genetic markers in plant breeding

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GENETIC MARKERS IN PLANT BREEDING

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GENETIC MARKERS IN PLANT BREEDING. Marker. Gene of known function and location, or a mutation within a gene that allows studying the inheritance of that gene Genetic information resides in the genome. Genetic Marker. - PowerPoint PPT Presentation

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Page 1: GENETIC MARKERS  IN PLANT BREEDING

GENETIC MARKERS IN PLANT BREEDING

Page 2: GENETIC MARKERS  IN PLANT BREEDING

MarkerGene of known function and location, or a mutation within a gene that allows studying the inheritance of

that geneGenetic information resides in the genome

Genetic MarkerAny phenotypic difference controlled by the genes, that can be used for studying

recombination processes or selection of a more or less closely associated target gene

Page 3: GENETIC MARKERS  IN PLANT BREEDING

Genetic Marker Morphological marker Molecular marker

Readily detectable sequence of protein or DNA that are closely linked to a gene locus and/or a morphological or other characters of a plantReadily detectable sequence of protein or DNA whose inheritance can be monitored and associated with the trait inheritance independently from the environment

1. Protein marker 2. DNA marker

Page 4: GENETIC MARKERS  IN PLANT BREEDING

Molecular markersMolecular markersR

eso l

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allozymes (protein-allozymes (protein-electrophoresiselectrophoresis))

chloroplastDNA PCR-chloroplastDNA PCR-RFLPRFLP

RAPDRAPD(random amplified polymorphic (random amplified polymorphic DNA)DNA)

AFLPAFLP(Amplified Fragment Length (Amplified Fragment Length Polymorphism)Polymorphism)

Microsatellites (SSRs)Microsatellites (SSRs)

SequencingSequencing (SNPs)(SNPs)

Page 5: GENETIC MARKERS  IN PLANT BREEDING

non-waxy waxy

Morphological markerMorphological marker (phenotypic/naked eye marker)(phenotypic/naked eye marker)

hulled naked

2-rowed 6-rowed Black white

Page 6: GENETIC MARKERS  IN PLANT BREEDING

Karl Von Linne (1707-1778)

Page 7: GENETIC MARKERS  IN PLANT BREEDING

Molecular markers

Important aspect:Polymorphism

The existence of two or more forms that are genetically distinct from one another but contained within the same interbreeding population Pattern of inheritance

The pattern of genetic information transmission from parents to progeny

Page 8: GENETIC MARKERS  IN PLANT BREEDING

Polymorphism

Page 9: GENETIC MARKERS  IN PLANT BREEDING

Polymorphism-Parent 1 : one band-Parent 2 : a smaller band-Offspring 1 : heterozygote =

both bands-Offspring 2 : homozygote

parent 1

Polymorphism

Parent 1 : one band

-Parent 2 : no band

-Offspring 1 : homozygote parent 1

-Offspring 2 : ????

P 2P 1 O 2O 1

Gel configuration

Co-dominant marker

P 2

Gel configurationP 1 O 1 O 2

Dominant marker

Page 10: GENETIC MARKERS  IN PLANT BREEDING

Dominant Dominant versusversus Co-dominant Co-dominant

Dominant

No distinction between homo- and heterozygotes possibleNo allele frequencies availableNo allele frequencies available

RAPD

Co-dominantCo-dominant

Homozygotes can be distinguished from Homozygotes can be distinguished from heterozygotes;heterozygotes;

Allele frequencies can be calculatedAllele frequencies can be calculated

microsatellites, SNP, RFLPs

Page 11: GENETIC MARKERS  IN PLANT BREEDING

High PolymorphicCo-dominant inheritance

Occurs throughout the genomeReproducible

Easy, fast and cheap to detectSelectivity neutral

High resolution with large number of samplesNondestructive assayNondestructive assay

Random distribution throughout the genome Random distribution throughout the genome Assay can be automatedAssay can be automated

