific laboratory techniques. With the advent of marker-assisted selection (MAS), a new breeding tool is now available to make more accurate and useful selections in breeding populations.
The different types of external stresses that influence the plant growth and development.
These stresses are grouped based on their characters
Biotic
Abiotic
Almost all the stresses, either directly or indirectly, lead to the production of reactive oxygen species (ROS) that create oxidative stress in plants.
This damages the cellular constituents of plants which are associated with a reduction in plant yield.
Molecular markers for measuring genetic diversity Zohaib HUSSAIN
Molecular markers for measuring genetic diversity
Introduction:
The molecular basis of the essential biological phenomena in plants is crucial for the effective conservation, management, and efficient utilization of plant genetic resources (PGR).
Determining genetic diversity can be based on morphological, biochemical, and molecular types of information. However, molecular markers have advantages over other kinds, where they show genetic differences on a more detailed level without interferences from environmental factors, and where they involve techniques that provide fast results detailing genetic diversity
Comparison of different methods
Morphological characterization does not require expensive technology but large tracts of land are often required for these experiments, making it possibly more expensive than molecular assessment. These traits are often susceptible to phenotypic plasticity; conversely, this allows assessment of diversity in the presence of environmental variation.
Biochemical analysis is based on the separation of proteins into specific banding patterns. It is a fast method which requires only small amounts of biological material. However, only a limited number of enzymes are available and thus, the resolution of diversity is limited.
Molecular analyses comprise a large variety of DNA molecular markers, which can be employed for analysis of variation. Different markers have different genetic qualities (they can be dominant or co-dominant, can amplify anonymous or characterized loci, can contain expressed or non-expressed sequences, etc.).
Genetic marker
The concept of genetic markers is not a new one; in the nineteenth century, Gregor Mendel employed phenotype-based genetic markers in his experiments. Later, phenotype-based genetic markers for Drosophila melanogaster led to the founding of the theory of genetic linkage. A genetic marker is an easily identifiable piece of genetic material, usually DNA that can be used in the laboratory to tell apart cells, individuals, populations, or species. The use of genetic markers begins with extracting proteins or chemicals (for biochemical markers) or DNA (for molecular markers) from tissues of the plant (for example, seeds, foliage, pollen, sometimes woody tissues).
Molecular markers In genetics, a molecular marker (identified as genetic marker) is a fragment of DNA that is associated with a certain location within the genome. Molecular markers which detect variation at the DNA level such as nucleotide changes: deletion, duplication, inversion and/or insertion. Markers can exhibit two modes of inheritance, i.e. dominant/recessive or co-dominant. If the genetic pattern of homozygotes can be distinguished from that of heterozygotes, then a marker is said to be co-dominant. Generally co-dominant markers are more informative than the
Role of Marker Assisted Selection in Plant Resistance RandeepChoudhary2
Topic Role of Marker Assisted Selection in Plant Resistance is described in detail including some case studies.
Types of markers used in genetic engineering and biotechnology are described in detail.
Marker assisted selection is a process whereby a marker (morphological, biochemical or one
based on DNA/RNA variation) is used for indirect selection of a genetic determinant of a trait
of interest. Since the first reported linkage of an agronomically important trait (a quantitative
trait locus affecting seed weight) to a simply controlled gene (seed colour) in common bean by
Sax (1923), it has taken more than 60 years for genetic markers to become a qualified tool for
plant breeding programs. In rice, the Xieyou 218 hybrid was the first to be developed through
MAS to select individuals carrying a bacterial blight-resistant gene. Marker-assisted selection
(MAS) can be applied at the seedling stage, with high precision and reductions in cost. Genetic
mapping of major genes and quantitative traits loci (QTLs) for agricultural traits is increasing
the integration of biotechnology with the conventional breeding process. Traits related to
disease resistance to pathogens and to the quality of some crop products are offering some
important examples of a possible routinary application of MAS. For more complex traits, like
yield and abiotic stress tolerance, a number of constraints have severe limitations on an efficient
utilization of MAS in plant breeding. However, the economic and biological constraints such
as a low return of investment in small-grain cereal breeding, lack of diagnostic markers, and
the prevalence of QTL-background effects hinder the broad implementation of MAS but over
the past 2 decades, a number of R-genes conferring resistance to a diverse range of pathogens
have been mapped in many crops using molecular markers.
