The document describes several classes of molecular markers used in genetic analysis, including isozymes, RFLPs, RAPDs, AFLPs, microsatellites, and SNPs. Isozymes analyze differences in protein mobility on a gel, while RFLPs, RAPDs, AFLPs detect DNA fragment length polymorphisms. Microsatellites analyze differences in repeat number, and SNPs detect single nucleotide differences. Each method has advantages and disadvantages related to factors like technical requirements, costs, reproducibility, and amount of polymorphism detected. The choice of marker depends on the application and study objectives.
This document discusses different types of molecular markers used in genetics including RFLP, RAPD, AFLP, STS, and microsatellites. It provides details on each technique such as how they work, their advantages and disadvantages. Some key applications of molecular markers mentioned are in forensics, disease detection, animal breeding through marker-assisted selection, and studying genetic diversity. The document aims to introduce molecular markers and their wide-ranging uses in fields like genetics, biotechnology, forensics and agriculture.
This document defines molecular markers and describes two main types - biochemical markers and molecular genetic markers. Biochemical markers involve studying gene expression products like proteins through electrophoresis. Isoenzymes are variant enzymes that catalyze the same reaction but differ in properties, while alloenzymes are different alleles that can be detected through electrophoresis. Molecular genetic markers involve DNA polymorphisms detected by probes and include RAPD, RFLP, AFLP, SSR, and DNA fingerprinting techniques. These markers allow genome profiling and studying genetic variation.
The document provides information about AFLP (Amplified Fragment Length Polymorphism) and RFLP (Restriction Fragment Length Polymorphism) techniques. It explains that AFLP involves selectively amplifying a subset of restriction fragments using restriction enzymes and PCR primers, while RFLP detects polymorphisms by probing restricted DNA on a Southern blot. The procedures for both techniques are described in detail, along with their applications and differences. AFLP is more efficient at detecting polymorphisms but also more expensive than RFLP.
This document discusses different types of molecular markers used in genetics research including RFLP, RAPD, AFLP, SSR, and SNP. It provides details on each technique, including how they work, their applications in areas like genetic mapping and disease analysis, and their advantages and disadvantages. The key molecular markers covered are RFLP (restriction fragment length polymorphism), RAPD (random amplification of polymorphic DNA), AFLP (amplified fragment length polymorphism), SSR (simple sequence repeats), and SNP (single nucleotide polymorphisms).
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
Molecular markers can be used to characterize plant genetic resources and assist in pre-breeding for climate change. Various marker techniques are described, including hybridization-based RFLP and PCR-based RAPD, ISSR, SSR, AFLP, EST, and SCoT. Molecular markers reflect heritable DNA differences and have advantages like being ubiquitous, stable, and not affecting phenotypes. Data from markers can be analyzed to construct genetic similarity matrices and dendrograms to study genetic diversity and relationships. Molecular markers have applications in fingerprinting, diversity studies, marker-assisted selection, genetic mapping, and gene tagging.
This document discusses genetic and molecular markers. It defines genetic markers as any phenotypic difference controlled by genes that can be used to study inheritance or selection of linked genes. Molecular markers are readily detectable DNA sequences whose inheritance can be monitored. The document describes different types of molecular markers including protein markers, RFLP, RAPD, AFLP, microsatellites, SNPs, VNTRs and STRs. It provides details on how each marker type is detected and analyzed, their advantages and disadvantages, and applications in genetics research.
This document discusses different types of molecular markers used in genetics including RFLP, RAPD, AFLP, STS, and microsatellites. It provides details on each technique such as how they work, their advantages and disadvantages. Some key applications of molecular markers mentioned are in forensics, disease detection, animal breeding through marker-assisted selection, and studying genetic diversity. The document aims to introduce molecular markers and their wide-ranging uses in fields like genetics, biotechnology, forensics and agriculture.
This document defines molecular markers and describes two main types - biochemical markers and molecular genetic markers. Biochemical markers involve studying gene expression products like proteins through electrophoresis. Isoenzymes are variant enzymes that catalyze the same reaction but differ in properties, while alloenzymes are different alleles that can be detected through electrophoresis. Molecular genetic markers involve DNA polymorphisms detected by probes and include RAPD, RFLP, AFLP, SSR, and DNA fingerprinting techniques. These markers allow genome profiling and studying genetic variation.
The document provides information about AFLP (Amplified Fragment Length Polymorphism) and RFLP (Restriction Fragment Length Polymorphism) techniques. It explains that AFLP involves selectively amplifying a subset of restriction fragments using restriction enzymes and PCR primers, while RFLP detects polymorphisms by probing restricted DNA on a Southern blot. The procedures for both techniques are described in detail, along with their applications and differences. AFLP is more efficient at detecting polymorphisms but also more expensive than RFLP.
This document discusses different types of molecular markers used in genetics research including RFLP, RAPD, AFLP, SSR, and SNP. It provides details on each technique, including how they work, their applications in areas like genetic mapping and disease analysis, and their advantages and disadvantages. The key molecular markers covered are RFLP (restriction fragment length polymorphism), RAPD (random amplification of polymorphic DNA), AFLP (amplified fragment length polymorphism), SSR (simple sequence repeats), and SNP (single nucleotide polymorphisms).
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
Molecular markers can be used to characterize plant genetic resources and assist in pre-breeding for climate change. Various marker techniques are described, including hybridization-based RFLP and PCR-based RAPD, ISSR, SSR, AFLP, EST, and SCoT. Molecular markers reflect heritable DNA differences and have advantages like being ubiquitous, stable, and not affecting phenotypes. Data from markers can be analyzed to construct genetic similarity matrices and dendrograms to study genetic diversity and relationships. Molecular markers have applications in fingerprinting, diversity studies, marker-assisted selection, genetic mapping, and gene tagging.
This document discusses genetic and molecular markers. It defines genetic markers as any phenotypic difference controlled by genes that can be used to study inheritance or selection of linked genes. Molecular markers are readily detectable DNA sequences whose inheritance can be monitored. The document describes different types of molecular markers including protein markers, RFLP, RAPD, AFLP, microsatellites, SNPs, VNTRs and STRs. It provides details on how each marker type is detected and analyzed, their advantages and disadvantages, and applications in genetics research.
Biochemical and molecular markers for characterizationmithraa thirumalai
This document discusses various biochemical and molecular markers that can be used for plant genetic characterization. It begins by explaining that genetic resources can be characterized based on genotypes, phenotypes, and various molecular traits. It then discusses different types of molecular markers including DNA fingerprinting using microsatellites, RAPDs, AFLPs, allozymes, and other protein markers. It provides details on how many of these techniques work, such as the PCR process for RAPDs and AFLPs. The document also discusses using these molecular markers for assessing population structure, assembling core collections, and addressing various taxonomic and phylogenetic questions.
RAPD (Random Amplification of Polymorphic DNA) is a PCR-based molecular marker technique that involves using short, arbitrary nucleotide primers to randomly amplify genomic DNA fragments. These fragments can then be analyzed as genetic markers. RAPD works by using a single short primer to amplify random DNA sequences from a complex template. Variations in priming sites between individuals result in presence or absence of bands that can be used to analyze genetic relationships. The technique is fast, inexpensive and does not require prior DNA sequence knowledge, but results can lack reproducibility between laboratories.
Methods for characterization of animal genomes(snp,str,qtl,rflp,rapd)Dr Vijayata choudhary
The document discusses several methods for characterizing animal genomes, including Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD), Single Nucleotide Polymorphisms (SNPs), Short Tandem Repeats (STRs), and Quantitative Trait Loci (QTL). It provides detailed descriptions of each method, including how they are detected and analyzed as well as their applications in areas like genetic mapping, diversity studies, and phylogenetic analysis.
