You are on page 1of 14

Review of Genetic Analysis

Genetic and Physical Mapping


Genetic mapping is based on the use of genetic techniques to construct linkage maps showing the positions of genes and other sequence features in a genome Physical mapping uses molecular biology techniques to examine DNA molecules directly in order to construct maps showing the exact position of genes and other sequences

Phenotype: 3:1 Genotype: 1:2:1

Lecture 2, 3/31/2011 Plant Pathology 703: Agricultural Genomics


Guo-Liang Wang Department of Plant Pathology, The Ohio State University Email: wang.620@osu.edu
1
TA Brown, p 135

Mendels Laws enable the outcome of genetic crosses to be predicted

Unlinked, linked and partial linked genes

A three-point mapping cross involving the yellow (y), white (w) and echinus (ec) genes on the X chromosome of Drosophila (Concept of Genetics, Klug and Cummings, p123)

TA Brown, p 136

How to make a linkage map for a selfpollinated plant


P1
1. Making a cross Parents

Genetic Linkage Maps


P2
1. A genetic linkage map shows the relative locations of specific DNA markers along the chromosome. Any inherited physical or molecular characteristic that differs among individuals and is easily detectable in the laboratory is a potential genetic marker. Markers can be expressed DNA regions (genes) or DNA segments that have no known coding function but whose inheritance pattern can be followed. DNA sequence differences are especially useful markers because they are plentiful and easy to characterize precisely. Markers must be polymorphic to be useful in mapping; that is, alternative forms must exist among individuals so that they are detectable among different members in family studies. Polymorphisms are variations in DNA sequence that occur on average once every 300 to 500 bp. Variations within exon sequences can lead to observable changes, such as differences in eye color, blood type, and disease susceptibility.
6

P1 X P2 1 3
F1 F2

F1

F2

2.

2. Marker genotype analysis

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

4.
3. Linkage analysis

MAPMAKER: a linkage map construction software developed by Eric


Lander. Website: http://linkage.rockefeller.edu/soft/mapmaker/
5

Barley Linkage Map

High-density molecular linkage map of rice chromosome 1

1990

2000

1. Restriction fragment length polymorphisms (RFLP)

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)

TA Brown, p 131

10

2. RAPD-Random amplified polymorphic DNA

3. Microsatellite Markers
1. Microsatellites are short segments of DNA that have a repeated sequence such as CACACACA, and they tend to occur in non-coding DNA. In some microsatellites, the repeated unit (e.g. CA) may occur four times, in others it may be seven, or two, or thirty. 2. The most common way to detect microsatellites is to design PCR primers that are unique to one locus in the genome and that base pair on either side of the repeated portion (See the Figure below). Therefore, a single pair of PCR primers will work for every individual in the species and produce different sized products for each of the different length microsatellites.

The PCR reaction performed on genomic DNA with an arbitrary oligonucleotide primer (10 bp) and low annealing temperature (35-38 C) results in the amplification of several discrete DNA products.
11

12

4. Single nucleotide polymorphisms (SNPs)

5. AFLP- Amplified fragment length polymorphism -- a DNA fingerprinting technique that combines features of RFLP and RAPD techniques for amplification of a subset of genomic restriction fragments using selective primers

Hybridization method ( the incubation temperature just below the melting temp or Tm)

13
TA Brown, p 132-134

14

AFLP- Amplified fragment length polymorphism

Quantitative trait locus/loci (QTL)


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 one gene, 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).
15

See more in page 155-158, Gibson and Muse

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

Association mapping
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 link phenotypes (observable characteristics) to genotypes (the genetic constitution of organisms. 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 original evolutionary ancestor, or in other words, will more often be found within a given haplotype, than outside of it. Association mapping thus asks if a particular genetic 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, in many cases are spotted onto glass slides to create SNP chips) and a particular phenotype. These associations must then be independently verified in order to show 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)
18

