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Planning breeding programs for impact QTL analysis and Marker aided selection.

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Presentation on theme: "Planning breeding programs for impact QTL analysis and Marker aided selection."— Presentation transcript:

1 Planning breeding programs for impact QTL analysis and Marker aided selection

2 IRRI: Planning Breeding Programs for Impact 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

3 IRRI: Planning Breeding Programs for Impact  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 Can anyone describe what a QTL is?

4 aa AA Phenotypic value 1 Leibowitz et al., 1987 QTL = underlying genes controlling quantitative traits Measured with large error effects resulting Result is continuous phenotypic distributions Mapping quantitative trait loci (QTL)

5 IRRI: Planning Breeding Programs for Impact 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 QTL mapping

6 IRRI: Planning Breeding Programs for Impact  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)? QTL effect

7 IRRI: Planning Breeding Programs for Impact DNA markers can be used to map useful genes using recombination frequencies of linked genes: Markers near QTLs co-segregate with them Markers tightly linked to QTL detected by ANOVA Most gametes from this F 1 = AM or am. If crossover between marker & QTL, Am & aM gametes will be produced A a M m QTL Marker Single-marker analysis

8 IRRI: Planning Breeding Programs for Impact Recombination between M and A is R  In RILs derived from MmAa F 1, 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 Effect of a marker linked to a QTL

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

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)

11 IRRI: Planning Breeding Programs for Impact DNA markers used to map useful genes using recombination frequencies of linked genes: M1M1 A m1m1 a M2M2 m2m2 Markers near QTLs co-segregate with them Markers tightly linked to QTL detected by ANOVA QTL mapping with molecular markers

12 IRRI: Planning Breeding Programs for Impact 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 F 2 - 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 QTL mapping strategies

13 IRRI: Planning Breeding Programs for Impact 1.Select parents that differ for a trait 2.Screen the two parents for polymorphic marker loci 3.Generate recombinant inbred lines (can use F 2 -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

14 Map position (cM) Mean of MM – mm lines (kg/ha) QTL? ANOVA done for each marker QTL declared if t significant QTL mapping strategy: single-marker analysis

15 IRRI: Planning Breeding Programs for Impact (taken from Kearsey and Pooni, pp ) 25 RILs produced from an F 1 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 Single-marker analysis: example Is there a QTL in this region?

16 IRRI: Planning Breeding Programs for Impact Measure of QTL contribution to σ P 2  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

17 IRRI: Planning Breeding Programs for Impact Measure of marker contribution to σ P 2 Y = m + G + e = m + G (M) + M + e

18 IRRI: Planning Breeding Programs for Impact 1.Select parents that differ for a trait 2.Screen the two parents for polymorphic marker loci 3.Generate recombinant inbred lines (can use F 2 - 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

19 IRRI: Planning Breeding Programs for Impact F-test for the difference between marker genotype classes = highly significant at locus B Therefore, there is a QTL at or near marker B Single-marker analysis example

20 IRRI: Planning Breeding Programs for Impact Measure of marker contribution to σ P 2 Y = m + G + e = m + G (M) + M + e

21 IRRI: Planning Breeding Programs for Impact σ 2 G σ2Pσ2P =H σ 2 G(QTL) + σ 2 A σ 2 G(QTL) + σ 2 A + (σ 2 e /r) = Broad-sense heritability for a trial in which 1 QTL is detected

22 IRRI: Planning Breeding Programs for Impact σ 2 A σ 2 G(QTL) + σ 2 A + (σ 2 e /r) = σ 2 A σ2Pσ2P = R2R2 R 2 is the proportion of σ 2 P explained by the QTL A

23 IRRI: Planning Breeding Programs for Impact 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) QTL mapping strategy: single-marker analysis

24 IRRI: Planning Breeding Programs for Impact 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 QTL mapping strategy: Interval mapping

25 DNA markers can be used to map useful genes using recombination frequencies of linked genes: M1M1 A m1m1 a M2M2 m2m2 Recombinant gametes: M 1 a, m 1 A, Parental gametes: M 1 A, m 1 a, Frequency of recombinants is map distance Finding the position of QTL with molecular markers

26 IRRI: Planning Breeding Programs for Impact 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) What are the problems with interval mapping?

27 IRRI: Planning Breeding Programs for Impact Double crossover products look like parental types, leading to map distance underestimates: M1M1 A m1m1 a M2M2 m2m2 Haldane and Kosambi mapping functions used to correct recombination frequencies Linkage mapping with molecular markers

28 IRRI: Planning Breeding Programs for Impact 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 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

29 IRRI: Planning Breeding Programs for Impact 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 Fine mapping

30 IRRI: Planning Breeding Programs for Impact 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 Marker-assisted backcrossing

31 IRRI: Planning Breeding Programs for Impact 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 How is QTL mapping best used?

32 IRRI: Planning Breeding Programs for Impact Does anyone else have other advice on QTL usage?

33 IRRI: Planning Breeding Programs for Impact 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) Some guidelines for successful QTL mapping

34 IRRI: Planning Breeding Programs for Impact 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 Using QTL in breeding

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

36 IRRI: Planning Breeding Programs for Impact 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

37 IRRI: Planning Breeding Programs for Impact 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 Summary


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