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Paras Yadav1, Aarti Bhardwaj3, Shalini Jain2 and Hariom Yadav2
1Animal Biotechnology Division, National Dairy Research Institute, Karnal-132001, Haryana, India
2College of Applied Education and Health Sciences, Meerut, U.P.
What is What is Marker?Marker?
`̀
Marker is a Marker is a
piece of DNA piece of DNA molecule that is molecule that is associated with a associated with a certain trait of a certain trait of a
organismorganismMorphologicMorphologicalal
BiochemicBiochemicalal
ChromosomChromosomalal
GenetiGeneticcTypes of Types of
MarkersMarkers
Animals are selected based on Animals are selected based on appearanceappearance
Eg. PIGMENTATIONEg. PIGMENTATION
Disadvantage: lack of polymorphismDisadvantage: lack of polymorphism
Animals are selected based on Animals are selected based on biochemical propertiesbiochemical properties
Eg. Hb, AMYLASE, BLOOD GROUPS ETC.Eg. Hb, AMYLASE, BLOOD GROUPS ETC.
Disadvantage:Disadvantage: Sex limitedSex limitedAge dependentAge dependentInfluenced by environmentInfluenced by environmentIt covers less than 10% of genomeIt covers less than 10% of genome
Animals are selected based on Animals are selected based on structural & numerical structural & numerical
variationsvariationsEg. Structural and Numerical VariationsEg. Structural and Numerical VariationsStructural- Structural- Deletions, Insertions etc.Deletions, Insertions etc.Numerical-Numerical- Trisomy, Monosomy, Nullysomy Trisomy, Monosomy, Nullysomy
Disadvantage: low polymorphismDisadvantage: low polymorphism
Molecular MarkerMolecular Marker
Revealing variation at a Revealing variation at a DNA levelDNA level
Characteristics:Characteristics: Co-dominant expressionCo-dominant expression Nondestructive assayNondestructive assay Complete penetranceComplete penetrance Early onset of phenotypic Early onset of phenotypic
expression expression High polymorphism High polymorphism Random distribution Random distribution
throughout the genome throughout the genome Assay can be automatedAssay can be automated
DNA isolated from any tissue eg. Blood, hair etc.
DNA isolated at any stage even during foetal life
DNA has longer shelf-life readily exchangeable b/w labs
Analysis of DNA carried out at early age/ even at the embryonic
Stage irrespective of sex.
Molecular MarkersMolecular Markers
Single locus markerSingle locus marker
Multi-locus markerMulti-locus marker
RFLP
Microsatellite
STS
DNA Fingerprinting
AFLP
RAPD
Randomly Amplified Randomly Amplified Polymorphic DNA (RAPD)Polymorphic DNA (RAPD)
PCR based marker with 10-12 base pairsPCR based marker with 10-12 base pairs Random amplification of several fragmentsRandom amplification of several fragments Amplified fragments run in agarose gel Amplified fragments run in agarose gel
detected by EtBrdetected by EtBr Unstable amplification leads to poor Unstable amplification leads to poor
repeatabilityrepeatability
Restriction Fragment Length Restriction Fragment Length Polymorphism (RFLP)Polymorphism (RFLP)
Genomic DNA digested with Genomic DNA digested with Restriction Enzymes Restriction Enzymes
DNA fragments separated via DNA fragments separated via electrophoresis and transfer to nylon electrophoresis and transfer to nylon membranemembrane
Membranes exposed to probes labelled Membranes exposed to probes labelled with Pwith P3232 via southern hybridization via southern hybridization
Film exposed to X-RayFilm exposed to X-Ray
Amplified Fragment Length Amplified Fragment Length Polymorphism (AFLP)Polymorphism (AFLP)
Restriction endonuclease digestion of Restriction endonuclease digestion of DNADNA
Ligation of adaptorsLigation of adaptors Amplification of ligated fragmentsAmplification of ligated fragments Separation of the amplified fragments Separation of the amplified fragments
via electrophoresis and visualizationvia electrophoresis and visualization AFLPs have stable amplification and AFLPs have stable amplification and
good repeatabilitygood repeatability
SSR: SSR: SSimple imple SSequence equence RRepeat epeat or Microsatelliteor Microsatellite
PCR based markers with 18-25 base PCR based markers with 18-25 base pair primerspair primers
SSR polymorphisms are based on no. SSR polymorphisms are based on no. of repeat units and are of repeat units and are hypervariablehypervariable
