this is a presentation on molecular markers that include what is molecular marker, it's types, biochemical markets (alloenzyme), it's classification, data analysis and it's applications
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.
Association mapping, also known as "linkage disequilibrium mapping", is a method of mapping quantitative trait loci (QTLs) that takes advantage of linkage disequilibrium to link phenotypes to genotypes.Varioius strategey involved in association mapping is discussed in this presentation
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.
Association mapping, also known as "linkage disequilibrium mapping", is a method of mapping quantitative trait loci (QTLs) that takes advantage of linkage disequilibrium to link phenotypes to genotypes.Varioius strategey involved in association mapping is discussed in this presentation
SNP (Single Nucleotide Polymorphic), SNP mapping, SNP profile, SNP types, SNP analysis by gel electropherosis and by mass spectrometry, SNP effects, single strand conformation polymorphism, SNP advantages and disadvantages and application of SNP profile in drug choice
Differences in DNA occur within genes, the differences have the potential to affect the function of the
gene and hence the phenotype of the individual. Genetic markers which have been used a lot in the past
include blood groups and polymorphic enzymes. We have relatively few such markers, but this has been
overcome with the advent of new types of markers. However, most molecular markers are not associated
with a visible phenotype.
Single Nucleotide Polymorphism Genotyping Using Kompetitive Allele Specific ...MANGLAM ARYA
Single Nucleotide Polymorphism
Single nucleotide polymorphism (SNP) refers to a single base change in a DNA sequence
SNP: Commonly biallelic
Two types(Based on presence in genome)
Synonymus
Non-synonymus
SNPs have largely replaced simple sequence repeats (SSRs)
Advantage of using SNPs
Low assay cost
High genomic abundance
Locus specificity
co-dominant inheritance
Simple documentation
Potential for high-throughput Analysis
Relatively low genotyping error rates
SNP genotyping platforms
BeadXpressTM,GoldenGateTM and Infinium from Illumina
GeneChipTM and GenFlexTM Tag array from Affimetrix
SNaPshotTM and TaqManTM from the Applied Biosystems
SNPWaveTM from KeyGene
iPLEX GoldTM Assay and Mass-RRAYTM from Sequonome
Variables to be considered
Throughput
Data turnaround
Time
Ease of use
Performance (sensitivity, reliability, reproducibility, and accuracy),
Flexibility (genotyping few samples with many snps or many samples with few snps),
Number of markers generated per run (uniplex versus multiplex assay capability)
Assay development requirements and genotyping cost per sample or data point.
KASP
KBioscience Competitive Allele-Specific PCR
Homogenous, Fluorescence-based genotyping technology, based on
Allele-specific oligo extension (primer)
Fluorescence resonance energy transfer
KASP Applications
Genotyping a wide range of species for various purposes.
KASP for Quality analysis, QTL mapping, MARS, and allele mining
Quality Control Analysis
QC analysis should be done for two reasons by genotyping the parents and F1s with the same subset of SNPs, in order to
confirm if F1s contains true-to-type alleles from their parents
check the genetic purity of the inbred parents.
F1s with true-to-type parental alleles for at least 90 % of the SNPs that were polymorphic between the parents should be advanced, while those with less than 10 % nonparental alleles should be discarded.
QTL Mapping
QTL mapping identifies a subset of markers that are significantly associated with one or more QTL influencing the expression of the trait of interest.
1) Select or develop a bi-parental mapping population.
2) Phenotype the population for a trait under greenhouse or field conditions.
3) Choose a molecular marking system – genotype parents of the mapping population and F1s with large numbers of markers, then select 200-400 markers exhibiting polymorphism between the parents.
4) Choose a genotyping approach, then generate molecular data for polymorphic markers
5) Identify the molecular markers associated with major QTL using statistical programs.
Large-scale allele mining
Allele mining is a promising approach to dissecting naturally occurring allelic variation at candidate genes controlling key agronomic traits.
KASP platform at CIMMYT has been used for the systematic mining of large germplasm collections for specific functional polymorphisms.
SNPs or small indels that
Biotechnology for Crop Improvement.
Molecular Plant Breeding-Marker Assisted Breeding/Selection.
Comparison between three main and commonly discussed marker systems- RFLP, RAPD and AFLP.
