1. QTL and QTL allele validation in cherry Amy Iezzoni Cameron Peace, Audrey Sebolt, Nnadozie Oraguzie, Umesh Rosyara, Travis Stegmeir 25 July 2013 ASHS Palm Desert, CA

2.  What is QTL Validation? What is QTL allele Validation?  QTL Validation: FW_G2 for Fruit Size  QTL Allele Validation for FW_G2  Other Jewels for Cherry Outline of Presentation

3. What is QTL Validation? What is QTL Allele Validation?

4. Definitions QTL validation is confirming that the QTL really exists in breeding germplasm using breeding-friendly DNA tests. QTL allele validation is detecting and determining the relative values of the alleles present in breeding germplasm detected by the breeding-friendly DNA tests.

5. What is Required for QTL Validation? Segregating germplasm derived from important breeding parents, representative of a program Breeding-relevant phenotypic data Genotypic data for breeding-friendly marker(s) at the QTL region QTL characterization software for multiple generations & various family sizes (FlexQTL™)

6. Pedigree linked germplasm Schneiders Rube Empress Eugenie Unknown Lambert EF Napoleon Bopparder Kracher Unknown V-160140 Unknown Drogana Zholtaya Valeriy Chkalov NY Regina Van JI2420 Namati Sam Krupno. NY x EF Stella Summit Namati x Krupno. Lapins Namati x Summit Regina x Lapins (n= 101) (n= 76) (n= 80) (n= 167) Fruit weight Data not available Pedimap software

7. Standardized Phenotyping Reference Germplasm Sets Standardized phenotyping at multiple locations, esp. for fruit quality (www.rosbreed.org) Evaluations done for 2 – 3 years Available at Genome Database for Rosaceae (www.rosaceae.org/breeders_toolbox)

8. Genome-scanning SNP arrays developed and utilized for apple (9K), peach (9K) and cherry (6K) by international RosBREED-led efforts Genome Wide SNP arrays

9. The statistical analyses must take advantage of the family structure in the breeding program. HiDRAS: European Apple Project FlexQTL™ Statistical Software: Capabilities: Identify and quantify QTLs in different genetic backgrounds (~allele mining) Strategy: Ties together many segregating crosses through the common ancestors in the pedigree utilizing the Identity by Descent concept.

10. What’s Required for QTL Allele Validation? A closer examination of the validated QTL in the breeding germplasm, to quantify, describe, and visualize: -number of alleles present -effect of each allele (or genotype, preferably) -frequency of alleles -origins of alleles -distribution of alleles in important breeding parents and other potential parents

11. Allele Validation Mendelian/major Quantitative trait loci (MTLs) trait loci (QTLs) Genes with allelic variation in available germplasm that explain/predict most or all of the phenotypic variation …that explain/predict some of the phenotypic variation frequency trait level mm MM, Mm frequency trait level qq QQ, Qq Useful for enriching for superior alleles Still valuable!! Definitive

12. QTL Validation - confirming the FW_G2 QTL really exists in cherry breeding germplasm

13. Example of a Valuable QTL Discovered: FW_G2 for fruit size (Zhang et al. 2010) Trait has value to stakeholders QTL explains a significant amount of trait variation Achieving the desired phenotype with breeding is very difficult

14. Breeding-Friendly DNA Markers Used 2 SSRs CPSCT038 BPPCT034 These 2 SSRs defined 3 functional alleles for FW_G2 in the bi-parental cross where the QTL was discovered

15. Utility assessment on fruiting seedlings 22 populations (219 seedlings), 0 cultivars Validation: Sweet Cherry Example

16. Simple Validation and Functional Genotype Effects for the Sweet Cherry Success Story Phenotypic data collection www.rosbreed.org/resources/fruit-evaluation

17. Simple Validation and Functional Genotype Effects for the Sweet Cherry Success Story Ran the DNA tests (2 flanking SSRs)

18. Zhang G, Sebolt AM, Sooriyapathirana SS, Wang D, Bink MCAM, Olmstead JW, Iezzoni AF (2009). Tree Genetics & Genomes 6:1614-1642 The previously identified G2 fruit weight QTL was significant

19. Marker polymorphism Sweet Cherry FW_G2 Example BPPCT034 CPSCT038 FW_G2 4 7 QTL Markers No. Alleles (SSR)

20. Alleles in important breeding parents BPPCT034 223225235237255257250 common Lambert rare NY54 Cristobalina Windsor common Bing rare Glacier Tieton Kiona Burlat common Napoleon rare Schneiders rare Schmidt Ulster

21. QTL Validation - Summary The G2 QTL for fruit size was identified in sweet cherry breeding germplasm The DNA test using the flanking SSR markers was still associated with the trait Origin and distribution of alleles in important breeding parents were determined

22. QTL Allele Validation - detecting and valuing FW_G2 alleles in cherry breeding germplasm

23. Inheritance of functional alleles in pedigree- linked germplasm Regina × Lapins progeny classes Mean fruit size for each progeny class (g)

