Nahla Bassil, B. Gilmore, D. Main, C. Peace, T. Mockler, L. Wilhelm, E. van de Weg, T. Davis, D. Chagne, S. Gardiner, R. Crowhurst, I. Verde, B. Sosinski, S. Scalabrini, K. Folta, V. Shulaev, P. Arus, R. Velasco, M.

Troggio, A. Cestaro, G. Fazio, J. Norelli, J. Rees and A. Iezzoni

RosBREED Deploys Genome-Wide Scans in Peach, Apple,

and Cherry

Integration of modern genomics tools with traditional breeding approaches will transform crop improvement in

Rosaceae, significantly improving profitability and sustainability of U.S. rosaceous crop industries & contribute to the increased consumption and enjoyment of these fruit,

nut and floral products.Overview Oral Presentation, Fruit Breeding and Genetics,

Monday at 12:15 pm, Cameron Peace

4 yrs$7.2M federal$7.2M matching

Amy Iezzoni, Michigan State UnivCameron Peace, WA State Univ

This project is supported by the Specialty Crop Research Initiative of USDA’s National Institute of Food and Agriculture

RosBREED - Vision

Reference Genomes Now AvailableApple, peach & diploid strawberry genome

Image courtesy of NSF

Images courtesy of www.beakandskiff.com, www.ehow.com, Jim Hancock

RosBREEDHigh Throughput SNP Genome Scans

G1

G4

G3G2

G6

G7

G8G5

≤5cM

QT

L

Markers for any trait of interest for MAB

3 posters

Peach #144Apple #145

Cherry #146

RosBREED MAB postersTues 1:15-2:00, Kona Ballroom

RosBREED

Re-sequencing of SNP Detection Panel: Illumina

Alignment to reference genome: apple, peach

SNP detection: SOAP, CLC BIO

30 57 16 8

Jasper Rees, ARC, SADavid Chagne, PFR, NZ

Ignazio Verde, ItalyPere Arus, Spain

Reference-Scaffold 1

Elberta

O’Henry

Bolinha

80 bp PE reads

Infinium Chip Development

RosBREED

SNP validation: GoldenGate

SNP Consortium Community chooses parameters for Infinium

chip: SNP Summit

Final filtering for Infinium chips

ASOs

384 SNPs480 DNA samples

Infinium Chip Development

Criteria for SNP Choice for GoldenGate Assay

RosBREEDG1

Genic-40%Intronic-20%Promoter-20%

Intergenic-20%

Equidistant SNPs

ApplePeachSweet CherryTart Cherry

144

96

9648

Elberta

O’HenryAccession-specific-20%

Bolinha

Zoomed-in-20%

RosBREED

GoldenGate Assay Validation Results

Failed 32%

Monomorphic 17%

Polymorphic 51%

Apple

Failed 26%

Monomorphic 21%

Polymorphic 53%

Peach

RosBREED

GoldenGate Assay Validation Results

Failed 40%

Monomorphic 31%

Polymorphic 29%

Sweet Cherry

Failed 27%

Monomorphic 42%

Polymorphic 31%

Tart Cherry

RosBREED

Effect of SNP Location

Exon Intron Promoter Intergenic0

5

10

15

20

25

Failed MonomorphicPolymorphic

Cindy Lawley, Illumina

RosBREED

Effect of Number of Peach Accessions Containing Sequence Data

2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 19 200

1

2

3

4

5

6

7

8

9

Failed Monomorphic Polymorphic

RosBREED

Haplotype Diversity at Zoomed-in Regions

Haplogroup 6

Haplogroup 3

Haplogroup 4Haplogroup 1

Haplogroup 5

Haplogroup 2

Yes No

160 accessions35 unique

haplotypes 6 haplogroups

Haplotype Diversity at Ma Locus

RosBREED

Infinium iSelect® HD Custom Genotyping

3,000 1 MAttempted Beadtypes

• 24 samples per chip

Sample Table

SNP Table

SNP Graph

AA AB BB

• Fully customizable• Low DNA input, 200 ng

iScan

RosBREED

Choosing SNPs for Infinium Chips

Pea

ch c

hrom

osom

e

1. Infinium II SNPs (one SNP per beadtype)2. Validated SNPs, RosCOS, candidate genes

9 K chips, publicly available

SNPSNP

SNP

RosBREED

Choosing SNPs for Infinium Chips

  Position of SNP-Peach genome

Tarts: no.

