1. QTL and QTL allele validation in apple Sujeet Verma, Cameron Peace, Jim Luby, Kate Evans, Susan Brown, Matt Clark, Cari Schmitz, Yingzhu Guan, Ben Orcheski, Umesh Rosyara, Nahla Bassil, Eric van de Weg 25 July 2013 ASHS Palm Desert, CA

2.  What to Target First?  QTL Validation: LG16Ma for acidity (and crispness and other traits)  QTL Allele Validation for LG16Ma (Ma-indel DNA test)  Other Jewels for Apple Outline of Presentation

3. What to Target First? - deciding which of the known QTLs is worth attempting to validate for breeding

4. Some Apple QTLs Known Previously (focusing on Mendelian Trait Loci and QTLs with candidate genes) Vf locus – scab resistance Firmness QTL – Md-Exp7 gene Md-PG1 gene for firmness Second major acidity locus 2MBAc “Apple flavor” locus Ma locus – “high” vs “medium” acidity, also major QTL for crispness, juiciness, consumer liking, others Md-MYB1 gene for red skin vs. non-red skin Md-MYB10 gene for red flesh Md-ACS1 gene for storability etc… Md-ACO1 gene for storability

5. Trait Values Breeder survey 2010 Producer survey Fall 2011- Winter 2012 Market Intermediary survey Fall 2010- Winter 2011 Household data analysis 2010; consumer survey 2012 What trait is my next breeding target? We like disease resistant apple trees! I like tasty strawberries! We like firm tart cherries that pit well! Processors RosBREED’s Socio-Economics Surveys

6. And the survey says... What Do People Really Want in Fruit of a New Apple Cultivar?

7. Some Apple QTLs Known Previously Vf locus – scab resistance Firmness QTL – Md-Exp7 gene Md-PG1 gene for firmness 2MBAc “Apple flavor” locus Ma locus – “high” vs “medium” acidity, also major QTL for crispness, juiciness, consumer liking, others Md-MYB1 gene for red skin vs non-red skin Md-MYB10 gene for red flesh Md-ACS1 gene for storability Md-ACO1 gene for storability Second major acidity locus

8. Some Apple QTLs Known Previously Ma locus – “high” vs “medium” acidity, also major QTL for crispness, juiciness, consumer liking, others

9. The Ma Locus Monogenic inheritance of apple acidity (Wellington 1924), designated as Ma locus (Visser & Verhaeg 1978) Detected as a QTL (~40% phen variance) in single F 1 populations (King et al. 2001, Leibhard et al. 2003), also QTL for crispness, juiciness, and others More recently, detected as a QTL for acidity via association mapping with SNPs (Kumar et al. 2012) = Malic acid transporter gene (Xu et al. 2012, Khan et al. 2013) Ma = Malic acid Ma locus – “high” vs “medium” acidity, also major QTL for crispness, juiciness, consumer liking, others

10. The Ma Locus Ma = Malic acid Ma locus – “high” vs “medium” acidity, also major QTL for crispness, juiciness, consumer liking, others Is this QTL, LG16Ma, detectable in U.S. breeding germplasm? Is it associated with other socio-economically valuable traits in addition to acidity? If so, how can breeders exploit it?

11. QTL Validation - confirming the LG16Ma QTL really exists in apple breeding germplasm

12. Germplasm from 3 major U.S. breeding programs -total n = 960 -all pedigree-linked -multiple families & ancestors -chosen to represent alleles of 57 important breeding parents Fruit quality standardized phenotyping for 3 years (at harvest and after 10 & 20 wks storage) Genome scanning with 8K SNP array and a nearby SSR QTL Validation Dataset WAMNNY

13. QTL Validation Analysis Use Pedigree-Based Analysis software

14. QTL Validation Results at harvest after 10 wks storage after 20 wks storage Expected LG16Ma locus position etc. Validated position of LG16Ma for acidity! Evidence for bitter pit QTL there too Some, but less, evidence for crispness/juiciness malic acid transporter gene

15. QTL Validation Results LG16Ma for acidity consistent over: - storage durations - years - families - breeding programs (second acidity QTL on LG8 also consistent) Results shown for the FlexQTL™ analysis of WSU RosBREED data

16. QTL Validation Results So, the QTL is there, but... o What functional alleles are present in breeding germplasm? o What are the effects and value of those alleles? (alone or in combination with others) o What is their occurence? - frequencies - sources (founders, important parents) And besides with genome-scan SNPs, can all this be captured with a simple DNA test?

17. QTL Allele Validation - detecting and valuing LG16Ma alleles in apple breeding germplasm

18. Functional Alleles Present With SNP and SSR haplotyping: o 11 alleles in Washington State Univ. apple breeding program germplasm o 15 more in Univ. Minnesota and Cornell Univ. breeding germplasm Are these all functionally unique?

