2015 International FruitBreedomics Conference

1. YOUR LOGO
Construction and Application
of a Multi-parental High Density
SNP Linkage Map of Apple
Erica A. Di Pierro, Luca Gianfranceschi, Johannes W. Kruisselbrink, Marco C.A.M. Bink, Roeland E. Voorrips,
Mario Di Guardo, Herma Koehorst van Putten, Sara Longhi, Luca Bianco, Michela Troggio, Diego Micheletti,
Riccardo Velasco, Larisa Gustavsson, Stefano Tartarini, Giulia Pagliarani, Hélène Muranty, François Laurens,
Eric van de Weg

2. YOUR LOGO
WHY THE NEED FOR A VERY RELIABLE
GENETIC MAP IN APPLE?

3. YOUR LOGO
Very final goal
THE NEED FOR A VERY RELIABLE
GENETIC MAP IN APPLE

4. YOUR LOGO
➧Reliable and consistent assessment of
correlation trait ↔ molecular marker
• Discovery & Characterization
• Validation
– Reproducibility on multiple families, pedigrees
NEEDS FOR SUCCESSFUL MAB STRATEGIES

5. YOUR LOGO
➧Reliable and consistent assessment of
correlation trait ↔ molecular marker
• Discovery & Characterization
• Validation
– Reproducibility on multiple families, pedigrees
NEEDS FOR SUCCESSFUL MAB STRATEGIES
➧Power and accurateness of QTL mapping and PBA approaches

6. YOUR LOGO
➧Reliable and consistent assessment of
correlation trait ↔ molecular marker
• Discovery & Characterization
• Validation
– Reproducibility on multiple families, pedigrees
NEEDS FOR SUCCESSFUL MAB STRATEGIES
➧Power and accurateness of QTL mapping and PBA analysis
• Correct linkage group (LG) assignment
• Correct markers succession within LG

7. YOUR LOGO
➧Approaches reducing size data sets
• High-density SNP arrays
• Computer memory limitations, increase computation time
• Genotyping by SNP haplotypes
– Use as multi-allelic markers, more informative than single di-allelic SNPs
NEEDS FOR SUCCESSFUL MAB STRATEGIES

8. YOUR LOGO
➧Approaches reducing size data sets
• High-density SNP arrays
• Computer memory limitations, increase computation time
• Genotyping by SNP haplotypes
– Use as multi-allelic markers, more informative than single di-allelic SNPs
NEEDS FOR SUCCESSFUL MAB STRATEGIES

9. YOUR LOGO
➧NEED
THE NEED FOR A VERY RELIABLE
GENETIC MAP IN APPLE

10. YOUR LOGO
➧NEED
➧APPROACH
THE NEED FOR A VERY RELIABLE
GENETIC MAP IN APPLE

11. YOUR LOGO
1. HIGH DENSITY SNPs ARRAY:
Illumina Infinium 20 K SNPs array Luca Bianco et al. 2014
FUNDAMENTAL TOOLS

12. YOUR LOGO
1. HIGH DENSITY SNPs ARRAY:
Illumina Infinium 20 K SNPs array Luca Bianco et al. 2014
2. RELIABLE CALLS OF INFORMATIVE SNP
ASSIsT software Mario Di Guardo et al.
FUNDAMENTAL TOOLS

13. YOUR LOGO
1. HIGH DENSITY SNPs ARRAY:
Illumina Infinium 20 K SNPs array Luca Bianco et al. 2014
2. RELIABLE CALLS OF INFORMATIVE SNP
ASSIsT software Mario Di Guardo et al.
3. RELIABLE SINGLE FAMILY MAPS FOR 21 FAMILIES
(ca. 1600 individuals)
FUNDAMENTAL TOOLS

14. YOUR LOGO
1. SNPs ORGANIZED in Focal Points (FPs)
• Exploiting FPs design introduced in Chagné et al. 2012
and Bianco et al. 2014
STRATEGY STEPS

15. YOUR LOGO
1. SNPs ORGANIZED in Focal Points (FPs)
• Exploiting FPs design introduced in Chagné et al. 2012
and Bianco et al. 2014
2. INNOVATIVE MAPPING APPROACH
• FPs strategy
• Backcross strategy in outcrossing species and data
integration across families prior to map construction
STRATEGY STEPS