This document provides information about molecular marker techniques RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA). RFLP is a non-PCR based technique that involves digesting genomic DNA with restriction enzymes, separating fragments via gel electrophoresis, and detecting variations. RAPD is a PCR-based technique that uses random primers to amplify random DNA segments, which are then separated and visualized. Both techniques can be used for genetic mapping, germplasm characterization, and other applications. Key differences between the two techniques include RFLP detecting fewer loci but being more reliable, requiring larger DNA quantities, and using species-specific probes.
Molecular marker analysis of A few Capsicum annum varietiesAnkitha Hirematha
The hybrid variety and parental varieties among the 3 chilly varieties were identified by finding out the genetic polymorphism between them. It helps to identify different plant varieties, disputed plant varieties, genetic polymorphism between intraspecific crosses of plants and also to protect Plant Breeder’s Rights (PBR). Based on banding pattern on gel, identification of KA, KS and HK chilly varieties using SSR & ISSR markers was successfully carried out.
TYPES OF MOLECULAR MARKERS,ITS ADVANTAGES AND DISADVANTAGESANFAS KT
Types of molecular markers (genetics)
ITS ADVANTAGES AND DISADVANTAGES
What is a genetic marker?
RFLP: Restriction fragment length polymorphism
AFLP: Amplified fragment length polymorphism
RAPD: Random amplification of polymorphic DNA
ISSR: Inter simple sequence repeat
STR: Short tandem repeats
SCAR: Sequence characterized amplified region
SNP: Single nucleotide polymorphism
SSR: Simple sequence repeat
Molecular markers are DNA sequences that can be easily detected and whose inheritance can be monitored. They are based on natural polymorphisms and allow studying the inheritance of genes. Common types of molecular markers include RFLPs, RAPDs, AFLPs, SSRs, and SNPs. RFLPs use restriction enzymes to detect differences in fragment lengths. RAPDs use random primers to detect sequence polymorphisms. AFLPs selectively amplify restriction fragments to detect length differences. SSRs detect variability in simple sequence repeats. Molecular markers are useful for applications like gene mapping, phylogenetic studies, and analyzing genetic diversity.
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
Genetic markers are sequences of DNA located at specific positions on chromosomes that can be associated with particular traits. Common types of genetic markers include RFLP, AFLP, RAPD, SNP, and VNTR. These markers allow researchers to locate genes associated with diseases and trace inheritance patterns. While harmless themselves, genetic markers enable the positioning of disease genes along chromosomes to better understand inheritance of traits and diseases.
Markers: Based on hybridization and PCRSachin Kumar
This document discusses different types of molecular markers used in genetics. It describes markers based on nucleic acid hybridization, such as restriction fragment length polymorphism (RFLP), which involves isolating DNA, digesting it with restriction enzymes, separating fragments via electrophoresis, and hybridizing with probes. It also describes polymerase chain reaction (PCR)-based markers like random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP), which allow amplification of genomic DNA regions. Sequence characterized amplified regions (SCARs) and cleaved amplified polymorphic sequences (CAPS) are also PCR-based marker techniques discussed.
The document provides information on RAPD (Random Amplified Polymorphic DNA) and RFLP (Restriction Fragment Length Polymorphism) molecular marker techniques. RAPD uses short random primers to amplify random DNA segments via PCR. RFLP involves digesting DNA with restriction enzymes, separating fragments by size, and detecting variants by probing fragmented DNA attached to membranes. Both techniques are used for applications like genetic diversity analysis, but RAPD requires less DNA and is quicker while RFLP has higher reproducibility and can detect allelic variants.
molecular marker and their types by gauravRoxxgaurav
DNA markers are used to identify genetic variations between individuals. There are several types of DNA markers including morphological markers, biochemical markers, cytological markers, and molecular markers. Molecular markers, also known as DNA markers, are the most widely used due to their abundance. There are two main types of molecular markers - non PCR-based markers like RFLPs, and PCR-based markers like RAPDs, AFLPs, SSRs, CAPs, SNPs, and SCARs. These marker techniques allow researchers to analyze genetic diversity and construct genetic maps to aid in plant breeding and disease identification.
This document provides an overview of molecular markers that can be used for crop improvement. It discusses different types of markers such as morphological, cytological, biochemical, DNA-based markers. DNA-based markers are further classified into hybridization-based markers like RFLP and PCR-based markers like RAPD, AFLP, SSR, ISSR, SNP. The document compares various marker techniques and provides their principles, strengths, weaknesses and applications in crop breeding programs. Molecular markers can be useful for tasks like hybrid purity testing, genetic diversity analysis, linkage mapping and marker-assisted selection.
RAPD markers are unstable and need to be converted to more reliable PCR-based markers like SCAR markers. This is done by isolating the polymorphic RAPD band, re-PCRing it, cloning and sequencing the product to design new specific primers with a higher annealing temperature, improving reliability. AFLP markers use restriction enzymes and linkers to amplify subsets of DNA fragments, allowing analysis of many loci simultaneously, though they are technically demanding. Marker assisted selection in plant and animal breeding can reduce time and costs by detecting traits at the DNA level early, before phenotypes appear.
The document discusses various types of genetic markers that can be used to measure genetic diversity, including random amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR), amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), microsatellites, minisatellites, and mitochondrial DNA markers. It provides details on how each type of marker works and its applications in studying genetic variation, relationships, and evolution.
1. Genetic markers are broadly grouped into classical markers (morphological, cytological, biochemical) and DNA/molecular markers. Molecular markers detect polymorphisms through techniques like RFLP, SSLP, AFLP, etc.
2. Molecular markers are DNA sequences that can detect polymorphisms through insertions, deletions, point mutations, and other variations. An ideal marker is co-dominant, evenly distributed in the genome, highly reproducible, and detects high levels of polymorphism.
3. Molecular markers are used for backcrossing, genetic mapping, QTL mapping, marker-assisted selection (MAS), and genome editing with CRISPR. They help solve
DNA markers can be used in plant breeding to identify plant varieties and track genetic inheritance. There are several types of DNA markers, including morphological markers, protein markers, RFLPs, RAPDs, AFLPs, SSRs, CAPS, SCARs, ISSRs, ESTs, STSs, and SNPs. DNA markers have advantages over morphological markers in that they are abundant, not influenced by environment, and can precisely track inheritance. The document discusses various DNA marker techniques and their applications in plant breeding, including genetic mapping, marker-assisted selection, and germplasm characterization.
This document discusses microsatellites and molecular markers. It begins by defining microsatellites as simple sequence repeats between 1-6 base pairs long. It then discusses different types of markers including morphological, biochemical, chromosomal, and genetic markers. The main part of the document focuses on different molecular markers used in genetics including RFLP, RAPD, AFLP, microsatellites, DNA fingerprinting, and SNPs. It provides details on what each marker is and how it is detected. The document concludes by discussing applications of molecular markers in areas like gene mapping, disease diagnosis, evolution studies, and animal breeding/selection.
1. Molecular markers are powerful tools that can detect genetic variation between individuals, populations, and species. They have revolutionized research on evolution, conservation, natural resource management, and genetic improvement programs.
2. There are two main types of molecular markers - those based on DNA hybridization and those based on PCR amplification. Examples include restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD).
3. Molecular markers have a variety of applications in fisheries, including identifying fish species, studying genetic variation and population structure in natural populations, comparing genetic variation between wild and hatchery populations, and marker-assisted selection in aquaculture.
The Matrix metalloproteinase-9 is involved in several pathologies. Its strong presence in ocular pathologies explains our interest for its genetic variation in cataract, glaucoma and retinoblastoma in Senegal. MMP9 is highly polymorphic with cataract and glaucoma. 77 mutations were noted with 21 haplotypes for the entire population. The haplotype diversity Hd is 0.831 and the nucleotide diversity Pi is 0.016; k = 17.395. The polymorphism of the Matrix metalloproteinase-9 gene is associated with all three diseases and SNP 3918249 is found in both cataract and glaucoma.
