RosBREED: Using DNA Markers as a Strategy to

Assure Plant Identity 

Nahla Bassil, Cameron Peace, Umesh Rosyara, Jim Luby and Amy Iezzoni

Take Home Messages

RosBREED

DNA tests can give us lots of useful info

May be “fingerprinting” or “trait-predictive”

Different techniques, always advancing

Commercial services are available for your DNA diagnostics needs!

Outline of Presentation

RosBREED

The RosBREED Project

DNA Diagnostics in Plants

Case Studies: Identity Confirmation

Preliminary Survey of U.S. Service Providers

The RosBREED

Project

RosBREED

• PI: Amy Iezzoni, Michigan State University

• 4 years, Sep 2009 – Aug 2013

• Funded by SCRI(USDA-NIFA’s Specialty Crop Research Initiative)

• $14.4 M ($7.2 M SCRI, $7.2 M in-kind Partners)

• Collaboration U.S.-wide and international

The RosBREED Project

• Centered on breeders & breeding programs

The RosBREED Project

Project Director,Amy Iezzoni

-breeder (tart cherry)-geneticist-genomicist-leader

RosBREED

1) Enhance new cultivar adoption, enlarge market potential, increase consumption= socio-economics knowledge of trait values

2) Establish sustainable infrastructure for MAB in Rosaceae= genome scanning capability, new QTLs (“jewels”)

3) Integrate socio-econ and genomics resources into breeding= pedigree-based breeding information management system

4) Conduct MAB in demo breeding programs, fruit quality focus= MAB implementation

5) Enhance sustainability of cultivar development= tech transfer to more breeders & engage other stakeholders

RosBREED’s Objectives

DNA-trait association discovery

(looks promising...)

MAB Pipelining(polishing...)

Breeding(assembling into masterpieces)

RosBREED

“Jewels in the Genome”- discovering, polishing, applying

Project Assistant

Extension Team Leader: Cholani Weebadde

Genomics Team Leader: Dorrie Main

Genotyping Team Leader: Nahla Bassil

BIMS Team Leader: Gennaro Fazio

Socio- Economics Team

Leader: Chengyan Yue

MAB Pipeline Team Leader

Cameron Peace

Pedigree- Based Analysis Team Leader: Eric van de Weg

Breeding Team Leader

Jim Luby

Apple· Jim Luby· Susan Brown· Kate Evans

Strawberry· Chad Finn· Jim Hancock· Tom Davis

Peach· Ksenija Gasic· John Clark· Dave Byrne· Tom Gradziel

Cherry· Amy Iezzoni· Nnadozie Oraguzie

Executive CommitteeCameron Peace Nahla BassilGennaro FazioJim Luby Dorrie MainJim McFersonEric van de WegCholani WeebaddeChengyan Yue

Project DirectorAmy Iezzoni

RosBREED’s Management

WSU

U Minn

Cornell U

UC Davis

Clemson U

U Arkansas

Texas A&M

MSU

UNHUSDA-ARS

Corvallis

WSU MSU

Rosaceae

Demonstration Breeding Programs

Demonstration Breeding Programs

RosBREED

DNA Diagnostics

in Plants

science.howstuffworks.com

RosBREED

Can use any living plant tissue

DNA Diagnostics

RosBREED

1. DNA Fingerprinting- Identity confirmation- Parentage verification- Intellectual Property protection

2. Performance-Predictive DNA Tests- Efficient parental combinations- Selection of superior seedlings- Confidence in new cultivar attributes

DNA Diagnostics

any marker

trait locus

markers

RosBREED

uses variations in DNA among individuals at

Simple Sequence Repeats (SSRs)

DNA Fingerprinting

What are SSRs?

♀

♂

ATTGTATTACAGG AGG AGG AGG AGG AGG AGGGAAATTGCAG (AGG/TCC)7

5’-TAACATAATG-3’ Forward Primer (P1) P2TAACATAATGTCC TCC TCC TCC TCC TCC TCCCTTTAACGTC

ATTGTATTACAGG AGG AGG AGG AGG AGG --- GAAATTGCAG (AGG/TCC)6

P1 Reverse Primer (P2) 3’-GAAATTGCAG-5’ TAACATAATGTCC TCC TCC TCC TCC TCC --- CTTTAACGTC

Genotype: (AGG)7 x (AGG)6

♀ ♂Size: 133 bp 130 bp

Progeny: (AGG)7/(AGG)6

Size: 133/130

Thermocycler for Polymerase Chain Reaction (PCR)

Mendelian inheritance

Other DNA Markers: SNPs

Single Nucleotide Polymorphisms (SNPs)‘Genetic variation in a DNA sequence that occurs when a single nucleotide in a genome is altered’ (Webster’s dictionary)

• Most abundant type of variation

• Many detection methods, advances

• Can detect 1-1000s at same time (chips)Apple 9,000Peach 9,000

Cherry 6,000 Strawberry 90,000

(ordering this month)

Other DNA marker detection techniques/methods

DNA Diagnostics Steps

RosBREED

1. DNA extraction

2. DNA amplification

(PCR)3. Size separation

(capillary electrophoresis)

4. Data analysis

Case Studies: Identity

Confirmation

RosBREED

• 299 apple cultivars and selections• 37 breeder populations: 759

U.S. Apple Breeding Germplasm

Incorrect fa-ther

Incorrect mother

Correct parentage

0

200

400

600

800

1000

1200

Apple Identity Confirmation

Nu

mb

er

of

Ind

ivid

ua

ls

2 SSR markers

CH05c06

& Hi04e04

WHAT DO I SEE ?