Desirable properties for a good molecular marker

Page 12: GENETIC MARKERS  IN PLANT BREEDING

Protein markers

a. Allozyme isoenzymes of proteins nature whose synthesis is usually controlled by codominant alleles and inherited by monogenic ratios. They show a specific banding pattern if separated by electrophoresisb. IsozymeA species of enzyme that exists in two or more structural form, which are easily identified by electrophoretic methods

Genetic markers which based on protein polymorphisms

Page 13: GENETIC MARKERS  IN PLANT BREEDING

Seed storage proteins

Isozymes

Proteins Polymorphisms

Page 14: GENETIC MARKERS  IN PLANT BREEDING

Isozyme Isozyme

Page 15: GENETIC MARKERS  IN PLANT BREEDING

Starch gel of the isozyme malate dehydrogenase (MDH). Starch gel of the isozyme malate dehydrogenase (MDH). The numbers indicate first the MDH locus, and next the The numbers indicate first the MDH locus, and next the

allele present (ie. 3-18 is locus 3 allele 18). Some bands are allele present (ie. 3-18 is locus 3 allele 18). Some bands are heterodimers (intralocus or interlocus).heterodimers (intralocus or interlocus).

Isozyme Isozyme

Page 16: GENETIC MARKERS  IN PLANT BREEDING

DNA markerSegments of DNA with an identifiable

physical location on a chromosome and whose inheritance can be followed

Types of DNA Marker can be differentiated based on molecular technique used to develop the marker1.Restriction enzymes2.Hybridization3.PCR4.Sequencing

A marker can be a gene, or it can be some section of DNA with no known function

Page 17: GENETIC MARKERS  IN PLANT BREEDING

Chromosome to DNA

DNA structure

1 ccacgcgtcc gtgaggactt gcaagcgccg cggatggtgg gctctgtggc tgggaacatg 61 ctgctgcgag ccgcttggag gcgggcgtcg ttggcggcta cctccttggc cctgggaagg 121 tcctcggtgc ccacccgggg actgcgcctg cgcgtgtaga tcatggcccc cattcgcctg 181 ttcactcaga ggcagaggca gtgctgcgac ctctctacat ggacgtacag gccaccactc 241 ctctggatcc cagagtgctt gatgccatgc tcccatacct tgtcaactac tatgggaacc 301 ctcattctcg gactcatgca tatggctggg agagcgaggc agccatggaa cgtgctcgcc 361 agcaagtagc atctctgatt ggagctgatc ctcgggagat cattttcact agtggagcta 421 ctgagtccaa caacatagca attaaggtag gaggagggat ggggatgttg tgtggccgac 481 agttgtgagg ggttgtggga agatggaagc cagaagcaaa aaagagggaa cctgacacta 541 tttctggctt cttgggttta gcgattagtg cccctctctc atttgaactc aactacccat 601 gtctccctag ttctttctct gcctttaaaa aaaaatgtgt ggaggacagc tttgtggagt 661 ctgaaatcac catctacctt tacttaggtt ctgagtgcca aacccaaggc accaggcatg 721 cgtccttgac tccggagcca tcaggcaggc tttcctcagc cttttgcagc caagtctttt 781 agcctattgg tctgagttca gtgtggcagt tggttaggaa agaaggtggt tcttcgacca 841 ctaacagttt ggatttttta ggatgctagt cctttaaaa ……….

