WP6

Results achieved since the beginning of the project and plans for 2013

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....)

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)

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.

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

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)

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

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

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

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/.

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

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

Disequilibrium matrix for polymorphic sites

Genotyping by Sequencing

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

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

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

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)

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

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

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)