24. Simple Validation and Functional Genotype Effects for the Sweet Cherry Success Story Haplotyped and diplotyped every individual

25. Genotypes 4 5 6 7 8 9 10 Mean fruit wieght (g) 225223235 255237 1110892774 a a b bab BPPCT034 allele 225 235 223 235 223 255 235 255 235 237 235 225 255 237 255 396254253415412 a ab abc b b bc c BPPCT034 genotype Number of seedlings Alleles Validation: Sweet Cherry Example

26. 0 Simple Validation and Functional Genotype Effects for the Sweet Cherry Success Story Calculated functional genotype effects fruit size BNABAA AL BL firmness BN AA AB ALBL probability g g/mm

27. Functional Allele Distribution Empress Eugenie ? ? Regina ? Beaulieu Emperor Francis JI2420 Schmidt Early Burlat Ulster Lambert Sam Stella PMR-1 Glacier Tieton Cashmere PC7147-4 PC7146-11 Chelan GG Kiona Cowiche Napoleon Black Republican Van Bing Rainier Chinook Vic Summit Lapins Newstar Brooks DDEEBBCC Sweetheart P8-79 Selah Benton WindsorMIM17 maternal parent pollen parent ABAB ABAB ABAB ABAB ABAB AA ABAB AFAF AB’AB’ ACAC ABAB ACAC ABAB ABAB AHAH AHAH ACAC APAP AEAE AEAE AEAE AEAE ACAC AFAF ACAC BOBO ALALAOAO A mim Gil-Peck NY54 BNBN What crosses to make?

28. Cherry fruit size “How To” for Rosaceae Breeders

29. Functional Alleles for FW_G2 in the Michigan Sour Cherry Breeding Program N = 72N = 57N = 79 N = 36 N = 22

30. Phenotypic data – standardized phenotyping Standardized phenotyping protocols can be found at http://www.rosbreed.org/resources/fruit-evaluation

31. FW_G2 in tetraploid sour cherry 17 alleles for the G2 QTL region were identified in sour cherry using SNP markers 17 alleles likely an over- estimation of the number of functional SNP alleles in the sour cherry breeding materials

32. Gbrowse view of the peach sequence for the G2 fruit size QTL region

33. Tomato: fw2.2 Tomato and cherry fruit are both enlarged ovaries A fruit size gene was discovered in tomato that is a regulator of cell division

34. Breeding-Friendly DNA Markers Used 3 SSRs CPSCT038 SSR linked to the Cell Number Regulator candidate gene: PavCNR12 & PceCNR12 BPPCT034

35. 7 sour cherry G2 SSR-flanked QTL haplotypes hypothesized instead of 17 based on new SSR data 7 sour cherry CNR alleles (bp) – 2 (250)* – 4 (210) – 5 (212) – 6 (235) – 7 (239) – 8 (225) – 9 (228) * Same as in sweet cherry based on other marker data

36. Sour cherry - putative PavCNR12 & PcrCNR12 alleles 2/no 28/no 87+8/no 7 or 8 Number of progeny128/146139/13556/56 Mean fruit weight (g)5.64/5.305.11/5.814.66/5.74 P-value0.07000.00020.0004 Mean fruit weights based on the presence or absence of putative PavCNR12 & PcrCNR12 alleles (n=274)

37. FW-G2 exists in sweet cherry and sour cherry breeding germplasm! In sweet cherry, 9 ancestral haplotypes for the G2 region were identified In sour cherry, 8 ancestral haplotypes for the G2 region were identified

38. Other Jewels for Cherry

39. Other cherry “Jewels” available now! JUNJUL Bloom TimeMaturity Date Self-fertility Flesh Color Disease Resistance Fruit Size Firmness Acidity

40. Conclusion RosBREED has and continues to provide DNA tests for valuable traits that have been challenging to plant breeders’ efficiency

41. Sour cherry breeding program Prior to RosBREED, I had “no clue” about the inheritance of any fruit quality or disease resistance trait in sour cherry. Now I not only have an understanding of trait inheritance, but I have DNA markers for parent selection, cross design and seedling selection. The end result is increased breeding efficiency!

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

43. MSU Amy Iezzoni (PD) Jim Hancock Dechun Wang Cholani Weebadde WSU Cameron Peace Dorrie Main Kate Evans Karina Gallardo Vicki McCracken Nnadozie Oraguzie Former WSU Raymond Jussaume Mykel Taylor Cornell Susan Brown Kenong Xu Clemson Ksenija Gasic Gregory Reighard Texas A&M Dave Byrne Univ. of CA-Davis Tom Gradziel Carlos Crisosto Univ. of New Hamp. Tom Davis Univ. of Minnesota Jim Luby Chengyan Yue Oregon State Univ. Alexandra Stone Plant Research Intl, Netherlands Eric van de Weg Marco Bink USDA-ARS Nahla Bassil Gennaro Fazio Chad Finn Univ. of Arkansas John Clark