Stretch of nitrogen fixation gene in soybean

Page 18: GENETIC MARKERS  IN PLANT BREEDING

DNA

Gene A Gene B

AACCTGAAAAGTTACCCTTTAAAGGCTTAAGGAAAAAGGGTTTAACCAAGGAATTCCATCGGGAATTCCG

MFG

1 ccacgcgtcc gtgaggactt gcaagcgccg cggatggtgg gctctgtggc tgggaacatg 61 ctgctgcgag ccgcttggag gcgggcgtcg ttggcggcta cctccttggc cctgggaagg 121 tcctcggtgc ccacccgggg actgcgcctg cgcgtgtaga tcatggcccc cattcgcctg 181 ttcactcaga ggcagaggca gtgctgcgac ctctctacat ggacgtacag gccaccactc 241 ctctggatcc cagagtgctt gatgccatgc tcccatacct tgtcaactac tatgggaacc 301 ctcattctcg gactcatgca tatggctggg agagcgaggc agccatggaa cgtgctcgcc 361 agcaagtagc atctctgatt ggagctgatc ctcgggagat cattttcact agtggagcta 421 ctgagtccaa caacatagca attaaggtag gaggagggat ggggatgttg tgtggccgac 481 agttgtgagg ggttgtggga agatggaagc cagaagcaaa aaagagggaa cctgacacta 541 tttctggctt cttgggttta gcgattagtg cccctctctc atttgaactc aactacccat 601 gtctccctag ttctttctct gcctttaaaa aaaaatgtgt ggaggacagc tttgtggag

MFG

DNA marker

M1 M2

readily detectable sequence of DNA whose inheritance can be monitored and associated with

the trait inheritance

Page 19: GENETIC MARKERS  IN PLANT BREEDING

Image from UV light table

Image from computer screen

Page 20: GENETIC MARKERS  IN PLANT BREEDING

Basis for DNA marker technology

•Restriction Endonucleases

•DNA-DNA hybridization

•Polymerase chain reaction (PCR)

•DNA sequencing

Page 21: GENETIC MARKERS  IN PLANT BREEDING

RFLP techniques

Page 22: GENETIC MARKERS  IN PLANT BREEDING

3

6

2

61 2 43 5

4

5

1MFG

RFLP Polymorphisms interpretation

Page 23: GENETIC MARKERS  IN PLANT BREEDING

Advantages and disadvantages of RFLP

• Advantages– Reproducible– Co-dominant– Simple

• Disadvantages– Time consuming– Expensive– Use of probes

RFLP based markers

Examine differences in size of specific DNA restriction fragments

Require pure, high molecular weight DNAUsually performed on total cellular genome

Page 24: GENETIC MARKERS  IN PLANT BREEDING

AFLP Markers

Most complex of marker technologies Involves cleavage of DNA with two different

enzymes Involves ligation of specific linker pairs to the

digested DNA Subsets of the DNA are then amplified by PCR The PCR products are then separated on

acrylamide gel 128 linker combinations are readily available Therefore 128 subsets can be amplified

Page 25: GENETIC MARKERS  IN PLANT BREEDING
Page 26: GENETIC MARKERS  IN PLANT BREEDING
Page 27: GENETIC MARKERS  IN PLANT BREEDING
Page 28: GENETIC MARKERS  IN PLANT BREEDING

AFLP Markers

Technically demanding Reliable and stable Moderate cost Need to use different kits adapted to

the size of the genome being analyzed.

Like RAPD markers need to be converted to quick and easy PCR based marker

Page 29: GENETIC MARKERS  IN PLANT BREEDING

RAPD

• Domimant markers• Reproducibility

problems

Amplifies anonymous stretches of DNA using arbitrary primers

Fast and easy method for detecting polymorphisms

Page 30: GENETIC MARKERS  IN PLANT BREEDING

RAPD Polymorphisms among landraces of sorghum

M

Sequences of 10-mer RAPD primers

Name Sequence

OP A08 5’ –GTGACGTAGG- 3’OP A15 5’ –TTCCGAACCC- 3’OP A 17 5’ –GACCGCTTGT- 3’OP A19 5’ –CAAACGTCGG- 3’OP D02 5’ –GGACCCAACC- 3’RAPD gel configuration

Page 31: GENETIC MARKERS  IN PLANT BREEDING

RAPD MarkersRAPD Markers

There are other problems with RAPD markers associated with reliability

Because small changes in any variable can change the result, they are unstable as markers

RAPD markers need to be converted to stable PCR markers.

How?

