lect.2.2011. genetic physical mapping4 page

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How to make a linkage map for a self-

pollinated plant

P1 P2X1. Making a cross

2. Marker genotypeanalysis

3. Linkage analysis

Parents

F1

F2

MAPMAKER: a linkage map construction software developed by EricLander. Website: http://linkage.rockefeller.edu/soft/mapmaker/

1

2

3

Marker 1 and 2, and marker 3 and 4 are linked

P1 P2

F1

F2

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1. A genetic linkage map shows the relative locations of specific DNA markersalong the chromosome. Any inherited physical or molecular characteristic

that differs among individuals and is easily detectable in the laboratory is apotential genetic marker.

2. Markers can be expressed DNA regions (genes) or DNA segments that haveno known coding function but whose inheritance pattern can be followed.

DNA sequence differences are especially useful markers because they areplentiful and easy to characterize precisely.

3. Markers must be polymorphic to be useful in mapping; that is, alternativeforms must exist among individuals so that they are detectable among

different members in family studies.

4. Polymorphisms are variations in DNA sequence that occur on average onceevery 300 to 500 bp. Variations within exon sequences can lead toobservable changes, such as differences in eye color, blood type, and

disease susceptibility.

Genetic Linkage Maps

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Barley Linkage Map

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1990 2000

High-density molecular linkage map of rice chromosome 1

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DNA Markers for Genetic Mapping

1. Restriction fragment length polymorphisms(RFLP)

2. RAPD-Random amplified polymorphic DNA(RAPD)

3. Microsatellite, simple sequence repeat (SSR)markers or short tandem repeat (STR)

4. Single nucleotide polymorphisms (SNPs)5. Amplified fragment length polymorphism (AFLP)

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1. Restriction fragment length polymorphisms (RFLP)

TA Brown, p 13110

2. RAPD-Random amplified polymorphic DNA

The PCR reaction

performed on genomicDNA with an arbitrary

oligonucleotide primer

(10 bp) and low

annealing temperature

(35-38 C) results in the

amplification of several

discrete DNA products.

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3. Microsatellite Markers

1. Microsatellites are short segments of DNA that have arepeated sequence such as CACACACA, and they tend tooccur in non-coding DNA. In some microsatellites, therepeated unit (e.g. CA) may occur four times, in others itmay be seven, or two, or thirty.

2.

The most common way to detect microsatellites is todesign PCR primers that are unique to one locus in thegenome and that base pair on either side of the repeatedportion (See the Figure below). Therefore, a single pair ofPCR primers will work for every individual in the speciesand produce different sized products for each of thedifferent length microsatellites.

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4. Single nucleotide polymorphisms (SNPs)

TA Brown, p 132-134

Hybridization method ( the incubation

temperature just below the melting temp or Tm)

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5. AFLP- Amplified fragment

lengthpolymorphism -- a DNA

fingerprinting technique that combinesfeatures of RFLP and RAPD techniques

for amplification of a subset of

genomic restriction fragments using

selective primers

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AFLP- Amplified fragment lengthpolymorphism

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A quantitative trait locus/loci (QTL) is the location of

individual locus or multiple loci loci that affects a trait

that is measured on a quantitative (linear) scale.

Examples of quantitative traits are:

- Plant height (measured on a ruler)

- Grain yield (measured on a balance)

These traits are typically affected by more than onegene, and also by the environment. Thus, mapping

QTL is not as simple as mapping a single gene that

affects a qualitative trait (such as flower color).

Quantitative trait locus/loci (QTL)

See more in page 155-158, Gibson and Muse


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Chromosomal location of transcript-derived fragments (TDFs) and QTL likelihood maps forearliness (obtained by multiple QTL mapping) on linkage groups E5 and E12.

Fernndez-del-Carmen A et al. J. Exp. Bot.

2007;58:2761-2774

2007 The Author(s).18

See more in page 158-160, Gibson and Muse

Association mapping, also known as "linkage disequilibrium mapping",

or genome-wide association study, GWAS, is a method of mapping quantitative

trait loci (QTLs) that takes advantage of historic linkage disequilibrium to linkphenotypes (observable characteristics) to genotypes (the genetic constitution oforganisms.

GWAS is based on the idea that traits that have entered a population only

recently will still be linked to the surrounding genetic sequence of the originalevolutionary ancestor, or in other words, will more often be found within a given

haplotype, than outside of it. Association mapping thus asks if a particulargenetic marker (most often a SNP) is more common in a particular phenotype

than you would expect by chance. It is most often performed by scanning the

entire genome for significant associations between a panel of SNPs (which, inmany cases are spotted onto glass slides to create SNP chips) and a particular

phenotype. These associations must then be independently verified in order toshow that they either a. contribute to the trait of interest directly, or b. are linked

to/ in linkage disequilibrium with a quantitative trait locus (QTL) that contributes

to the trait of interest (From Wikipedia)

Association mapping

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QTL mapping

QTL and Association Mapping

Construction of a physical map whichconsists of continuous overlapping

fragments of cloned DNA that has the samelinear order as found on the chromosomes

from which they were derived. Usually large

insert genome clones such as BacterialArtificial Chromosome (BAC), Yeast Artificial

Chromosome (YAC) and cosmid clones are

used for construction of whole-genome or a

specific genomic region.