Biochemical and molecular markers for characterizationmithraa thirumalai
This document discusses various biochemical and molecular markers that can be used for plant genetic characterization. It begins by explaining that genetic resources can be characterized based on genotypes, phenotypes, and various molecular traits. It then discusses different types of molecular markers including DNA fingerprinting using microsatellites, RAPDs, AFLPs, allozymes, and other protein markers. It provides details on how many of these techniques work, such as the PCR process for RAPDs and AFLPs. The document also discusses using these molecular markers for assessing population structure, assembling core collections, and addressing various taxonomic and phylogenetic questions.
RAPD (Random Amplification of Polymorphic DNA) is a PCR-based molecular marker technique that involves using short, arbitrary nucleotide primers to randomly amplify genomic DNA fragments. These fragments can then be analyzed as genetic markers. RAPD works by using a single short primer to amplify random DNA sequences from a complex template. Variations in priming sites between individuals result in presence or absence of bands that can be used to analyze genetic relationships. The technique is fast, inexpensive and does not require prior DNA sequence knowledge, but results can lack reproducibility between laboratories.
Methods for characterization of animal genomes(snp,str,qtl,rflp,rapd)Dr Vijayata choudhary
The document discusses several methods for characterizing animal genomes, including Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD), Single Nucleotide Polymorphisms (SNPs), Short Tandem Repeats (STRs), and Quantitative Trait Loci (QTL). It provides detailed descriptions of each method, including how they are detected and analyzed as well as their applications in areas like genetic mapping, diversity studies, and phylogenetic analysis.
This document provides information about molecular marker techniques RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA). RFLP is a non-PCR based technique that involves digesting genomic DNA with restriction enzymes, separating fragments via gel electrophoresis, and detecting variations. RAPD is a PCR-based technique that uses random primers to amplify random DNA segments, which are then separated and visualized. Both techniques can be used for genetic mapping, germplasm characterization, and other applications. Key differences between the two techniques include RFLP detecting fewer loci but being more reliable, requiring larger DNA quantities, and using species-specific probes.
Molecular marker analysis of A few Capsicum annum varietiesAnkitha Hirematha
The hybrid variety and parental varieties among the 3 chilly varieties were identified by finding out the genetic polymorphism between them. It helps to identify different plant varieties, disputed plant varieties, genetic polymorphism between intraspecific crosses of plants and also to protect Plant Breeder’s Rights (PBR). Based on banding pattern on gel, identification of KA, KS and HK chilly varieties using SSR & ISSR markers was successfully carried out.
TYPES OF MOLECULAR MARKERS,ITS ADVANTAGES AND DISADVANTAGESANFAS KT
Types of molecular markers (genetics)
ITS ADVANTAGES AND DISADVANTAGES
What is a genetic marker?
RFLP: Restriction fragment length polymorphism
AFLP: Amplified fragment length polymorphism
RAPD: Random amplification of polymorphic DNA
ISSR: Inter simple sequence repeat
STR: Short tandem repeats
SCAR: Sequence characterized amplified region
SNP: Single nucleotide polymorphism
SSR: Simple sequence repeat
Molecular markers are DNA sequences that can be easily detected and whose inheritance can be monitored. They are based on natural polymorphisms and allow studying the inheritance of genes. Common types of molecular markers include RFLPs, RAPDs, AFLPs, SSRs, and SNPs. RFLPs use restriction enzymes to detect differences in fragment lengths. RAPDs use random primers to detect sequence polymorphisms. AFLPs selectively amplify restriction fragments to detect length differences. SSRs detect variability in simple sequence repeats. Molecular markers are useful for applications like gene mapping, phylogenetic studies, and analyzing genetic diversity.
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
Genetic markers are sequences of DNA located at specific positions on chromosomes that can be associated with particular traits. Common types of genetic markers include RFLP, AFLP, RAPD, SNP, and VNTR. These markers allow researchers to locate genes associated with diseases and trace inheritance patterns. While harmless themselves, genetic markers enable the positioning of disease genes along chromosomes to better understand inheritance of traits and diseases.
Markers: Based on hybridization and PCRSachin Kumar
This document discusses different types of molecular markers used in genetics. It describes markers based on nucleic acid hybridization, such as restriction fragment length polymorphism (RFLP), which involves isolating DNA, digesting it with restriction enzymes, separating fragments via electrophoresis, and hybridizing with probes. It also describes polymerase chain reaction (PCR)-based markers like random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP), which allow amplification of genomic DNA regions. Sequence characterized amplified regions (SCARs) and cleaved amplified polymorphic sequences (CAPS) are also PCR-based marker techniques discussed.
The document provides information on RAPD (Random Amplified Polymorphic DNA) and RFLP (Restriction Fragment Length Polymorphism) molecular marker techniques. RAPD uses short random primers to amplify random DNA segments via PCR. RFLP involves digesting DNA with restriction enzymes, separating fragments by size, and detecting variants by probing fragmented DNA attached to membranes. Both techniques are used for applications like genetic diversity analysis, but RAPD requires less DNA and is quicker while RFLP has higher reproducibility and can detect allelic variants.
molecular marker and their types by gauravRoxxgaurav
DNA markers are used to identify genetic variations between individuals. There are several types of DNA markers including morphological markers, biochemical markers, cytological markers, and molecular markers. Molecular markers, also known as DNA markers, are the most widely used due to their abundance. There are two main types of molecular markers - non PCR-based markers like RFLPs, and PCR-based markers like RAPDs, AFLPs, SSRs, CAPs, SNPs, and SCARs. These marker techniques allow researchers to analyze genetic diversity and construct genetic maps to aid in plant breeding and disease identification.
This document provides an overview of molecular markers that can be used for crop improvement. It discusses different types of markers such as morphological, cytological, biochemical, DNA-based markers. DNA-based markers are further classified into hybridization-based markers like RFLP and PCR-based markers like RAPD, AFLP, SSR, ISSR, SNP. The document compares various marker techniques and provides their principles, strengths, weaknesses and applications in crop breeding programs. Molecular markers can be useful for tasks like hybrid purity testing, genetic diversity analysis, linkage mapping and marker-assisted selection.
RAPD markers are unstable and need to be converted to more reliable PCR-based markers like SCAR markers. This is done by isolating the polymorphic RAPD band, re-PCRing it, cloning and sequencing the product to design new specific primers with a higher annealing temperature, improving reliability. AFLP markers use restriction enzymes and linkers to amplify subsets of DNA fragments, allowing analysis of many loci simultaneously, though they are technically demanding. Marker assisted selection in plant and animal breeding can reduce time and costs by detecting traits at the DNA level early, before phenotypes appear.
The document discusses various types of genetic markers that can be used to measure genetic diversity, including random amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR), amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), microsatellites, minisatellites, and mitochondrial DNA markers. It provides details on how each type of marker works and its applications in studying genetic variation, relationships, and evolution.
1. Genetic markers are broadly grouped into classical markers (morphological, cytological, biochemical) and DNA/molecular markers. Molecular markers detect polymorphisms through techniques like RFLP, SSLP, AFLP, etc.
2. Molecular markers are DNA sequences that can detect polymorphisms through insertions, deletions, point mutations, and other variations. An ideal marker is co-dominant, evenly distributed in the genome, highly reproducible, and detects high levels of polymorphism.
3. Molecular markers are used for backcrossing, genetic mapping, QTL mapping, marker-assisted selection (MAS), and genome editing with CRISPR. They help solve
DNA markers can be used in plant breeding to identify plant varieties and track genetic inheritance. There are several types of DNA markers, including morphological markers, protein markers, RFLPs, RAPDs, AFLPs, SSRs, CAPS, SCARs, ISSRs, ESTs, STSs, and SNPs. DNA markers have advantages over morphological markers in that they are abundant, not influenced by environment, and can precisely track inheritance. The document discusses various DNA marker techniques and their applications in plant breeding, including genetic mapping, marker-assisted selection, and germplasm characterization.
