QTL analysis

Map Distances
• Crossing over occurs at random along chromosome--means that the
closer 2 genes are, the less frequently recombination occurs. Basis for
mapping.
• Recombination Fraction (RF or theta or θ) is the percentage of
recombinant gametes produced.
– one complicating factor when looking at offspring: meiosis occurs
in both parents.
• RF is never more than 50%--due to only 2 of the 4 chromatids
recombining
• 1% recombination = 1 map unit = 1 centiMorgan (cM), but only for short
distances.
• for longer distances, double crossovers decrease observed
recombination frequency.
– two crossovers between marker genes leaves the markers in the
parental configuration: no way to tell there were any crossovers.
• Double crossovers should occur at frequency predictable from
distances between genes, but there is also interference, which affects
the chance for C-O in any interval.
– interference: one crossover inhibits the occurrence of another
nearby.
 Single-marker analysis
DNA markers can be used to map useful genes
using recombination frequencies of linked genes:
A M

QTL Marker
a m

• Markers near QTLs co-segregate with them

• Markers tightly linked to QTL detected by ANOVA
• Most gametes from this F1 = AM or am. If crossover
between marker & QTL, Am & aM gametes will be produced
 Effect of a marker linked to a QTL

Recombination between M and A is R

In RILs derived from MmAa F1, individuals with MM
marker genotype are made up of 2 QTL genotypes: AA
& aa

- If M and A are tightly linked, most = AA

- If M and A are far apart, as many as half = aa
 QTL mapping with molecular markers

DNA markers used to map useful genes using

recombination frequencies of linked genes:
M1 A M2

m1 a m2

• Markers near QTLs co-segregate with them

• Markers tightly linked to QTL detected by ANOVA
 QTL mapping strategies
All marker-based mapping experiments have same
basic strategy:
1. Select parents that differ for a trait
2. Screen the two parents for polymorphic marker loci
3. Generate recombinant inbred lines (can use F2-
derived lines)
4. Phenotype (screen in field)
5. Contrast the mean of the MM and mm lines at every
marker locus
6. Declare QTL where (MM-mm) is greatest
 Single-marker analysis

1. Select parents that differ for a trait

2. Screen the two parents for polymorphic marker loci
3. Generate recombinant inbred lines (can use F2-derived
lines)
4. Phenotype (screen in field)
5. Do a separate ANOVA on the effect of each marker
6. Declare QTL where F-test is significant
 QTL mapping strategy: single-
marker analysis
200
QTL? QTL?
Mean of
MM – mm • ANOVA done for each marker
lines (kg/ha) 100
• QTL declared if t significant

0 20 40 60 80 100 120

Map position (cM)

 Single-marker analysis: example
(Kearsey and Pooni, pp. 137-142)

• 25 RILs produced from an F1 between 2 homzygous

parents
• Parents differ at marker loci A, B, and C on 1
chromosome:
A-------------B------------------------------C
19 cM 51 cM
• Lines are evaluated in 4-rep trial

Is there a QTL in this region?

 Single-marker analysis example

1. Select parents that differ for a trait

2. Screen the two parents for polymorphic marker loci
3. Generate recombinant inbred lines (can use F2-
derived lines)
4. Phenotype (screen in field)
5. Do a separate ANOVA on the effect of each marker
6. Declare QTL where F-test is significant
Single-marker analysis example

F-test for the difference between marker genotype

classes = highly significant at locus B

Therefore, there is a QTL at or near marker B

Broad-sense heritability for a trial in
which 1 QTL is detected
σ2G
H =
σ2 P

σ2G(QTL) + σ2A
=
σ2G(QTL) + σ2A + (σ2e /r)
 R2 is the proportion of σ2P explained by
the QTL A

σ2A
R2 =
σ2P
 QTL mapping strategy: single-
marker analysis

Problems with single-marker analysis:

 Not very accurate at assigning QTL position because of
recombination between marker and QTL
 Doing a t-test at every marker results in many false
positives (this is a general problem with QTLs)
 Interval mapping

• Marker interval = the segment between 2 markers

• Interval mapping methods use information on values of 2
flanking markers to estimate QTL position
• The probability that the data could be obtained assuming
a QTL at several positions between the markers is
calculated
• QTL = declared where the probability of obtaining the
observed data is highest
 Finding the position of QTL with molecular markers

DNA markers can be used to map useful genes

using recombination frequencies of linked genes:
M1 A M2

m1 a m2

Recombinant gametes: M1a, m1A,

Parental gametes: M1A, m1a,
Frequency of recombinants is map distance
 What are the problems with interval
mapping?

• Can’t resolve 2 QTL in a marker interval

• Although the LOD thresholds seem very high, too many
QTLs are declared (all methods do)
• Ignores epitasis
• Not accurate for QTL with small effects (no methods are)
 Linkage mapping with molecular markers

Double crossover products look like parental types,

leading to map distance underestimates:
M1 A M2

m1 a m2

Haldane and Kosambi mapping functions used to correct

recombination frequencies
 QTL mapping strategy: interval mapping

Significance test:
 Logarithm of the odds ratio (LOD score):
probability of the data occurring with a QTL