Morphological, Cytological and Biochemical MarkersJay Khaniya
I've put a lot of effort for create this presentation. This'll help to lot of biotechnology and agricultural students for there assignments and exam study.
A genetic marker is a gene or DNA sequence with a known location on a chromosome and associated with a particular gene or trait. It can be described as a variation, which may arise due to mutation or alteration in the genomic loci that can be observed. A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change (single nucleotide polymorphism, SNP), or a long one, like mini & microsatellites.
All about molecular markers like - hybridization based markers (RFLP), pcr based markers (RAPD, SSR, SNP) etc. Morphological and biochemical markers also covered.
A powerful non-transgenic reverse genetics method that combines chemical mutagenesis with PCR based screening to identify point mutations in regions of interest.
EcoTILLING is a molecular technique that is similar to TILLING, except that its objective is to uncover natural genetic variation as opposed to induced mutations.
Speed Breeding is new technology to develop plants or breeding materials within a short possible time without affect seed viability and yield performance.
The shifted multiplicative model was developed by Cornelius and Seyedsadr in 1992.
SHMM is used to analyze the complete separability, genotypic separability, environmental separability, and inseparability of environment effects and genotypic effects.
Gregorius and Namkoong (1986) defined Separability as the property which is that cultivar effect is separable from environmental effect so that there is no rank.
The shifted multiplicative model (SHMM) is used in an exploratory step-down method for identifying subsets of environments in which genotypic effects are "separable" from environmental effects. Subsets of environments are chosen on the basis of a SHMM analysis of the entire data set. SHMM analyses of the subsets
may indicate a need for further subdivision and/or suggest that a different subdivision at the previous stage should be tried. The process continues until SHMM analysis indicates that a SHMM with only one multiplicative term and its "point of concurrence" outside (left or right) of the cluster of data points adequately fits the data in all subsets.
Crops undergo artificially DNA modifications for improvements are considered as genetically modified (GM) crops. These modifications could be in indigenous DNA or by the introduction of foreign DNA as transgenes. There are 29 different crops and fruit trees in 42 countries, which have been successfully modified for various traits like herbicide tolerance, insect/pest resistance, disease resistance and quality improvement. GM crops are grown worldwide and its area is significantly increasing every year. Many countries have very strict rules and regulations for GM crops and are also a trade barrier in some situations. Hence, identification and testing of crops for GM contents are important for the identity and legitimacy of the transgene to simplify the international trade. Normally, molecular identification is performed at three different levels, i.e., DNA, RNA and protein, and each level have its own importance in testing the nature and type of GM crops. In this chapter, the current scenario of GM crops and different molecular testing tools are described in brief.
Extranuclear inheritance or cytoplasmic inheritance is the transmission of genes that occur outside the nucleus. It is found in most eukaryotes and is commonly known to occur in cytoplasmic organelles such as mitochondria and chloroplasts or from cellular parasites like viruses or bacteria. Determining the contribution of organelle genes to plant phenotype is hampered by several factors, including the paucity of variation in the plastid and mitochondrial genomes. Mitochondria are organelles which function to transform energy as a result of cellular respiration. Chloroplasts are organelles which function to produce sugars via photosynthesis in plants and algae. The genes located in mitochondria and chloroplasts are very important for proper cellular function, yet the genomes replicate independently of the DNA located in the nucleus, which is typically arranged in chromosomes that only replicate one time preceding cellular division. The extranuclear genomes of mitochondria and chloroplasts however replicate independently of cell division. They replicate in response to a cell's increasing energy needs which adjust during that cell's lifespan. There is consistent difference between the results from reciprocal crosses; generally only the trait from female parent is transmitted. In most cases, there is no segregation in the F2 and subsequent generations.