16. YOUR LOGO
1. SNPs ORGANIZED in Focal Points (FPs)
• Exploiting FPs design introduced in Chagné et al. 2012
and Bianco et al. 2014
2. INNOVATIVE MAPPING APPROACH
• FPs strategy
• Backcross strategy in outcrossing species and data
integration across families prior to map construction
3. SOFTWARE DEVELOPMENT FOR DATA INTEGRATION AND
CONVERSION (FP-mapper by J. Kruisselbrink)
STRATEGY STEPS

17. YOUR LOGO
➧Focal Points design
Regions of max 10kb having up to 10 SNPs
➝ Suitable for building stable multi-allelic SNP-haplotypes
➝ Distribution across genome
1. SNPs ORGANIZED IN FPs

18. YOUR LOGO
➧Focal Points design
Regions of max 10kb having up to 10 SNPs
➝ Suitable for building stable multi-allelic SNP-haplotypes
➝ Distribution across genome
1. SNPs ORGANIZED IN FPs
SNP name FP segr phase segr phase segr phase
SNP_028768 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa <aaxab> {-0} -- aa ab ab -- aa ab ab
SNP_028769 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
SNP_028770 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa -- -- -- -- -- -- -- --
SNP_028771 FP_60 <abxab> {00} ab -- bb -- ab bb ab ab <abxaa> {0-} aa -- -- aa ab aa -- aa <aaxab> {-0} aa aa ab ab ab aa ab ab
SNP_028772 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <abxaa> {0-} aa aa aa aa ab aa aa aa <abxab> {10} ab ab ab ab bb aa ab ab
SNP_028773 FP_60 <aaxab> {-0} aa aa ab -- ab ab ab aa -- -- -- -- -- -- -- -- <abxab> {10} -- -- ab ab bb aa ab ab
Pop 1 Pop 2 Pop 3

19. YOUR LOGO
➧Focal Points design
Regions of max 10kb having up to 10 SNPs
➝ Suitable for building stable multi-allelic SNP-haplotypes
➝ Distribution across genome
1. SNPs ORGANIZED IN FPs
SNP name FP segr phase segr phase segr phase
SNP_028768 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa <aaxab> {-0} -- aa ab ab -- aa ab ab
SNP_028769 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
SNP_028770 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa -- -- -- -- -- -- -- --
SNP_028771 FP_60 <abxab> {00} ab -- bb -- ab bb ab ab <abxaa> {0-} aa -- -- aa ab aa -- aa <aaxab> {-0} aa aa ab ab ab aa ab ab
SNP_028772 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <abxaa> {0-} aa aa aa aa ab aa aa aa <abxab> {10} ab ab ab ab bb aa ab ab
SNP_028773 FP_60 <aaxab> {-0} aa aa ab -- ab ab ab aa -- -- -- -- -- -- -- -- <abxab> {10} -- -- ab ab bb aa ab ab
Pop 1 Pop 2 Pop 3

20. YOUR LOGO
➧Focal Points design
Regions of max 10kb having up to 10 SNPs
➝ Suitable for building stable multi-allelic SNP-haplotypes
➝ Distribution across genome
1. SNPs ORGANIZED IN FPs
SNP name FP segr phase segr phase segr phase
SNP_028768 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa <aaxab> {-0} -- aa ab ab -- aa ab ab
SNP_028769 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
SNP_028770 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa -- -- -- -- -- -- -- --
SNP_028771 FP_60 <abxab> {00} ab -- bb -- ab bb ab ab <abxaa> {0-} aa -- -- aa ab aa -- aa <aaxab> {-0} aa aa ab ab ab aa ab ab
SNP_028772 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <abxaa> {0-} aa aa aa aa ab aa aa aa <abxab> {10} ab ab ab ab bb aa ab ab
SNP_028773 FP_60 <aaxab> {-0} aa aa ab -- ab ab ab aa -- -- -- -- -- -- -- -- <abxab> {10} -- -- ab ab bb aa ab ab
Pop 1 Pop 2 Pop 3

