Mol Markers

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Introduction to Molecular Markers and

their Application in Biotechnology


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Molecular Markers 1. What are molecular markers?

Introduction to technology

Description of types of markers

2. Uses of molecular markers

Application as a genetic tool for plant genotyping and genemapping

Applications in Human Health

Association with genetic-based diseases

Forensic studies

Application in agriculture

Marker-assisted breeding

Plant variety protectionAssessing genetic diversity


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Molecular Markers

Heritable DNA sequence differences (polymorphisms)

1. What are molecular markers?

2. Types of Markers

Phenotypically neutral, developmentally and environmentally stable

Identified by techniques such as Southern hybridizations or PCR

RFLPs --Restriction Fragment Length Polymorphisms

VNTRs -- variable number of tandem repeats (minisatellites)

Those detected by Southern Hybridizations

Those detected by PCR-based methods

RAPD -- randomly amplified polymorphic DNA

AFLP -- amplification fragment length polymorphism

CAPS -- cleaved amplified polymorphic site

SSR -- simple sequence repeats (microsatellites)

SNP -- single nucleotide polymorphisms

The best molecular markers are those that distinguish

multiple alleles per locus (i.e. are highly polymorphic) and

are co-dominant (each allele can be observed)


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RFLP- a site in a genome where the distance between two restriction sitesvaries among different individuals. These sites are identified by restriction enzymedigests of chromosomal DNA, and the use of Southern blotting to identify the specificfragments. Requires a radioactive probe!

SNP-

a single nucleotide difference in the sequence of a gene or segment of thegenome. There are typically tens of 1,000s of SNPs and a variety of methods foranalyzing them, including highly automated/high throughput procedures withsimultaneous scoring of many markers. Detection of SNPs can be done without gels.

SSR-a site in the genome that contains many short tandem repeat sequences(microsatellites). These sites are usually in the size range of 100-500 base pairscomposed of dinucleotide and trinucleotide repeats. They are very polymorphic,scattered through out genomes. Genomes typically contain 1,000s of SSRs! They aredetected by PCR using primers flanking the repeats and resolved on gels.

CAPS-

a site in the genome polymorphic for the presence or absence of arestriction enzyme site. Detected by PCR and restriction enzyme digests on gels.


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RFLPs can arise through point mutations, deletions, insertions, etc.

Allele A Allele B

probe probe

RERE RERE RE

polymorphism

AA BB AB

RE RERE RE

A deletion between tworestriction enzyme sites

AA BB AB

RE RERE RE

An insertion between two restriction enzyme sites

transposon


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RFLPs

Restriction Fragment Length Polymorphism (RFLP)

probe binding site5 EcoRI EcoRI 3

CHR 1: NNNG|AATTCNNN-------NNNG|AATTCNNN

NNNCTTAA|GNNN-------NNNCTTAA|GNNN

3 5

probe binding site

5 EcoRI EcoRI 3

CHR 2: NNNG|AATTCNNN-------------------NNNG|AATTCNNN

NNNCTTAA|GNNN-------------------NNNCTTAA|GNNN

3 5

allele A

allele B

allele C

allele A

allele B

allele C

Genomic DNA cut by EcoRI, DNA run

on gel, blotted, and hybridized to probe

Polymorphism: fragments of different

lengths migrate differently in the gel


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RFLPs

probe probe

RE RE RERE RE

Allele A Allele B

polymorphism

Allele B (RFLP B) appears

associated with the phenotype!

But not completely! #12 must be arecombinant between the phenotypic locusand the RFLP B

BBAB

AA BB AB

Southern blot

P1 P2 1 2 3 4 5 6 7 8 9 10 11 12* * * ** **


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RFLPs

probe probe

RE RE RERE RE

Allele A Allele B

AA BB AB

Southern blot

P1 P2 1 2 3 4 5 6 7 8 9 10 11 12* * * ** **

RE RE

RE RERE

Allele A

*Allele B


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Review of Genetic Linkage and

Recombination

Genes that are close together on a

chromosome tend to be inherited together.

In the example shown at left, genes A and

B would tend to be inherited together

much more often than with gene C.

Gene C would be inherited with B slightlymore often than with A.

Recombination & Crossing Over

Exchange of alleles, but not gene order,

between the two homologouschromosomes in diploids during meiosis.

Frequency of crossing-over increases

with distance between loci.


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RFLPs

probe probe

RE RE RERE RE

Allele A Allele B

AA BB AB

Southern blot

P1 P2 1 2 3 4 5 6 7 8 9 10 11 12* * * ** **

RE RE

RE RERE

Allele A

*Allele B

Allele B

Allele A

RE RE

RE RERE

*

*


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CAPS (cleaved amplified polymorphic sequence) as a molecular marker.

Allele A Allele BRERE RERE RE

polymorphism

Sequence Specific Primers

Amplify by PCR

AA BB AB

Visualize on Agarose Gel

Digest with restriction enzymeThis procedure is much quicker thandoing a Southern hybridization, yetyields the same information!!


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SSR: simple sequence repeat(length polymorphism)

(CA) (CA)n n+4

PCR Primer

PCR yields products of different size:

variation between strains in number of repeatsat a given locus


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Polymorphism is based on the

number of times a simple

sequence of DNA, usually 2-3

base pairs, is repeated. Thevariant alleles are probably

generated by stuttering of DNA

polymerase or repair enzymes

during DNA replication of

repeated sequences.

Simple Sequence Repeat (SSR) or Microsatellite Markers

Male parent (red square) contains alleles 5 and 2

Female parent (black circle) contains alleles 6 and 3

Progeny segregate for alleles 2, 3, 5, and 6

One daughter is mated to a male containing another

allele (4), resulting in offspring heterozygous for

alleles 5 and 4.