Fernndez-del-Carmen A et al. J. Exp. Bot. 2007;58:2761-2774


2007 The Author(s).

See more in page 158-160, Gibson and Muse

QTL and Association Mapping QTL mapping

Physical Mapping
Construction of a physical map which consists of continuous overlapping fragments of cloned DNA that has the same linear order as found on the chromosomes from which they were derived. Usually large insert genome clones such as Bacterial Artificial Chromosome (BAC), Yeast Artificial Chromosome (YAC) and cosmid clones are used for construction of whole-genome or a specific genomic region.
19 20

Physical Map vs Genetic Map


Limitations of genetic maps
1. The resolution of a genetic map depends on the number of crossovers that have been scored. Limited resolution (1 cM is approx. 1 Mb) Genetic maps have limited accuracy. Certain genomic regions more sensitive to recombination Markers must be polymorphic for genetic mapping

Physical Map of a Chromosome


Contig: A series of overlapping clones or sequences that collectively span a particular chromosomal region

2.

3.

21

Depicts genetic markers and DNA sequences between the markers measured in base pairs (High Resolution)

22

Physical Mapping Methods


1. 2. 3. 4. 5. 6. Optical mapping Chromosome walking BAC end sequencing BAC fingerprinting Sequence tagged site (STS) mapping Fluorescent in situ hybridization (FISH)

1. Optical Mapping
Optical mapping is a technique for constructing ordered, genome-wide, high-resolution restriction maps from single, stained molecules of DNA, called "optical maps". By mapping the location of restriction 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.
23 24

Optical Mapping Methods

Optical Mapping Methods

Add fluorescent dye

TA Brown, p 151

Gel stretching and molecular combing

25

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 constructed directly from genomic DNA molecules, cloning or PCR artifacts are avoided. However, each OM process is still affected by false positive and negative sites because not all restriction sites are cleaved in each molecule and 26 some sites may be incorrectly cut. In practice, multiple optical maps are created from molecules of the same genomic region, and an algorithm is used to determine the best consensus map. (Wikipedia)

2. Chromosome walking
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 be overlapping. 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 region Disadvantages: labor intensive, repetitive sequence misleading, markers unevenly distributed in the genome.

Chromosome Walking
2

BACs

3 Targeted Gene

Marker A

Marker B

R S S R S S
27 28

3. BAC End Sequencing


Sequencing of the ends of a large number of BAC clones, which is then followed by a global search for an identical sequence between the nucleotide sequence of 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
29

4. Restriction fragment fingerprinting


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 collected and analyzed for contig assembly. Advantages: Fast and large-scale Disadvantages: labor intensive and difficult to fill gaps.
30

Procedure of genome-wide physical mapping using finger printing method


BAC DNA Purification BAC fingerprinting: digest and label DNAs Run a sequencing gel Autoradiography Fingerprint digitizing: Input to a computer via a scanner BAC Fingerprint Images Covert the fingerprint images into database. BAC Fingerprint Database Data analysis and contig assembly Physical Map & BAC Contigs
31

Autoradiography

BAC Fingerprinting

Fingerprint Image Ready for computer analysis

32

Modifications
1. 2. 3. 4. 5. Different enzyme digestion (4 or 6 bp endonuclease) Fluorescent dye labeling Multiplexing BAC digests (different pairs of restriction endonucleases, each labeled with a different fluorescent dye) A single restriction endonuclease pair employing a type IIS restriction endonuclease SNaPshot high-information contentfingerprinting (HICF) technology
33

SNaPshot high-information contentfingerprinting (HICF) technology


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 artificial chromosomes using the snapshot labeling kit and sizing of restriction fragments by capillary 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 physical maps 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-recognizing restriction endonucleases that generate 3' recessed ends and one 4-bp-recognizing restriction endonuclease that generates a blunt end. Each of the four recessed 3' ends is labeled with a different fluorescent dye, and restriction fragments are sized on a capillary DNA analyzer. The resulting fingerprints are edited with a fingerprintediting computer program and contigs are assembled with the FPC computer program. The technique was evaluated by repeated fingerprinting of several BACs included as controls in plates during routine fingerprinting of a BAC library and by reconstruction of contigs of rice BAC clones with known positions on rice chromosome 10. 34

