Comparative Mapping

Introduction
What is mapping? determining the location of elements within a genome, with respect to identifiable landmarks. Types of mapping genetic mapping physical mapping restriction mapping cytogenetic mapping somatic cell mapping radiation hybrid mapping comparative mapping

Introduction
Genetic mapping Utilize recombination events to estimate distance between genetic markers. RFLP STRP SNP Look at a population and estimate the recombination fraction = # recombinants / # total

Introduction
Physical mapping Relies upon observable experimental outcomes hybridization amplification May or may not have a distance measure.

Genetic Mapping
Requires informative markers -- polymorphic and a population with known relationships

Best if a measured between close markers.

Unit of distance in genetic maps = centimorgans, cM

1 cM = 1% chance of recombination between markers

Genetic Mapping
A2 A2 B2 B2 A2 A1 B2 B1 A1 A1 B2 B1 A2 A1 B 1 B1

A2 A1 B2 B1

A1 A1 B1 B1

A2 A1 B2 B1

A1 A1 B1 B1

A1 A1 B2 B1

A2 A1 B2 B1

A1A1 B 1 B1

A2 A1 B1 B1

A2 A1 B2 B 1

NR

NR

NR

NR

NR

= # recombinant / # total = 2/7 = 0.286

Genetic Mapping
Theta calculation with inbred population
bn det+

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x
bn det det

bn

bn bn

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bn

banded

detached banded, wild-type detached

483

512

= # recombinant # total = 5/1000 = 0.005

RFLP

Jack 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTCJill 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC-

Jack 2: -GAATTC--(1.8 kb)-CCCTTT--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)-GAATTCJill 2: -GAATTC--(1.8 kb)-GAATTC--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)-GAATTC-

Let's look at a simple example in fruit flies. Two RFLP loci with two RFLP bands possible at each locus:

In this example, 70% of the progeny were produce from parental genotype eggs and 30% were produced by recombinant genotype eggs. Therefore, these two RFLP loci are 30 centiMorgans apart from each other.

Comparative Mapping
Maps constructed in one species can be compared by means of common markers (or common single gene traits) with closely related species. These comparative maps can be used to study genome evolutionhow the genome has been rearranged through timeand to make inferences about gene organization, repeated sequences, etc. e.g., rice (with a small genome) vs. wheat (with a massive genome).

What are some questions that comparative genomics can address?

How has the organism evolved? What differentiates species?

Which non-coding regions are important?

Which genes are required for organisms to survive in a certain environment?

synteny: a similar linear order of markers in two different species (or on two different linkage groups, as in soybean or Brassica)

microsynteny: synteny at the sequence level, Conservation of the order of genes in DNA fragments that are bigger than 50 kb. Deletion, inversions and duplications are detected at this level. In different species

Organism A
1a 2a 3a 4a 5a 6a

Block of synteny
7b 2b 3b 4b 8b 9b

Organism B

Collinearity The order of the gene loci is preserved across species

Comparative Mapping
Sources of Information
sequence mapping potential orthologs BLAST sequence mapping

colocalization

Putative orthologs and syntenic segments

Homologoues can either be orthologues, paralogues or xenologues.

Orthologues are homologues that have evolved from a common ancestral gene by speciation. They usually have similar functions. Paralogues are homologues that are related or produced by duplication within a genome followed by subsequent divergence. They often have different functions. Xenologues are homologous that are related by an interspecies (horizontal transfer) of the genetic material for one of the homologues. The functions of the xenologues are quite often similar.

Comparative Mapping
Examples
0 100 900 200 600 300 300 400

RNO18

MMU18 1200

Comparative Mapping
Examples
RNO4
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HSA7
RNO12 { { { RNO12 { {

0
100

400

} HSA11 }

500
600 700

200
}

{ RNO5 {

300

} HSA7p } HSA4

Comparative Mapping Between Arabidopsis thaliana and Brassica nigra

No. of linkage groups=no. of chromosomes(n)

Evolution and sequence conservation

Genome comparisons are based on simple premise:

conservation = functional importance

If there are no constraints on DNA sequence, random mutations will occur

Over large evolutionary times (millions of years), these

random mutations make two related sequences different Sequences from different genomes will be conserved if:

They code for proteins

They are important for regulation (protein binding)

Finding DNA regulatory motifs (protein binding sites)

Experimental approaches
Promoter Trapping
DNA Footprinting In-vitro binding site selection (SELEX)

Computational approaches
Searching databases of known sites Finding over-represented motifs in a group of sequences (Gibbs sampling, Expectation Maximization)

In promoters of homologous genes

In promoters of functionally linked genes In promoters of interacting proteins Ab initio methods
Positional conservation of (pseudo)palindromic DNA motifs

Finding motifs in promoters of homologous genes

Perform all-versus-all proteomes BLAST search Pool together promoters of related genes Find conserved motifs

Only DNA motifs in related genes can be identified

Many potential binding sites are found ...

Sulfate metabolism

Transposons

GTP-binding ATPase

RNA Pol K Ribosomal proteins

Short hypothetical proteins

The role of found motifs is difficult to predict

Comparative genomics helps genome annotations

In prokaryotes, finding genes is relatively

easy based on open reading frames (ORFs)

In eukaryotes, we have to look for ORFs, exons, introns, splice sites, polyA sites
Bad news: Predicted exons sometimes do not exist More bad news: Pseudogenes Bad news keep coming: Alternative splicing

Good news: In different species, the genes

normally have similar exon-intron structure

Summary of Mapping Strategies

Mapping Strategy Genetic Requires Resolution How to increase resolution Medium to Increase High number of markers or people High Increase number of enzymes. Low to Medium High Increase number of deletion hybrids Use additional hybrids, or make a new panel. RHMap, MultiMap, RHMAPPER Software Polymorphic Markers, and Pedigrees Restriction Enzymes Somatic Hybrid Panel, and STSs Radiation Hybrid Panel, and STSs GeneHunter

Restriction

Somatic Cell Hybrid Radiation Hybrid