21. YOUR LOGO
➧Focal Points design
Regions of max 10kb having up to 10 SNPs
➝ Suitable for building stable multi-allelic SNP-haplotypes
➝ Distribution across genome
1. SNPs ORGANIZED IN FPs
SNP name FP segr phase segr phase segr phase
SNP_028768 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa <aaxab> {-0} -- aa ab ab -- aa ab ab
SNP_028769 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
SNP_028770 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa -- -- -- -- -- -- -- --
SNP_028771 FP_60 <abxab> {00} ab -- bb -- ab bb ab ab <abxaa> {0-} aa -- -- aa ab aa -- aa <aaxab> {-0} aa aa ab ab ab aa ab ab
SNP_028772 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <abxaa> {0-} aa aa aa aa ab aa aa aa <abxab> {10} ab ab ab ab bb aa ab ab
SNP_028773 FP_60 <aaxab> {-0} aa aa ab -- ab ab ab aa -- -- -- -- -- -- -- -- <abxab> {10} -- -- ab ab bb aa ab ab
Pop 1 Pop 2 Pop 3

22. YOUR LOGO
2. NEW MAPPING APPROACH: FPs STRATEGY
➧FPs strategy
SNP name FP segr phase segr phase segr phase
SNP_028768 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa <aaxab> {-0} -- aa ab ab -- aa ab ab
SNP_028769 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
SNP_028770 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa -- -- -- -- -- -- -- --
SNP_028771 FP_60 <abxab> {00} ab -- bb -- ab bb ab ab <abxaa> {0-} aa -- -- aa ab aa -- aa <aaxab> {-0} aa aa ab ab ab aa ab ab
SNP_028772 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <abxaa> {0-} aa aa aa aa ab aa aa aa <abxab> {10} ab ab ab ab bb aa ab ab
SNP_028773 FP_60 <aaxab> {-0} aa aa ab -- ab ab ab aa -- -- -- -- -- -- -- -- <abxab> {10} -- -- ab ab bb aa ab ab
Pop 1 Pop 2 Pop 3
FP-mapper by J. Kruisselbrink

23. YOUR LOGO
2. NEW MAPPING APPROACH: FPs STRATEGY
➧FPs strategy
SNP name FP segr phase segr phase segr phase
SNP_028768 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa <aaxab> {-0} -- aa ab ab -- aa ab ab
SNP_028769 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
SNP_028770 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa -- -- -- -- -- -- -- --
SNP_028771 FP_60 <abxab> {00} ab -- bb -- ab bb ab ab <abxaa> {0-} aa -- -- aa ab aa -- aa <aaxab> {-0} aa aa ab ab ab aa ab ab
SNP_028772 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <abxaa> {0-} aa aa aa aa ab aa aa aa <abxab> {10} ab ab ab ab bb aa ab ab
SNP_028773 FP_60 <aaxab> {-0} aa aa ab -- ab ab ab aa -- -- -- -- -- -- -- -- <abxab> {10} -- -- ab ab bb aa ab ab
Pop 1 Pop 2 Pop 3
FP-mapper by J. Kruisselbrink
• Stable SNP haplotypes

24. YOUR LOGO
2. NEW MAPPING APPROACH: FPs STRATEGY
➧FPs strategy
SNP name FP segr phase segr phase segr phase
SNP_028768 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa <aaxab> {-0} -- aa ab ab -- aa ab ab
SNP_028769 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
SNP_028770 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa -- -- -- -- -- -- -- --
SNP_028771 FP_60 <abxab> {00} ab -- bb -- ab bb ab ab <abxaa> {0-} aa -- -- aa ab aa -- aa <aaxab> {-0} aa aa ab ab ab aa ab ab
SNP_028772 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <abxaa> {0-} aa aa aa aa ab aa aa aa <abxab> {10} ab ab ab ab bb aa ab ab
SNP_028773 FP_60 <aaxab> {-0} aa aa ab -- ab ab ab aa -- -- -- -- -- -- -- -- <abxab> {10} -- -- ab ab bb aa ab ab
Pop 1 Pop 2 Pop 3
Female meiosis ab aa ab ab aa ab ab ab aa aa aa aa ab aa aa aa ab ab aa aa ab aa aa aa
Male meiosis aa aa ab ab ab ab ab aa aa ab ab ab ab ab ab aa aa aa ab ab ab aa ab ab
FP-mapper by J. Kruisselbrink
• Stable SNP haplotypes
• Haplotype data integration
 bi-parental genotypes split into single parent datasets

