1. WP3
Results achieved since the beginning
of the project and plans for 2013

2. Main objectives of WP3
1. Extend software for pedigree based QTL
analyses
2. Discovery of QTL for fruit quality traits and
disease resistance.
3. Validation of QTL-alleles over climatic regions
and harvest dates
4. Assessment of genetic diversity as exploited
within European breeding programs

3. Results: PBA software – T3.1
Intro:
• FlexQTLTM software
– Used to search for markers linked to traits.
– Designed for low marker densities
• SSR, AFLP...
• High densities (SNP): computation time too
long
Needs:
– Approaches reducing computation time

4. Results: FlexQTLTM
Approaches:
• More efficient algorithm implemented: -25%
• Option to sample marker info less frequently: -10x
• Windows -> version Linux platform : - 5x

5. Results: PBA –software II
Intro-II
• SNP calling is performed using Genome Studio software
• Easily creates > million data points
• Issue: preparation of input data files for PBA software
Approach
• Script developed that converts Genome Studio output to
a Pedimap (FlexQTLTM) input file

6. Intro III
• Visual interpretation easier than inspection of
large tables
Approach
• Important QTL results are presented in figures
– R-scripts have been added or improved for
• Trace plots of QTL positions along the genome
• Bin-wise posterior probabilities of QTL
• Posterior mean estimates of QTL effects
• Posterior probabilities of QTL genotypes of individuals
Results: PBA –software III

7. Intro IV
• FlexQTLTM results are presented in a large
series of output files. Not user friendly for
occasional users.
Approach
• A Interface is under development. Test version
is un use.

8. PBA – software: Visual FlexQTL

9. PBA – software: Visual FlexQTL

10. PBA-Software: Integrated maps
Intro V
• QTL mapping requires markers to be in correct order.
– LG, succession in within Linkage group
– Physical map not adequate: ± 15% SNP misplaced
Need
– Markers correctly positioned on linkage map
Approach
– Protocols developed for the generation of integrated
genetic linkage maps over multiple families

11. PBA-software
Summary
- Better adopted to high marker densities
- Reductions in computation time
- Additional new functionalities
- Increased user friendliness
- Interface
Acknowledgements
• RosBREED project
– USDA-SCRI
– www.RosBREED.org Amy
Iezzoni
Cameron
Peace

12. Results: Training on PBA software – T3.7
Workshop
• Aim: Intro to FlexQTLTM & Pedimap
• Participants: Partners WP3-Peach
• Date: Feb 17-18, 2013
• Instructors: IRTA: Diego Micheletti
DLO: Marco Bink
Eric van de Weg

13. Results: Discovery QTL T3.2-T3.2
Background
1. Fruit related Traits
– Maturity time
– Fruit Size
– Flesh color
– Firm/soft melting
– Flesh adherence,
– Titrable acidity
– Total sugar
– Flowering time
– Flowering intensity
– White anthers
– Monilinia resistance
3. Phenotypic data
– Historic: EU-Projects, National projects
2. Segregating germplasm
– 18 families , ±1500 individuals
– 5 breeding programs, 3 countries
4. Marker Data: next slide
Peach
3. Phenotypic data
– New : – 2011 & 2012 season
– Uploaded to DataBase
Results

14. Peach – Marker data
• 9K SNP array
• Filtered for robust SNP
• Marker maps generated for each family

15. Genotyping performance
• Assayed SNPs = 8144
• Polymorphic: 67.1%
– Robust and informative 53.8%
– Null Allele/preferencial annealing 2.9%
– Paralogous regions 10.1%
• Non-Polymorphic 32.9%
– False SNP 9.6%
– Failed 23.3%

16. Genetic mapping
• Comparison of the genetic map of
chromosome 3 for two crosses
– Belbinette x Nectalady
– (IF7319828 x P. ferganensis) x IF7310828
• High colinearity between genetic map
and genome  No need to create an
integrated genetic for the PBA

17. PBA Preliminary results Peach
 FlexQTL confirms known QTLs both
for Flowering intensity & time
 Peaks in Green
 FlexQTL gives some evidence for
additional minor QTL
 peaks in black
G 8
Flowering
Time
G 4
Flower
intensity

18.  Fruit Quality
– Taste
• Firmness
• Juiciness
• Crispness
• Total Sugar
• Acidity
– Storability
– Shelf life
– Harvest date
– Fruit size
– Skin Color
 Resistance to Nectria
Apple
1. Fruit Quality Traits: > 10
3. Phenotypic data
– HiDRAS
• 3 Years, Multiple time moments
– Harvest, Storage 2 & 4M
– Shelf Life: 2wks - 2M storage
– National projects
2. Segregating germplasm
 24 families , ±1600 individuals
 6 breeding programs, 6 countries
4. Genotyping: next slide
Results: Discovery QTL T3.2-T3.2
Results
Background