Physical Mapping

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Physical Map vs Genetic Map

1. The resolution of a geneticmap depends on the numberof crossovers that have been

scored. Limited resolution (1cM is approx. 1 Mb)

2. Genetic maps have limitedaccuracy. Certain genomicregions more sensitive to

recombination3. Markers must be polymorphic

for genetic mapping

Limitations of

genetic maps

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Physical Map of a Chromosome

Depicts genetic markers and DNA sequences between

the markers measured in base pairs (High Resolution)

Contig:A series of overlapping clones or sequences that collectively

span a particular chromosomal region

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Physical Mapping Methods

1. Optical mapping2. Chromosome walking3. BAC end sequencing4. BAC fingerprinting5. Sequence tagged site (STS) mapping6. Fluorescent in situ hybridization (FISH)

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Optical mapping is a technique for constructing

ordered, genome-wide, high-resolution restrictionmaps from single, stained molecules of DNA,

called "optical maps". By mapping the location ofrestriction enzyme sites along the unknown DNA

of an organism, the spectrum of resulting DNA

fragments collectively serve as a unique"fingerprint" or "barcode" for that sequence.

1. Optical Mapping


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Gel stretching and molecular combing

Add fluorescent dye

Optical Mapping Methods

TA Brown, p 15125 26

Optical Mapping Methods

The advantage of OM over traditional mapping techniques is that it preserves the order of the DNA fragment,

whereas the order needs to be reconstructed using restriction mapping. In addition, since maps are constructeddirectly from genomic DNA molecules, cloning or PCR artifacts are avoided. However, each OM process is stillaffected by false positive and negative sites because not all restriction sites are cleaved in each molecule and

some sites may be incorrectly cut. In practice, multiple optical maps are created from molecules of the samegenomic region, and an algorithm is used to determine the best consensus map. (Wikipedia)

Markers with known map position are used as probe to

screen the large insert library. Clones hybridizing with

the same single copy marker are considered to beoverlapping. PCR amplification of DNA pools using

primers derived from DNA markers with known position

was also used for physical map construction.

Advantages: Good for a small regionDisadvantages: labor intensive, repetitive sequence

misleading, markers unevenly distributed in the

genome.

2. Chromosome walking

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ChromosomeWalking

BACs

R

S

Marker A Marker BTargeted Gene

S

S

1

S

R

3

2

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3. BAC End Sequencing

Sequencing of the ends of a large number of

BAC clones, which is then followed by a global search foran identical sequence between the nucleotide sequenceof a seed BAC and the BAC end sequences. Contigs are

built in a stepwise fashion as sequencing proceeds along

a chromosome

Advantages: Fast and large-scale

Disadvantages: Difficult to sequence some BAC ends,repetitive sequences affect accurate contig construction

leading to many gaps

Individual clones are digested with different

restriction enzymes. The digested DNA is labeled

with radioactive or fluorescent dye and run on a

sequencing gel. The fingerprint data is collectedand analyzed for contig assembly.

Advantages: Fast and large-scaleDisadvantages: labor intensive and difficult to fill

gaps.

4. Restriction fragment fingerprinting

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BAC DNA PurificationBAC fingerprinting:digest and label DNAsRun a sequencing gel

Autoradiography

BAC Fingerprinting

Fingerprint digitizing:Input to a computervia a scanner

BAC Fingerprint Images

Covert the fingerprintimages into database.

BAC Fingerprint Database

Data analysis and

contig assembly

Physical Map & BAC Contigs

Procedure of genome-wide physical mapping

using finger printing method

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Autoradiography

Fingerprint

Image

Ready for

computer

analysis

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Modifications

1. Different enzyme digestion (4 or 6 bpendonuclease)

2. Fluorescent dye labeling3. Multiplexing BAC digests (different pairs of

restriction endonucleases, each labeled

with a different fluorescent dye)

4. A single restriction endonuclease pairemploying a type IIS restriction

endonuclease5. SNaPshot high-information content-

fingerprinting (HICF) technology34

Luo MC, Thomas C, You FM, Hsiao J, Ouyang S, Buell CR, Malandro M, McGuire

PE, Anderson OD, Dvorak J. High-throughput fingerprinting of bacterial artificialchromosomes using the snapshot labeling kit and sizing of restriction fragments bycapillary electrophoresis. Genomics. 82(3):378-89.