SSRs have stable amplification and SSRs have stable amplification and good repeatabilitygood repeatability
SSR are easy to run and automateSSR are easy to run and automate
DFP: DNA finger DFP: DNA finger printing printing
DNA extraction from individualDNA extraction from individual Amplification of markersAmplification of markers Electrophoresis separation of Electrophoresis separation of
markersmarkers Visualization of markersVisualization of markers Scoring of markers for each Scoring of markers for each
individual individual Data analysisData analysis
FeaturesFeatures RFLPRFLP PCR-PCR-RFLPRFLP
DFPDFP RAPDRAPD MicrosatelliteMicrosatellite SNPSNP
Detection methodDetection method HybridizationHybridization PCRPCR HybridizationHybridization PCRPCR PCRPCR PCRPCR
Type of Type of probe/primer probe/primer
usedused
g DNA/g DNA/cDNA sequence of cDNA sequence of structural genesstructural genes
Sequence Sequence specific specific primersprimers
Mini satellite Mini satellite synthetic synthetic
oligosoligos
Arbitrarily Arbitrarily design design primerprimer
Sequence Sequence specific primersspecific primers
Sequence Sequence specific specific primersprimers
Requirement of Requirement of radioactivityradioactivity
YesYes No/YesNo/Yes YesYes No/YesNo/Yes No/YesNo/Yes No/YesNo/Yes
Extant of genomic Extant of genomic coveragecoverage
LimitedLimited LimitedLimited ExtensiveExtensive ExtensiveExtensive ExtensiveExtensive ExtensiveExtensive
Degree of Degree of polymorphismspolymorphisms
LowLow LowLow HighHigh Medium to Medium to HighHigh
HighHigh HighHigh
Phenotype Phenotype expressionexpression
Co dominantCo dominant Co Co dominantdominant
Co dominantCo dominant Co Co dominant/Ddominant/D
ominantominant
DominantDominant Co Co dominantdominant
Possibility of Possibility of automationautomation
NoNo YesYes NoNo YesYes YesYes YesYes
Gene mappingGene mapping Pre and post natal Pre and post natal
diagnosis of diseasesdiagnosis of diseases Anthropological and Anthropological and
molecular evolution molecular evolution studiesstudies
Contd…Contd…
Animal breedingAnimal breedingA. A. Conventional breeding Conventional breeding
strategiesstrategies1.1. Short rangeShort range2.2. Long rangeLong range
B. B. Transgenic breeding strategiesTransgenic breeding strategies
Short Range ApplicationShort Range Application Parentage determinationParentage determination Genetic distance estimationGenetic distance estimation Determination of twin zygosity & Determination of twin zygosity &
freemartinsfreemartins Sexing of pre-implanted embryosSexing of pre-implanted embryos Identification of disease carriesIdentification of disease carries
Long Range ApplicationsLong Range Applications
Gene mapping & mapping Gene mapping & mapping of QTL by linkageof QTL by linkage
Marker assisted selectionMarker assisted selection
TRANSGENIC BREEDING TRANSGENIC BREEDING STRATEGIESSTRATEGIES
IDENTIFICATION OF ANIMALS IDENTIFICATION OF ANIMALS CARRYING THE TRANSGENESCARRYING THE TRANSGENES
CONCLUSIONSCONCLUSIONS
The genetic improvement of animals is a continuous and The genetic improvement of animals is a continuous and complex process. Ever since the domestication of animals complex process. Ever since the domestication of animals by man, he has always remained busy in improving hisby man, he has always remained busy in improving hisanimals. In this pursuit many methods have been developedanimals. In this pursuit many methods have been developedand tested. In recent years, the demonstration of geneticand tested. In recent years, the demonstration of geneticpolymorphism at the DNA sequence level has provided a polymorphism at the DNA sequence level has provided a large number of marker techniques with variety oflarge number of marker techniques with variety ofapplications. This has, in turn, prompted furtherapplications. This has, in turn, prompted furtherconsideration for the potential utility of these markers inconsideration for the potential utility of these markers inanimal breeding. However, utilization of marker-basedanimal breeding. However, utilization of marker-basedinformation for genetic improvement depends on the choice information for genetic improvement depends on the choice of an appropriate marker system for a given application. of an appropriate marker system for a given application.