Integrating Molecular Tools and Conventional Approaches in the OSU Hazelnut Breeding Program Shawn A. Mehlenbacher Dept. of Horticulture Oregon State University Workshop on the Genetics of Host-Parasite Interactions in Forestry Eugene, OR 31 July – 5 August 2011 Integrating Molecular Tools and Conventional Approaches in the OSU Hazelnut Breeding Program • Hazelnut, a model for the Betulaceae • Breeding program - the driving force • Eastern filbert blight resistance • DNA markers, marker-assisted selection, mapping • Microsatellite markers and uses • Map-based cloning (‘Gasaway’ EFB resistance gene) • Sequencing the hazelnut genome (and transcriptome) • HRM for marker-assisted selection • Quantitative EFB resistance Taxonomy of Hazelnut (Corylus) Angiosperms, Eudicots, Core Eudicots, Rosids Rosids: Order Fagales: from tolweb.org Family Betulaceae Betuloideae Betula = birch (35-50 species) Alnus = alder (35 species) Coryloideae Corylus = hazelnut, filbert (11-15 species) Ostrya = hophornbeam (10 species) Ostryopsis = hornbeam (2 species) There are 11 commonly recognized Corylus species, all native to the northern hemisphere. All species produce edible kernels, but only the European hazelnut is commercially important. Corylus species Leafy-husked shrubs C. avellana = European hazel Grown commercially C. americana = American hazel C. heterophylla = Siberian hazel Bristle-husked shrubs native to C. cornuta = beaked hazel North America C. californica = California hazel C. sieboldiana = Manchurian hazel Tree hazels C. colurna = Turkish tree hazel C. jacquemontii = Indian tree hazel C. chinenesis = Chinese tree hazel C. fargesii = paperbark tree hazel Tree hazel with spiny husk (like chestnut bur) C. ferox = Tibetan tree hazel Why sequence hazelnut? European hazelnut (Corylus avellana) – a model for the Betulaceae • relatively small stature • small genome (~380 Mb) • diploid (2n = 2x = 22) • relatively short life cycle, ~5 years to first flowering • OSU breeding program • diverse collection of ~900 Corylus accessions • genetic linkage map • BAC library • amenity to transformation with Agrobacterium Expectations based on other sequenced plant genomes 30,000 -35,000 genes each gene ~2500 bp with ~3 introns 400 NBS sequences European hazelnut (Corylus avellana) At Corvallis Repository, 825 accessions of Corylus, 429 of C. avellana www.plantyfolia.com/photos106/corylus_ens.jpg ~ 100 more at OSU http://caliban.mpiz-koeln.mpg.de/~stueber/thome/band2/tafel_005.jpg www.funghiitaliani.it/Alberi/nocciolo/Corylus%2520avellana1.jpg The distribution of Corylus avellana includes many climatic zones, but commercial production regions are limited. (Mehlenbacher, 2003) Qualitative Traits in Hazelnut – single recessives Contorted growth Yellow leaves Yellow styles Cutleaf http://ace.acadiau.ca/science/biol/E vans/Biology www.cnr.vt.edu/dendro/dendrology/ syllabus/maps/corylus_cornuta.jpg Beaked hazelnut Corylus cornuta Smaller bush, smaller nuts, and longer husk than C. californica. California hazelnut Corylus californica www.canadianbiodiversity.mcgill.ca/ ww1.clunet.edu/wf/nca/flowers/picts/nca-222.jpg American hazelnut Corylus americana native to eastern North America http://project.bio.iastate.edu/trees/campustrees/images/Corylus/ Corylus.jpeg www.horticopia.com/hortpix/pix/U6HEH0.jpg Hazelnut Production (MT) 1. Turkey 504,000 71.2% 2. Italy 116,500 16.4% 3. Azerbaijan (est.) 35,000 4.9% 4. United States 27,000 3.8% 5. Georgia (est.) 25,000 3.5% 6. Spain 18,000 2.5% Hazelnuts in Turkey are grown on the Black Sea Coast. Tombul is an important cultivar. It has long, clasping husks and small nuts, for kernel market. Hazelnuts in Oregon are mechanically harvested. Nuts of ‘Barcelona’ fall free of the husk at maturity. The large nuts are suited to the in-shell market. Hazelnut Breeding Objectives A. Blanched kernel market (for chocolate, baked goods) 1. Bud mite resistance 5. Easy pellicle removal 2. Round nut shape 6. Few defects 3. High percent kernel 7. Early maturity 4. Precocity 8. Free-falling nuts 5. High yield B. Resistance to eastern filbert blight (EFB) 1. Simply inherited resistance (‘Gasaway’ & others) 2. Quantitative resistance (e.g. ‘Tonda di Giffoni’) Hazelnut Breeding Flow Chart 1. Choose parents, make crosses 2. Grow in greenhouse 10. Plant replicated trials 3. Seedlings in field 11. (trees grow) 4. “ 12. Evaluate nuts 5. “ 13. Evaluate nuts 6. Evaluate a few nuts 14. Evaluate nuts 7. Evaluate nuts 15. Evaluate nuts 8. Layer, Evaluate nuts 16. Evaluate nuts, 9. Nursery, Evaluate nuts summarize data 17. Release new cultivar Breeding cycle: 8 years from seed to seed. Release of new cultivar: 16-17 yrs after cross. Obtaining Hybrid Seed Growing Hybrid Seedlings (4000 planted per year) First Stage of Evaluation – Original Seedlings with removal of discards Propagation of Selections by Tie-Off Layerage; suckers form every year Harvested layers are weak. They are held in the nursery for one year, and then planted in the orchard. Second Stage of Evaluation – in Replicated Trials Releases Cultivars with Quantitative EFB Resistance: Willamette - 1990 cross made in 1973 Lewis - 1997 cross made in 1981 Clark - 1999 cross made in 1982 Sacajawea - 2006 cross made in 1990 Cultivars with Very High Resistance: Santiam - 2005 cross made in 1989 Yamhill - 2008 cross made in 1990 Jefferson - 2009 cross made in 1993 Pollinizers with Very High Resistance: VR4-31, VR11-27, VR20-11, VR23-18 - 1990 Gamma, Delta, Epsilon, Zeta - 2002 Eta, Theta - 2009 Cultivar susceptible to EFB: Tonda Pacifica - 2010 ‘Yamhill’ (OSU 542.102) was released for the kernel market in 2008. Buyers are interested. Jefferson (OSU 703.007) was released in 2009 as an EFB-resistant replacement for ‘Barcelona’. It is now the most widely planted cultivar in Oregon. Late-shedding pollinizers ‘Eta’ and ‘Theta’ released at same time. Micropropagation is now routine 44 days liquid media added ‘Yamhill’ Proebsting & Meneghelli Eastern Filbert Blight Fungus Anisogramma anomala, 2-year life cycle. Cankers girdle and kill branches. http://oregonstate.edu/dept/botany/epp/EFB/ DiseaseDisease InoculationsInoculations inin thethe GreenhouseGreenhouse 3 scions per selection DiseaseDisease InoculationsInoculations inin thethe GreenhouseGreenhouse Cankers 13-16 months after inoculation. Very High Resistance to EFB in Corylus avellana Genotype Origin Gasaway Washington Zimmerman Oregon OSU 408.040 Minnesota "Weschcke Sdlg" Ratoli Spain OSU 759.010 Republic of Georgia OSU 495.072 Russia COR 157 Finland Culpla Spain Medium Long Geneva, NY, USA Very High Resistance to EFB in Corylus avellana (cont’d) