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QTL and QTL allele validation in cherry

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. Outline of Presentation. What is QTL Validation? What is QTL allele Validation?

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QTL and QTL allele validation in cherry

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  1. QTL and QTL allele validation in cherry Amy IezzoniCameron Peace, Audrey Sebolt, NnadozieOraguzie, UmeshRosyara, Travis Stegmeir • 25 July 2013 • ASHS Palm Desert, CA

  2. Outline of Presentation • 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

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

  7. Standardized Phenotyping • Reference Germplasm Sets • Standardized phenotypingat 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) RosBREED

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

  9. HiDRAS: European Apple Project The statistical analyses must take advantage of the family structure in the breeding program. 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/majorQuantitative trait loci (MTLs) trait loci (QTLs) …that explain/predict some of the phenotypic variation Genes with allelic variation in available germplasmthat explain/predict most or all of the phenotypic variation Useful for enriching for superior alleles Still valuable!! Definitive MM, Mm QQ, Qq mm qq frequency frequency trait level trait level

  12. QTL Validation - confirming the FW_G2 QTL really existsin 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. Validation: Sweet Cherry Example Utility assessment on fruiting seedlings 22 populations (219 seedlings), 0 cultivars

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

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

  20. Alleles in important breeding parents BPPCT034 223 225 235 237 250 255 257 common Lambert common Bing common Napoleon rare NY54 Cristobalina Windsor rare Glacier Tieton Kiona Burlat rare Schmidt Ulster rare Schneiders

  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 allelesin 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. Validation: Sweet Cherry Example Alleles Genotypes 10 a 9 (g) ab a a abc abc abc b b 8 wieght bc b ab b c 7 bc 6 Mean fruit 5 Number of 4 77 92 108 11 2 41 15 34 25 4 5 2 6 39 seedlings 4 237 255 225 235 223 225 223 223 223 235 235 235 225 255 237 255 255 237 255 255 255 235 235 223 235 BPPCT034 allele BPPCT034 genotype

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

  27. Functional Allele Distribution AB BO AB AL AO Amim Emperor Francis Empress Eugenie Black Republican ? Napoleon ? Regina Windsor MIM17 AB AC AC AA AB AH BN Van Lambert JI2420 Gil-Peck Sam Bing Schmidt NY54 What crosses to make? AH AE AB AB AA AF AB Ulster Early Burlat AA Chinook Beaulieu Rainier Vic Stella PMR-1 Summit ? AA P8-79 Brooks AC AE AE Benton PC7147-4 AC Glacier PC7146-11 AA GG Tieton AA AB’ AA AF AC Chelan Selah Cashmere DD EE BB CC Newstar AB AE Lapins AP AB Kiona Cowiche Sweetheart maternal parent pollen parent

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

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

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

  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 & PcrCNR12alleles Mean fruit weights based on the presence or absence of putative PavCNR12& PcrCNR12alleles (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! JUN JUL Bloom Time Maturity Date Disease Resistance Self-fertility Flesh Color 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. Cornell Susan Brown KenongXu Clemson KsenijaGasic Gregory Reighard Texas A&M Dave Byrne Univ. of CA-Davis Tom Gradziel Carlos Crisosto Univ. of New Hamp. Tom Davis MSU Amy Iezzoni (PD) Jim Hancock Dechun Wang CholaniWeebadde WSU Cameron Peace Dorrie Main Kate Evans Karina Gallardo Vicki McCracken Nnadozie Oraguzie Former WSU Raymond Jussaume Mykel Taylor Univ. of Arkansas John Clark USDA-ARS Nahla Bassil Gennaro Fazio Chad Finn Univ. of Minnesota Jim Luby Chengyan Yue Oregon State Univ. Alexandra Stone Plant Research Intl, Netherlands Eric van de Weg Marco Bink

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