Plant genetic engineering is one of the key technologies for crop improvement as well as an emerging approach for producing recombinant proteins in plants. Both plant nuclear and plastid genomes can be genetically modified, yet fundamental functional differences between the eukaryotic genome of the plant cell nucleus and the prokaryotic-like genome of the plastid will have an impact on key characteristics of the resulting transgenic organism. So, which genome, nuclear or plastid, to transform for the desired transgenic phenotype? In this paper we compare the advantages and drawbacks of engineering plant nuclear and plastid genomes to generate transgenic plants with the traits of interest, and evaluate the pros and cons of their use for different biotechnology and basic research applications. The chloroplast is a pivotal organelle in plant cells and eukaryotic algae to carry out photosynthesis, which provides the primary source of the world’s food. The expression of foreign genes in chloroplasts offers several advantages over their expression in the nucleus: high-level expression, no position effects, no vector sequences allowing stable transgene expression. In addition, transgenic chloroplasts are generally not transmitted through pollen grains because of the cytoplasmic localization. In the past two decades, great progress in chloroplast engineering has been made.
Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
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June 3, 2024 Anti-Semitism Letter Sent to MIT President Kornbluth and MIT Cor...Levi Shapiro
Letter from the Congress of the United States regarding Anti-Semitism sent June 3rd to MIT President Sally Kornbluth, MIT Corp Chair, Mark Gorenberg
Dear Dr. Kornbluth and Mr. Gorenberg,
The US House of Representatives is deeply concerned by ongoing and pervasive acts of antisemitic
harassment and intimidation at the Massachusetts Institute of Technology (MIT). Failing to act decisively to ensure a safe learning environment for all students would be a grave dereliction of your responsibilities as President of MIT and Chair of the MIT Corporation.
This Congress will not stand idly by and allow an environment hostile to Jewish students to persist. The House believes that your institution is in violation of Title VI of the Civil Rights Act, and the inability or
unwillingness to rectify this violation through action requires accountability.
Postsecondary education is a unique opportunity for students to learn and have their ideas and beliefs challenged. However, universities receiving hundreds of millions of federal funds annually have denied
students that opportunity and have been hijacked to become venues for the promotion of terrorism, antisemitic harassment and intimidation, unlawful encampments, and in some cases, assaults and riots.
The House of Representatives will not countenance the use of federal funds to indoctrinate students into hateful, antisemitic, anti-American supporters of terrorism. Investigations into campus antisemitism by the Committee on Education and the Workforce and the Committee on Ways and Means have been expanded into a Congress-wide probe across all relevant jurisdictions to address this national crisis. The undersigned Committees will conduct oversight into the use of federal funds at MIT and its learning environment under authorities granted to each Committee.
• The Committee on Education and the Workforce has been investigating your institution since December 7, 2023. The Committee has broad jurisdiction over postsecondary education, including its compliance with Title VI of the Civil Rights Act, campus safety concerns over disruptions to the learning environment, and the awarding of federal student aid under the Higher Education Act.
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The Roman Empire’s society was hierarchical, with a rigid class system. At the top were the patricians, wealthy elites who held significant political power. Below them were the plebeians, free citizens with limited political influence, and the vast numbers of slaves who formed the backbone of the economy. The family unit was central, governed by the paterfamilias, the male head who held absolute authority.
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TESDA TM1 REVIEWER FOR NATIONAL ASSESSMENT WRITTEN AND ORAL QUESTIONS WITH A...
Molecular markers
1. 09‐May‐17
1
INTRODUCTION
Those characters which can be easily identified are called marker characters.
Any genetic element (locus, allele, DNA sequence or chromosome feature) which can be readily detected by
phenotype, cytological or molecular techniques, and used to follow a chromosome or chromosomal segment
during genetic analysis is referred to as marker.