SNaPshot BAC fingerprinting


Restriction cleavage

Fluorescent labeling
5 3 TC AGATC

5 3

GGCC

TCTAGA AGATCT

CCGG GGCC

3 5 CTAGA CT 3 5

CCGG HaeIII

Xba I

HaeIII Luo M., UCDavis

Xba I

Restriction cleavage and fluorescent labeling


5 3 5 3

Bam HI

Characteristics of restriction sites and labeling of fragments


3 5 3 5
XbaI T^CTAGA C^TCGAG GG^CC C T none dTAMRA dROX Yellow Red XhoI HaeIII
Restriction endonuclease Restriction site ddNTP Fluorescent dye Color of fragment

G GATC C C CTAG G

Eco RI

EcoRI

G^AATTC G^GATTC

A G

dR6G dR110

Green Blue

G AAT T C C T TAAG

BamHI

5 3

Xho I

CTCGAG G A G CT C

3 5

Portion of multi-color fingerprinting profile of a BAC clone

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

BamHI

EcoRI

XbaI

XhoI

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

Liz-1200 Size standard

10

The DNA fingerprinting approach to building a whole genome physical map

5. Sequence Tagged Sites (STSs).


An STS is a short region of DNA about 200-300 bases long whose exact sequence is found nowhere else in the genome. Two or more clones containing the same STS must overlap and the overlap must include the STS. Advantages: Rapid and simple Disadvantages: Still very labor intensive and high expensive for primer synthesis.
Meyers BC, et al.41 Rev Nat Genet. 2004, 5:5578-88.

42

PCR confirmation of STS markers in the genome Each STS contains a unique sequence

STS mapping of linked markers and BAC clones


43 44

11

6. Fluorescence in situ hybridization (FISH)


This technique uses synthetic polynucleotide strands or short DNA fragment that bear sequences known to be complementary to specific target sequences at specific chromosomal locations. The polynucleotides are bound via a series of link molecules to a fluorescent dye that can be detected by a fluorescence microscope. Advantages: Direct visualization of a chromosomal region and accurate Disadvantages: Technically challenging and difficult for some species
45 46

TA Brown, p 152

Human metaphase chromosomes hybridized to fluorescent probes from two overlapping microdissection libraries. Probes specific to chromosome regions 1p34 35 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/
.(

47

servlets/photohigh?fileid=g001276)

48

12

Application of FISH in physical mapping

BAC vector 7.4-kb

4-kb

2.5-kb


2-kb


vector
J. Jiang, UWM
49

vector

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


50

FISH mapping of BACs (bacterial articial chromosomes)

Combination of STS markers, BACs and FISH Combination of fingerprinting, molecular linkage map, STS, end sequencing and FISH mapping.

Human genome anatomy: BACs integrating the genetic and cytogenetic maps for bridging genome and biomedicine. Genome Res. 1999 9(10):994-1001 872 unique STSs and 957 BACs were used

51

52

13

Integrated genetic, physical and sequence map

53 Integrated genetic map of human chromosome 11

54

Summary
1. A genetic linkage map shows the relative locations of specific DNA markers along the chromosome. 2. Five major DNA markers for genetic mapping: RFLP, RAPD, SNP, AFLP, Microsatellites 3. A physical map shows the exact position of genes and other sequences on the chromosome 4. Six physical mapping methods: optical mapping, BAC end sequencing, BAC fingerprinting, chromosome walking, STS and FISH 55
1.

Additional reading materials


Meyers BC, Scalabrin S, Morgante M. 2004. Mapping and sequencing complex genomes: let's get physical! Nat Rev Genet. 5:5578-88. Luo M. et al. 2010. Feasibility of physical map construction from fingerprinted bacterial artificial chromosome libraries of polyploid plant species. BMC Genomics 2010, 11:122 TA. Brown. 2002. Genomics, Second Edition. Wiley-Liss, Chapter 5, pp 125-159.

2.

3.

56

14