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 Membranes exposed to probes labelled with Plabelled with P3232 via southern via southern hybridizationhybridization

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 breeding

A. A. Conventional breeding Conventional breeding strategiesstrategies

1.1. Short rangeShort range

2.2. 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 animals. In this pursuit many methods have been developeddevelopedand 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 information for genetic improvement depends on the choice choice of an appropriate marker system for a given application. of an appropriate marker system for a given application.