Quantitative Trait Loci

MAPPING
Non Credit Seminar
NISHAT
2009BS122M
CONTENT
Advantages and limitation of QTL mapping
Methods of QTL mapping and processing of this
Data using computer Software
Mapping of QTL and different Approaches
QTL & its Trait identification
Future Aspects: a wish from current research
QTLs are genes whose phenotypic effects show a continuous
range of variation in a population and is more or less normally
distributed


A quantitative trait locus/loci (QTL) is the location of individual
locus or multiple loci in the genome 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)

Definition of QTL
Trait effects
In this example genome have 3 loci, one associated with
decreased yield, and one associated with higher yield. The
phenotype, depending on the size of the effect of each QTL
and how they work together, may be low yield.
A variety may have some QTL that increase a trait (for
example, increase yield) and others that decrease the
trait. These work together to create the phenotype of
the plant.
Finding good QTL
So the key is identifying the good QTL those that affect
the trait in the direction you want, and then separating
those from the negative ones. This is where QTL
identification techniques are important. e.g.

Positive QTL: Grain Yield, Disease resistance, Oil content,
Protein or Mineral linked.

Negative QTL: Plant Height, Environment effected traits.

Note that these techniques are simply statistical
correlations, just like genetic mapping and any marker-
trait correlations; however, because we are looking for
many markers that correlate with a single trait, it is
somewhat more complex statistically.
QTL Mapping
QTL mapping is the statistical study of the alleles that occur in
a locus and the phenotypes (physical forms or traits) that they
produce.

The process of constructing linkage maps and conducting QTL
analysisto identify genomic regions associated with traitsis
known as QTL mapping (McCouch & Doerge, 1995)
QTL mapping is based on the segregation of genes and
markers via chromosome recombination (called
crossing-over) during meiosis (i.e. sexual reproduction),
thus allowing their analysis in the progeny (Paterson,
1996).

Partition of mapping population into different genotypic
classes based on genotype Marker and apply correlative
statistics to determine the significant difference of one
genotype with another with respect to trait being
measured.
Principles of QTL Mapping
Prerequisites for QTL
mapping
Availability of a good linkage map (this can be done at
the same time the QTL mapping)

A segregating population derived from parents that
differ for the trait(s) of interest, and which allow for
replication of each segregant, so that phenotype can
be measured with precision (such as RILs or DHs)

A good assay for the trait(s) of interest

Software available for analyses
The purpose of the phenotyping experiment is to assign a trait
value to each mapping population member. This value is then
combined with the allele score at the set of marker loci distributed
throughout the genome. A data file is then created which includes
all the trait data and all the marker data for the entire population.
various software applications can be applied to this data file to
identify statistical associations between the presence of
alternative alleles and the trait value.
Overview of QTL mapping
Approaches to mapping
1. Experimental crosses (Segregating Population)
- Backcrosses
- F2 intercrosses
- Recombinant inbred (RI) lines
- Double Haploids

2. Pedigree analysis

3. Association studies (Linkage disequilibrium, LD mapping)
- With candidate genes (direct approach)
- Localized association studies (chromosomal region)
- Whole-genome association studies
Need of Segregating Population
o In natural population consistent association between QTL
and Marker genotype will not usually exists (Except where
marker is completely linked to QTL, which is very rare). So
to study the recombination b/w QTL and marker
segregating population is useful.
Types and Size of population
A segregating population using parental lines that are highly
contrasting phenotypic ally for the trait.

The parent lines should be genetically divergent.

The size of mapping population depend upon type of
mapping population employed for analysis, genetic nature
of target trait.
Note: The choice of mapping population could vary based
upon the objectives of experiment and timeframe
as well as resources available for undertaking QTL
analysis.
Backcross (BC)
Advantages: It is easier to identify QTL as there
are less epistatic and linkage drag effects;
especially useful for crosses with wild species.

Disadvantages: Difficult or impossible in species
that are highly heterozygous and outcrossing.

Use: best when inbred lines are available

Huang et al.( 2003)
Advantage: Fast and easy to construct

Disadvantage: F3 families are still very
heterozygous, so the precision of the estimates
can be low (because of the high standard error);
cant be replicated
F2 populations
Jampatong et al. (2002)
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design is more powerful in
cases of partial dominance,
since heterozygous effects can
be identified
Backcross design is more efficient
in cases of complete dominance.
Comparison ..
True breeding or homozygous
Immortal collection
Replicate experiments in different
environments
Molecular Marker database can be
updated
Recombinant inbred (RI) lines
Advantages: fixed lines so can be replicated
across many locations and/or years; can
eliminate problem of background
heterozygosity

Disadvantages: Can take a long time to
produce. (Some species are not amenable).
He P et al.(2001)
Recombinant inbred (RI) lines
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Basic method

Start with inbred parental
lines, homozygous at every
locus

Make F1s, heterozygous at
every locus

Inbreed different F1 lines

These recombinant inbred
lines are homozygous at
each locus
Advantages: 1)Spontaneous chromosome doubling of
Haploid microspores in in vitro culture
2)Homozygosity achieved in a single step Plants.

