2. Main objectives of the WP6
• Task 6.1: Development of a low-density SNP-based genotyping array in apple.
– low-density array of highly informative 384 SNPs
• Task 6.2: Development of a medium-density SNP-based genotyping array in apple (3-9 -> 20K)
– Re-sequencing of 14 apple accessions and 2 double-haploid (30x coverage each) for SNP discovery
– Construction of an integrated map to identify a set of validated SNPs to be included in the second generation
apple chip (20k)
– Improvement of the apple genome assembly (version 3.0) to provide a more solid reference genome for reads
alignment and SNP calling
– Development of a pipeline for SNP calling and SNP selection in collaboration with DLO, CNAG in Barcelona and
PFR in New Zealand
– Use of the validated SNPs from the 8k apple chip to test the SNP calling pipeline and to avoid problems with
paralogous regions
• Task 6.3: Development of species-specific high-density genome-wide SNP arrays suitable for genome-
wide association mapping in apple and peach
– Evaluation of GBS vs high density chip for GWA mapping in apple (20-50 -> 400-600K ?)
– re-sequencing of 48(96) genotypes
• Task 6.4: Testing protocols for marker-assisted selection
– Testing different protocols for low-cost DNA extraction with different techniques
– Testing different methods for low-cost SNP genotyping: minisequencing and Sequenome. Evaluation of other
genotyping platforms available (Fluidigm, KASPar....)
3. Deliverables due in the project
• D6.1 – Development of the first 384 SNP array for apple (M4)
• D6.2 – Development of a 20K SNP chip array for apple (M22,
delayed from M12)
• D6.3 – Peach SNP array to be made available and screened
for all genotypes required (M12)
• D6.4 – Apple GWA genotyping array (over 400K) will be
made available (delayed from M24 to M36)
• D6.5 – Protocols for MAS/MAB should be made available
(due to M36, but several already tested and made available)
4. Results achieved since the beginning
of the project
• D6.1 – Developed the first 384 SNP array for apple (M4)
– The sequence information for the 384 SNPs are made available
to the partners together with their position in the seventeen LG
of an apple integrated linkage map.
– Transferability of SNPs included in the array based on FEM’s
internal data is high. These markers are those valuable
resources for quick genotyping of germplasm and progenies to
build framework linkage maps.
5. 20K Illumina chip development
• Capacity re-sequencing: 1 full run on Illumina HiSeq2000, 8 lanes
• Illumina paired-end (2x100 bp)
• 16 individuals, ~30x coverage each
• Read alignment to the reference genome (Primary assembly and
additional haplotypes) using GEM (CNAG in house development)
and BFAST
• Unique alignments (up to 4 mismatches allowed) 142Gb
• Variant calling pipeline (SAMtools + BCFtools)
16M SNPs+Indels predicted
D6.2 – Developed a 20 SNP
chip array for apple
6. Discovery panel: Composition
Major founders – Parents mapping populations
1 New founder: Malus micromalus – scab resistance
● Antonovka
● Braeburn
● Cox
● Delicious
● Dr.Oldenburg
● F2-26829-2-2
● Fuji
● Jonathan
● Lady Williams
● McIntosh
● Macoun
● Priscilla-NL
● Worcester
Pairmain
2 double haploids – SNP quality check
CLEAR DISCOVERY OF PARALOGOUS
● Polymorphism >1 locus involved (ORTHOLOGOUS)
7. D6.2 – Developed a 20 SNP
chip array for apple
Filtering SNP Candidates
• Occurring > 10 reads for at least 50% of the accessions
• Not polymorphic in the 2 di-haploids
• Illumina quality score > 20 -> 2.6 M SNPs
• Not tri-allelic; No A/T; no C/G; No additional SNP 50nt up/down stream
• Illumina ADT score >0.7
• Primers for unique positions (Blast of 24mer, 2 mismatches)
– Blasted against genome sequences (all contigs)
– Primary assembly
– Additional haplotypes (‘in parallel’ to prim. Assembly)
• Filtered high quality SNPs: 750 K SNPs
8. Array Design (FEM + DLO)
1. Source SNP
– 3.7K SNP previous 8K-chip: informative & robust
– 0.1 K Customized
– 16.2 K New SNP
2. Organization: Focal Points
– Regions of max 10cM having up to 11 SNPs (100Kb-8K chip)
Supports design stable multi-allelic SNP-haplotypes
3. Distribution across genome
– Genetic interval ~1 cM
– Physical interval < 400Kb
– Chromosomes ends: higher density
– 2040 Focal Points
9. Array design II (FEM + DLO)
4. Uniqueness profile across discovery panel
• No SNP with identical pattern
5. Allele frequency - Heterozygosity
– No rare (highly specific) nor very common SNP
Increases informativeness – wide germplasm
• Up to 5 SNP polymorphic in 5-8 panel members
• Complemented to 11 SNP polymorphic in 2-4 panel members
• N<11 ? Complemented with unique SNP
6. Representation: Balanced across panel members
– Minimizes over/under representation of specific members / random
sampling
10. Results achieved since the beginning
of the project
• D6.3 – Peach SNP array made available and screened for
all genotypes required (M12)
– The IPSC peach SNP array v1 developed by an international
consortium was successfully used for genotyping both segregating
populations and germplasm collections of peach WP3 and WP4. An
average of 2,300 validated SNPs were obtained for each mapping
population. Genotypic data are currently being analyzed by IRTA and
DLO for Pedigree-Based Analysis and Genome-wide association
studies.