This document discusses microsatellites and molecular markers. It begins by defining microsatellites as simple sequence repeats between 1-6 base pairs long. It then discusses different types of markers including morphological, biochemical, chromosomal, and genetic markers. The main part of the document focuses on different molecular markers used in genetics including RFLP, RAPD, AFLP, microsatellites, DNA fingerprinting, and SNPs. It provides details on what each marker is and how it is detected. The document concludes by discussing applications of molecular markers in areas like gene mapping, disease diagnosis, evolution studies, and animal breeding/selection.
1. Molecular markers are powerful tools that can detect genetic variation between individuals, populations, and species. They have revolutionized research on evolution, conservation, natural resource management, and genetic improvement programs.
2. There are two main types of molecular markers - those based on DNA hybridization and those based on PCR amplification. Examples include restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD).
3. Molecular markers have a variety of applications in fisheries, including identifying fish species, studying genetic variation and population structure in natural populations, comparing genetic variation between wild and hatchery populations, and marker-assisted selection in aquaculture.
The Matrix metalloproteinase-9 is involved in several pathologies. Its strong presence in ocular pathologies explains our interest for its genetic variation in cataract, glaucoma and retinoblastoma in Senegal. MMP9 is highly polymorphic with cataract and glaucoma. 77 mutations were noted with 21 haplotypes for the entire population. The haplotype diversity Hd is 0.831 and the nucleotide diversity Pi is 0.016; k = 17.395. The polymorphism of the Matrix metalloproteinase-9 gene is associated with all three diseases and SNP 3918249 is found in both cataract and glaucoma.
The document discusses DNA markers for genetic variability studies in fish. It describes several types of genetic variation, including single nucleotide polymorphisms (SNPs) and insertions/deletions, that can be revealed using DNA marker technology. It also discusses different types of molecular markers, such as microsatellites and DNA barcoding using the CO1 gene, that can help characterize genetic variation within and among species.
DNA SEQUENCING METHODS AND STRATEGIES FOR GENOME SEQUENCINGPuneet Kulyana
This presentation will give you a brief idea about the various DNA sequencing methods and various strategies used for genome sequencing and much more vital information related to gene expression and analysis
This document discusses molecular genetic methods such as polymerase chain reaction (PCR), DNA sequencing, DNA fingerprinting, and single nucleotide polymorphisms. It provides details on how each method works, including how PCR amplifies DNA, the process of manual and automated DNA sequencing, using variable number tandem repeats as markers for DNA fingerprinting, and applications of these molecular genetic techniques.
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.
Microsatellites are tandemly repeated DNA sequences with repeat units of 1-6 base pairs. They are highly polymorphic due to variations in the number of repeats between individuals. Microsatellites can be analyzed using PCR and electrophoresis to differentiate alleles and study genetic diversity, population structure, and parentage. A genetic map of microsatellites was constructed for turbot fish to enable future quantitative trait locus identification and evolutionary studies. Microsatellites are a powerful tool for various areas of genetics research.
SAGE- Serial Analysis of Gene ExpressionAashish Patel
Serial Analysis of Gene Expression (SAGE) is a method to quantify gene expression in cells. It involves extracting short sequence tags from mRNA transcripts and concatenating them for efficient sequencing. This allows simultaneous analysis of thousands of transcripts. SAGE provides quantitative gene expression data without prior knowledge of genes and can identify differentially expressed genes between cell types or conditions. While powerful, it requires substantial sequencing and computational analysis of large datasets.
TILLING is a general reverse genetic technique that combines chemical mutagenesis with PCR based screening to identify point mutations in regions of interest.
Western Blotting Of Camkii Β And T 287Beth Salazar
1. Tomato production is affected by various bacterial, fungal and viral diseases which can cause considerable yield losses.
2. One of the most devastating diseases is tomato leaf curl disease (ToLCD), caused by geminiviruses, which is increasing worldwide and poses a major constraint to tomato production in India.
3. ToLCD causes serious yield losses according to studies from the 1940s and more recently. Effective management strategies are needed to control this and other diseases threatening tomato production.
This document discusses genomic and cDNA libraries. Genomic libraries are made from genomic DNA and represent all genes in an organism. They require a minimum number of clones to ensure all genes are captured. cDNA libraries are made from mRNA and represent expressed genes, avoiding introns. Key steps in making cDNA libraries include mRNA isolation, cDNA synthesis, addition of linkers, and ligation into a vector. Screening methods to identify clones of interest include hybridization, expression screening, and hybrid arrest/release.
This study investigated genetic variations among four sheep breeds related to fecundity and weight gain using microsatellite markers. Blood samples were collected from 40 female sheep from four breeds and were used to extract DNA. Five microsatellites linked to genes for litter size (Fec gene) and fat deposition/weight gain (Ob gene) were selected. Polymerase chain reaction and automated sequencing were used to analyze polymorphisms in the microsatellite markers. Serum was also isolated from blood samples and tested via ELISA to measure levels of LH and leptin hormones. The aim was to analyze the effects of genetic differences among the breeds for two traits: fecundity and weight gain.
Discover Therapeutic Aptamers For Vegf165 And EgfrJessica Myers
The document discusses discovering therapeutic aptamers for VEGF165 and EGFR through in vitro selection. Aptamers are ssDNA or RNA oligonucleotides that bind targets with high selectivity and specificity due to their well-defined tertiary structures. They have advantages over antibodies such as not requiring animals or cell culture for selection. The author aims to select aptamers for VEGF165 and EGFR to potentially use as therapeutic agents.
This document summarizes a study that identified single nucleotide polymorphisms (SNPs) in bread wheat. Researchers sequenced the genomes of 16 Australian wheat varieties and identified over 4 million intervarietal SNPs. SNP calling and validation was performed using various software and validation methods. Analysis of the SNPs showed transitions were more common than transversions, and SNP density varied across chromosomes with some genes located in low-SNP regions. The identified SNPs can be used for phylogenetic analysis, marker-assisted selection, and gene mapping in wheat.
This document provides an overview of the TILLING (Targeted Induced Local Lesions IN Genome) technique. TILLING combines chemical mutagenesis with PCR screening to identify point mutations in genes of interest. It has been used successfully in plants like Arabidopsis thaliana and Lotus japonicus to generate allelic series and study gene function. The document discusses the TILLING methodology, including EMS mutagenesis to generate populations, DNA pooling, PCR amplification of target regions, detection of mutations via CEL1 enzyme cleavage, and sequencing. Advantages of TILLING include its applicability to any organism and ability to saturate genes with mutations without excessive DNA damage. Eco-TILLING is also
Microsatellite are powerful DNA markers for quantifying genetic variations within & between populations of a species, also called as STR, SSR, VNTR. Tandemly repeated DNA sequences with the repeat/size of 1 – 6 bases repeated several times
TILLING is a non-transgenic method for identifying mutations in a gene of interest from a mutagenized population. It involves chemical mutagenesis followed by PCR amplification of the target region and cleavage of heteroduplexes formed between mutant and wildtype sequences by CEL I endonuclease. The cleavage products are then analyzed to detect mutations. EcoTILLING is a modification that detects natural polymorphisms between accessions without mutagenesis. TILLING is a high-throughput and cost-effective method for reverse genetics that does not require plant transformation.
