1. PBA workshop, MSU JUNE’ 10 1
QTL Discovery
& Perspectives of use of
markers in fruit breeding programs

2. Content
 Introduction
 QTL-Discovery
 Perspectives for use
 Example: Nectria (Canker) resistance
 Summary
PBA workshop, MSU JUNE’ 10 2

3. Introduction
 QTL discovery
 Assessment of correlation trait ↔ molecular marker
 Initial step towards Marker Assisted Breeding
 Discovery  Validation  High Throughput markers  Logistics
 Validation
 Reproducibility on multiple families, pedigrees
 Mode of action
– Additive, Dominance
– Gene x Gene interactions? Independent action?
 Allelic diversity
 Relative importance
PBA workshop, MSU JUNE’ 10 3
R R R S S S

4. Introduction
 QTL Discovery
 Assessment of correlation trait ↔ molecular marker
 Requirements:
1. Trait of interest
2. Phenotypic data
3. Segregating germplasm
4. Marker data, genome-wide
5. QTL-mapping software
PBA workshop, MSU JUNE’ 10 4

5. QTL-Discovery in FruitBreedomics - Peach
1. Fruit related Traits
PBA workshop, MSU JUNE’ 10 5
– Maturity time
– Fruit Size
– Flesh color
– Firm/soft melting
– Flesh adherence,
– Titrable acidity
– Total sugar
– Flowering time
– Flowering intensity
– White anthers
– Monilinia resistance
2. Phenotypic data
 Historic
• EU-Projects
• National projects
 New - FruitBreedomics
3. Segregating germplasm
 18 families , ±1500 individuals
 5 breeding programs, 2 countries
4. Marker Data
 9K array

6. QTL-Discovery in FruitBreedomics - apple
1. Fruit Quality Traits: > 10
PBA workshop, MSU JUNE’ 10 6
 Fruit Quality
– Taste
• Firmness
• Juiciness
• Crispness
• Total Sugar
• Acidity
– Storability
– Shelf life
– Harvest date
– Fruit size
– Skin Color
 Resistance to Nectria
2. Phenotypic data
 HiDRAS
• 3 Years, Multiple time moments
– Harvest, Storage 2 & 4M
– Shelf Life: 2wks - 2M storage
 National projects
3. Segregating germplasm
 24 families , ±1600 individuals
 6 breeding programs, 6 countries
4. Marker Data

7. Discovery: Marker data
 20K SNP array
 New: available 2013 Jan 16
 First marker data: Jan 18!
 Great performance
 Some SSR
 previous projects
 2013: Exciting year
PBA workshop, MSU JUNE’ 10 7

8. Perspectives:
PBA workshop, MSU JUNE’ 10 8
Great perspectives
• Many QTL discovered in EU-HiDRAS
• Large intervals due to low marker density (# individuals high!)
25
56
77
0.0
0.2
0.4
1
1
14
24
51
67
0.0
0.2
0.4
2
19
24
43
76
0.0
0.2
0.4
3
2
18
32
55
73
0.0
0.2
0.4
4
8
14
20
34
45
47
69
90
100
107
0.0
0.2
0.4
5
9
40
78
0.0
0.2
0.4
6
26
35
0.0
0.2
0.4
7
18
33
52
62
0.0
0.2
0.4
8
24
42
59
69
0.0
0.2
0.4
0.6
9
13
14
40
50
64
81
104
0.0
0.2
0.4
10
13
29
45
51
61
76
0.0
0.2
0.4
12
3
18
30
45
64
89
0.0
0.2
0.4
13
8
23
37
77
110
116
0.0
0.2
0.4
15
8
14
30
47
76
0.0
0.2
0.4
16
0
12
23
25
35
55
91
100
0.0
0.2
0.4
17

9. Validation & Marker development
 On-going within FruitBreedomics
 Development of SNP markers (WP1)
 Firmness & Storability
 2 genes involved in ethylene pathway
 Relevance confirmed in multiple studies
 Already in use for MAB
• WSU, Russian programs
PBA workshop, MSU JUNE’ 10 9

10. Validation & Marker development: RosBREED
 RosBREED project
 US, USDA-SCRI
 Multi State, International
 Apple, Peach, Cherry, Strawberry
 www.RosBREED.org
 Validation apple
 Acidity:
• 2 loci, including Ma locus
 Crispness, Juiciness (one LG)
 Skin colour: Rf locus
 Bitter pit: 3 loci
 and others...
 Validation Peach
 Acidity
 Sweetness
 Maturity date
 Fruit size
 and others...