25. YOUR LOGO
2. NEW MAPPING APPROACH: FPs STRATEGY
➧FPs strategy
SNP name FP segr phase segr phase segr phase
SNP_028768 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa <aaxab> {-0} -- aa ab ab -- aa ab ab
SNP_028769 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
SNP_028770 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa -- -- -- -- -- -- -- --
SNP_028771 FP_60 <abxab> {00} ab -- bb -- ab bb ab ab <abxaa> {0-} aa -- -- aa ab aa -- aa <aaxab> {-0} aa aa ab ab ab aa ab ab
SNP_028772 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <abxaa> {0-} aa aa aa aa ab aa aa aa <abxab> {10} ab ab ab ab bb aa ab ab
SNP_028773 FP_60 <aaxab> {-0} aa aa ab -- ab ab ab aa -- -- -- -- -- -- -- -- <abxab> {10} -- -- ab ab bb aa ab ab
Pop 1 Pop 2 Pop 3
Female meiosis ab aa ab ab aa ab ab ab aa aa aa aa ab aa aa aa ab ab aa aa ab aa aa aa
Male meiosis aa aa ab ab ab ab ab aa aa ab ab ab ab ab ab aa aa aa ab ab ab aa ab ab
FP-mapper by J. Kruisselbrink
• Stable SNP haplotypes
• Haplotype data integration
 bi-parental genotypes split into single parent datasets
• Missing values reduction

26. YOUR LOGO
2. NEW MAPPING APPROACH: FPs STRATEGY
➧FPs strategy
SNP name FP segr phase segr phase segr phase
SNP_028768 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa <aaxab> {-0} -- aa ab ab -- aa ab ab
SNP_028769 FP_60 <abxaa> {1-} ab aa ab ab aa ab ab ab -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
SNP_028770 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <aaxab> {-0} aa ab ab ab ab ab ab aa -- -- -- -- -- -- -- --
SNP_028771 FP_60 <abxab> {00} ab -- bb -- ab bb ab ab <abxaa> {0-} aa -- -- aa ab aa -- aa <aaxab> {-0} aa aa ab ab ab aa ab ab
SNP_028772 FP_60 <abxab> {00} ab aa bb bb ab bb ab ab <abxaa> {0-} aa aa aa aa ab aa aa aa <abxab> {10} ab ab ab ab bb aa ab ab
SNP_028773 FP_60 <aaxab> {-0} aa aa ab -- ab ab ab aa -- -- -- -- -- -- -- -- <abxab> {10} -- -- ab ab bb aa ab ab
Pop 1 Pop 2 Pop 3
Female meiosis ab aa ab ab aa ab ab ab aa aa aa aa ab aa aa aa ab ab aa aa ab aa aa aa
Male meiosis aa aa ab ab ab ab ab aa aa ab ab ab ab ab ab aa aa aa ab ab ab aa ab ab
FP-mapper by J. Kruisselbrink
• Stable SNP haplotypes
• Haplotype data integration
 bi-parental genotypes split into single parent datasets
• Missing values reduction

27. YOUR LOGO
2.NEW MAPPING APPROACH: BACKCROSS STRATEGY
➧Backcross strategy and
data integration across families (FP-mapper by J. Kruisselbrink)
Female meiosis ab aa ab ab aa ab ab ab aa aa aa aa ab aa aa aa ab ab aa aa ab aa aa aa
Male meiosis aa aa ab ab ab ab ab aa aa ab ab ab ab ab ab aa aa aa ab ab ab aa ab ab
Segr Pop 1 Pop 2 Pop 3