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdfamzonknr
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE
BACKGROUND CONTEXT Lab: Differential Expression Differential gene expression provides
the ability for a cell or organism to respond to a constantly changing external environment. The
specific constellation of proteins required for optimal function and growth varies with cellular
age and environmental context. Thus, protein production is carefully regulated by multiple
mechanisms that modulate both transcriptional and translational pathways. Control of
transcription initiation by RNA polymerase is a predominant mechanism for regulating
expression of specific proteins, presumably because it provides maximal conservation of energy
for the cell. We can often observe the consequence of differential transcription due to the
presence or absence of particular proteins or the growth in particular environments. Control can
also occur at translation; the mRNA is synthesized, but only in certain circumstances is it
translated. Control can also occur at the level of protein function; the protein is inactive, or
activity is not observed due to the lack of the substrate. In this lab we will observe differential
expression of two different genes encoded on plasmids. We will analyze transcriptional activity,
translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often
have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission)
and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to
stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to
maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori:
the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also
contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening
4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant.
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdfamzonknr
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE
BACKGROUND CONTEXT Lab: Differential Expression Differential gene expression provides
the ability for a cell or organism to respond to a constantly changing external environment. The
specific constellation of proteins required for optimal function and growth varies with cellular
age and environmental context. Thus, protein production is carefully regulated by multiple
mechanisms that modulate both transcriptional and translational pathways. Control of
transcription initiation by RNA polymerase is a predominant mechanism for regulating
expression of specific proteins, presumably because it provides maximal conservation of energy
for the cell. We can often observe the consequence of differential transcription due to the
presence or absence of particular proteins or the growth in particular environments. Control can
also occur at translation; the mRNA is synthesized, but only in certain circumstances is it
translated. Control can also occur at the level of protein function; the protein is inactive, or
activity is not observed due to the lack of the substrate. In this lab we will observe differential
expression of two different genes encoded on plasmids. We will analyze transcriptional activity,
translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often
have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission)
and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to
stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to
maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori:
the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also
contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening
4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant.
An honest effort to present molecular marker in easiest way both informative and conceptual. Hybridization based (non-PCR) and PCR based markers are discussed to the point with suitable diagram.
EWOCS-I: The catalog of X-ray sources in Westerlund 1 from the Extended Weste...Sérgio Sacani
Context. With a mass exceeding several 104 M⊙ and a rich and dense population of massive stars, supermassive young star clusters
represent the most massive star-forming environment that is dominated by the feedback from massive stars and gravitational interactions
among stars.
Aims. In this paper we present the Extended Westerlund 1 and 2 Open Clusters Survey (EWOCS) project, which aims to investigate
the influence of the starburst environment on the formation of stars and planets, and on the evolution of both low and high mass stars.
The primary targets of this project are Westerlund 1 and 2, the closest supermassive star clusters to the Sun.
Methods. The project is based primarily on recent observations conducted with the Chandra and JWST observatories. Specifically,
the Chandra survey of Westerlund 1 consists of 36 new ACIS-I observations, nearly co-pointed, for a total exposure time of 1 Msec.
Additionally, we included 8 archival Chandra/ACIS-S observations. This paper presents the resulting catalog of X-ray sources within
and around Westerlund 1. Sources were detected by combining various existing methods, and photon extraction and source validation
were carried out using the ACIS-Extract software.
Results. The EWOCS X-ray catalog comprises 5963 validated sources out of the 9420 initially provided to ACIS-Extract, reaching a
photon flux threshold of approximately 2 × 10−8 photons cm−2
s
−1
. The X-ray sources exhibit a highly concentrated spatial distribution,
with 1075 sources located within the central 1 arcmin. We have successfully detected X-ray emissions from 126 out of the 166 known
massive stars of the cluster, and we have collected over 71 000 photons from the magnetar CXO J164710.20-455217.
Mending Clothing to Support Sustainable Fashion_CIMaR 2024.pdfSelcen Ozturkcan
Ozturkcan, S., Berndt, A., & Angelakis, A. (2024). Mending clothing to support sustainable fashion. Presented at the 31st Annual Conference by the Consortium for International Marketing Research (CIMaR), 10-13 Jun 2024, University of Gävle, Sweden.
The debris of the ‘last major merger’ is dynamically youngSérgio Sacani
The Milky Way’s (MW) inner stellar halo contains an [Fe/H]-rich component with highly eccentric orbits, often referred to as the
‘last major merger.’ Hypotheses for the origin of this component include Gaia-Sausage/Enceladus (GSE), where the progenitor
collided with the MW proto-disc 8–11 Gyr ago, and the Virgo Radial Merger (VRM), where the progenitor collided with the
MW disc within the last 3 Gyr. These two scenarios make different predictions about observable structure in local phase space,
because the morphology of debris depends on how long it has had to phase mix. The recently identified phase-space folds in Gaia
DR3 have positive caustic velocities, making them fundamentally different than the phase-mixed chevrons found in simulations
at late times. Roughly 20 per cent of the stars in the prograde local stellar halo are associated with the observed caustics. Based
on a simple phase-mixing model, the observed number of caustics are consistent with a merger that occurred 1–2 Gyr ago.
We also compare the observed phase-space distribution to FIRE-2 Latte simulations of GSE-like mergers, using a quantitative
measurement of phase mixing (2D causticality). The observed local phase-space distribution best matches the simulated data
1–2 Gyr after collision, and certainly not later than 3 Gyr. This is further evidence that the progenitor of the ‘last major merger’
did not collide with the MW proto-disc at early times, as is thought for the GSE, but instead collided with the MW disc within
the last few Gyr, consistent with the body of work surrounding the VRM.
Authoring a personal GPT for your research and practice: How we created the Q...Leonel Morgado
Thematic analysis in qualitative research is a time-consuming and systematic task, typically done using teams. Team members must ground their activities on common understandings of the major concepts underlying the thematic analysis, and define criteria for its development. However, conceptual misunderstandings, equivocations, and lack of adherence to criteria are challenges to the quality and speed of this process. Given the distributed and uncertain nature of this process, we wondered if the tasks in thematic analysis could be supported by readily available artificial intelligence chatbots. Our early efforts point to potential benefits: not just saving time in the coding process but better adherence to criteria and grounding, by increasing triangulation between humans and artificial intelligence. This tutorial will provide a description and demonstration of the process we followed, as two academic researchers, to develop a custom ChatGPT to assist with qualitative coding in the thematic data analysis process of immersive learning accounts in a survey of the academic literature: QUAL-E Immersive Learning Thematic Analysis Helper. In the hands-on time, participants will try out QUAL-E and develop their ideas for their own qualitative coding ChatGPT. Participants that have the paid ChatGPT Plus subscription can create a draft of their assistants. The organizers will provide course materials and slide deck that participants will be able to utilize to continue development of their custom GPT. The paid subscription to ChatGPT Plus is not required to participate in this workshop, just for trying out personal GPTs during it.
The cost of acquiring information by natural selectionCarl Bergstrom
This is a short talk that I gave at the Banff International Research Station workshop on Modeling and Theory in Population Biology. The idea is to try to understand how the burden of natural selection relates to the amount of information that selection puts into the genome.
It's based on the first part of this research paper:
The cost of information acquisition by natural selection
Ryan Seamus McGee, Olivia Kosterlitz, Artem Kaznatcheev, Benjamin Kerr, Carl T. Bergstrom
bioRxiv 2022.07.02.498577; doi: https://doi.org/10.1101/2022.07.02.498577
PPT on Direct Seeded Rice presented at the three-day 'Training and Validation Workshop on Modules of Climate Smart Agriculture (CSA) Technologies in South Asia' workshop on April 22, 2024.
The technology uses reclaimed CO₂ as the dyeing medium in a closed loop process. When pressurized, CO₂ becomes supercritical (SC-CO₂). In this state CO₂ has a very high solvent power, allowing the dye to dissolve easily.
ESR spectroscopy in liquid food and beverages.pptxPRIYANKA PATEL
With increasing population, people need to rely on packaged food stuffs. Packaging of food materials requires the preservation of food. There are various methods for the treatment of food to preserve them and irradiation treatment of food is one of them. It is the most common and the most harmless method for the food preservation as it does not alter the necessary micronutrients of food materials. Although irradiated food doesn’t cause any harm to the human health but still the quality assessment of food is required to provide consumers with necessary information about the food. ESR spectroscopy is the most sophisticated way to investigate the quality of the food and the free radicals induced during the processing of the food. ESR spin trapping technique is useful for the detection of highly unstable radicals in the food. The antioxidant capability of liquid food and beverages in mainly performed by spin trapping technique.