GENETIC MARKERS
Any traits of an organism that can be identified with confidence and relative ease, and can be followed in a
mapping population is called genetic marker.
It can be detected with naked eye, as differences in electrophoretic mobility of specific proteins, or as differences
in specific DNA sequences.
Markers are of four types, viz:
(i) Morphological,
(ii) Biochemical,
(iii) Cytological, and
(iv) DNA markers.
1.Morphological marker
also called naked eye polymorphism.
In plant breeding, markers that are related to variation in shape, size, colour and surface of various plant parts are
called morphological markers. Such markers refer to available gene loci that have obvious impact on morphology
of plant. Genes that affect form, coloration, male sterility or resistance among others have been analyzed in many
plant species.
In rice, examples of this type of marker may include the presence or absence of awn, leaf sheath coloration, height,
grain color, aroma etc. In well-characterized crops like maize, tomato, pea, barley or wheat, tens or even hundreds
of such genes have been assigned to different chromosomes.
Advantages
a) NEP traits represents the actual phenotypes of importance to us, while protein and DNA markers are important only as
arbitrary loci used for linkage mapping and often do not Correspond directly to specific phenotypes.
b) NEPs factors can be scored quickly, simply, without laboratory equipment.
Disadvantages
a. They generally express late into the development of an organism. Hence their detection is dependent on the development
stage of the organism.
b. They usually exhibit dominance.
c. Sometimes they exhibit deleterious effects.
d. They exhibit pleiotropy.
e. They exhibit epistasis.
f. They exhibit less polymorphism.
g. They are highly influenced by the environmental factors.
3. 09‐May‐17
3
ii. Cytological markers
Markers that are related to variation in chromosome number, shape, size and banding pattern are referred to as
cytological markers. In other words, it refers to the chromosomal banding produced by different stains;
for example, G banding or Giemsa banding is a technique used in cytogenetics to produce a visible karyotype by staining
condensed chromosomes. It is useful for identifying genetic diseases through the photographic representation of the
entire chromosome complement..
advantages of cytological marker
1. Readily available.
2. Requires small equipments.
Disadvantages of cytological marker
1. Limited in number
2. They exhibit less polymorphism, need experts.
iii. Biochemical markers
Monoterpenes
Monoterpenes are a subgroup of the terpenoid substances found in resins and essential
oils of plants ,Although the metabolic functions of monoterpenes are not fully understood, they probably play an important
role in resistance to attack by diseases and insects (Hanover, 1992). The concentrations of different monoterpenes, such as
alpha‐pinene, beta‐pinene, myrcene, 3‐carene and limonene are determined by gas chromotography and are useful as
genetic markers.
Advantage
1. Monoterpene genetic markers have been applied primarily to taxonomic and evolutionary
studies.
2. Although, monoterpenes were the best available genetic markers for forest trees in the 1960s and
early 1970s.
Demerits
they require specialized and expensive equipment for assay.
there are relatively few monoterpene marker loci available and most express some form of
dominance in their phenotypes.
5. 09‐May‐17
5
iv. Molecular marker
DNA is the genetic material. Each chromosome has about 108‐1010 base pairs but only 10% of the genome is actively engaged in
translation. The rest of the genetic material remains unnoticed in terms of character expression. The assessment of variation at
the DNA level provides a chance to map the genome. Specific regions on the DNA molecule both in the coding as well as non‐
coding regions can be identified as markers.
DNA markers are such polymorphic sequences seen in different individuals.
A considerable part of the DNA shows highly repetitive sequences in the non‐coding regions of DNA. The comparison of these
regions can also provide valuable clues of genetic and evolutionary relationships. The DNA sequences can be cut with the help of
restriction endonucleases, analyzed with the help of DNA probes and electrophoretic procedures.
Definition
Any unique DNA sequence which can be used in DNA hybridization, PCR or restriction mapping experiments to identify that
sequence is called DNA marker.