Disadvantages: Less recombination between linked markers
Not all systems are amenable to in vitro
culture
Doubled haploid Lines(DHL)
Advantage: Very precise and
statistically strong, as background
is constant; especially useful for
validation experiments

Disadvantage: Can take time to
construct; only useful for specific
target QTL
Near Isogenic Lines (NILs)
Szalma SJ et al.
Single Marker approach
The simplest, and probably most common method.
Distribution of trait values is examined separately for each marker
locus.
Each marker-trait association test is performed independent of
information from all other markers, so that a chromosome with n
markers offers n separate single marker tests.

Uses: This technique is good choice when the goal is simple detection of
a QTL linked to a marker, rather than estimation of its position
and effects.

Limitations: This method can not Determine Whether the marker
are associated with one or more QTLs.
The effect of QTL are likely to be under estimated because
they are confound with recombination frequencies.
Methods to detect QTLs
This technique use the two Flanking marker.
A separate analysis is performed for each pair of adjacent marker
loci.
The use of such two-locus marker genotypes results in n
-1

separate tests of marker-trait associations for a chromosome
with n markers (one for each marker interval).
Both single-marker and interval mapping approaches are biased
when multiple QTLs are linked to the marker/interval being
considered. Methods simultaneously using three or more
marker loci attempt to reduce or remove such bias.

Advantages: Interval mapping offers increased of power of
detection and more precise estimates of QTL effects and
position.

WITH INTERVAL MAPPING
Lander and Botstein 1989
Flanking marker analysis
It evaluates the association between the trait values and the expected
contribution of a QTL (the target QTL) at multiple analysis points between
each pair of adjacent marker loci (the target interval).

The expected QTL genotype is estimated from the genotypes of flanking
marker loci and their distance from the QTL. Since there is usually uncertainty
in the QTL genotype, the like- lihood is a sum of terms, one for each possible
QTL genotype, weighted by the probability of that genotype given the
genotypes of the flanking markers. The analysis point that yields the most
significant association may be taken as the location of a putative QTL.
Simple Interval Mapping (SIM)
CIM evaluates the possibility of a target QTL at multiple analysis points across
each inter-marker interval (same as SIM). However at each point it also
includes the effect of one or more background markers.
composite interval mapping
Background markers: That have been shown to be associated with the trait
and therefore lie close to other QTLs (background QTLs) affecting the
trait.
Multiple QTL mapping (Jansen 1993)
The inclusion of a background marker in the analysis helps in one of two
ways, Based upon the linkage of Background marker and the target
interval
1) If they are linked, inclusion of the background marker may help to
separate the target QTL from other linked QTLs.
2) If they are not linked, inclusion of the background marker makes the
analysis more sensitive to the presence of a QTL in the target interval.
(Zeng 1993, 1994).
INTERVAL MAPPING
Advantages
Takes proper account of
missing data
Interpolate positions
between markers
Provide a support interval
Provide more accurate
estimate of QTL effect
Disadvantages
Intense computation
Rely on a genetic map with
good quality
Difficult to incorporate
covariate
The power of a QTL-detection experiment, defined as the probability
of detecting a QTL at a given level of statistical significance,
depends upon the strength of the QTL and the number of progeny
in the population. If we consider the strength of the QTL in terms
of the fraction of the total trait variance that it explains, we can
define three categories of QTLs. Those which explain over 20% of
the variance are strong QTLs; traits controlled by such QTLs can
be considered almost Mendelian. Strong QTLs can be detected
with a power greater than 80% even with the AXB/BXA set of
recombinant inbred strains. At the other extreme, weak QTLs,
which explain 1% or less of the trait variance, require at least a
thousand progeny to detect them with high power. Detection of
such QTLs is not routinely feasible.
The number of progeny required to detect a QTL is, roughly
speaking, proportional to the variance of the nongenetic
(environmental) contributions and inversely proportional to the
square of the strength of the QTL.
Name of Software URL of the site
Mapmaker/QTL ftp://genome.wi.mit.edu/pub/mapmaker3
QTL Cartographer http://statgen.ncsu.edu/qtlcart/
Map Manager QT http://mcbio.med.buffalo.edu/mapmgr.html
QGene qgene@clarityconnect.com
MapQTL http://www.cpro.dlo.nl/cbw/
PLABQTL http://www.unihohenheim.de/~ipspwww/soft.html
MQTL ftp://gnome.agrenv.mcgill.ca/pub/genetics/software/M
QTL/
Multimapper http://www.RNI.Helsinki.FI/~mjs/
The QTL Cafe http://sun1.bham.ac.uk/g.g.seaton/
Epistat http://www.larklab.4biz.net/epistat.htm
Manly et al. (1999)
MULTIPLE REGRESSION ANALYSIS
To obtain the final estimates of the effects of the QTL
detected with CIM

proportion of phenotypic variation accounted for by an
individual QTL


The mapping approaches described so far require crosses.
Mapping with experimental crosses requires organisms that
can be manipulated and that have sufficiently short generation
times to be tractable.