– The IPSC chip is commercially available from Illumina. SNP markers
included in the IPSC peach 9K array are available to download in excel
and view in GBrowse at http://www.rosaceae.org/.
11. Main challenges for 2013
• Re-sequencing of 48 (96?) apple genotypes for
SNPs development (Affimetrix Axiom 400-600k
SNPs)
OR
• Participate on GBS for GWA of WP4 (sequencing
and bioinformatic analysis)
• Updating of protocols for MAB
12. Evaluation of the 20K Illumina chip
48 old cultivars tested
(INRA sub-CC)
LD decay: r2 <0,2 for a
distance > 55 kb
Also with GBS at Cornell
(S. Myles)
1536 progenies/germplasm
WP3 preliminary results
(Eric Van de Weg)
• 1369 monomorphics
• 185 failed
• 232 missing class AB
• 1390 freq AB<0.1
• 416 freq AB>0.7
• 14,427 (80%) freq 0.1>AB<0.7
ADDITIONAL FILTERS
- GenTrain>0.4
- All three classes of genotypes observed
- MAF >0.05
- <10% missing data
• 10,363 (56%)
• D6.4 – Apple GWA
genotyping array will be
made available
15. Pros & Cons of GBS
Sean Myles personal communication and FEM preliminary analysis
Pros
- obtain large amount of data very quickly
- inexpensive (one enzyme, $30/sample)
- relatively free of ascertainment bias
Cons
- large amount of missing data/ uneven coverage across samples
- low read depth – many genotype calling errors (undercall
heterozygosity)
- allelic dropout when sequence are too divergent from reference
genome or polymorphism in restriction sites
- technically missing confounded with biologically missing
16. Pros & Cons of SNP array
Pros
- no complex bioinformatic analyses
- few missing data
- easy to perform
- Cheap quotation 135€ x sample for 420K (1,248 minimum order,
further discount 125€ minimum 192 samples)
Cons
- design time
- suffer of ascertainment bias (SNPs discovered in a small set of
samples), prior selection of SNPs to be assayed
- higher cost/sample
- unreliable genotypes call in highly divergent samples because of
reduced probe-sequence hybridization
17. SNP genotyping technologies – MAS/MAB
• KASP™-SNP Genotyping technology -
KBioscience Ltd.
• MassARRAY Analyzer- Sequenom, Inc.
• TaqMan® OpenArray® Genotyping – APPLIED
BIOSYSTEMS
• SNPtype™ Assays – FLUIDIGM
• BeadXpress® Reader – ILLUMINA
D6.5 – Protocols for MAS/MAB
are made available
18. SNP genotyping technologies – MAS/MAB
D6.5 – Protocols for MAS/MAB
are made available
• KASP™-SNP – KBioscience UK Itd.
Pro: Low price, flexibility of no. of SNPs, low quality DNA requested
Cons: NO Multiplex
• Sequenome – University of Bologna
Pro: Low price, solid approach, low quality DNA requested
Test of different extraction protocol
Reproducible results between Qiagen-CTAB-Sigma-Nucleospin
Cons: Convenient with a min. of 30 SNPs assay
• TaqMan® OpenArray® Genotyping
Pro: Solid approach, different design of the multiplex (16-256 SNPs)
Cons: high quality DNA requested, (1.5 € for DNA extraction at UniPD)
19. Publications
• Antanaviciute L et al. (2012) An evaluation of the Malus Infinium whole
genome genotyping array in an apple rootstock mapping progeny. BMC
Plant Biol. 13:203
• Chagné D et al. (2012) Genome-wide SNP detection, validation and
development of a SNP Infinium II assay for apple. PLoSONE 7(2): e31745
• Verde I et al. (2012) Development and evaluation of a 9K SNP array for
peach by internationally coordinated SNP detection and validation in
breeding germplasm. PLoSONE 7(4): e35668
• Eduardo I et al. (2012) Genetic dissection of aroma volatile compounds
from the essential oil of peach fruit: QTL analysis and identification of
candate genes using dense SNP maps. TGG 9: 189-204
20. Action Plan for 2013
WHAT WHO HOW DEADLINE
Re-sequencing of 48(96) genotypes FEM Illumina M28
Analysis of re-sequenced genotypes FEM Self-made
pipeline
M32
Development of Axiom SNP chip FEM-Affimetrix Axiom®
myDesign™
M36
(alternative to above SNP chip: GBS
genotypes for GWA in WP4)
FEM + WP4
partners
Illumina M36
Regular update of protocol for MAB FEM, EWD Lab testing M36
21. Interactions between your WP and the
rest of the project
• Interactions planned with other WPs of the
project:
– From us: WP1, WP3, WP4, WP5, WP7, WP8
– To us: WP3, WP4, WP8
• Interactions planned with other EU projects or
stakeholders of the project (none)