Properties of DNA Marker:
An ideal DNA marker should have some properties or characteristics.
Important properties of an ideal DNA markers
i. Polymorphism:
Markers should exhibit high level of polymorphism. In other words, there should be variability in the markers. It
should demonstrate measurable differences in expression between trait types and/or gene of interest.
ii. Co-Dominant:
Marker should be co-dominant. It means, there should be absence of intra-locus interaction. It helps in identification
of heterozygotes from homozygotes.
Advantages
They are not influenced by the environment.
They are more reliable (marker should be tightly linked to target loci, preferably less than 5cM genetic distance).
Level of polymorphism is high.
Easily excessable marker sequences.
Highly reproducible.
Multiple alleles for each markers.
They abundantly occur throughout the genome
They have simple inheritance (often co‐dominant).
They are easy and fast to detect.
They exhibit minimum pleiotropic effect.
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iii. Multi-Allelic:
The marker should be multi-allelic. It useful in getting more variability/ polymorphism for a character.
iv. No Epistasis:
There should be absence of epistasis. It makes Identification of all phenotypes (homo- and heterozygotes)
easy.
v. Neutral:
The marker should be neutral. The substitution of alleles at the marker locus should not alter the
phenotype of an individual. This property is found in almost all the DNA markers.
vi. No Effect of Environment:
Markers should be insensitive to environment. This property is also found in almost all the DNA markers.
Type of molecular markers
RFLP (or Restriction fragment length polymorphism)
AFLP (or Amplified fragment length polymorphism)
RAPD (or Random amplification of polymorphic DNA)
SSR Microsatellite polymorphism, (or Simple sequence repeat)
SNP (or Single nucleotide polymorphism)
STS (or sequence taged sites)
CAPS (Cleaved amplified polymorphic sequence )
SCAR (Sequence Characterized Amplified Region )
1.Restriction fragment length polymorphism (RFLP):
RFLP was the very first technology employed for the detection of polymorphism, based on the DNA sequence
differences. RFLP is mainly based on the altered restriction enzyme sites, as a result of mutations and re-combinations
of genomic DNA. An outline of the RFLP analysis is given in Fig.1, and schematically depicted in Fig.2.
The procedure basically involves the isolation of genomic DNA, its digestion by restriction enzymes, separation by
electrophoresis, and finally hybridization by incubating with cloned and labeled probes.
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Advantages of RFLPs
• Highly robust methodology with good transferability between laboratories
• Codominantly inherited and, as such, can estimate heterozygosity
• No sequence information required, Because based on sequence homology.
• Highly recommended for phylogenetic analysis between related species
• Well suited for constructing genetic linkage maps
• Locus‐specific markers, which allow synteny studies( physical co‐localization
of genetic loci on the same chromosome within an individual or species)
• Discriminatory power—can be at the species and/or population levels (single‐
locus probes), or individual level (multi‐locus probes)
• Simplicity—given the availability of suitable probes, the technique can readily be
applied to any plant
• Disadvantages of RFLPs
• Large amounts of DNA required
• Automation not possible
• Low levels of polymorphism in some species
• Few loci detected per assay
• Need a suitable probe library
• Time consuming, especially with single‐copy probes
• Costly
• Distribution of probes to collaborating laboratories required
• Moderately demanding technically
• Different probe/enzyme combinations may be needed
Applications
• Genetic diversity
• Genetic relationships
• History of domestication
• Origin and evolution of species
• Genetic drift and selection
• Whole genome and comparative mapping
• Gene tagging
• Unlocking valuable genes from wild species
• Construction of exotic libraries
2.Random amplified polymorphic DNA (RAPD) markers:
RAPD is a molecular marker based on PCR amplification. An outline of RAPD is depicted in fig.3. The DNA isolated
from the genome is denatured the template molecules are annealed with primers, and amplified by PCR.