Basic Understanding for Simple Sequence Repeats, SCAR and CAPS.
Strategies to overcome food shortages using molecular plant breeding approaches, Application of various molecular marker systems and examples.
Reference List.
Presenter: Brenda Chong
Random amplified polymorphic DNA-RAPD.pptxNusrat Sheikh
Random Amplified Polymorphic DNA is a type of PCR in which the segments of DNA that are amplified are random.
Williams et al. (1990) developed this technique using very short primers to generate random fragments from template DNAs.
RAPD fragments can be separated and used as genetic markers or a kind of DNA fingerprinting.
Principle
The standard RAPD technology utilizes short synthetic oligonucleotides (10 bases long) of random sequences as primers to amplify nanogram amounts of total genomic DNA under low annealing temperatures by PCR. Amplification products are generally separated on agarose gels and stained with ethidium bromide.
At an appropriate annealing temperature during the thermal cycle, oligonucleotide primers of random sequence bind several priming sites on the complementary sequences in the template genomic DNA and produce discrete DNA products if these priming sites are within an amplifiable distance of each other.
The profile of amplified DNA primarily depends on nucleotide sequence homology between the template DNA and oligonucleotide primer at the end of each amplified product. Nucleotide variation between different sets of template DNAs will result in the presence or absence of bands because of changes in the priming sites.
Procedure
1. DNA is made single stranded by raising the temperature to 940C.
2. In second step, temperature is lowered to 40- 650C which results in annealing of the primer to their target sequences on the template DNA.
3. Temperature is chosen where the activity of the thermostable Taq DNA polymerase is optimal.
4. The polymerase now extends the 3’ ends of the DNA primer hybrids towards the outer primer binding site.
5. Repeating these three step cycles 40 to 50 times results in the exponential amplification of the target between the 5’ ends of the two primer binding sites.
6. Amplification products are separated by gel electrophoresis and visualized by ethidium bromide staining.
Taxonomy is the branch of science concerned with the classification of organisms. A taxonomic designation is more than just a name. Ideally, it reflects evolutionary history and the relationship between organisms. Traditionally, taxonomic classification has relied upon morphological features and physiological characteristics. However, for bacterial taxonomy, phenotypic approaches have proven insufficient. Unrelated bacteria can exhibit identical traits, closely related bacteria can have divergent features, and methods for accurate identification may be too cumbersome for routine use. In contrast, molecular taxonomy approaches use data derived from hereditary material and provide a robust view of genetic relatedness. Advances in technology have been accompanied by improvements in the cost, speed, and availability of molecular methods. Here, we provide a brief history of approaches to prokaryotic classification and describe how molecular taxonomy is redefining our understanding of bacterial evolution and the tree of life.
SNP (Single Nucleotide Polymorphic), SNP mapping, SNP profile, SNP types, SNP analysis by gel electropherosis and by mass spectrometry, SNP effects, single strand conformation polymorphism, SNP advantages and disadvantages and application of SNP profile in drug choice
Differences in DNA occur within genes, the differences have the potential to affect the function of the
gene and hence the phenotype of the individual. Genetic markers which have been used a lot in the past
include blood groups and polymorphic enzymes. We have relatively few such markers, but this has been
overcome with the advent of new types of markers. However, most molecular markers are not associated
with a visible phenotype.
Single Nucleotide Polymorphism Genotyping Using Kompetitive Allele Specific ...MANGLAM ARYA
Single Nucleotide Polymorphism
Single nucleotide polymorphism (SNP) refers to a single base change in a DNA sequence
SNP: Commonly biallelic
Two types(Based on presence in genome)
Synonymus
Non-synonymus
SNPs have largely replaced simple sequence repeats (SSRs)
Advantage of using SNPs
Low assay cost
High genomic abundance
Locus specificity
co-dominant inheritance
Simple documentation
Potential for high-throughput Analysis
Relatively low genotyping error rates
SNP genotyping platforms
BeadXpressTM,GoldenGateTM and Infinium from Illumina
GeneChipTM and GenFlexTM Tag array from Affimetrix
SNaPshotTM and TaqManTM from the Applied Biosystems
SNPWaveTM from KeyGene
iPLEX GoldTM Assay and Mass-RRAYTM from Sequonome
Variables to be considered
Throughput
Data turnaround
Time
Ease of use
Performance (sensitivity, reliability, reproducibility, and accuracy),
Flexibility (genotyping few samples with many snps or many samples with few snps),
Number of markers generated per run (uniplex versus multiplex assay capability)
Assay development requirements and genotyping cost per sample or data point.