28. YOUR LOGO
2.NEW MAPPING APPROACH: BACKCROSS STRATEGY
➧Backcross strategy and
data integration across families (FP-mapper by J. Kruisselbrink)
FP_60 unique marker
Female meiosis ab aa ab ab aa ab ab ab aa aa aa aa ab aa aa aa ab ab aa aa ab aa aa aa
Male meiosis aa aa ab ab ab ab ab aa aa ab ab ab ab ab ab aa aa aa ab ab ab aa ab ab
Segr Pop 1 Pop 2 Pop 3
Merging single parent datasets in a single backcross-type population
➝ Twice the individual of the original population ➝ 3200 meiosis

29. YOUR LOGO
2.NEW MAPPING APPROACH: BACKCROSS STRATEGY
➧Backcross strategy and
data integration across families (FP-mapper by J. Kruisselbrink)
FP_60 unique marker
Female meiosis ab aa ab ab aa ab ab ab aa aa aa aa ab aa aa aa ab ab aa aa ab aa aa aa
Male meiosis aa aa ab ab ab ab ab aa aa ab ab ab ab ab ab aa aa aa ab ab ab aa ab ab
Segr Pop 1 Pop 2 Pop 3
Merging single parent datasets in a single backcross-type population
➝ Twice the individual of the original population ➝ 3200 meiosis

30. YOUR LOGO
ADVANTAGES

31. YOUR LOGO
ADVANTAGES

32. YOUR LOGO
Families
Individuals:
Focal Points (FPs):
Total SNPs:
Average SNPs/FP:
Average mv%/FP:
Average distance: cM
max d. (LG6) : cM
min d. (many LGs): cM
Total Map Length: cM
21
~1600
~3000
~15000
~5
~40
0.40
3.30
0.00
~1267
HIGH DENSITY FPs GENETIC MAP
Join Map (V4.1)

33. YOUR LOGO
LG1 LG2 LG3 LG4 LG5 LG6 LG7 LG8 LG9 LG10 LG11 LG12 LG13 LG14 LG15 LG16 LG17
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110
HIGH DENSITY FPs GENETIC MAP Join Map (V4.1)

34. YOUR LOGO
LG1 LG2 LG3 LG4 LG5 LG6 LG7 LG8 LG9 LG10 LG11 LG12 LG13 LG14 LG15 LG16 LG17
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110
HIGH DENSITY FPs GENETIC MAP Join Map (V4.1)
63 cM
112 cM

35. YOUR LOGO
LG 3
Join Map (V4.1)
Test for alternative positions
(1000 iterations)
0%
50%
100%
o ROBUSTNESS OF MARKERS POSITIONS
QUALITY OF THE MAP
THE PROBABILITY OF
EACH MARKER (FP) TO
BE ASSIGNED TO ITS
POSITION IN THE MAP

36. YOUR LOGO
Join Map (V4.1)
Estimated Plausible Positions
0%
50%
100%
THE PROBABILITY OF
EACH MARKER (FP) TO
BE ASSIGNED TO ITS
POSITION IN THE MAP
0%
50%
100%
LG 3
QUALITY OF THE MAP
o ROBUSTNESS OF MARKERS POSITIONS

37. YOUR LOGO
Join Map (V4.1)
Estimated Plausible Positions
0%
50%
100%
THE PROBABILITY OF
EACH MARKER (FP) TO
BE ASSIGNED TO ITS
POSITION IN THE MAP
LG 3
0%
50%
100%
LG3_FP_1140
o ROBUSTNESS OF MARKERS POSITIONS
QUALITY OF THE MAP

38. YOUR LOGO
0%
50%
100%
THE PROBABILITY OF
EACH MARKER (FP) TO
BE ASSIGNED TO ITS
POSITION IN THE MAP
Join Map (V4.1)
Test for alternative positions
(1000 iterations)
LG 3
QUALITY OF THE MAP
o ROBUSTNESS OF MARKERS POSITIONS

39. YOUR LOGO
IMPACTS

40. YOUR LOGO
IMPACTS
• MARKERS TO BE MAPPED ON THE CORRECT LG
• MARKER POSITIONING through FPs VALIDATION

41. YOUR LOGO
IMPACTS
• Special approach elaborated by Voorrips et al. see
POSTER SESSION

42. YOUR LOGO
B
A
B
A
B
B
A
B
A
B
B
A
B
A
B
B
B
B
A
B
Haplotype24
B
A
B
A
A
A
A
B
A
A
Haplotype12
Haplotype13
10 SNP markers
HAPLOTYPING
Special approach elaborated by Voorrips et al. see POSTER SESSION
Joint genotypes of multiple
successive SNPs
• single multi-allelic marker