Single short oligonucleotide primers (usually a 10-base primer) can be arbitrarily selected and used for the
amplification DNA segments of the genome (which may be in distributed throughout the genome). The amplified
products are separated on electrophoresis and identified.
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Advantages
• It can be used with uncharacterized genomes
• can be applied to cases in which only small quantities of DNA are available
• Absolutely no knowledge of the target genome is required
• It can be used on any DNA sample.
• RAPD is an inexpensive yet powerful tool for typing of bacterial species.
• Even if the same primer is used with different samples, they will produce different results and different bands
patterns that may allow for recognition of the various strains.
• Can be used to study genetic polymorphism between closely related species.
• Can be used to select variants of microbial isolates.
Limitations
• The primer sequence has to be right to produce the right results.
• Since the primer targeting is random, it is absolutely essential to have a large genome template.
• Minute quantities of available DNA or degraded DNA samples cannot be subjected to RAPD (unlike PCR).
• It is very likely that the primer will not be able to find complementary sequences on the target.
• It has a low power of resolution unlike other methods of DNA analysis.
• The quality of the DNA used in the reaction will affect the outcome.
• The concentration of PCR reagents, their purity, and the conditions in which the reaction is carried out will all
affect the results.
• RAPD requires knowledgeable and careful operation for good results and for these results to be reproducible.
• If there is a mismatch between the primer and the template DNA, there will be reduced product or even no PCR
product. Hence, reading the results can be a little tricky sometimes.
• If the 3' ends of the primer are not facing each other there will be little or no product.
• Mutation of target DNA can give wrong profile.
3.Amplified fragment length polymorphism
(AFLP):
AFLP is a novel technique involving a combination of RFLP and RAPD. AFLP
is based on the principle of generation of DNA fragments using restriction
enzymes and oligonucleotide adaptors (or linkers), and their amplification by
PCR. Thus, this technique combines the usefulness of restriction digestion
and PCR.
The DNA of the genome is extracted. It is subjected to restriction digestion by two
enzymes (a rare cutter e.g. Msel; a frequent cutter e.g. EcoRI). The cut ends on both
sides are then ligated to known sequences of oligonucleotides.
PCR is now performed for the pre‐selection of a fragment of DNA which has a single
specific nucleotide. By this approach of pre‐selective amplification, the pool of
fragments can be reduced from the original mixture. In the second round of
amplification by PCR, three nucleotide sequences are amplified.
This further reduces the pool of DNA fragments to a manageable level (< 100).
Autoradiography can be performed for the detection of DNA fragments. Use of
radiolabeled primers and fluorescently labeled fragments quickens AFLP.
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Advantages of AFLPs
• AFLPs allow a quick scan of the whole genome for polymorphisms
• Because of the large number of bands generated, each marker gives a highly informative fingerprint
• They are also highly reproducible
• No prior sequence information or probe generation is needed
• Extremely useful in creating quick genetic maps.
Disadvantages of AFLPs
• AFLPs generate huge quantities of information, which may need automated analysis and therefore computer technology
• AFLP markers display dominance
• In genetic mapping, AFLPs often cluster at the centromeres and telomeres
• They are technically demanding in the laboratory and, especially, in data analysis
Applications
• Genetic diversity assessment
• Genetic distance analysis
• Genetic fingerprinting
• Analysis of germplasm collections
• Genome mapping
• Monitoring diagnostic markers
4. Sequence‐Tagged Site (STS)
is a relatively short, easily PCR‐amplified sequence (200 to 500 bp) which can be specifically amplified by PCR and detected in
the presence of all other genomic sequences and whose location in the genome is mapped.
The STS concept was introduced by Olson et al. (1989).
In assessing the likely impact of the Polymerase Chain Reaction (PCR) on human genome research, they recognized that single‐
copy DNA sequences of known map location could serve as markers for genetic and physical mapping of genes along the
chromosome.
The advantage of STSs over other mapping landmarks is that the means of testing for the presence of a particular STS can be
completely described as information in a database: anyone who wishes to make copies of the marker would simply look up the
STS in the database, synthesize the specified primers, and run the PCR under specified conditions to amplify the STS from
genomic DNA.