Sexuality - Issues, Attitude and Behaviour - Applied Social Psychology - Psyc...PsychoTech Services
A proprietary approach developed by bringing together the best of learning theories from Psychology, design principles from the world of visualization, and pedagogical methods from over a decade of training experience, that enables you to: Learn better, faster!
ESA/ACT Science Coffee: Diego Blas - Gravitational wave detection with orbita...Advanced-Concepts-Team
Presentation in the Science Coffee of the Advanced Concepts Team of the European Space Agency on the 07.06.2024.
Speaker: Diego Blas (IFAE/ICREA)
Title: Gravitational wave detection with orbital motion of Moon and artificial
Abstract:
In this talk I will describe some recent ideas to find gravitational waves from supermassive black holes or of primordial origin by studying their secular effect on the orbital motion of the Moon or satellites that are laser ranged.
Gadgets for management of stored product pests_Dr.UPR.pdf
Classes of-molecular-markers
1. -1-
Classes of Molecular Markers
Isozymes
Gel-based approach
Protein analyzed
Allelic difference detected
o Alleles migrate at different rate in starch gel
Locus-by-locus approach
RFLP – Restriction Fragment Length Polymorphism
Hybridization-based approach
RFLP recognized by specific clone/enzyme combinations
Locus-by-locus approach
RAPD – Randomly Amplified Polymorphic DNA
Gel-based approach
PCR amplification of fragment
10mer oligonucleotides recognize inverted repeats
o Fragment between the inverted repeats is amplified
Genome-wide approach
AFLP – Amplified Fragment Length Polymorphism
2. -2-
Gel- or capillary-based approach
Combines restriction disgestion with PCR amplification
Selectively amplifies a subset of possible genomic fragments
Genome-wide approach
Microsattelites
Gel- or capillary-based approach
Mostly based on differences in number of di- or tri nucleotide
repeats at a specific locus
Locus-by-locus approach
SNP – Single Nucleotide Polymorphism
Multiple detection procedures
Based on nucleotide differences between two alleles
Locus-by-locus approach
3. -3-
Isozymes
Enzyme Systems
Rbcs – Ribulose bisphosphate carboxylase, small subunit
Skdh – Shikimate dehydrogenase
Prx – Peroxidase
Me – Malic enzyme
Mdh – Malate dehyrogenase
Diap – Diaphorase
Lap – Leucine aminopeptidase
Matrix
Starch
Typically five samples per gel
Scoring Protocol
Most frequent allele is given the designation “100”
Other alleles designated by distance in millimeters from the
most frequent allele
Allele nomenclature examples from P. vulgaris
o Me98
– 2 millimeters slower in the gel
o Me100
– most frequent allele
o Me105
– 5 millimeters faster in the gel
4. -4-
RFLP Clones, Enzymes, and
Informative Hybridizations
1. Source of clones
a. Random clones – poor choice because they often contain
repetitive DNA
b. cDNA clones – contain only expressed sequences; often
low copy number
c. PstI clones – based on the concept that expressed (low
copy number) genes are undermethylated; therefore the
methylation sensitive enzyme PstI will only cut in regions
where expressed genes reside
2. Enzymes – need to screen parents by digestions with a series
of enzymes to find polymorphic hybridizations
3. Informative hybridizations – those specific restriction
enzyme/clone combinations that are polymorphic will be
informative for mapping a segregating population
5. -5-
Restriction-modification system
Restriction enzyme - cleaves DNA at a specific sequence
Methylase - protects the host DNA from being cleaved
The E. coli Type RI (EcoRI) restriction enzyme site is:
m
5' - G A A T T C - 3'
3' - C T T A A G - 5'
m
(Remember, the enzyme will not cut
if the 3' A is methylated.)
5' - G - 3' 5' - A A T T C - 3'
3' - C T T A A - 5' 3' - G - 5'
6. -6-
Restriction enzyme = EcoRI cuts between the G and A. in the
sequence GAATTC
Methylase = EcoRI methylase protects the EcoRI by adding a
methyl group to the 3'-adenine
How the system works
1. Growth of foreign DNA (such as virus DNA) is restricted in the
cell by the restriction enzyme
2. The bacterial DNA is modified by the methylase to prevent
cleavage by the restriction enzyme.
7. -7-
Methylation of Plant DNA and its Effect on Restriction
Digestion
Grenbaum et al. Methylation of cytosines in higher plants
Nature 297:86 August 1981
5-methyl cytosine (5mC) is found to be a component of plant DNA
much more frequently than animal DNA
% Cytosine as 5mC
Animals 2-7
Plants >25
Why???
1. 5mC occurs at 70-80% of the 5'-CG-3' dinucleotides. What are
the occurrences of the dinucleotide in the two kingdoms?
% dinucleotides
as 5-'CG-3
Animals 0.5-1.0
Plants 3.4
2. 5mC also occurs at the 5'-CXG-3' sequence in plants but
does not occur in animals. How often are these sequences
methylated in plants?
Sequence % C methylated
5'-CAG-3 80
5'-CCG-3 50
8. -8-
Do Not Use These Enzymes
to Analyze Plant DNA
1. Site has 5'-CXG-3'
PstI 5'-C*TGCAG-3'
PvuII 5'-CAGC*TG-3'
MspI 5'-C*CGG-3
EcoRII 5'-CC*WGG-3'
(W = A or T)
2. C at or near end of site;if
next base in DNA is G, it will
be methylated
BamHI 5'-GGATC*C-3
KpnI 5'-GGTACC*-3
Use These Enzymes Instead
DraI 5'-TTTAAA-3'
EcoRV 5-'GA*TATC-3
EcoRI 5-GAA*TTC*-3'
HindIII 5'-A*AGC*TT-3'
XbaI 5'-TC*TAGA*-3'
9. -9-
RAPD Markers
A PCR-based marker system
Amplifies inverted repeats in the genome
Can generate larger number of markers than RFLP in a
short period of time
Popular because easy to do and does not require
radioisotopes
10. -10-
AFLP Procedure
1. Digest sample DNA with restriction enzymes EcoRI and
MseI.
5'----GAATTCN------------------------NTTAA----3'
3'----CTTAAGN------------------------NAATT----5"
AATTCN------------------------NT
GN------------------------NAAT
2. Anneal EcoRI and MseI adaptors to restriction products.
??????AATTCN-------------------------NTTA??????
??????TTAAGN-------------------------NAAT??????
(?????? = unknown sequences that are unique for the
companies primers)
3. Preselect products by PCR amplification with "EcoRI + A"
and "MseI +C" oligonucleotide primers.
EcoRI Primer+A
????????AATTCN---------------------NTTA????????
????????TTAAGN---------------------NAAT????????
C+MseI Primer
11. -11-
4. Selectively amplify preselect PCR products by using "EcoRI +
3" and "MseI +3" oligonucleotide primers.
EcoRI Primer+AAC
????????AATTCA----------------------GTTA????????
????????TTAAGT----------------------CAAT????????
AAC+MseI Primer
5. Separate fragments by denaturing polyacrylamide gel
electrophoresis
12. -12-
Microsatellites
Also called:
SSR = Simple Sequence Repeats
SSLP = Simple Sequence Length Polymorphisms
STMS = Sequence Tagged Microsatellites)
What are they?
Typically repeated di-, tri or tetranucleotide sequences
Examples:
AG4 (AG AG AG AG)
CGA3 (CGA CGA CGA)
GATA5 (GATA GATA GATA GATA)
13. -13-
How are they discovered?