Humans cannot be crossed for ethical reasons, and many other
organisms (perhaps most other organisms?) cannot be studied
this way for practical reasons (e.g. elephants, whales, giant
sequoias, many insects, many rodents,.....).
Pedigree Analysis
The basic idea in pedigree mapping is to follow affected
individuals and markers in related families. Markers that are
co-inherited with disease status are linked to the causative
gene.
Mapping with pedigrees
Two unrelated pedigrees
Haplotypes in each pedigree are
identified by numbers (different
for each family), and
determined from five linked
markers
Dark blue - affected
White - unaffected
Light blue - status uncertain
In each case one haplotype, in
red, co-segregates with the
disease
Linkage Disequilibrium (LD)
Non random association of alleles (nucleotides) at different genes
(sites)

The non-independence of alleles at different loci. This occurs
when certain combinations of alleles across loci occur more often
than expected by chance alone, based on their respective allele
frequencies in the population.
1 2
COMPLETE LINKAGE DISEQUILIBRIUM
Adapted from Rafalski (2002) Curr
Opin Plant Biol 5:94-100.
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Same mutational history
and no recombination.

No resolution
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LINKAGE DISEQUILIBRIUM
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Different mutational history
and no recombination.

Some resolution
3
1 2
LINKAGE EQUILIBRIUM
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Locus 1
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2

Same mutational history
with recombination.

Resolution
3
3
Markers are used to partition the mapping population into
different genotypic groups and to determine whether
significant differences exist between groups with respect to the
trait being measured.

A significant difference between phenotypic means and marker
system in mapping population indicates that the marker locus
being used to partition the mapping population is linked to a
QTL controlling the trait.

Therefore, the QTL and marker will be usually be inherited
together in the progeny, and the mean of the group with the
tightly-linked marker will be significantly different (P<0.05) to
the mean of the group without the marker.
Detection of LD
A difference in mean phenotype indicates that linkage disequilibrium
present in the population and marker is linked to a QTL. But this does
not mean that every marker that is linked to a QTL is expected to show
a mean difference in phenotype. This vary with the extent of LD

Linkage between markers is usually calculated using odds ratios

logarithm of odds:- the ratio of linkage versus no linkage

This ratio is generally called LOD score. LOD values of >3 are typically
used to construct linkage maps. A LOD value of 3 between genes and
marker indicates that linkage is 1000 times more likely (i.e. 1000:1)
than no linkage (null hypothesis). LOD values may be lowered in order
to detect a greater level of linkage or to place additional markers within
maps constructed at higher LOD value.
Extent of LD
Risch et al. 1992


o Mutation

o Drift

o Population structure (admixture)

o Gene flow

o Population contraction

o Selection (hitchhiking or epistasis)
What causes LD?
o Recombination

o Gene conversion

o Recurrent mutation
What reduces LD?
The amount and extent of LD that exists in the populations
that are used for genetic improvement is the net result of all
the forces that create and break-down LD and is therefore,
the result of the breeding and selection history of each
population, along with random sampling.

On this basis, populations that have been closed for many
generations are expected to be in linkage equilibrium, except
for closely linked loci. Thus, in those populations, only
markers that happen to be tightly linked to QTL may show an
association with phenotype, and even then there is no
guarantee because of the chance effects of random
sampling.
Implications of the extent of LD
With candidate genes
Candidate genes is the genes that are associated with the trait
of interest.

This technique evaluate markers that are inside the genes or
close to genes that are thought to be associated with the trait of
interest.

The candidate gene approach utilizes knowledge from species
that are rich in genome information (e.g. human, mouse),
effects of mutations in other species, previously identified QTL
regions, and/or knowledge of the physiological basis of traits to
identify genes that are thought to play a role in the physiology
of the trait.

Following mapping and identification of polymorphisms within
the gene in the plants, the association of genotype at the
candidate gene with phenotype can be estimated in a closed
breeding population.
child et al. (2003)
Whole Genome scan
The whole genome scan approach to QTL detection uses
random genetic markers spread over the genome to
identify genomic regions that harbor QTL. The QTL
regions are detected by following the co-segregation of
markers with phenotype in structured populations using
interval mapping .
(Haley et al. 1994)
Summary of QTL analysis
Recombinant Inbred Lines
(RILs,F2,F3,Doubled 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
Parent 1 Parent 2
QTL
Create a
Linkage
map with
molecularm
arkers
Merits of QTL Mapping
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
e.g.Flowering time QTL,EDI was the CRY2 gene
Natural variation studies provide insight into the origins of
plant evolution
Identification of novel genes
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
FUTURE PROSPECTS
Constant improvements of Molecular platforms

New Types of genetic materials( e.g. introgression lines:
small effect QTLs can be detected)

Advances in Bioinformatics