STS‐based PCR produces a simple and reproducible pattern on agarose or polyacrylamide gel. In most cases STS markers are co‐
dominant, i.e., allow heterorozygotes to be distinguished from the two homozygotes.
The DNA sequence of an STS may contain repetitive elements, sequences that appear elsewhere in the genome, but as long as
the sequences at both ends of the site are unique and conserved, researches can uniquely identify this portion of genome
using tools usually present in any laboratory.
in broad sense, STS include such markers as microsatellites , SCARs, CAPs, and ISSRs.
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Microsatellites
Polymorphic loci present in nuclear DNA and organellar DNA that consist of repeating units of 1‐10 base pairs,
most typically, 2‐3 bp in length, also called Simple Sequence Repeats (SSR), Sequence‐Tagged Microsatellite
Sites (STMS) or Simple Sequence Repeats Polymorphisms (SSRP). SSRs are highly variable and evenly
distributed throughout the genome. This type of repeated DNA is common in eukaryotes. These
polymorphisms are identified by constructing PCR primers for the DNA flanking the microsatellite region. The
flanking regions tend to be conserved within the species, although sometimes they may also be conserved in
higher taxonomic levels.
Sequence Characterized Amplified Region (SCAR)
DNA fragments amplified by the Polymerase Chain Reaction (PCR) using specific 15-30 bp primers, designed from
nucleotide sequences established in cloned RAPD (Random Amplified Polymorphic DNA) fragments linked to a trait of
interest. By using longer PCR primers, SCARs do not face the problem of low reproducibility generally encountered
with RAPDs. Obtaining a co-dominant marker may be an additional advantage of converting RAPDs into SCARs.
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Cleaved Amplified Polymorphic Sequences (CAPS)
polymorphisms are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction
endonuclease recognition sites in PCR amplicons produced by locus‐specific oligonucleotide primers.
The CAPS assay uses amplified DNA fragments that are digested with a restriction endonuclease to display RFLP.
Unique sequence primers are used to amplify a mapped DNA sequence from two related individuals (for example, from two
different inbred ecotypes), A/A and B/B, and from the heterozygote A/B. The amplified fragments from A/A and B/B contain
two and three RE recognition sites, respectively. In the case of the heterozygote A/B, two different PCR products will be
obtained, one which is cleaved three times and one which is cleaved twice. When fractionated by agarose or acrylamide gel
electrophoresis, the PCR products digested by the RE will give readily distinguishable patterns. Some bands will appear as
doublets.
Advantages of CAPS
• Most CAPS markers are co‐dominant and locus‐specific.
• Most CAPS genotypes are easily scored and interpreted.
• CAPS markers are easily shared between laboratories.
• CAPS assay does not require the use of radioactive
isotopes, and it is more amenable, therefore, to
analyses in clinical settings.
single-nucleotide polymorphism (SNP)
It is a DNA sequence variation occurring when a single nucleotide adenine (A), thymine (T), cytosine (C), or guanine
(G]) in the genome(or other shared sequence) differs between members of a species or paired chromosomes in an
individual.
For example, two sequenced DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in
a single nucleotide. In this case we say that there are two alleles C and T.
Almost all common SNPs have only two alleles.
advantages
• PCR products can be very small: – Markers will work with extremely degraded DNA samples
• More common in genome
• May possibly multiplex hundreds or thousands on one chip
• Sample processing may be completely automated
• No stutter products
• disadvantages
• • Less alleles – Each marker is less informative
• • Therefore have to genotype many more SNPs to get same level of information about DNA sample
• • Mixture interpretation is more difficult
• • Multiplexes don’t actually work yet – Currently uses more of the DNA sample than STRs use
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Mapping and tagging of genes: Generating tools for marker-assisted selection
in plant breeding
Plant improvement, either by natural selection or through the efforts of breeders, has always relied upon creating,
evaluating and selecting the right combination of alleles. The manipulation of a large number of genes is often
required for improvement of even the simplest of characteristics With the use of molecular markers it is now a
routine to trace valuable alleles in a segregating population and mapping them. These markers once mapped
enable dissection of the complex traits into component genetic units more precisely, thus providing breeders with
new tools to manage these complex units more efficiently in a breeding programme.