19. Effects of Functional Alleles Ma1 Ma2 Ma3 Ma4 Ma5 ma1 ma2 ma3 ma4 ma5 Titratable Acidity (TA, mEq/L) above the season average of 0.70 (2011 at harvest, WSU seedlings, n=214) xx1 For acidity, can simplify to two types: “Ma” (high acid) and “ma” (low acid) High-acid alleles Low-acid alleles

20. Effects of Functional Alleles Crispness rating (1-5) above season average of 2.90 (2011 at harvest, WSU seedlings, n=214) Bitter pit incidence rating (1-5) below season average of 1.50 Ma1 Ma2 Ma3 Ma4 Ma5ma1 ma2 ma3 ma4 ma5xx1 (As in other graphs, high here is good) But various effects on other traits...

21. Values of Functional Alleles So, each of the 11 alleles can be +ve or -ve for effect on acidity, crispness, bitter pit incid. - often trade-offs Which ones are most desirable? Overall value of each allele can be determined as socio-economic value of its relative effect on each trait

22. (Example of Socio-Economics Results – Market Intermediary $ Values) TraitThresholdValue* * Willingness-to-pay of market intermediaries per lb of a 42 lb box Shelf life Crispness Flavor Appearance Firmness Size $0.134 $0.129 $0.128 $0.123 $0.009 $0.002 1 week not  very 3% defects 14 lb 100-count weak/mild  full/intense

23. Values of Functional Alleles Using $ values from RosBREED’s survey results of market intermediaries... Ma1 Ma2 Ma3 Ma4 Ma5 xx1 ma1 ma2 ma3 ma4 ma5 Bitter pit incidence Crispness Acidity Most valuable allele because of valuable contribution to all 3 traits In contrast, these only valuable for bitter pit (low incidence) High-acid alleles tend to be most valuable overall Total socio- economic value of allele

24. Values of Functional Alleles Strategy: recombination to get even more valuable alleles (assuming three different genes) Ma1 Ma2 Ma3 Ma4 Ma5 xx1 ma1 ma2 ma3 ma4 ma5 Bitter pit incidence Crispness Acidity Total socio- economic value of allele Can we combine best of each into a single allele?

25. Frequencies of Functional Alleles

26. ma2 ma3 ma4 ma5 ma - other Ma1 Ma2 Ma3 Ma4 Ma5 xx1 ma1 Ma - other Shown for parent pool (58 cultivars and selections) of WSU apple breeding program

27. Sources of Functional Alleles Shown for parent pool (58 cultivars and selections) of WSU apple breeding program Ma1 Ma2 Ma3 Ma4 Ma5 xx1 ma1 ma2 ma3 ma4 ma5 Ma - other ma - other Parents ( founder sources ) (Red Delicious, Golden Delicious) Arlet, Aurora, Arlet, Cameo, Enterprise, Fuji, Gala, Splendour, WA 2 (Granny Smith) (Lady Williams) Cripps Pink (Rome Beauty) WA5 (NJ90) WA7 (Duchess of Oldenburg) Honeycrisp Arlet, Cameo, Cripps Pink, Splendour (Golden Delicious) Honeycrisp (Frostbite) Enterprise (McIntosh) Braeburn (Lady Williams, Granny Smith) Aurora, Gala, Sansa, WA 2 (Red Delicious)

28. Simple Locus-Specific DNA Test All previous effects, values, frequencies were defined by 8K-array SNP haplotypes Converted to a simple PCR marker – “Ma-indel” – captured most information Can now use in high-throughput MAB Already used to screen many parents and >10,000 seedlings (WSU & UMN) in spring 2013

29. Ma-indel DNA Test: a “Jewel in the Apple Genome”

30. Other Jewels for Apple

31. Other Apple Jewels Available Now

32. The LG8A Locus LG16Ma : additivity LG8A : partial dominance Together : some epistasis  Best to consider together 7 Med- High 9 Very High 6 High Relative acidity level 8 High 5 Med- High 1 Very Low 3 Low 2 Med. 4 Med. 2011 at-harvest TA All WSU populations 9 = 0.94 ±0.15 7 = 0.67 ±0.06 5 = 0.67 ±0.14 3 = 0.53 ±0.11 1 = 0.43 ±0.09 Cat TA ±sd – as big as LG16Ma for acidity

33. The Bp13 Locus – combines well with Bp16 (Ma) Homozygous “high” for both loci Homozygous “high” for Bp16 (Ma) Homozygous “high” for Bp13 Not homozygous for either locus

34. Cover amount over apple skin surface Allele & Genotype frequencies in 192 U.S. cvsrf Rf rfrf RfRf Rfrf probability The Rf locus – skin color amount, type, hue

35. 4 6 8 10 12 14 Both genes - additive 0 2 4 6 8 10 12 14 Md-ACS1 & Md-ACO1 - the ethylene synthesis genes Change in firmness (Newtons, M2 of Mohr Digi-Test) from harvest to 20 weeks storage plus 1 week at room temperature 0 2 4 6 8 10 12 14 some softening more softening ACS:FF FF FF FN FN FN ACO:NN FN FF NN FN FF FF FN NN ACS1ACO1 allele frequencies in U.S. cultivars No N/N N/N U.S. cultivars found F=favorable N=non-favorable ACS1 ACO1

36. Md-ACS1 & Md-ACO1 www.rosbreed.org/breeding/jewel-use/apple

37. Md-ACS1 & Md-ACO1

38. Apple Jewels = QTLs & Their Alleles Validated, with DNA Tests Developed...and Used in MAB

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

40. 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