Abstract: We have developed an automated, high-throughput fingerprinting

technique for large genomic DNA fragments suitable for the construction of physicalmaps of large genomes. In the technique described here, BAC DNA is isolated in a

96-well plate format and simultaneously digested with four 6-bp-recognizingrestriction endonucleases that generate 3' recessed ends and one 4-bp-recognizingrestriction endonuclease that generates a blunt end. Each of the four recessed 3'

ends is labeled with a different fluorescent dye, and restriction fragments are sizedon a capillary DNA analyzer. The resulting fingerprints are edited with a fingerprint-

editing computer program and contigs are assembled with the FPC computerprogram. The technique was evaluated by repeated fingerprinting of several BACsincluded as controls in plates during routine fingerprinting of a BAC library and by

reconstruction of contigs of rice BAC clones with known positions on ricechromosome 10.

SNaPshot high-information content-

fingerprinting (HICF) technology

TC T A G A

A G A T C T

Xba I

5

3

3 5

G G C C

G G C CC C G G

C C G G

Restriction cleavage

SNaPshot BAC fingerprinting

HaeIII HaeIII

Luo M., UCDavis

T

A G A T C

Xba I

5

3

C T A G A

T

3

5

Fluorescent labeling

C

C


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G G A T C C

C C T A GG

Bam HI5 3

3 5

GAA T T C

C T T A AG

Eco RI5 3

3 5

CTC G A G

G A G CTC

Xho I5 3

3 5

Restriction cleavage and fluorescent labeling Characteristics of restriction sites

and labeling of fragments

noneGG^CCHaeIII

dROXTC^TCGAGXhoI

YellowdTAMRACT^CTAGAXbaI

dR110GG^GATTCBamHI

GreendR6GAG^AATTCEcoRI

Color offragment

Fluorescentdye

ddNTPRestrictionsite

Restrictionendonuclease

Blue

Red

Portion of multi-color fingerprinting profile of a BAC clone

BamHI

EcoRI

XbaI

XhoI

Liz-1200

Size standard

A sample contig: 424 BAC clones, ca. 3.7 Mb

Advantages: Robust, reliable and good for large complex

genome

Disadvantages: Labor intensive

Luo et al. 2010. Feasibility of physical map construction from fingerprinted bacterial

artificial chromosome libraries of polyploid plant species. BMC Genomics 2010,11:122


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The DNA fingerprinting approach to building a whole genome physical map

Meyers BC, et al. Nat Rev

Genet. 2004, 5:5578-88.

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An STS is a short region of DNA about 200-300bases long whose exact sequence is foundnowhere else in the genome. Two or more

clones containing the same STS must overlapand the overlap must include the STS.

Advantages: Rapid and simpleDisadvantages: Still very labor intensive and

high expensive for primer synthesis.

5. Sequence Tagged Sites (STSs).

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STS mapping of linked markers and BAC clones43

PCR confirmation of STS markers in the genome

Each STS contains a unique sequence

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6. Fluorescence in situ hybridization (FISH)

This technique uses synthetic polynucleotide strands orshort DNA fragment that bear sequences known to be

complementary to specific target sequences at specificchromosomal locations. The polynucleotides are bound

via a series of link molecules to a fluorescent dye that

can be detected by a fluorescence microscope.

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Advantages: Direct visualization of a chromosomal

region and accurate

Disadvantages: Technically challenging and difficultfor some species

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TA Brown, p 15247

Human metaphase chromosomes hybridized to fluorescent probes from twooverlapping microdissection libraries. Probes specific to chromosome regions 1p3435 and 1p36 were labeled using the ULYSIS Oregon Green 488 (U21659) and Alexa

Fluor 594 (U21654) Nucleic Acid Labeling Kits, respectively.(http://www.probes.com/servlets/photohigh?fileid=g001276) 48


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4-kb

2.5-kb2-kb

BAC vector 7.4-kb

FISH mapping of BACs (bacterial artificial chromosomes)

vector

vector49

J. Jiang, UWM

Application of FISH in physical mapping

Jackson S. et al. 2000. Genetics, 156: 833-838

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Combination of fingerprinting, molecular

linkage map, STS, end sequencing and FISHmapping.

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Human genome anatomy: BACs integratingthe genetic and cytogenetic maps for bridging

genome and biomedicine.

Genome Res. 1999 9(10):994-1001

Combination of STS markers, BACs and FISH

872 unique STSs and 957 BACs were used

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Integrated genetic map of human chromosome 1153

Integrated genetic, physical and sequence map

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Summary

1.A genetic linkage map shows the relativelocations of specific DNA markers along thechromosome.

2. Five major DNA markers for genetic mapping:RFLP, RAPD, SNP, AFLP, Microsatellites

3.A physical map shows the exact position ofgenes and other sequences on thechromosome

4. Six physical mapping methods: opticalmapping, BAC end sequencing, BAC

fingerprinting, chromosome walking, STS and

FISH55

Additional reading materials

1. Meyers BC, Scalabrin S, Morgante M. 2004. Mapping andsequencing complex genomes: let's get physical! Nat Rev

Genet. 5:5578-88.

2. Luo M. et al. 2010. Feasibility of physical map constructionfrom fingerprinted bacterial artificial chromosome libraries of

polyploid plant species. BMC Genomics 2010, 11:122

3. TA. Brown. 2002. Genomics, Second Edition. Wiley-Liss,Chapter 5, pp 125-159.

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