?

Apple Parentage Verification

?

RosBREED

• 47 populations (650 individuals), cultivars, selections, wild accessions (380)

U.S. Strawberry Breeding Germplasm

Parent RH50   ARSFL007 Populations

Fort Laramie1149 $$ 161 164 170 182   216 216 216 218 221 221 246 254

MSU_9-8; 9-9; 9-10; 9-11

Fort Laramie2$$ $$ 161 161 170 140   216 216 216 216 221 232 246 246

MSU_9-1; 18; ORUS_3314, 3315, 3316

Tillamook1 149 $$ 161 164 176 182   212 212 216 212 221 221b 246 246ORUS_3305, 3304, 3321

Tillamook2 149 $$ $$ 164 176 170   212 212 216 216 221 221 246 246 ORUS_3324, 3325

Puget Reliance1 $$ $$ 161 164 140 176   212 212 212 216 221b 221 246 246ORUS_3323, 3326, 3315, 3317

Puget Reliance2 $$ $$ $$ 164 140 176   212 212 212 216 221 221 246 260 ORUS_3318

FRA 1702-1 $$ $$ 155 161 170 $$   212 216 216 216 221 221 246 256 MSU_9-1, 4, 5, 12

FRA 1702-2179 $$ 161 161 140 $$   220 220 210 218 221 $$ 254 258

ORUS_3277, 3304, 3306, 3317

RosBREED

• Mixed pollen for ORUS_2427- eliminate 3 populations

• 36 progeny have incorrect father (outcrosses)

Strawberry Parentage Verification

Preliminary Surveyof U.S.

Service Providers

RosBREED Experiences

with DNA Diagnostics Service Providers

1. Identified 6 U.S. Service Providers

2. Surveyed services: Questionnaire (5 replies)

3. Surveying services: Test jobs

Questionnaire Results: Receiving Tissue

1. TissueType, (leaf, any)Format (fresh, lyophilized, frozen)Min (1, 24, 96), no max

2. CollectionSupplies (possible NOT encouraged)Labeling (samples and excel)

3. Shipping

Standard if lyophilizedOvernight if fresh/frozen-keep frozen

Questionnaire Results: Extracting DNA

1. Plant tissue preferred Type (leaf preferred)Amount (2-3 leaves; 10-500 mg)Min (1, 12, 24, 96), no max

2. Extraction method (in house, bead-based)

3. DNA obtainedShelf life (3 m – 10 yrs)Quantity (10-500 ng/µL)

4. Cost ($2-35 per sample depending on number)

Questionnaire Results: Test Techniques1. Platform

2. Marker type (SSR, SNP [single, multiple], PCR-based)

PAGE

CapillaryElectrophoresis

HRM

(GAAG)12

.

.

CTA[A/C]GTA

GAT[T/G]CAT

SNP

Questionnaire Results: Costs &Timelines

3. Fingerprinting $2.2/sample-15 marker in humans$8.9, 60, 80, depends on crop

4. Marker development (inquire on cost)

5. Genetic marker ($1-2 per sample; Inquire on volume for discount)

6. Timeline: 1-4 weeks from sample submission

9-STR profile in humanshttp://ars.els-cdn.com/content/image/1-s2.0-S0160932703000681-gr4.jpg

Test Jobs: Total Cost

Markers 2x SSR 1x SCAR 2x SNP(SSR)

24x SNP

Company Samples 10 20 10 192

1 2,080 2,312 2,208 25,000

2 130 230 350 (1,000) 5,213

3 3,140

4 800 360 1,555 29,520

5 574 395 165 10,216

6 197 204 675 2,980

Test Jobs: Sample Cost

Company 2x SSR

1x SCAR 2x SNPSSR

24x SNP 1 SNP

1 208.0 115.6 220.8 128.2 5.32 13.0 11.5 3.5 (10.0) 26.7 1.13 16.1 0.74 80.0 18.0 155.5 151.4 6.35 57.4 19.8 16.5 52.4 2.26 19.7 10.2 67.5 15.3 0.6

Test Jobs: Preliminary Results

• Commercial DNA diagnostics services are available

• Technologies are improving and getting cheaper

• Important to conduct a pilot test and include known controls

• Reliability and costs are variable: Beware of hidden costs (management fee, minimum sample no.)

• User feedback is useful

• Commercial services need improvement…

Take Home Messages

RosBREED

DNA tests can give us lots of useful info

May be “fingerprinting” or “trait-predictive”

Different techniques, always advancing

Commercial services are available for your DNA diagnostics needs!

Acknowledgements

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

Questions?

www.rosbreed.org