19. Genotyping
• Infinium 20K SNP
– Available Jan 16, First 2x96 individuals tested Jan 19
• Jonathan x Prima
– High quality data!
– High number of polymorphic markers

20. Genotyping: 1st SNP results
• High number of polymorphic markers
• 70% (1820/2598) excluded: same segregation pattern
• 700 – 800 Unique segregation patterns, ±45/LG
• ± 3 SNP / Focal Point
Jonathan
# # Left
Prima
# # Left
J+P
#
J+P
# Left
Total Filtered 3000 2958 2400
Similarity -JM -1549 1409
Ungrouped -33 1376
Excluded 1081 -271 1105
Similarity map -317 764 -303 764
Unique patterns 764 802

21. Mapped easily
J1 P1 J2 P2 J3 P3 J4 P4 J5 P5 J6 P6 J7 P7 J8 P8 J9 P9 J10 P10 J11 P11 J12 P12 J13 P13 J14 P14 J15 P15 J16 P16 J17 P17
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105 Some ends of chromosomes need to be checked +P7

22. Graphical genotyping: Error added

23. • Graphical genotyping: High quality maps!

24. Results genetic diversity EU breeding programs
T3.4
• Peach:
– SSR study
– Presentation Maria Jose – Stakeholdersday
• Apple
– Results for 2 breeding programs (Y1)
– Y2: No activities
– Urgent need to progress

25. Publications Peach: Planned
Title WP Responsible Participants
Application of PBA in peach: PBA to identify
major genes*
3 IRTA All WP3-Peach
Genetic diversity as exploited in
EU apple and peach breeding.
3 DLO DLO+IRTA
AG and PBA: characters related to climate
change *(blooming/flowering/maturity)
3-4 INRA tbd
AG? and PBA to detect QTLs of characters
related to disease resistance
3-4 INRA tbd
AG and PBA to detect QTLs of characters
related to fruit quality
3-4 UNIMI-CRA tbd

26. Publications Apple: Published
1. Development of a dense SNP-based linkage map of an apple
rootstock progeny using the Malus Infinium whole genome
genotyping array.
– 2012, BMC Genomics 25;13:203.
– Antanaviciute L, Fernández-Fernández F, Jansen J, Banchi E, Evans KM,
Viola R, Velasco R, Dunwell JM, Troggio M, Sargent DJ
2. PEDIMAP: Software for the visualization of genetic and
phenotypic data in pedigrees.
– 2012, Journal of Heredity.
– Voorrips RE, Bink MCAM, Van de Weg WE

27. Publications Apple: Planned ‘13 - ‘14
1. Procedures for construction of Integrated Genetic linkage
using multiple families.
– Jansen et al.
– DLO, IASMA, PFR
2. Procedures for filtering and calling of SNP markers.
– DiGuardo et al.
– DLO, IASMA
3. Integrated genetic linkage map of apple using ± 24 families.
– DiGuardo et al.
– DLO, IASMA, .....?
4. QTLs for Nectria resistance in apple
– Koehorst et al.
– DLO, IASMA

28. Main challenges for 013 - Peach
Extend PBA to the 18 subpopulations (starting from March)
– Normalization of phenotypic data from 5 different
location in Europe:
• INRA - Avignon (F), INRA- Bordeaux (F), IRTA (E), CRA-
FRU (I), and University of Milan (I)
 Evaluation of the construction of SNP haplotypes (to use as
multi-allelic markers, more informative than single SNPs)
 Correction of the genetic/physic position of some
misplaced SNPs

29. Main challenges for 2013 - Apple
1. Apple: Integrated genetic linkage map
– Needed to get started with QTL analyses
2. Development of a sustained/ ‘agreed’ vision on type
and format of QTL & marker of use to breeders
– Joined activity WP1.5, WP3, WP7
3. Involve stakeholders in validation of QTL in their
breeding germplasm.
4. Report on the delivery of genetic diversity in Breeding
programs

30. Interactions between WP3 ↔ other WP
• Interactions needed with other WPs:
– WP1: Keep informed on discovery Fruit quality QTL
– WP4: Comparison on marker-trait-associations
– WP5: Phenotyping protocols + Phenotypic data
– WP6: SNP genotyping, Genetic <> Physical map
– WP7: Development conceptual model for WP3 data
– WP8: Organization of PBA workshop for stakeholders
– WP9: management of the project, reports
• Interactions planned with other projects or
stakeholders of the project
– RosBREED
– Apple Breeders