reads

Tarts: freq of rare base

(AAAB, AABC)

Tarts: skew from 1:1 (Chi2 value)

Tarts: suspected

sub-genome

SNP bases

(common-rare)

Chromosome 1 

         

SNP97 1,900,073 29 0.03 25.1 fru A-G

SNP98 9,112,138 23 0.17 5.3 av G-A

SNP99 16,224,137 12 0.17 5.3 fru C-GSNP100 23,498,747 50 0.04 42.3 av C-T

Sweet cherry (4,180), tart cherry (1,551)

RosCOS (227), candidate genes (2)

RosBREED

Peach, Cherry and Apple SNPs: Downloadable from GDR

RosBREED

Deploying 9K Infinium Chip in Peach

Failed10%

Monomorphi

c13%

Polymorphic77%

• 953 peach samples• 1400 SNP markers added to map• QTLs for bacterial spot resistance

in peach leaf (LEA) and fruit (FRU) in NC and SC, in 2008, 2009 and 2011

G1

RosBREED

Deploying 6K Infinium Chip in Cherry

No call; 217

Call; 5479

RosBREED

Deploying 9K Infinium Chip in Apple

1

3

5

7

9

11

13

15

17

0 100 200 300 400 500 600

Evaluation in Plant & Food Research dataset of 1619 accessions

Number of Polymorphic SNPs

Lin

ka

ge

Gro

up

5554 (72.2%) pol. SNPs

Chagné et al. 2011, manuscript in prep.

RosBREED

Infinium Chips will be used via Pedigree-Based Analysis to identify and validate

marker-locus-trait associations for application in breeding

RosBREED Use of SNP Genome Scans

DNA INFORMATION

SOCIO-ECONOMICS

INFORMATION

ROUTINE BREEDING

OPERATIONS

1 2 3

4

5 6

7 8

2. Choose efficient genetic screening technologies and service providers

1. Choose valuable, impactful targets

3. Adapt reported genetic tests to local genetic screening approach

4. Validate genetic tests across crop

5. Assess utility of genetic tests for breeding germplasm

7. Identify efficient seedling selection schemes

8. Trial seedling selection schemes

6. Inform parent selection and crossing

Pipeline Stages

MARKER-ASSISTED BREEDING PIPELINERosBREED’s

RosBREED

To Achieve Our Dream

Ultra-crisp tasty applessweet juicy peaches flavorful cherries luscious strawberries

Enjoyed by consumers, regularly

Consistent quality, available & affordable

Sustainably produced throughout the U.S. & worldwide

OSU

Todd Mockler

Larry Wilhelm

Chris Sullivan

Mark Dasenko

MSU

Jeff Langraff

Rutgers

David Sidote

Intuitive GenomicsDoug BryantTodd Mockler

GDR, WSUStephen Ficklin

IGA, ItalySimone Scalabrin

PFR, NZRoss Crowhurst

IlluminaMark Hansen

RosBREED

Sequencing and Bioinformatic Support

Acknowledgements

This project is supported by the Specialty Crop Research Initiative of USDA’s National Institute of Food and Agriculture

MSUAmy Iezzoni (PD)

Jim HancockDechun Wang

Cholani Weebadde

WSUCameron Peace

Dorrie MainKate Evans

Karina Gallardo Raymond Jussaume

Vicki McCracken Nnadozie Oraguzie

Mykel Taylor

CornellSusan BrownKenong Xu

Clemson Ksenija Gasic

Gregory Reighard

Texas A&MDave Byrne

Univ. of CA-DavisTom Gradziel

Carlos Crisosto 

Univ. of New Hamp.Tom Davis

Univ. of MinnesotaJim Luby

Chengyan Yue

Oregon State Univ.Alexandra Stone

Plant Research Intl, Netherlands

Eric van de WegMarco Bink

USDA-ARSNahla Bassil

Gennaro FazioChad Finn

Univ. of ArkansasJohn Clark