43. YOUR LOGO
B
A
B
A
B
B
A
B
A
B
B
A
B
A
B
B
B
B
A
B
Haplotype24
B
A
B
A
A
A
A
B
A
A
Haplotype12
Haplotype13
LG1
HaploBlock 2
10 SNP markers
HAPLOTYPING
Special approach elaborated by Voorrips et al. see POSTER SESSION
Joint genotypes of multiple
successive SNP
• single multi-allelic marker
• converted into haploblocks

44. YOUR LOGO
Joint genotypes of multiple
successive SNP
• single multi-allelic marker
• converted into haploblocks
• allow exploring entire allelic
variation
B
A
B
A
B
B
A
B
A
B
B
A
B
A
B
B
B
B
A
B
Haplotype24
B
A
B
A
A
A
A
B
A
A
Haplotype12
Haplotype13
LG1
HaploBlock 2
3 haplotype variants
10 SNP markers
HAPLOTYPING
Special approach elaborated by Voorrips et al. see POSTER SESSION

45. YOUR LOGO
➧ Flow of haplotype alleles along pedigrees
– LG1 apple, region flanking the Vf gene for scab
resistance
– Pedigree 1 commercial cv:
• Galarina Vf-resistant
HAPLOTYPING
Special approach elaborated by Voorrips et al. see POSTER SESSION

46. YOUR LOGO
➧ Single HaploBlock
locus / marker
• 5 generations
• Flow of Haplotype 13
Delicious
11 12
8 4
2 2
3 2
GoldenDel
2 4
2 4
2 4
3 2
F2_26829-2-2
13 4
9 5
8 7
3 3
Jonathan
5 2
4 2
4 2
3 2
Cox
4 5
4 4
12 4
2 3
PRI14-126
4 13
4 9
4 8
2 3
KidsOrRed
11 5
8 4
2 4
3 3
PRI612-1
12 13
4 9
2 8
2 2
Gala
5 4
4 4
4 2
3 3
Florina
13 2
9 2
8 *
2 2
Galarina
13 5
9 4
8 4
2 3
4 55 213 42 411 12
11 54 13
12 13 5 4
13 2
13 5
HAPLOTYPE FLOW

47. YOUR LOGO
HAPLOTYPE FLOW
➧ Single HaploBlock
locus / marker
• 5 generations
• Flow of Haplotype 13
Delicious
11 12
8 4
2 2
3 2
GoldenDel
2 4
2 4
2 4
3 2
F2_26829-2-2
13 4
9 5
8 7
3 3
Jonathan
5 2
4 2
4 2
3 2
Cox
4 5
4 4
12 4
2 3
PRI14-126
4 13
4 9
4 8
2 3
KidsOrRed
11 5
8 4
2 4
3 3
PRI612-1
12 13
4 9
2 8
2 2
Gala
5 4
4 4
4 2
3 3
Florina
13 2
9 2
8 *
2 2
Galarina
13 5
9 4
8 4
2 3
4 55 213 42 411 12
11 54 13
12 13 5 4
13 2
13 5

48. YOUR LOGO
HAPLOTYPE FLOW
➧ Single HaploBlock
locus / marker
• 5 generations
• Flow of Haplotype 13
Delicious
11 12
8 4
2 2
3 2
GoldenDel
2 4
2 4
2 4
3 2
F2_26829-2-2
13 4
9 5
8 7
3 3
Jonathan
5 2
4 2
4 2
3 2
Cox
4 5
4 4
12 4
2 3
PRI14-126
4 13
4 9
4 8
2 3
KidsOrRed
11 5
8 4
2 4
3 3
PRI612-1
12 13
4 9
2 8
2 2
Gala
5 4
4 4
4 2
3 3
Florina
13 2
9 2
8 *
2 2
Galarina
13 5
9 4
8 4
2 3
4 55 213 42 411 12
11 54 13
12 13 5 4
13 2
13 5