KASP
KBioscience Competitive Allele-Specific PCR
Homogenous, Fluorescence-based genotyping technology, based on
Allele-specific oligo extension (primer)
Fluorescence resonance energy transfer
KASP Applications
Genotyping a wide range of species for various purposes.
KASP for Quality analysis, QTL mapping, MARS, and allele mining
Quality Control Analysis
QC analysis should be done for two reasons by genotyping the parents and F1s with the same subset of SNPs, in order to
confirm if F1s contains true-to-type alleles from their parents
check the genetic purity of the inbred parents.
F1s with true-to-type parental alleles for at least 90 % of the SNPs that were polymorphic between the parents should be advanced, while those with less than 10 % nonparental alleles should be discarded.
QTL Mapping
QTL mapping identifies a subset of markers that are significantly associated with one or more QTL influencing the expression of the trait of interest.
1) Select or develop a bi-parental mapping population.
2) Phenotype the population for a trait under greenhouse or field conditions.
3) Choose a molecular marking system – genotype parents of the mapping population and F1s with large numbers of markers, then select 200-400 markers exhibiting polymorphism between the parents.
4) Choose a genotyping approach, then generate molecular data for polymorphic markers
5) Identify the molecular markers associated with major QTL using statistical programs.
Large-scale allele mining
Allele mining is a promising approach to dissecting naturally occurring allelic variation at candidate genes controlling key agronomic traits.
KASP platform at CIMMYT has been used for the systematic mining of large germplasm collections for specific functional polymorphisms.
SNPs or small indels that
Biotechnology for Crop Improvement.
Molecular Plant Breeding-Marker Assisted Breeding/Selection.
Comparison between three main and commonly discussed marker systems- RFLP, RAPD and AFLP.
Basic Understanding for Simple Sequence Repeats, SCAR and CAPS.
Strategies to overcome food shortages using molecular plant breeding approaches, Application of various molecular marker systems and examples.
Reference List.
Presenter: Brenda Chong
Random amplified polymorphic DNA-RAPD.pptxNusrat Sheikh
Random Amplified Polymorphic DNA is a type of PCR in which the segments of DNA that are amplified are random.
Williams et al. (1990) developed this technique using very short primers to generate random fragments from template DNAs.
RAPD fragments can be separated and used as genetic markers or a kind of DNA fingerprinting.
Principle
The standard RAPD technology utilizes short synthetic oligonucleotides (10 bases long) of random sequences as primers to amplify nanogram amounts of total genomic DNA under low annealing temperatures by PCR. Amplification products are generally separated on agarose gels and stained with ethidium bromide.
At an appropriate annealing temperature during the thermal cycle, oligonucleotide primers of random sequence bind several priming sites on the complementary sequences in the template genomic DNA and produce discrete DNA products if these priming sites are within an amplifiable distance of each other.
The profile of amplified DNA primarily depends on nucleotide sequence homology between the template DNA and oligonucleotide primer at the end of each amplified product. Nucleotide variation between different sets of template DNAs will result in the presence or absence of bands because of changes in the priming sites.
Procedure
1. DNA is made single stranded by raising the temperature to 940C.
2. In second step, temperature is lowered to 40- 650C which results in annealing of the primer to their target sequences on the template DNA.
3. Temperature is chosen where the activity of the thermostable Taq DNA polymerase is optimal.
4. The polymerase now extends the 3’ ends of the DNA primer hybrids towards the outer primer binding site.
5. Repeating these three step cycles 40 to 50 times results in the exponential amplification of the target between the 5’ ends of the two primer binding sites.