Sequenced genes in databases searched for repeats
Repeat olignucleotide probes used to screen random clone
library
Rice example [Mol Gen Genet (1996) 252:597]:
Library:
300-600 bp fragments generated by shearing DNA
fragments cloned into plasmid vector
Probes:
GA13
CGG9
ATC9
ATT9
14. -14-
The Microsatellite Application
Primers highly specific to sequences flanking the repeat are
designed
Individual DNA samples are amplified
Products compared by polyacrylamide or agarose gels using
staining producedures
Recently fluorescently labeled primers are used and products
are analyzed with laser technology (gel or capillary)
Differences in two samples are represented by size differences
of amplified fragments
The size difference is the polymorphism
Single-base pair polymorphisms can be detected
15. -15-
Advantages of Microsatellites
Genetic
Many loci in the genome (goal: 30,000 in humans)
Randomly distributed in a genome
Extensive polymorphism within a species
Many act as codominant markers
Technical
Generally reproducible from lab to lab
Small amounts of target DNA needed
Can be automated
Mulitplexing possible
Disdvantages of Microsatellites
Genetic
Null alleles at a specific locus result in a dominant marker
and heterozygotes can not be scored
Technical
Very high development costs
16. -16-
Arabidopsis Microsatellite Summary
Bioinformatics (2004) 20:1081-1086
93% of microsattelites are trinucleotides
Repeats less frequent in coding than non-coding regioins
Microsatellite more abundant in 5’ region of gene
AG and AAG were the most frequent repeats
Studied 1140 full length genes with an AG or AAG repeat in 5”
region
o These repeats often associated with function of gene
o Based on proximity to beginning of first exon
These genes more often performed a molecular
activitythan genes without a repeat
17. -17-
Modern Approach to Microsatellite Discovery
Collect sequence data
EST
Will be in genes that are in the low copy region of the
genome
Random genome reads
May be low copy or high copy regions of the genome
Soybean EST Discovery
Song et al. Crop Science 50:1950 (2010)
Screened
Whole genome sequence
ESTs
Minimum of 5 copies of the repeat
Genome sequence EST sequences
Total 210,990 33,327
Dinucleotide 168,625 20,161
Trinucleotide 38,411 12,440
Tetranucleotide 3,954 636
Most abundant in genome sequence
(AT)n, (ATT)n, (AAAT)n
o 61,458
Testing the approach
1034 primer pairs developed
Screened 7 diverse genotypes
o 94.6% (978) amplified a single PCR product
77.2% (798) were polymorphic
18. -18-
Single Nucleotide Polymorphisms (SNPs)
Single nucleotides are the smallest unit of mutation
Single nucleotides therefore are the smallest polymorphic unit
SNP = single nucleotide polymorphisms
Uses:
Detect level of variation within a species
Follow patterns of evolution
Mark genes
Distinguish alleles of “disease” genes
Create designer pharmaceuticals
The SNP Consortium, Ltd
Goal
Identify 300,000 human SNPs by Dec. 31, 2001
Progress
7,365 (Dec. 31, 1999)
1.2 million detected (Jan 26, 2002) and mapped in one
population
1.1 high quality mapped in three populations; 1 SNP every
5 kilobases
19. -19-
Detecting DNA Polymorphisms
DNA molecules greater than 10 base pairs contains essentially
the same mass-to-charge ratio
Procedure that separates the molecules based on mass alone will
uncover DNA polymorphisms
Current Procedure
Gel electrophoresis
Emerging Procedures
Capillary array electrophoresis
Matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF MS)
New technology in 2002 that has not really caught on
Advantage of New Procedures
SPEEEEEEEED
20. -20-
Gel Electrophoresis
Most widely adapted technique for detecting polymorphism.
Principle
Samples loaded into a gel and allowed to migrate in an
electric field
DNA is positively charged and samples migrate toward
the negative pole
Separation of the molecules is strictly based on size
smallest fragments move farther in the gel
They can navigate through the small pores in the gel
faster than large molecules.
Types of Gel Matrices
Agarose
Polyacrylamide
Separation is function of the polymer concentration
Agarose Polyacrylamide
% Resolution (kb) % Resolution (bp)
0.9 0.5 - 7.0 3.5 1000 - 2000
1.2 0.4 - 6.0 5.0 80 - 500
1.5 0.2 - 3.0 8.0 60 - 400
2.0 0.1 - 2.0 12.0 40 - 200
21. -21-
What polymer do you choose?
Polymer choice is based on the size range of fragment
Agarose
RFLP
RAPD
Polyacrylamide
Microsatellites
AFLPs
Observing the Polymorphisms
Dye Detection
Ethidium bromide (Agarose)
RAPD
Silver nitrate (Polyacrylamide)
Microsatellites
AFLPs
Authoradiography Detection
RFLP (more later)
Microsatellites
AFLP
22. -22-
Laser Technology Detection
Applied to the microsatellites
Procedure
PCR primer is labelled with a fluorescent dye
Samples are separated in a polyacrylamide gel
Fragments are detected by a laser as they flow through
the bottom of the gel
Computer programs output data as size difference
RFLPs and Southern Hybridizations
Restriction digested DNA separated in agarose gel
DNA transferred to a nylon membrane
Membrane contains a faithful representation of the
fragment distribution found in the gel
Probe is hybridized to the membrane
Unbound probe is removed by a series of stringent
washes
Membrane is exposed autoradiographic film
Polymorphisms observed
23. -23-
Capillary Array Electrophoresis
Small DNA fragments are rapidly separated in narrow capillaries.
Heat is lost rapidly from thin capillaries
Thus, molecules can be separated rapidly using high voltage.
High voltage separation greatly speeds sample processing
Capillaries arrays have reached the market
Eight capillary array (Beckman-Coulter)
96 capillary array (Applied Biosystems)
Advantage
Multipe samples simultaneously introduce into each
arrays
Size detected by laser technology
24. -24-
Advantages and Disadvantages of Different
Marker Systems
Marker Advantages Disadvantages
Classical Easily scored; new
equipment not needed;
traits of economic
importance
Time to score;
environmental influences;
limited for most species;
mostly dominant
Isozyme Easy to apply; codominant;
multiple alleles
Limited number of assays
available; scored at only one
developmental stage
RFLP Codominant; highly
repeatable; maps available
for major species
Limited polymorphisms, time
consuming; relatively
expensive; requires
radioactivity
RAPD Easy technique; rapidly
screening; no radioactivity
Low lab-to-lab repeatability;
mostly dominant
AFLP Large number of
polymorphisms per
sample; easy to apply
(once mastered)
Difficult to learn; radioactivity
might be used; expensive;
time-consuming; mostly
dominant
Microsatellites Highly polymorphic; easy
to apply; large number of
loci available; mostly co-
dominant
Slow and expensive
development costs
SNP High accuracy; potentially
a very large number; co-
dominant
Expensive to discover;
assays are currently
expensive
25. -25-
What to consider when choosing a marker
system
Genetic nature of the system
Radioactivity
Cost
Reproducibility
Time (Discovery and application)
Efficiency (Time invested vs. breadth of results)
Is a system in place??? (Map available; technique defined)
Reason for experiments (Marker development vs. application)
Amount of polymorphisms
Technical skill needed
26. -26-
Types of Nucleic Acid Hybridizations
1. Southern hybridization - hybridization of a probe to filter
bound DNA; the DNA is typically transferred to the filter from a
gel
2. Northern hybridization - hybridization of a probe to filter
bound RNA; the RNA is typically transferred to the filter from a
gel
Probe - a single-stranded nucleic acid that has been radiolabelled
and is used to identify a complimentary nucleic acid sequence
that is membrane bound
The following steps describe the Southern transfer procedure.
1. Digest DNA with the restriction enzyme of choice.
2. Load the digestion onto a agarose gel and apply an electrical
current. DNA is negatively charged so it migrates toward the "+"
pole. The distance a specific fragment migrates is inversely
proportional to the fragment size.