49. YOUR LOGO
HAPLOTYPE FLOW
➧ Single HaploBlock
locus / marker
• 5 generations
• Flow of Haplotype 13
Delicious
11 12
8 4
2 2
3 2
GoldenDel
2 4
2 4
2 4
3 2
F2_26829-2-2
13 4
9 5
8 7
3 3
Jonathan
5 2
4 2
4 2
3 2
Cox
4 5
4 4
12 4
2 3
PRI14-126
4 13
4 9
4 8
2 3
KidsOrRed
11 5
8 4
2 4
3 3
PRI612-1
12 13
4 9
2 8
2 2
Gala
5 4
4 4
4 2
3 3
Florina
13 2
9 2
8 *
2 2
Galarina
13 5
9 4
8 4
2 3
4 55 213 42 411 12
11 54 13
12 13 5 4
13 2
13 5

50. YOUR LOGO
HAPLOTYPE FLOW
➧ Single HaploBlock
locus / marker
• 5 generations
• Flow of Haplotype 13
 came from F2-26829:Vf-resistant
selection, 2nd generation from
M.foribunda 821
Delicious
11 12
8 4
2 2
3 2
GoldenDel
2 4
2 4
2 4
3 2
F2_26829-2-2
13 4
9 5
8 7
3 3
Jonathan
5 2
4 2
4 2
3 2
Cox
4 5
4 4
12 4
2 3
PRI14-126
4 13
4 9
4 8
2 3
KidsOrRed
11 5
8 4
2 4
3 3
PRI612-1
12 13
4 9
2 8
2 2
Gala
5 4
4 4
4 2
3 3
Florina
13 2
9 2
8 *
2 2
Galarina
13 5
9 4
8 4
2 3
4 55 213 42 411 12
11 54 13
12 13 5 4
13 2
13 5

51. YOUR LOGO
Delicious
11 12
8 4
2 2
3 2
GoldenDel
2 4
2 4
2 4
3 2
F2_26829-2-2
13 4
9 5
8 7
3 3
Jonathan
5 2
4 2
4 2
3 2
Cox
4 5
4 4
12 4
2 3
PRI14-126
4 13
4 9
4 8
2 3
KidsOrRed
11 5
8 4
2 4
3 3
PRI612-1
12 13
4 9
2 8
2 2
Gala
5 4
4 4
4 2
3 3
Florina
13 2
9 2
8 *
2 2
Galarina
13 5
9 4
8 4
2 3
4 55 213 42 411 12
11 54 13
12 13 5 4
13 2
13 5
HAPLOTYPE FLOW
➧ Single HaploBlock
locus / marker
• 5 generations
• Flow of Haplotype 13
 came from F2-26829:Vf-resistant
selection, 2nd generation from
M.foribunda 821
• Flow of Haplotype 5
 came from Cox

52. YOUR LOGO
HAPLOTYPE FLOW
➧ Multiple HaploBlocks
• Consistent flow
Delicious
11 12
8 4
2 2
3 2
GoldenDel
2 4
2 4
2 4
3 2
F2_26829-2-2
13 4
9 5
8 7
3 3
Jonathan
5 2
4 2
4 2
3 2
Cox
4 5
4 4
12 4
2 3
PRI14-126
4 13
4 9
4 8
2 3
KidsOrRed
11 5
8 4
2 4
3 3
PRI612-1
12 13
4 9
2 8
2 2
Gala
5 4
4 4
4 2
3 3
Florina
13 2
9 2
8 *
2 2
Galarina
13 5
9 4
8 4
2 3

53. YOUR LOGO
HAPLOTYPE FLOW
➧ Multiple HaploBlocks
• Consistent flow
• Tracing recombination events
Delicious
11 12
8 4
2 2
3 2
GoldenDel
2 4
2 4
2 4
3 2
F2_26829-2-2
13 4
9 5
8 7
3 3
Jonathan
5 2
4 2
4 2
3 2
Cox
4 5
4 4
12 4
2 3
PRI14-126
4 13
4 9
4 8
2 3
KidsOrRed
11 5
8 4
2 4
3 3
PRI612-1
12 13
4 9
2 8
2 2
Gala
5 4
4 4
4 2
3 3
Florina
13 2
9 2
8 *
2 2
Galarina
13 5
9 4
8 4
2 3