6. Amplification products are separated by gel electrophoresis and visualized by ethidium bromide staining.
Taxonomy is the branch of science concerned with the classification of organisms. A taxonomic designation is more than just a name. Ideally, it reflects evolutionary history and the relationship between organisms. Traditionally, taxonomic classification has relied upon morphological features and physiological characteristics. However, for bacterial taxonomy, phenotypic approaches have proven insufficient. Unrelated bacteria can exhibit identical traits, closely related bacteria can have divergent features, and methods for accurate identification may be too cumbersome for routine use. In contrast, molecular taxonomy approaches use data derived from hereditary material and provide a robust view of genetic relatedness. Advances in technology have been accompanied by improvements in the cost, speed, and availability of molecular methods. Here, we provide a brief history of approaches to prokaryotic classification and describe how molecular taxonomy is redefining our understanding of bacterial evolution and the tree of life.
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
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.
Molecular markers are the DNA sequences that are used to tag or highlight specific genes or nucleotide sequence in the genome. In the past, diagnosis, and monitoring of infectious disease, there many conventional methods like Biotyping, Ribotyping and Protein analyses are used but these methods are not so reliable and exact. So by the invention of the molecular markers, the detection of diseases was started by using these molecular markers. In this review, we discussed different types of molecular markers that are used to detect diseases and many purposes as diagnostic tools. The Molecular markers include Restriction fragment length polymorphism (RFLP), Random amplification of polymorphic DNA (RAPD), Amplified fragment length polymorphism (AFLP), Simple sequence repeats (SSR), Inter-Simple Sequence Repeat Amplification (ISSR), Cleaved Amplified Polymorphic Sequences (CAPS) and Sequence Characterized Amplified Region (SCAR) are discussed in the article.
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
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Molecular markers by tahura mariyam ansari
1. TOPIC- MOLECULAR MARKERS
PRESENTED BY:
TAHURA MARIYAM
MSc. MICROBIOLOGY
PRENENTED TO: Dr. Gurudayal Ram (Assistant prof. Sr. Grade)
DEPARTMENT OF INDUSTRIAL MICROBIOLOGY
JACOB INSTITUTE OF BIOTECHNOLOGY AND BIO-ENGINEERING
SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE,TECHNOLOGY, AND SCIENCES,
PRAYAGRAJ
2. CONTENT
MOLECULAR MARKERS
DESIRABLE PROPERTIES OF MOLECULAR MARKERS
TYPES OF MOLECULAR MARKERS
Biochemical markers
HYBRIDIZATION BASED (non-PCR) TECHNIQUE
RFLP (Restriction fragment length polymorphism analysis)
PCR based techniques
(RAPD, ISSR, SSR, AFLP, EST, SCoT, Microsatellite)
DATA ANLYSIS
DNA marker applications
3. MOLECULAR MARKER
A molecular marker is a molecule contained within a sample taken from an
organism (biological markers) or other matter. It can be used to reveal certain
characteristics about the respective source. DNA, for example, is a molecular
marker containing information about genetic disorders, genealogy and the
evolutionary history of life. Specific regions of the DNA (genetic markers) are
used for diagnosing the autosomal recessive genetic disorder cystic fibrosis,
taxonomic affinity (phylogenetics) and identity (DNA barcoding). Further, life
forms are known to shed unique chemicals, including DNA, into
the environment as evidence of their presence in a particular location.
Other biological markers, like proteins, are used in diagnostic tests for
complex neurodegenerative disorders, such as Alzheimer’s disease. Non-
biological molecular markers are also used, for example, in environment studies.
4. DESIRABLE PROPERTIES OF
MOLECULAR MARKERS
High level polymorphism
Co-dominant inheritance
Unambiguous designation of alleles
Frequent occurrence in the genome
Even distribution throughout the genome
Selectively neutral behaviour (no pleiotropic effect)
Easy access (no cloning)
Easy and fast assay 9amenable to automation
High reproducibility
Easy exchange of data between laboratories
Development at reasonable cost
5. TYPES OF MOLECULAR MARKERS
Due to rapid developments in the field of molecular genetics, a variety of
molecular markers has emerged during the last few decades.
6. Biochemical markers
Biochemical markers are generally the protein marker. These are based on the change in the
sequence of amino acids in a protein molecule. The most important protein marker
is alloenzyme. Alloenzymes are variant forms of an enzyme that are coded by different
alleles at the same locus and this alloenzymes differs from species to species. So for
detecting the variation alloenzymes are used. These markers are type-i markers.
