Quantitative Trait LocusQTL Mapping An alternative approach to gene identification

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Quantitative Trait Locus(QTL) Mapping An
alternative approach to gene identification
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Introduction Underlying Principles and objectives
of QTL mapping Experimental data Brief overview
in animal systems Limitations
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Height - An example of a Quantitative Trait
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Definition..... Shows a continuous range of
variation in a population and is more or less
normally distributed
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Characteristic Features....
Continuous distribution
Involvement of multiple loci (polygenic) Effect
of each can be small Environmental effects can
be large
Analysis has been challenging
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Natural Genetic Variation
Accumulation of mutations during evolution
-natural allelic variation Developmen
tal traits - flowering time, seed size, plant
size. Physiological traits - seed dormancy,
phosphate uptake Biochemical traits -
enzymatic activities , oil content Resistance
- biotic and abiotic
stresses pathogens
temperature, metals, drought
Important for adaptation Natural Genetic
Variation exploited for QTL Mapping
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Requirements For QTL Mapping
Measureable trait Molecular Markers
Restriction Fragment Length Polymorphism(RFLP)
Amplified Fragment Length Polymorphism(AFLP)
Cleaved Amplified Polymorphic
Sequences(CAPS) Simple Sequence Length
Polymorphism(SSLP) Segregating Population
F2,F3,
Recombinant Inbred Lines (RILs)
Double Haploid Lines QTL Mapping
Software
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Recombinant Inbred Lines Parent A X Parent B
Double haploid Lines(DHL) Spontaneous chromosome
doubling of Haploid microspores
in in vitro culture Homozygosity achieved in a
single step Not all systems are amenable to in
vitro culture
F1
AB
F2
F3
True breeding or homozygous Immortal
collection Replicate expts. in different
environments Molecular Marker database can be
updated
F8
RILs
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Flow Chart explaining QTL analysis
Create a Linkage map with molecularmarkers
Parent 1
Parent 2
Recombinant Inbred Lines (RILs,F2,F3,Double
Haploid Lines)
Genotype with molecular markers
Analyse trait data for each line
Link trait data with marker data - Mapping
software
Trait QTL mapped at bottom of small chromosome
QTL
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Principles of QTL Mapping
Parent allele A
Parent allele B
Plant height8cm
Plant height8cm
QTL ! Allele B increases by 2.0 cm
M
Plant height10cm
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QTLs for root elongation 2,4-D sensitivity
120 Ler x Cvi RILs analysed in Arabidopsis
thaliana Seeds germinated on MS media and
transferred to MS and MS 4 x 10-8M 2,4-D
plates. Roots measured on the 4th day after
transfer.
Cvi
Ler
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QTLs for root elongation 2,4-D sensitivity
120 Ler x Cvi RILs analysed 3
QTLs identified
Ler
Ler
Cvi
Cvi
Linkage group 5
Linkage group 3
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Map Positions for Putative
2,4-D QTLs
cM
1
2
3
4
5
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2,4D2
42
60
2,4D1
78
104
2,4D3
117
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The Way Forward .
Characterisation of an Individual QTL Use of
Near Isogenic Lines (NILs)
Parent A
Parent B
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Confirmation of QTL 2,4-D1
3 NILs used
QTL
3-13
3-14
3-15
Ler
Cvi allele decreases root elongation in 2,4-D
media at this locus
No
Yes
Yes
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Steps towards cloning the QTL
Hurrah!
Cloning gene characterisation
Candidate gene Knockouts Complementation
Locus Identification
NIL X parent, phenotype, genotype
Fine Mapping
Broad mapping
NILs
Trait analysis QTL mapping
RILs
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Objectives...Functional Analysis of genes
Laboratory -induced mutants
Forward genetic screens
Reverse genetic screens Assign a function to
all the genes using these methods ?
Redundant and lethal genes
Labour intensive mutant screens Mutant phenotype
undetected if WT allele is weak or null
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QTL analysis finds application in both plant and
animal systems
Plants
Animals
Dicots
Monocots Rice Maize Wheat
Vertebrates
Invertebrates
Mice Humans
Drosophila
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Recombinant Inbred Lines in mice
Requires 20 generations of sib-mating to reach
homozygosity Viability of the inbred strains is
reduced Maintenance can be difficult Hence, RI
families are generally small Data from other
families is combined RI lines were first
developed for mapping in mice (Taylor 1975) Over
25 mouse RI families exist
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In Mice....
Commonly used Lab strains C57BL/6 ,
BALB/c F2 and RI lines Putative QTLs confirmed
by use of Congenic lines Identify underlying
genes by fine mapping or candidate gene
approach Examples include the identification of
circadian rhythm QTLs(Hofstetter,J.R.et al.1995)

In Humans.... RI lines are similar to CEPH
families(centre detude du polymorphism
humaine) Immortalized white cell lines from large
human families Inheritance of new markers can be
compared to previously analysed ones
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Merits of QTL Mapping
Identification of novel genes
Where mutant approaches fail to detect
genes with phenotypic functions , QTL
mapping can help
Good alternative when mutant screening is
laborious and expensive e.g circadium rhythm
screens
Can identify New functional alleles of known
function genes (El-Assal,S.E. et.al(2001)
Natural variation studies provide insight into
the origins of plant evolution (Mauricio,R.(2001)
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Limitations....
Mainly identifies loci with large effects
Less strong ones can be hard to pursue
No. of QTLs detected, their position and effects
are subjected to statistical error
Small additive effects / epistatic loci are not
detected and may require further analyses
Cloning can be challenging but not impossible
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Future is orange.....

• Constant improvements of Molecular
  platforms
• New Types of genetic materials
• Progress in bioinformatics
• Advances in testing candidate genes
• Can facilitate QTL cloning!