11. Perspectives on use in breeding
 Discovery : great potential
 Validation : completed for 1st genes
 Marker Development : started
 Logistics : started
 Enhancement through intercontinental collaborations?
 RosBREED
 Plant & Food Research
PBA workshop, MSU JUNE’ 10 11

12. Nectria Canker

13. Introduction on Nectria
PBA workshop, MSU JUNE’ 10 13
 Causes canker in apple
 Problem in NW-Europe (Humid climates)
 Many (popular) cvs extremely susceptible
 Braeburn, Cox, Gala, Kanzi, Rubens, Prima
 Fungicides for Scab act also to Nectria
 Problems with Nectria may increase
 Scab resistance
 Susceptible parents

14. Introduction to Nectria
 Breeding for resistance is hard
 R – highly quantitative
 Only few cultivars show reasonable resistance
• Jonathan, Golden Delicious, Lombart’s Calvillle, Santana
 Resistance tests are laborious and time-lasting
 Availability of markers: highly useful
 Difficult to develop
PBA workshop, MSU JUNE’ 10 14

15. Discovery
 Germplasm
 Jonathan x Prima
• Jonathan: cv with highest level of resistance
• Prima: highly susceptible: chopped down in biological production
PBA workshop, MSU JUNE’ 10 15

16. Discovery: National Project
 Germplasm
 Jonathan x Prima
PBA workshop, MSU JUNE’ 10 16
 Phenotyping
 Artificial inoculation (1994)
 Greenhouse: no 2nd infection
 Colonization rate: Length of cankers in time (1996)

17. Phenotypic data: Frequency Distribution
Size of cankers (in mm)
#progeny
0
5
10
15
20
25
30
35
56
4
8
12
16
20
24
28
32
36
40
44
48
52
60
64
68
72
76
1
28
25
16
12
5
4
2
1
13
30
18
8
6
3
1
4
5
3
1 11 1 11 1
• Prima: Large experimental variation
Jonathan x Prima
Jonathan
Prima
• JP-Population: sub-groups

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J13 P13 J14 P14
***
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********
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E35M59-228-2 -
E32M52-113-12-Pf -63.7
E35M49-108-16 +65.8
E32M52-330-3-Pf +65.9
E34M51-276-4 -67.0
E34M48-140-21 +
E34M48-138-32 +67.4
E33M60-107-15-fD +67.9
E34M48-130-27 -79.0
J16 P16
E31M48-150-18 -79.6
E32M58-316-2- 0.0
CH02g01-217+ 0.9
E31M52-127-15- 2.8
E31M52-127-10- 3.6
E34M51-164-6- 4.3
E32M52-120-9- 13.3
E31M48-100-23+ 26.7
E34M51-159-7+ 27.0
E33M47-140-12+ 31.6
E34M53-191-6 +0.0
E34M48-283-8 +5.2
E32M58-92-10 +5.8
E34M59-255-5-Pf +7.2
CH02g01-0/226 -9.1
E34M60-272-3 -26.5
E35M54-137-8 -35.7
E31M48-165-17 +50.7
E35M50-219-4 -
E31M49-139-8 -62.7
E35M54-207-6 -63.0
CH03h03-91/103 +-63.4
E33M47-348-2 -66.6
CH05f04-162/158 +-
E34M59-118-12 -
66.9
E35M58-134-5 -
E31M52-158-8 -
E33M47-165-10 -
E34M48-419-4-fD -
67.1
E31M52-158-13 -67.7
E34M48-275-9- 0.0
E31M56-328-1- 3.9
E32M47-209-3+ 5.0
E34M48-182-15-pF-Fd- 10.2
E34M50-265-3- 11.0
E34M55-106-12- 20.7
E31M52-75-14- 22.0
CH05g11-243/233+- 22.7
CH04c07-112/114-+ 24.6
CH05d03-158/173+- 33.0
E31M56-188-3+ 33.3
E34M57-250-7-Fd+ 36.2
E34M60-290-2a-Fd+ 37.8
E34M54-340-19- 41.8
E34M55-167-8+ 43.1
E34M53-225-4- 43.6
E32M52-148-8- 46.9
E31M48-228-10 -0.0
E31M48-195-12 -10.9
CH04f06-175/178 -+ ***13.6
E34M48-179-16-Pf +15.1
E32M47-92-19 +16.1
E33M51-112-15 +16.7
E33M47-172-9 +19.7
E33M47-275-4 -27.3
CH05g11-243/0 +-32.8
CH04c07-106/108 -+33.8
CH05d03-152/179 -+43.2
E34M60-247-6 +44.9
E34M49-170-4 -45.6
E35M47-149-16-Pf-fD +47.3
E34M55-166-9 +47.8
E35M58-95-8 -58.6
J13 P13 J14 P14
***
+-*****
********
+-********
*
E34M50-177-8- 0.0
CH05c06-104/118 16.3
E35M47-287-6- 17.1
CH05e04a-159/167 18.6
LY05b- 22.4
E32M52-264-4+ 34.1
CH05e04a-159/0 -+0.0
CH05c06-104/110 -+ ******0.2
E35M50-99-15 + ******3.4
E34M57-292-5 + *****10.4
E33M47-357-1 - ***21.8
E31M49-81-9 + ****26.0
CH05a04-163/183 +- ***27.8
J16 P16
Marker data
 AFLP & SSR
 1998-2000