Odds ratio =
probability of the data occurring with no QTL

• LOD of 2 means that it is 100x more likely that a QTL exists in the
interval than that there is no QTL
• LOD of 3 means that it is 1000x more likely
 Fine mapping
• To be useful in breeding applications, gene of interest
must be tightly linked to marker
• Ideally, gene itself is used as marker
• Process of “tagging” gene means it must be cloned
through:
1. Fine-mapping
2. Assigning to a cloned fragment in a DNA library
3. Sequencing
 Marker-assisted backcrossing
• Main application of gene-tagging is marker-
assisted backcrossing of recessive genes
• Permits “pyramiding” of resistance genes with
similar phenotypic effects in a screen, e.g Pi1
and Pi2
• Permits rapid recovery of recurrent-parent
genome
How is QTL mapping best used?
1. QTL mapping = very inaccurate for detecting,
localizing, and estimating the effect size of genes
with a small effect
2. If repeatability QTL phenotyping experiment = low
QTL map very unreliable

3. QTL mapping works very well to find single genes

with large effects
4. QTL mapping requires a phenotypic screening
system with high H
 Some guidelines for successful QTL mapping

• Focus on lines that are easy to see in a good screen

• Derive traits where difference between susceptible and
resistant mapping populations from crosses between
highly resistant and highly susceptible lines
• Use highly reliable screening systems, and that are
known to differentiate resistant from susceptible lines
• Do analysis on the means of repeated screens rather
than single trials
• Ensure that repeatability of your screen is as high as
possible (0.7 or higher)
 Using QTL in breeding
• QTLs with small effects = hard to accurately map

• Only QTLs that are localized to very small chromosome

segments can be successfully used in marker-aided
backcrossing

• Fine-mapped QTLs with big effects in most genetic

backgrounds and most environments are most useful
e.g. disease resistance genes, Sub1
 Summary
• QTL mapping = process of locating genes with effects on
quantitative traits using molecular markers

• QTL mapping strategies = based on measuring the mean

difference between lines with contrasting marker alleles

• QTL mapping = preliminary step in the discovery of

useful genes for marker-aided backcrossing
 Summary
• So far, only successful with disease resistance and
stress tolerance genes having very large effects

• QTL mapping = basic research activity requiring careful

planning of crosses and high-precision phenotyping
Linkage Analysis and Map
Construction
 Testing Mendelian segregation
Consider marker A with two alleles A and a

Backcross F2
Aa aa AA Aa aa
Observation n1 n0 n2 n1 n0
Expected frequency ½ ½ ¼ ½ ¼
Expected number n/2 n/2 n/4 n/2 n/4

The x2 test statistic is calculated by

x2 =  (obs – exp)2 /exp
= (n1-n/2)2/(n/2) + (n0-n/2)2/(n/2) =(n1-n0)2/n ~x2df=1, for BC,
(n2-n/4)2/(n/4)+(n1-n/2)2/(n/2)+(n0-n/4)2/(n/4)~x2df=2, for F2
 Examples
Backcross F2
Aa aa AA Aa aa
Observation 44 59 43 86 42
Expected frequency ½ ½ ¼ ½ ¼
Expected number 51.5 51.5 42.75 85.5 42.75

The x2 test statistic is calculated by

x2 =  (obs – exp)2 /exp
= (44-59)2/103 = 2.184 < x2df=1 = 3.841, for BC,
(43-42.75)2/42.75+(86-85.5)2/85.5+(42-42.75)2/42.75=0.018 < x2df=2 =5.991, for F2

The marker under study does not deviate from

Mendelian segregation in both the BC and F2.
Genetic design
 Linkage analysis
Backcross
Parents AABB x aabb

AB ab
F1 AaBb x aabb
AB Ab aB ab ab

BCAaBb Aabb aaBb aabb

Obs n11 n10 n01 n00  n = nij
Freq ½(1-r) ½r ½r ½(1-r)

r is the recombination fraction between two markers A and B.

The maximum likelihood estimate (MLE) of r is
r^ = (n10+n01)/n. r has interval [0,0.5]: r=0 complete linkage, r=0.5, no linkage
 Map function

• Transfer the recombination fraction (non-additivity)

between two genes into their corresponding
genetic map distance (additivity)
• Map distance is defined as the mean number of
crossovers
• The unit of map distance is Morgan (in honor of T.
H. Morgan who obtained the Novel prize in 1930s)
• 1 Morgan or M = 100 centiMorgan or cM
 The Haldane map function (Haldane 1919)
Assumptions:
• No interference (the occurrence of one crossover is independent of that of
next)
• Crossover events follow the Poisson distribution.
– Probability of an event occurring x times in a particular time period

Consider three markers with an order A-B-C

A triply heterozygous F1 ABC/abc backcrossed to a pure parent abc/abc

Event Gamete Frequency

No crossover ABC or abc (1-rAB)(1-rBC)
Crossover between B&C ABc or abC (1-rAB)rBC
Crossover between A&B Abc or aBC rAB(1-rBC)
Crossovers between A&B and B&C AbC or aBc rABrBC

The recombination fraction between A and C is expected to be

rAC = (1-rAB)rBC + rAB(1-rBC) = rAB+rBC-2rABrBC

 (1-2rAC)=(1-2rAB)(1-2rBC)
Map distance:

A genetic length (map distance) of a chromosome is

defined as the mean number of crossovers.
 The Kosambi map function (Kosambi 1943)

The Kosambi map function is an extension of the

Haldane map function

For gene order A-B-C

[1] rAC = rAB + rBC – 2rABrBC
[2] rAC  rAB + rBC, for small r’s
[3] rAC  rAB + rBC – rABrBC, for intermediate r’s

The Kosambi map function attempts to find a general

expression that covers all the above relationships