Page 32: GENETIC MARKERS  IN PLANT BREEDING

RAPD MarkersRAPD Markers

The polymorphic RAPD marker band is isolated from the gel

It is used a template and re-PCRed The new PCR product is cloned and

sequenced Once the sequence is determined,

new longer and specific primers can be designed

Page 33: GENETIC MARKERS  IN PLANT BREEDING

VNTRVNTRVariable Number of Tandem RepeatsVariable Number of Tandem Repeats

Tandem repeats (TR): DNA sequences which are existed in

repeated numbers in the genome• Satellite DNA• Minisatellites• Microsatellites

Variable Number (VN)High polymorphism in number of repeats

Page 34: GENETIC MARKERS  IN PLANT BREEDING

VNTRVNTRVariable Number of Tandem RepeatsVariable Number of Tandem Repeats

• Satellite DNA 2-250 bp repeat unit size Constitutes 1- 60% of the genome Some can be separated in CsCl

• ‘satellite band’

• Minisatellites 9-50 bp repeat unit size 100 – 1000 x repeated

• Microsatellites 2-6 bp repeat unit size 10s – 100 x repeated

Page 35: GENETIC MARKERS  IN PLANT BREEDING

Microsatellites Short tandem repeats (simple sequence repeat)

• 2 – dinucleotides• 3 – trinucleotides• 4 – tetranucleotides

Randomly distributed in genome Non-coding

• Some within coding sequences Especially trinucleotides

• Some related to diseases Nomenclature

• PerfectGCTAGCCACACACACACACATGCATC

• InterruptedGCTAGCCACACGTCACACACTGCATC

• CompoundGCTAGCCACACATATATGTGTGCATC

Page 36: GENETIC MARKERS  IN PLANT BREEDING

Sequence

GCGCCGAGTTCTAGGGTTTCGGAATTTGAACCGTC

ATTGGGCGTCGGTGAAGAAGTCGCTTCCGTCGTTTGATTCCGGTCGTCAGAATCAGAATCAGAATCGATATGGTGGCAGTGGTGGTGGTGGTGGTGGTTTTGGTGGTGGTGAATCTAAGGCGGATGGAGTGGATAATTGGGCGGTTGGTAAGAAACCTCTTCCTGTTAG

ATTCTGGAATGGAACCAGATCGCTGGTCTAGAGGTTCTGCTGTGGAACCA…..

Repeat

GGT(5)

SSR repeats and primers

Page 37: GENETIC MARKERS  IN PLANT BREEDING

AATCCGGACTAGCTTCTTCTTCTTCTTCTTTAGCGAATTAGGP1

AAGGTTATTTCTTCTTCTTCTTCTTCTTCTTCTTAGGCTAGGCGP2

P1 P2

SSR polymorphisms

Gel configuration

Page 38: GENETIC MARKERS  IN PLANT BREEDING

SNP (Single Nucleotide Polymorphisms)

• Any two unrelated individuals differ by one base pair every 1,000 or so, referred to as SNPs.

• Many SNPs have no effect on cell function and therefore can be used as molecular markers.

Hybridization using fluorescent dyesSNPs on a DNA strand

Page 39: GENETIC MARKERS  IN PLANT BREEDING

Genetic marker characteristicsCharacteri

sticsMorpholog

ical markers

Protein markers

RFLP markers

RAPD markers

SSR markers

Number of Number of lociloci

LimitedLimited LimitedLimited Almost Almost unlimitedunlimited

UnlimitedUnlimited HighHigh

InheritanceInheritance DominantDominant CodominanCodominantt

CodominanCodominantt

DominantDominant CodominanCodominantt

Positive Positive featuresfeatures

VisibleVisible Easy to Easy to detectdetect

Utilized Utilized before the before the latest latest technologitechnologies were es were availableavailable

Quick Quick assays with assays with many many markersmarkers

Well Well distributed distributed within the within the genome, genome, many many polymorphipolymorphismsm

Negative Negative featuresfeatures

Possibly Possibly negative negative linkage to linkage to other other characterscharacters

Possibly Possibly tissue tissue specificspecific

RadioactiviRadioactivity ty requiremerequirements, rather nts, rather expensiveexpensive