Advantages:
Co-dominant markers.
Less price.
Disadvantages:
Require prior information.
Low polymorphism power.
Applications:
Linkage mapping.
Population studies.
8. RFLP (Restriction fragment length
polymorphism analysis)
Genetic markers resulting from the variation or
change in the length of defined DNA fragments
produced by digestion of the DNA sample with
restriction endonucleases.
9. RFLP contd…
Electrophoretic comparison of the size of defined restriction fragments
derived from genomic DNA
1. Isolate high quality DNA
2. Digest with a combination of restriction enzymes
3. Fractionate digested samples by electrophoresis
4. Transfer fragments to membrane
5. Hybridize with radioactively labeled DNA probe(s); detect by
autoradiography. Can also use non-radioactive labeling systems
12. Considerations for use of RFLPs
Relatively slow process
Use of radioisotopes has limited RFLP use to certified laboratories ( but
non - radioactive labeling systems are now in wide use )
Co - dominant markers ; often species - specific
Need high quality DNA - Need to develop polymorphic probes
expensive
13. Applications
Intraspecific level or among closely related taxa
Presence and absence of fragments resulting from changes in recognition
sites are used for identifying species or populations
Estimating genetic distance and fingerprinting
Forensic - biological parentage , paternity cases
Disease status
Genetic mapping
16. Random Amplified Polymorphic DNA (
RAPD )
Randomly Amplified Polymorphic DNA ( RAPDs ) are genetic markers
resulting from PCR amplification of genomic DNA sequences recognized by
ten - mer random primers of arbitrary nucleotide sequence ( Williams et al .,
1990 ).
RAPDs are dominant markers that require no prior knowledge of the DNA
sequence , which makes them very suitable for investigation of species that
are not well known ( Williams et al . 1993 ) .
17.
18. Inter - Simple Sequence Repeats (ISSE)
The generation of ISSR markers involve PCR amplification of DNA using a
single primer composed of a microsatellite repeated sequence and in
some cases primer also contains 1 4 base anchor at either 3 ' or 5 ' or at
both ends, which target a subset of ' simple sequence repeats ' ( SSRs )
and amplify the region between two closely spaced and oppositely
oriented SSRs ( Fang et al . , 1997 ; Fang and Roose , 1997 ; Moreno et al . ,
1998 ) .
ISSR technique permits the detection of polymorphisms in microsatellites
and inter - microsatellites loci without previous knowledge of the DNA
sequence ( Moreno et al . , 1998 )
19.
20. Simple Sequence Repeats ( SSR )
Are DNA sequences with repeat lengths of a few base pairs . Variation in
the number of repeats can be detected with PCR by developing primers for
the conserved DNA sequence flanking the SSR . As molecular markers ,
SSR combine many desirable marker properties including high levels of
polymorphism and information content , unambiguous designation of
alleles , even dispersal , selective neutrality , high reproducibility , co -
dominance , and rapid and simple genotyping assays . Microsatellites have
become the molecular markers of choice for a wide range of applications
in genetic mapping and genome , genotype identification and variety
protection , seed purity evaluation and germplasm conservation ,
diversity studies , paternity determination and pedigree analysis ,
gene and quantitative trait locus analysis , and marker - assisted
breeding .
21. Advantages
Require very little and not necessarily high quality DNA
Highly polymorphic
Evenly distributed throughout the genome
Simple interpretation of results
Easily automated , allowing multiplexing
Good analytical resolution and high reproducibility
Codominant marker ("New allozyme”)
24. Amplified Fragment Length
Polymorphism (AFLP)
AFLP technology is a DNA fingerprinting technique
that combines RFLP and PCR . It is based on the
selective amplification of a subset of genomic
restriction fragments using PCR .
AFLP process
1. Digest genomic DNA with restriction enzymes
2. Ligate commercial adaptors ( defined sequences )
to both ends of the fragments
3. Carry out PCR on the adaptor - ligated mixture ,
using primers that target the adaptor , but that
vary in the base ( s ) at the 3 ' end of the primer .
25.