19. QTL –mapping
PBA workshop, MSU JUNE’ 10 19
 Effect QTL:
 Additive
 Q1+Q2 -> Cankers as small as
for Jonathan
• Prima 47 mm
• Jonathan: 23 mm
• JxP_Q1Q2: 23 mm
 Highly effective!
6 . 7 . 8 . 9 . 10 . 11 . 12 . 13 . 14 . 15 .P-Q38 . 9 . 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 .
J-Q2. 12 . 13 . 14 . 15 . 16 . 17 .
J-Q1
 3 QTL identified
 2x Jonathan, 1x Prima

20. Fruitbreedomics
PBA workshop, MSU JUNE’ 10 20
6 . 7 . 8 . 9 . 10 . 11 . 12 . 13 . 14 . 15 .P-Q38 . 9 . 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 .
J-Q2. 12 . 13 . 14 . 15 . 16 . 17 .
J-Q1
 Position?
 J-Q1
• No markers for distal part
• Sub-optimum?
 J-Q2
• 1 marker / 35 cM
• Position?

21. Fruitbreedomics
Progresses on these historic data
 Fine-mapping
 Increase marker density
PBA workshop, MSU JUNE’ 10 21
J01 J02 J03 J04 J05 J06 J07 J08 J09 J10 J11 J12 J13 J14 J15 J16 J17
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
• SNP-array: ±8000 markers for JxP
• ±3000 for Jonathan

22. Fruitbreedomics
Progresses on these historic data
 Fine-mapping
 Increase marker density
 Validation:
 Reproducibility: 2nd mapping population
 Extension
 Stability over environments
• Phenotype JxP in Sweden
 Other parameters for resistance
• Infection percentage
Larisa
Gustavsson
Hilde
Nybom

23. Summary
 Title: QTL Discovery & Perspectives of use of
markers in fruit breeding programs
 QTL Discovery 1st step to application
 Discovery  Validation  High Throughput markers  Logistics
 Assessment of correlation trait ↔ molecular marker
 Potential - couple of years
 Apple > 10 fruit quality traits + Nectria resistance
 Peach > Flowering + Fruit traits + Monilinia resistance
 Potential - short term (from / building on other projects)
 Apple: Fruit firmness (ACS, ACO), Skin Color (Rf), Acidity (Ma), Bitter pit
 Peach: Sub-acid/Acid, Flat/round (Peach/Nectarine, Yellow/white flesh)
 Pipeline-Logistics: under development
PBA workshop, MSU JUNE’ 10 23