54. YOUR LOGO
HAPLOTYPE FLOW
➧ Multiple HaploBlocks
• Consistent flow
• Tracing recombination events
• Consistent results validated
with SSR-Hidras data
Delicious
11 12
8 4
2 2
3 2
GoldenDel
2 4
2 4
2 4
3 2
F2_26829-2-2
13 4
9 5
8 7
3 3
Jonathan
5 2
4 2
4 2
3 2
Cox
4 5
4 4
12 4
2 3
PRI14-126
4 13
4 9
4 8
2 3
KidsOrRed
11 5
8 4
2 4
3 3
PRI612-1
12 13
4 9
2 8
2 2
Gala
5 4
4 4
4 2
3 3
Florina
13 2
9 2
8 *
2 2
Galarina
13 5
9 4
8 4
2 3

55. YOUR LOGO
CONCLUSION
ACCURATE AND RELIABLE ORDER OF THE
HIGHEST POSSIBLE NUMBER OF MARKER LOCI

56. YOUR LOGO
CONCLUSION
ACCURATE AND RELIABLE ORDER OF THE
HIGHEST POSSIBLE NUMBER OF MARKER LOCI

57. ACKNOWLEDGEMENTS
Eric Van de Weg
Johannes Kruisselbrink
Herma Koehorst
Sara Longhi
Stefano Tartarini
Giulia Pagliarani
Luca
Gianfranceschi
Mario Di Guardo
Diego Micheletti
Luca Bianco
Michela Troggio
Hélène Muranty
Larisa
Gustavsson

58. YOUR LOGO
THANKS FOR YOUR
ATTENTION !

59. YOUR LOGO
Roeland E. Voorrips
Marco C.A.M. Bink
Johannes W. Kruisselbrink
Herma J.J. Koehorst - van Putten
W. Eric Van de Weg
POSTER SESSION

60. YOUR LOGO
ADVANTAGES
➧Advantages of the FPs strategies
• Reduced number of missing values
• Increased robustness of marker scores
• Complete exploitation of genetic information
• Fully informative markers

61. YOUR LOGO
LG1 LG2 LG3 LG4 LG5 LG6 LG7 LG8 LG9 LG10 LG11 LG12 LG13 LG14 LG15 LG16 LG17
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110
CORE DATASET in Black
• at least 800 meiosis present
• 25% of present data
in Violet
• at least 300 meiosis present
• 10% of present data
THE FINAL INTEGRATED GENETIC MAP

62. YOUR LOGO
➧Advantages of the backcross strategy
NEW MAPPING APPROACH
Standard outcrossers
integrated map
• Integration of the 2 parental maps
• Integration across families
Integration of DATASETS prior to map
construction
Novel approach
VS

63. YOUR LOGO 63
HAPLOBLOCKS
HB 1
HB 2
HB 3
HB 4
HB 5
HB 6
HB 7
HB 8
Galarina Florina Gala • Tightly linked sets of SNPs
• Recombination occurs only
between haploblock and
NOT WITHIN
multi-allelic markers
based on correctly
assigned haplotypes
Voorrips et al. POSTER SESSION
LG1

64. YOUR LOGO
➧Approaches reducing size data sets
• High-density SNP arrays
• Computer memory limitations, increase computation time
• Genotyping by SNP haplotypes
– Use as multi-allelic markers, more informative than single di-allelic SNPs
NEEDS FOR SUCCESSFUL MAB STRATEGIES
Complexity of Apple Genome
➾ Apple Physical Map V2 with uncertainties

65. YOUR LOGO
NEXT STEP
➧PUBLICATION of the Integrated Genetic Map
➧ORIGINAL DATA will become publicly AVAILABLE on the
FB-database
➝ Allow USERS to FURTHER IMPROVE REGION of INTEREST by
 further DATA SCRUTINIZING
 adding data (e.g. additional families)
➝ MOST RELIABLE marker-loci ORDER for the HIGHEST
POSSIBLE NUMBER of MARKERS