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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? QTL Validation: FW_G2 for Fruit Size QTL Allele Validation for FW_G2 Other Jewels for Cherry What is QTL Validation? What is QTL Allele Validation? 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. 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™) Pedigree linked germplasm Fruit weight Schneiders Rube Regina Empress Eugenie Unknown Van Stella Lambert EF Lapins Data not available JI2420 Napoleon Namati Summit Regina x Lapins Bopparder Kracher Unknown (n= 101) Sam Namati x Summit V-160140 Unknown Krupno. NY (n= 76) (n= 80) Drogana Zholtaya Valeriy Chkalov Namati x Krupno. NY x EF (n= 167) Pedimap software 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) Genome Wide SNP arrays • Genome-scanning SNP arrays developed and utilized for apple (9K), peach (9K) and cherry (6K) by international RosBREED-led efforts The statistical analyses must take advantage of the family structure in the breeding program. FlexQTL™ Statistical Software: HiDRAS: European Apple Project 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. 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 Allele Validation Mendelian/major trait loci (MTLs) Genes with allelic variation in available germplasm that explain/predict most or all of the phenotypic variation Quantitative trait loci (QTLs) …that explain/predict some of the phenotypic variation Useful for enriching for superior alleles Still valuable!! mm trait level MM, Mm frequency frequency Definitive qq trait level QQ, Qq QTL Validation - confirming the FW_G2 QTL really exists in cherry breeding germplasm 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 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 Validation: Sweet Cherry Example Utility assessment on fruiting seedlings EmpressEugenie F_Van Van EmperiorFrancis Lambert Stella Newstar Sweetheart PopSw-17 Pop-SwxSw-4 22 populations (219 seedlings), 0 cultivars J12420 Beaulieu Lapins PopSwxAm-11 F_Bing Kordia Bing Selah PopSwxCh-3 PopSwxPM-4 EarlyBurlat PMR-1 PopLaxAm-26 PopSwxRe-51 Regina PopLaxCh-41 PopSwxTi-5 Dzherlo Linda PopGoxDz-10 Rube Chelan PopLaxLa-4 Gold PopKo-8 Schneiders Ambrunes Kiona Napoleon PopSwxKi-1 PopLi-3 PopLaxRe-4 Pop-SexSe-7 PopKa-2 Rainier Tieton PopBi-2 Cashmere Pop-LaxTi-11 PopSexPM-1 Katalin PopRaxLa-1 PopSexAm-1 P8-79 PopLaxPM-3 PopSexVa-6 PopSe-16 Simple Validation and Functional Genotype Effects for the Sweet Cherry Success Story Phenotypic data collection www.rosbreed.org/resources/fruit-evaluation Simple Validation and Functional Genotype Effects for the Sweet Cherry Success Story Ran the DNA tests (2 flanking SSRs) 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 Marker polymorphism Sweet Cherry FW_G2 Example QTL FW_G2 Markers CPSCT038 (SSR) BPPCT034 (SSR) No. Alleles 7 4 Alleles in important breeding parents BPPCT034 223 common Lambert 225 235 rare NY54 Cristobalina Windsor common Bing 237 250 rare Glacier Tieton Kiona Burlat rare Schmidt Ulster 255 common Napoleon 257 rare Schneiders 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 QTL Allele Validation - detecting and valuing FW_G2 alleles in cherry breeding germplasm Inheritance of functional alleles in pedigreelinked germplasm Regina × Lapins progeny classes Mean fruit size for each progeny class (g) Simple Validation and Functional Genotype Effects for the Sweet Cherry Success Story Haplotyped and diplotyped every individual Validation: Sweet Cherry Example Alleles Genotypes Mean fruit wieght (g) 10 9 a a 8 ab b bc b b abc b abc 34 25 abc ab a c 7 bc 6 5 4 77 92 108 11 2 41 15 4 5 2 6 39 4 225 235 223 255 237 BPPCT034 allele 225 223 223 223 235 235 235 225 237 255 235 235 223 255 255 235 237 255 255 255 BPPCT034 genotype Number of seedlings Simple Validation and Functional Genotype Effects for the Sweet Cherry Success Story Calculated functional genotype effects probability BL BN AB AA AL fruit size probability 0 g BN AA AB AL BL firmness g/mm Functional Allele Distribution AB Empress Eugenie Napoleon AC Lambert BO AB Emperor Francis JI2420 AH ? AB Van Schmidt Black Republican ? AL AO Amim Regina Windsor MIM17 AA AB AC Sam Bing Gil-Peck BN NY54 AE AB AH Early Burlat Stella AB Ulster Beaulieu AA