26. Advantages of AFLP's
Very sensitive
Good reproducibility but technically demanding
Relatively expensive technology
Discriminating homozygotes from heterozygotes
Requires band quantitation ( comparison of pixel density in images from a gel
scanner )
Bands are anonymous
27. Applications
Monitoring inheritance of agronomic traits
Diagnostic in genetically inherited disease
Pedigree analysis
Forensic typing - Parentage analysis
Identifying hybrids
Species level relationship
Also in some case at higher level relationship
28. Microsatellites
Microsatellites or simple sequence repeated (SSR) loci, which have been referred to in the
literature as variable number of tandem repeats (VNTRs) and simple sequence length
polymorphisms (SSLPs), are found throughout the nuclear genomes of most eukaryotes
and to a lesser extent in prokaryotes.
Microsatellites range from one to six nucleotides in length and are classified as mono-, di-
, tri-, tetra-, penta- and hexanucleotide repeats.
The sequences of di-, tri- and tetranucleotide repeats are the most common choices for
molecular genetic studies.
They are tandemly repeated (usually 5-20 times) in the genome with a minimum repeat
length of 12 base-pairs.
The number of repeats is variable in populations of DNA and within the alleles of an
individual.
The sequence below has a 20 dinucleotide repeat (40bp) stretch of CA that is shown in
bold.
30. Microsatellites can be used as markers in genetic studies of linkage in families and linkage
disequilibrium studies of populations. In linkage studies one can examine large number of
families and see when the alleles of specific markers are inherited together with a phenotype
in more cases than not. Microsatellite repeat are amplified with fluorescently labeled primers
and then the alleles from each individual in a family are separated by size and the
marker tested for linkage with another as shown in Figure.
Raw of Genotyping Data
31. This approach assumes that a certain quantitative trait was affected by many unknown
genes. So, this approach is looking for associations between the variation of allele and
quantitative traits at the neutral DNA markers. The DNA marker is located on a
chromosome and its inheritance can be monitored [48]. Microsatellites are the most
commonly applied molecular marker in ecological research
Number of publications (selected
biological subject) between 1970
and 2007 employing mtDNA,
Allozymes, Microsatellites, RFLPs,
RAPDs and AFLPs found via ISI
web of knowledge
32. Start Codon Targeted ( SCOT )
Polymorphism analysis
SCoT is a novel method for generating plant DNA markers
.
This method was developed based on the short conserved
region flanking the ATG start codon in plant genes
SCoT uses single 18 - mer primers in polymerase chain
reaction (PCR) and an annealing temperature of 50 ° C .
PCR amplicons are resolved using standard agarose gel
electrophoresis.
This method was validated in rice using a genetically
diverse set of genotypes and a backcross population.
36. Steps for EST's
cDNA libraries (containing many of the expressed genes of an organism)
pick cDNA clones randomly
rapidly determine some of the sequence of nucleotides from the end of
each clone.
These ESTs could then be compared to all known sequences using a
program called BLAST.
37. Steps of ESTs contd…
An exact match to a sequenced gene means that the gene encoding that
EST is already known.
If the match was close but not exact one could conclude that the EST is
derived from a gene with a function similar to that of the known gene.
The EST sequences with their putative identification are then deposited in the
GenBank and the clones from which they were derived are kept in a freezer
for later use.
38. Overview of the EST sequencing
process
Clones are picked from petri dishes into microtitre plates, and archived for later use. All
subsequent manipulations (PCR, clean up and sequencing) are carried out in microtitre
plates to yield medium-throughput.
42. Similarity
Simple matching coefficient ( Sneath and Sokal , 1973 ) : measures the
proportion of shared band presence and absences
Jaccard's coefficient ( Jaccard , 1908 ) : Proportion of shared bands
Nei and Li coefficient ( Nei and Li , 1979 ) : Probability a band being
amplified in one sample being amplified in another sample (biological
perspective : inherited from a common ancestor
Major problem : False positive – similar in RAPD
43. Genetic Similarity matrix calculated according to
Jaccard’s coefficient based on marker data
2 4 7 3 5 6
2 100
4 94.1 100
7 93.3 87.5 100
3 87.5 94.1 80.0 100
5 66.7 62.5 71.4 53.3 100
6 57.1 53.3 61.5 57.1 92.3 100
