1. RosBREED: Using DNA Markers as a Strategy to Assure Plant Identity Nahla Bassil, Cameron Peace, Umesh Rosyara, Jim Luby and Amy Iezzoni

2. Take Home Messages  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!

3. Outline of Presentation  The RosBREED Project  DNA Diagnostics in Plants  Case Studies: Identity Confirmation  Preliminary Survey of U.S. Service Providers

4. The RosBREED Project

5. 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

6. Centered on breeders & breeding programs The RosBREED Project Project Director, Amy Iezzoni -breeder (tart cherry) -geneticist -genomicist -leader

7. 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

8. DNA-trait association discovery (looks promising...) MAB Pipelining (polishing...) Breeding (assembling into masterpieces) “Jewels in the Genome” - discovering, polishing, applying

9. 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 Committee Cameron Peace Nahla Bassil Gennaro Fazio Jim Luby Dorrie Main Jim McFerson Eric van de Weg Cholani Weebadde Chengyan Yue Project Director Amy Iezzoni RosBREED’s Management WSU U Minn Cornell U UC Davis Clemson U U Arkansas Texas A&M MSU UNH USDA-ARS Corvallis WSU MSU Rosaceae Demonstration Breeding Programs

11. DNA Diagnostics in Plants science.howstuffworks.com

12. Can use any living plant tissue DNA Diagnostics

13. 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

14. uses variations in DNA among individuals at Simple Sequence Repeats (SSRs) DNA Fingerprinting

15. What are SSRs? ♀♂♀♂ ATTGTATTACAGG AGG AGG AGG AGG AGG AGGGAAATTGCAG (AGG/TCC) 7 5’-TAACATAATG-3’ Forward Primer (P1)P2 TAACATAATGTCC 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

16. 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 9,000 Peach 9,000 Cherry 6,000 Strawberry 90,000 (ordering this month)

17. Other DNA marker detection techniques/ methods DNA Diagnostics Steps 1. DNA extraction 2. DNA amplification (PCR) 3. Size separation (capillary electrophoresis) 4. Data analysis

18. Case Studies: Identity Confirmation

19. 299 apple cultivars and selections 37 breeder populations: 759 U.S. Apple Breeding Germplasm 2 SSR markers CH05c06 & Hi04e04

20. ? Apple Parentage Verification ?

21. 47 populations (650 individuals), cultivars, selections, wild accessions (380) U.S. Strawberry Breeding Germplasm ParentRH50 ARSFL007Populations Fort Laramie1 149$$161164170182 216 218221 246254 MSU_9-8; 9-9; 9-10; 9-11 Fort Laramie2 $$ 161 170140 216 221232246 MSU_9-1; 18; ORUS_3314, 3315, 3316 Tillamook1149$$161164176182 212 216212221221b246 ORUS_3305, 3304, 3321 Tillamook2149$$ 164176170 212 216 221 246 ORUS_3324, 3325 Puget Reliance1$$ 161164140176 212 216221b221246 ORUS_3323, 3326, 3315, 3317 Puget Reliance2$$ 164140176 212 216221 246260 ORUS_3318 FRA 1702-1 $$ 155161170$$ 212216 221 246256 MSU_9-1, 4, 5, 12 FRA 1702-2 179$$161 140$$ 220 210218221$$254258 ORUS_3277, 3304, 3306, 3317

22. Mixed pollen for ORUS_2427- eliminate 3 populations 36 progeny have incorrect father (outcrosses) Strawberry Parentage Verification

23. Preliminary Survey of U.S. Service Providers

24. 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

25. Questionnaire Results: Receiving Tissue 1.Tissue Type, (leaf, any) Format (fresh, lyophilized, frozen) Min (1, 24, 96), no max 2.Collection Supplies (possible NOT encouraged) Labeling (samples and excel) 3.Shipping Standard if lyophilized Overnight if fresh/frozen-keep frozen

26. 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 obtained Shelf life (3 m – 10 yrs) Quantity (10-500 ng/µL) 4.Cost ($2-35 per sample depending on number)

27. Questionnaire Results: Test Techniques 1.Platform 2. Marker type (SSR, SNP [single, multiple], PCR- based) PAGE Capillary Electrophoresis HRM (GAAG) 12.. CTA[A/C]GTA GAT[T/G]CAT SNP

28. 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 humans http://ars.els-cdn.com/content/image/1-s2.0-S0160932703000681-gr4.jpg

29. Test Jobs: Total Cost Markers2x SSR1x SCAR2x SNP (  SSR) 24x SNP Company Samples102010192 12,0802,3122,20825,000 2130230350 (1,000)5,213 33,140 48003601,55529,520 557439516510,216 61972046752,980

30. Test Jobs: Sample Cost Company2x SSR 1x SCAR2x SNP  SSR 24x SNP1 SNP 1208.0115.6220.8128.25.3 213.011.53.5 (10.0)26.71.1 316.10.7 480.018.0155.5151.46.3 557.419.816.552.42.2 619.710.267.515.30.6

31. 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…

32. Take Home Messages  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!

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

34. Questions? www.rosbreed.org