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Bottom line:interpretation ofdata for RFLP markers and

microsatellites is the same, eventhough the data is generated bymeans of different techniques(Southern blotting for RFLP, PCR

for microsatellite)


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Molecular Markers 1. What are molecular markers?

Introduction to technology

Description of types of markers

2. Uses of molecular markers

Application as a genetic tool for plant genotyping and genemapping

Applications in Human Health

Association with genetic-based diseases

Forensic studies

Application in agriculture

Marker assisted breeding

Plant variety protection

Assessing genetic diversity


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Genotyping Progeny in a Standard Genetic Cross

Prepare DNA from leaves of each type and use molecular

marker to determine genotype!

An example of a genetic cross

wild type X teosinte branched mutant

(+/+) X (tb1/tb1)

all wild type (+/tb1)F1

Normal : tb1

3 : 1

(+/+ or +/tb1) : (tb1/tb1)

F2

Normals are either +/+ or +/tb1, butone can not distinguish between the twogenotypes because + is dominant to therecessive tb1!

BUT, a molecular marker can easilydistinguish between the genotypes!


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Molecular markers can be mapped relative to one another

This leads to a physical map that illustrates the linkage relationshipsbetween physical markers on each of the chromosomes

Now, one can map a new mutation (morphological marker) relative to the

molecular markers by following linkage between the mutant phenotype andpolymorphisms in molecular markers.

Physical Maps


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Lets use molecular markers to find themap location of a newly isolated mutant!


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First, make a mapping population:

New mutant: tb1-like. This mutant was found in an EMS

screen for new mutants in maize inbred B73

tb

tb

Map your mutant by comparing its inheritance relative

to the inheritance of mapped molecular markers

In a B73 background x Mo17

B1 B2 tb B3 B4

B1 B2 tb B3 B4

M1 M2 TB M3 M4

M1 M2 TB M3 M4x

B1 B2 tb B3 B4

M1 M2 TB M3 M4F1


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If a molecular marker is very closely linked to the mutation(tb), then those progeny homozygous for the mutation will

also be homozygous for the B73 polymorphism

B73

Mo17

tb1

tb2

tb3

tb4

tb5

tb6

tb7

tb8

tb9

tb10

tb11

tb12

tb13

tb14

tb15

tb16

tb17

tb18

tb19

tb20


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If a molecular marker is unlinked to the mutation (tb), then

those progeny homozygous for the mutation are just as likelyto inherit the MO17 polymorphism as the B73 polymorphism

B73

Mo17

tb1

tb2

tb3

tb4

tb5

tb6

tb7

tb8

tb9

tb1

0

tb1

1

tb1

2

tb1

3

tb1

4

tb1

5

tb1

6

tb1

7

tb1

8

tb1

9

tb2

0

20 recombinant chromosomes out of a total of 40 scored

This marker is unlinked to the mutation!


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If a molecular marker is linked to the mutation (tb), then those

progeny homozygous for the mutation are more likelyto

inherit the B73 polymorphism then the MO17 polymorphism

B1 B2 tb B3 B4

B73

Mo17

tb1

tb2

tb3

tb4

tb5

tb6

tb7

tb8

tb9

tb10

tb11

tb12

tb13

tb14

tb15

tb16

tb17

tb18

tb19

tb20


8/40 = 0.2 or 20% recombination or 20 map units (20 cM betweenthis marker and the mutation).

You have found a linked molecular marker!!


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If a molecular marker is linked to the mutation (tb), then those

progeny homozygous for the mutation are more likelyto

inherit the B73 polymorphism then the MO17 polymorphism

B1 B2 tb B3 B4

B73

Mo17

tb1

tb2

tb3

tb4

tb5

tb6

tb7

tb8

tb9

tb10

tb11

tb12

tb13

tb14

tb15

tb16

tb17

tb18

tb19

tb20


8/40 = 0.2 or 20% recombination or 20 map units (20 cM betweenthis marker and the mutation).

You have found a linked molecular marker!!


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If a molecular marker is linked

to the mutation

Bulked Segregant Analysis

B73 Mo17

Pooled

(bulked)mutants

B

73

M

o17 mutants

If a molecular marker is

unlinked to the mutationB73

Mo17 mutants

B73 Mo17

Pooled(bulked)

mutants

F1

Pooled

(bulked)mutants

Pooled

(bulked)normals


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SNP: single nucleotidepolymorphisms

Outgrowth of sequencing projects

Detection of SNPs can be done without gels:

highly automated/high throughput and/or

highly parallel (simultaneous scoring ofMANY markers)


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AlleleAllele--Specific Extension & Identification in CE:Specific Extension & Identification in CE:

MinisequencingMinisequencing (ABI(ABI SNaPShotSNaPShotTMTM))


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dR6G

dR110

Degree of Multiplexing Depends on ResolutionDegree of Multiplexing Depends on Resolution

ABI SNaPshot on 3130xl


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PCR Amplification

Single Base Extension

SAP Treatment

MALDI-TOF Mass Spec

Spot on 384-place Chips

Genotyping by SBE and Mass SpectrometryGenotyping by SBE and Mass Spectrometry


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Other uses for Molecular Markers

Plant Variety Protection

Verify varietal identity, purity and stability

Estimation of Genetic Variation

For phylogenetic analysis

For preservation of rare plant and animal species

For management of wild species

Plant Pathogen Identification

Marker-Assisted BreedingFor accelerated trait/transgene introgressions

For introgression of quantitative traits


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Molecular Markers in Human Health

Identification of human genetic

diseasesSickel cell anemia

Huntingtons disease

Tay Sachs disease

Cystic Fibrosis (and many more)

Forensic Science

Paternity Determinations

I t d ti t M l l M k d


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Introduction to Molecular Markers and

their Application in Biotechnology

Mol Markers

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