Lect.2.2011. Genetic Physical Mapping4 Page
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Transcript of 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/
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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)
23 24
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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