1. Planning breeding programs for impact
QTL analysis and
Marker aided selection
quantitative trait loci (QTL)

2. Marker aided selection and QTL analysis
References:
Kearsey, M.J. and Pooni, H.S The genetical analysis of quantitative traits. Chapter 7
Bernardo, R Breeding for quantitative traits in plants. Chapters 13 and 14
quantitative trait loci (QTL)
IRRI: Planning Breeding Programs for Impact

3. Can anyone describe what a QTL is?
A gene or chromosomal region that affects a quantitative trait
Must be polymorphic (have allelic variation) to have an effect in a population
Must be linked to a polymorphic marker allele to be detected
quantitative trait loci (QTL)
Question to the group to get a feel of their general knowledge of QTL
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4. Mapping quantitative trait loci (QTL)
QTL = underlying genes controlling quantitative traits
Measured with large error effects resulting
Result is continuous phenotypic distributions aa AA
Phenotypic value
1Leibowitz et al., 1987

5. IRRI: Planning Breeding Programs for Impact
QTL mapping
Example: In progeny derived from cross AA x aa:
Mean of AA lines is 3100 ± s.e.m
Mean of aa lines is 2900 ± s.e.m
BUT, AA and aa individuals can’t be visually distinguished
Some AA lines will have low yield due to e’s or other genes
Some aa lines will have high yield due to e’s or other genes
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6. IRRI: Planning Breeding Programs for Impact
QTL effect
Additive effect of a QTL allele = a
Average value of random lines from a cross between AA and aa parents = P
Mean of AA lines is P + a
Mean of aa lines is P – a
From previous example, what is the effect of the QTL (a)?
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7. Single-marker analysis
DNA markers can be used to map useful genes using recombination frequencies of linked genes: A a M m
QTL
Marker
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
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8. 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
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9. IRRI: Planning Breeding Programs for Impact
So, the effect of marker M is a function of:
(i) distance from the QTL
(ii) size of the QTL effect
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10. IRRI: Planning Breeding Programs for Impact
MM lines are easily distinguished from mm lines, but AA lines can’t be distinguished from aa lines
If M and A are linked, average of MM lines will differ from average of mm lines
Size of difference can be between 0 and a, depending on marker-QTL distance
Means of MM and mm recombinant inbred lines
MM = P + a(1-2R)
mm = P – a(1-2R)
R = 2r/(1+2r)
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11. QTL mapping with molecular markers
DNA markers used to map useful genes using recombination frequencies of linked genes: M1 A m1 a M2 m2
Markers near QTLs co-segregate with them
Markers tightly linked to QTL detected by ANOVA
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12. QTL mapping strategies
All marker-based mapping experiments have same basic strategy:
Select parents that differ for a trait
Screen the two parents for polymorphic marker loci
Generate recombinant inbred lines (can use F2-derived lines)
Phenotype (screen in field)
Contrast the mean of the MM and mm lines at every marker locus
Declare QTL where (MM-mm) is greatest
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13. Single-marker analysis
Select parents that differ for a trait
Screen the two parents for polymorphic marker loci
Generate recombinant inbred lines (can use F2-derived lines)
Phenotype (screen in field)
Do a separate ANOVA on the effect of each marker
Declare QTL where F-test is significant
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14. QTL mapping strategy: single-marker analysis
Map position (cM)
Mean of MM – mm lines (kg/ha)
200
100
QTL?
ANOVA done for each marker
QTL declared if t significant

15. Single-marker analysis: example
(taken from Kearsey and Pooni, pp )
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 cM
Lines are evaluated in 4-rep trial
Is there a QTL in this region?
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16. IRRI: Planning Breeding Programs for Impact
Measure of QTL contribution to σP2
 Recall that the simplest QTL model divides the genotypic effect into a QTL effect (A) and an effect of all other genes within QTL classes (G(QTL)):
Y = m + G e
= m + G(QTL) + A + e
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17. IRRI: Planning Breeding Programs for Impact
Measure of marker contribution to σP2
Y = m + G e
= m + G(M) M + e
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18. Single-marker analysis example
Select parents that differ for a trait
Screen the two parents for polymorphic marker loci
Generate recombinant inbred lines (can use F2-derived lines)
Phenotype (screen in field)
Do a separate ANOVA on the effect of each marker
Declare QTL where F-test is significant
IRRI: Planning Breeding Programs for Impact

19. 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
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20. IRRI: Planning Breeding Programs for Impact
Measure of marker contribution to σP2
Y = m + G e
= m + G(M) M + e
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21. Broad-sense heritability for a trial in which 1 QTL is detected
σ2G
σ2P = H
σ2G(QTL) + σ2A
σ2G(QTL) + σ2A + (σ2e /r) =
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22. R2 is the proportion of σ2P explained by the QTL A= R2
σ2A
σ2G(QTL) + σ2A + (σ2e /r) =
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23. 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)
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24. QTL mapping strategy: 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
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25. 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 m1 a M2 m2
Recombinant gametes: M1a, m1A,
Parental gametes: M1A, m1a,
Frequency of recombinants is map distance

26. 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)
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27. Linkage mapping with molecular markers
Double crossover products look like parental types, leading to map distance underestimates: M1 A m1 a M2 m2
Haldane and Kosambi mapping functions used to correct recombination frequencies
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28. 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
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29. IRRI: Planning Breeding Programs for Impact
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:
Fine-mapping
Assigning to a cloned fragment in a DNA library
Sequencing
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30. 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
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31. How is QTL mapping best used?
QTL mapping = very inaccurate for detecting, localizing, and estimating the effect size of genes with a small effect
If repeatability QTL phenotyping experiment = low QTL map very unreliable
QTL mapping works very well to find single genes with large effects
QTL mapping requires a phenotypic screening system with high H
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32. Does anyone else have other advice on QTL usage?
IRRI: Planning Breeding Programs for Impact

33. 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)
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34. IRRI: Planning Breeding Programs for Impact
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
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35. IRRI: Planning Breeding Programs for Impact
Can anyone briefly explain QTL mapping strategy? (single-marker analysis & interval mapping)
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36. IRRI: Planning Breeding Programs for Impact
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
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37. IRRI: Planning Breeding Programs for Impact
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
IRRI: Planning Breeding Programs for Impact