• first genome maps in plants was reported in maize , followed by rice, Arabidopsis etc. using RFLP markers.
have since then been constructed for several other crops like potato, barley, banana, members of
Brassicaceae, etc.
• RFLP markers have proved their importance as markers for gene tagging and are very useful in locating and
manipulating quantitative trait loci (QTL) in a number of crops.
• The very first reports on gene tagging were from tomato, availing the means for identification of markers
linked to genes involved in several traits like water use efficiency, resistance to Fusarium oxysporum (the 12
gene), leaf rust resistance genes LR 9 and 24, and root knot nematodes (Meliodogyne sp.).
Phylogeny and evolution
Most of the early theories of evolution were based on morphological and geographical variations
between organisms. However, it is becoming more and more evident that the techniques from
molecular biology hold a promise of providing detailed information about the genetic structure of
natural population, than what we have been able to achieve in the past. RFLP, DNA sequencing, and
a number of PCR‐based markers are being used extensively for reconstructing phylogenies of various
species.
RFLPs have been used in evolutionary studies for deducing the relationship between the hexaploid
genome of bread wheat and its ancestors.
Diversity analysis of exotic germplasm
genetic variation in crop plants has continued to narrow due to continuous selection pressure for specific traits i.e.
yield, . This risk was brought sharply into focus in 1970 with the outbreak of southern corn leaf blight which
drastically reduced corn yield in USA, and was attributed to extensive use of a single genetic male sterility factor
which was unfortunately linked to the disease susceptibility.
Genotyping of cultivars
The repetitive and arbitrary DNA markers are markers of choice in genotyping of cultivars. Microsatellites like (CT)10,
(GAA)5, (AAGG)4, (AAT)6 (GATA)4, (CAC)5 and minisatellites have been employed in DNA fingerprinting for the
detection of genetic variation, cultivar identification and genotyping. This information is useful for quantification of
genetic diversity, characterization of accessions in plant germplasm collections and taxonomic studies.
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Indian scenario for development of molecular markers in crop
improvement programmes
• Agriculture is one of the most important occupations in India with almost 55% of the population being dependent
on it. A noteworthy research in conventional breeding for several years has made this country self-sufficient in
many respects. However, the ever-increasing population has alarmed food security in India and attempts have
been initiated to integrate modern biotechnology tools in conventional breeding to improve the most important
crops such as rice, wheat and legumes.
• Extensive research using DNA markers is in progress in many institutions all over India. Markers tagged and
mapped with specific genes have been identified
• such examples- include resistance genes for blas and gall midge using RFLP- and PCR-based approaches in
rice.
• Similarly, in wheat, leaf rust resistance gene LR 28 , and -harvest sprout tolerance gene have been tagged. QTLs
such as protein content in wheat and heterosis in rice142 have also been identified.
While efforts for tagging genes providing resistance to BPH, WBPH, sheath rot and drought are going on,
many attempts are also being made towards pyramiding different resistance genes for a specific disease or pest
attack like blast, bacterial blight, gall midge, BPH, WBPH, etc. in rice in order to increase the field life of the crop.
• Germplasm analysis to study genetic diversity is another important area in which a lot of efforts have been put in.
Fingerprinting of crops like rice, wheat, chickpea, pigeonpea, pearlmillet etc. is being carried out extensively. This
information has potential in strategic planning of future breeding towards crop sustainability in India.
• Apart from use of molecular markers in crop plants, efforts are also underway in other horticultural plants. Early
identification of sex in dioecious papaya using molecular marker is one such example.