High basic High basic investmentinvestment

Long Long developmedevelopment of the nt of the markers, markers, expensiveexpensive

Page 40: GENETIC MARKERS  IN PLANT BREEDING

Developing a Marker

Best marker is DNA sequence responsible for phenotype i.e. gene

If you know the gene responsible and has been isolated, compare sequence of wild-type and mutant DNA

Develop specific primers to gene that will distinguish the two forms

Page 41: GENETIC MARKERS  IN PLANT BREEDING

Developing a Marker

If gene is unknown, screen contrasting populations

Use populations rather than individuals Need to “blend” genetic differences

between individual other than trait of interest

Page 42: GENETIC MARKERS  IN PLANT BREEDING

Developing Markers Cross individual differing in trait you wish to

develop a marker Collect progeny and self or polycross the

progeny Collect and select the F2 generation for the

trait you are interested in Select 5 - 10 individuals in the F2 showing

each trait Extract DNA from selected F2sExtract DNA from selected F2s Pool equal amounts of DNA from each Pool equal amounts of DNA from each

individual into two samples - one for each traitindividual into two samples - one for each trait Screen pooled or “bulked” DNA with what Screen pooled or “bulked” DNA with what

method of marker method you wish to usemethod of marker method you wish to use

Page 43: GENETIC MARKERS  IN PLANT BREEDING

Single gene trait: seed shape Multigenic trait; ex: plant growth =Quantitative Trait

Loci

Types of traits (types of markers)

MFG

MFG

Page 44: GENETIC MARKERS  IN PLANT BREEDING

USES OF MOLECULAR MARKER Clonal identityClonal identity

Parental analysisParental analysis

Family structureFamily structure

Population structurePopulation structure

Gene flowGene flow

HybridisationHybridisation

PhylogenyPhylogeny

Measure genetic diversity

Mapping

Tagging

Page 45: GENETIC MARKERS  IN PLANT BREEDING

Genetic DiversityGenetic Diversity Define appropriate geographical scales for Define appropriate geographical scales for

monitoring and management (epidemology)monitoring and management (epidemology) Establish gene flow mechanismEstablish gene flow mechanism

identify the origin of individual (mutation identify the origin of individual (mutation detection)detection)

Monitor the effect of management practicesMonitor the effect of management practices manage small number of individual in ex situ manage small number of individual in ex situ

collectioncollection Establish of identity in cultivar and clones Establish of identity in cultivar and clones

(fingerprint)(fingerprint) paternity analysis and forensicpaternity analysis and forensic

Page 46: GENETIC MARKERS  IN PLANT BREEDING

Genetic Diversity

Page 47: GENETIC MARKERS  IN PLANT BREEDING

Mapping

The determination of the position and The determination of the position and relative distances of gene on chromosome relative distances of gene on chromosome

by means of their linkage by means of their linkage

Genetic mapA linear arrangement of genes or genetic markers

obtained based on recombinationAn ordering of genes and markers in a linear An ordering of genes and markers in a linear arrangement corresponding to their physical arrangement corresponding to their physical

order along the chromosome, based on order along the chromosome, based on linkagelinkage.. Physical map

A linear order of genes or DNA fragmentsAn ordering of landmarks on DNA, regardless of An ordering of landmarks on DNA, regardless of

inheritance, measured in base pairs.inheritance, measured in base pairs.

Page 48: GENETIC MARKERS  IN PLANT BREEDING

Physical Mapping

It contains ordered overlapping cloned DNA fragment

The cloned DNA fragments are usually obtained using restriction enzyme digestion

Page 49: GENETIC MARKERS  IN PLANT BREEDING

QTL MappingQTL Mapping

A set of procedures for detecting genes controlling quantitative traits (QTL) and estimating their genetics

effects and location

To assist selection

Page 50: GENETIC MARKERS  IN PLANT BREEDING

Fundamental Genetics Fundamental Genetics (Background for Linkage Analysis)(Background for Linkage Analysis)