3. Stain the gel with EtBr, a fluorescent dye which intercalates into
the DNA molecule. The DNA can be visualized with a UV light
source to assess the completeness of the digestion.
4. Denature the double-stranded fragments by soaking the gel in
alkali (>0.4 M NaOH)
5. Transfer the DNA to a filter membrane (nylon or nitrocellulose)
by capillary action.
27. -27-
Steps in Southern Hybridization Procedure
1. Prepare a probe by nick translation or random, oligo-primed
labelling.
2. Add the probe to a filter (nylon or nitrocellulose) to which
single-stranded nucleic acids are bound. (The filter is
protected with a prehybridization solution which contains
molecules which fill in the spots on the filter where the nucleic
acid has not bound.
3. Hybridize the single-stranded probe to the filter-bound nucleic
acid for 24 hr. The probe will bind to complementary
sequences.
4. Wash the filter to remove non-specifically bound probe.
5. Expose the filter and determine:
a. Did binding occur?
b. If so, what is the size of hybridizing fragment?
28. -28-
Hybridization Stringency
Temperature and salt concentrations of hybridization
conditions directly affect hybridization results
The degree of homology required for binding to occur can be
controlled by these factors
Results are directly related to the “degrees below the Tm” at
which the hybridizations and washes are performed
Tm is the melting temperature of the DNA
29. -29-
Tm = 69.3oC + 0.41(% G + C)oC
From this formula you can see that the GC content has a direct
effect on Tm. The following examples, demonstrate the point.
Tm = 69.3oC + 0.41(45)oC = 87.5oC (for wheat germ)
Tm = 69.3oC + 0.41(40)oC = 85.7oC
Tm = 69.3oC + 0.41(60)oC = 93.9oC
Hybridizations though are always performed with salt. This
requires another formula which considers this fact. This formula
is for the Effective Tm (Eff Tm).
Eff Tm = 81.5 + 16.6(log M [Na+]) + 0.41(%G+C) - 0.72(%
formamide)
30. -30-
Na+ ion concentration of different strengths of SSC
SSC Content [Na+] M
20X 3.3000
10X 1.6500
5X 0.8250
2X 0.3300
1X 0.1650
0.1X 0.0165
Another relevant relationship is a that 1% mismatch of two
DNAs lowers the Tm 1.4oC. So in a hybridization with wheat
germ that is performed at Tm - 20oC (=67.5oC), the two DNAs
must be 85.7% homologous for the hybridization to occur.
100% - (20oC/1.4oC) = 85.7% homology
31. -31-
Let's now look at an actual experiment.
Wheat DNA
Hybridization at 5X SSC at 65o
C
Non-stringent wash: 2X SSC at 65o
C
Stringent wash: 0.1X SSC at 65o
C
The first step is to derive the Eff Tm.
Eff Tm = 81.5 + 16.6(log 0.825) + 18.5 = 98.6
Next figure out hybridization homology
100 - [(98.6-65.0)/1.4] = 100 - (23.6/1.4) = 83.1%.
Next figure out Eff Tm and hybridization homology for non-stringent wash
Eff Tm = 81.5 + 16.6[log(0.33)] + 0.41(45%) = 92.0oC
% Homology = 100 - [(92-65)/1.4] = 80.7%
Next figure out Eff Tm and hybridization homology for stringent wash
Eff Tm = 81.5 + 16.6[log(0.0165)] + 0.41(45%) = 70.4oC
% Homology = 100 -[(70.4-65)/1.4] = 96.1%
Stringency - a term used in hybridization experiments to denote the
degree of homology between the probe and the filter bound nucleic acid;
the higher the stringency, the higher percent homology between the probe
and filter bound nucleic acid
32. -32-
Mapping and Mapping Populations
Types of mapping populations
a. F2
b. Backcross
c. Recombinant Inbred Lines (RILs; F2-derived lines)
Homozygosity of Recombinant Inbred Lines
RI population % within-line homozygosity at each
locus
F23 75.0
F24 87.5
F25 92.25
F26 96.875
F27 98.4375
F28 99.21875
Value of Recombinant Inbred Population
1. Eternal source materials
2. Phenotypic data collection can be replicated to ensure
accuracy
3. Large field trials can be performed to collective quantitative trait
data
4. Problem: dominance and epistasis can not be measured
because no heterozygotes
33. -33-
Segregation Ratio of Mapping Populations
Population Codominant
loci
Dominant
loci
F2 population 1:2:1 3:1
Backcross population 1:1 1:1*
Recombinant inbred population 1:1 1:1
*To score a dominant maker in a backcross population, you must
cross the recessive parent with the F1 plant. Therefore to score
RAPD loci you would need to create two populations, each one
developed by backcrossing to one of the two parents. For this
reason, backcross populations have not been used for mapping
RAPD loci.
34. -34-
Other Mapping Populations
Association Mapping (AM) Population
Limits of traditional bi-parental populations
Limited number of recombination events
o F2: one round of recombination
o RI populations: maximum of two rounds of
recombination
Allele richness
o Poor
Only alleles of parents sampled
Limits discover of all factors controlling a quantitative trait in a
species
But the advantage is:
o With limited recombination
Fewer markers needed to discover relevant
genetic factors
35. -35-
What is an association mapping population?
Collection of genotypes from a species
o Represent the genetic background for which you want
to make inferences
Arabidopsis
o Collection of wild samples from throughout the world
25 regions, four populations each
PLoS Biology (2005) 3:e196
Maize
o 92 inbred
12 stiff stalk, 45 non-stiff stalk, 35 tropical,
semitropical
Nature Genetics (2001) 28:286
Major benefit
o Samples many more recombination events
Great resolution
Resolution depends upon linkage
disequilibrium in sample
But the disadvantage
o Need many markers to find meaningful associations
Size for population used today
o Several hundreds (200-300 individuals)
36. -36-
Nested Association Mapping (NAM) Population
How is a NAM population created?
Developed from crossing
o One common parent to
o Multiple parents of diverse origin
Parents contain relevant diversity specific to the trait(s) of
interest
Often a single populations is developed per species
o High resource cost, so
Choice of parents is important
Maize example
o B73: common parent
Sequenced by maize genome project
o 25 other parents
Represent diversity in maize
200 lines per cross (5,000 total)
o Science (2009) 325:714
Large number of populations give better resolution
o Genetics (2009) 183:1525
Advantages
Like bi-parental population
o Uses current recombination events created by the cross
o Fewer markers than AM will discover the QTL
Like AM population
o Uses many recombination events
o Based on the many crosses used to make the
population members
37. -37-
Diversity or Phylogenetic Populations
Used to determine the relationship among individuals
Define patterns of relatedness
Selection of parents for breeding
Determine ancestral origin
Define gene tree
o Origin of a gene in a lineage
Define a species tree
o What is the relationships of members of a species
Within a species
Within a genera
Within a family
Considerations
Should represent the lineage that is of concern to the study
38. -38-
Specialized Mapping Topics
Bulk Segregant Analysis
Useful for targeting a specific genomic region
Create two DNA bulks
- one contains homozygous dominant individuals
- other contains homozygous recessive individuals
Perform a molecular marker analysis
The bulks are equally random for all regions of the genome
except that which contains your gene of interest
Any difference should be linked to the gene controlling the trait
you bulked upon
39. -39-
Sequence Tagged Sites (STS)
Defined by a pair of PCR sites obtained from DNA sequencing
Each site is usually 18-20 nucleotides long
Amplification is more specific because of the size of the primer
that anneals to one end of the STS
May require subsequent restriction digestion to define a
polymorphism
Reduces lab to lab variability seen with RAPDs
Arabidopsis STS Primers (called CAPS)
- two per chromosome
- allows a rapid mapping of new mutant to a specific
chromosome