Rainier Summit ? Glacier AE Tieton AC PC7147-4 PC7146-11 Vic Chinook Kiona PMR-1 P8-79 Brooks AC AA AC Selah GG AA AB’ AF AA DD Newstar AE AF Benton Chelan Cashmere AB What crosses to make? AA AE AA EE BB CC AB AP Cowiche Lapins AB Sweetheart maternal parent pollen parent “How To” for Rosaceae Breeders Cherry fruit size Functional Alleles for FW_G2 in the Michigan Sour Cherry Breeding Program Pandy38 UjfehertoiF NY-6935 Montmorency RhSchatten ErdiBotermo M172 EnglaiseTimp Surefire 25-02-29 25-14-20 RSxET UFxSurfire 25x25 M172x25 Montx25 N = 79 N = 36 N = 72 N = 57 N = 22 Phenotypic data – standardized phenotyping • Standardized phenotyping protocols can be found at http://www.rosbreed.org/resources/fruit-evaluation 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 overestimation of the number of functional SNP alleles in the sour cherry breeding materials Gbrowse view of the peach sequence for the G2 fruit size QTL region 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 Breeding-Friendly DNA Markers Used 3 SSRs • CPSCT038 • SSR linked to the Cell Number Regulator candidate gene: PavCNR12 & PceCNR12 • BPPCT034 7 sour cherry G2 SSR-flanked QTL haplotypes hypothesized instead of 17 based on new SSR data RB_S_2_14926622 CPSCT038 RB_S_2_15084429 RB_S_2_15127760 RB_S_2_15129278 RB_S_2_15162260 RB_S_2_15172649 RB_S_2_15337787 RB_S_2_15372418 RB_S_2_15492297 RB_S_2_15598480 CNR RB_S_2_15658996 LG2+18K_SSR RB_S_2_15747822 RB_S_2_15778222 RB_2_15846482 RB_T_2_15863936 RB_S_2_15873315 RB_S_2_15873418 RB_S_2_15894385 RB_S_2_15894441 RB_T_2_16005866 RB_T_2_16111179 RB_S_2_16118423 RB_S_2_16142700 RB_S_2_16229065 BPPCT034 d A 204 A A B B B B B B B b A 185 A B A A A A B B A l A 185 A B A A A A B B A e A 190 A B A A A A B B A h A 190 A B A A A A B B A o A 190 A B A A A B B B A k A 192 A B A A A B B B A m A null A B A A A B B B A a A 185 A B A A A A B B A q A 192 A B A A A A B B A c A 192 A B A B B A B A B g A 192 B A B B B A B A B p A 190 B A B B B B B A B j A 192 B A B B B B B A B n f i A B A 192 190 null B B A A A A B B B B B B B B B B B B B A B A B B B B B A B B B B 235 B A A A A 218 B A A B A B A A A B 241 228 A A A A A 206 A A A A A 206 A A A A A 206 A A A A A 206 B A A A A 210 A A A A A A A A A B B B B B B B A B B B B B A B A A A A A A B A A B B B B B B B 235 237 237 225 237 237 255 228 7 sour cherry CNR alleles (bp) – 2 (250)* – 4 (210) – 5 (212) B A A B B B B B A A B B B B B B B 250 210 210 210 210 210 210 212 235 239 239 239 225 225 225 228 228 – 6 (235) A A A A A A A A A A A A B B A B A A B B A B B B B B B A A A A A A A B B B B B B B B B B B B B B B B B – 7 (239) B A A B A A A A A A B B B B B B B B A A B A A A A A A B B B B B B B – 8 (225) B A A B A A A A A A B B B A B B B B A A A A A A A A A B B B A B B A A B B A B B B B B B A A A A A A A – 9 (228) * Same as in sweet cherry based on other marker data Sour cherry - putative PavCNR12 & PcrCNR12 alleles Mean fruit weights based on the presence or absence of putative PavCNR12 & PcrCNR12 alleles (n=274) 2/no 2 8/no 8 7+8/no 7 or 8 Number of progeny 128/146 139/135 56/56 Mean fruit weight (g) 5.64/5.30 5.11/5.81 4.66/5.74 0.0700 0.0002 0.0004 P-value 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 Other Jewels for Cherry Other cherry “Jewels” available now! JUN Bloom Time JUL Maturity Date Self-fertility Flesh Color Disease Resistance Fruit Size Firmness Acidity Conclusion RosBREED has and continues to provide DNA tests for valuable traits that have been challenging to plant breeders’ efficiency 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! Acknowledgements This project is supported by the Specialty Crop Research Initiative of USDA’s National Institute of Food and Agriculture MSU Amy Iezzoni (PD) Jim Hancock Dechun Wang Cholani Weebadde Univ. of Arkansas John Clark WSU Cameron Peace Dorrie Main Kate Evans Univ. of Minnesota Karina Gallardo Jim Luby Vicki McCracken Chengyan Yue Nnadozie Oraguzie Former WSU Oregon State Univ. Raymond Jussaume Alexandra Stone Mykel Taylor Cornell Susan Brown Kenong Xu Clemson Ksenija Gasic Gregory Reighard Texas A&M Dave Byrne USDA-ARS Nahla Bassil Gennaro Fazio Univ. of CA-Davis Chad Finn Tom Gradziel Carlos Crisosto Plant Research Intl, Netherlands Univ. of New Hamp. Eric van de Weg Tom Davis Marco Bink