Rule of SegregationRule of Segregation• offspring receive ONE allele (genetic offspring receive ONE allele (genetic

material) from the pair of alleles material) from the pair of alleles possessed by BOTH parentspossessed by BOTH parents

Rule of Independent AssortmentRule of Independent Assortment• alleles of one gene can segregate alleles of one gene can segregate

independently of alleles of other genesindependently of alleles of other genes• (Linkage Analysis relies on the violation (Linkage Analysis relies on the violation

of Independent Assortment Rule)of Independent Assortment Rule)

Page 51: GENETIC MARKERS  IN PLANT BREEDING

Linkage AnalysisLinkage Analysis

Goal: Goal:

find a marker “linked” to a disease gene.find a marker “linked” to a disease gene. LOD score = log of likelihood ratioLOD score = log of likelihood ratio LR[θ;data] == k P[data; θ]LR[θ;data] == k P[data; θ] θθ = = estimate of genetic distance estimate of genetic distance

(recombination fraction) between marker and (recombination fraction) between marker and

quantitative traitsquantitative traits

= proportion of recombinant gametes/total = proportion of recombinant gametes/total gametesgametes

Page 52: GENETIC MARKERS  IN PLANT BREEDING

Linkage AnalysisLinkage Analysis

Genes near each other on a chromosome tend to be inherited together, that is, they are linked.

Linkage analysis are the techniques used to identify such linkages among genes

Linkage groups which include genetic markers and genes determinative of phenotype allow the identification of determinative alleles (and therefore prediction)

Page 53: GENETIC MARKERS  IN PLANT BREEDING

Linkage

Mendel showed that alleles segregate independently. Then he tested genes

Sometimes inheritance of two genes are independent of another, that is phenotype ratios are 9:3:3:1

Sometimes inheritance of two genes are linked together, showing a ratio of 3:0:0:1

Linkage can vary continuously from perfectly correlated to uncorrelated.

Page 54: GENETIC MARKERS  IN PLANT BREEDING

Why genes are linked

Alleles are arranged linearly Each parent passes only one of its two

chromosomes to an offspring. Recombination periodically switches

which chromosome in the parent is passed along

Alleles near each other are more likely to be passed along than ones further apart

Alleles on different chromosomes are always inherited independently.

Page 55: GENETIC MARKERS  IN PLANT BREEDING

Marker Assisted Selection

Breeding for specific traits in plants and animals is expensive and time consuming

The progeny often need to reach maturity before a determination of the success of the cross can be made

The greater the complexity of the trait, the more time and effort needed to achieve a desirable result.

Page 56: GENETIC MARKERS  IN PLANT BREEDING

MASMAS

The goal to MAS is to reduce the The goal to MAS is to reduce the time needed to determine if the time needed to determine if the progeny have traitprogeny have trait

The second goal is to reduce costs The second goal is to reduce costs associated with screening for traitsassociated with screening for traits

If you can detect the distinguishing If you can detect the distinguishing trait at the DNA level you can trait at the DNA level you can identify positive selection very early. identify positive selection very early.

Page 57: GENETIC MARKERS  IN PLANT BREEDING

Marker Assisted Breeding

MAS allows for gene pyramiding - incorporation of multiple genes for a

trait Prevents development of biological

resistance to a gene Reduces space requirements - dispose of unwanted plants and

animal early

Page 58: GENETIC MARKERS  IN PLANT BREEDING

Trait2.58.47.12.54.52.3

P.1P.2I.1I.2I.3I.4

M. 1133221

M. 2131131

M. 3131123

QTL study

Statistical programs used in molecular marker studies* SAS* ANOVA* Mapmaker* Cartographer

Types of population used for molecular markers studies: F2, RILs, Backcrosses (MILs), DH.

Page 59: GENETIC MARKERS  IN PLANT BREEDING

QTL MappingQTL Mapping

Page 60: GENETIC MARKERS  IN PLANT BREEDING

Recombination picture

Crossover is the alternation of allele generating chromatid (half of chromosome)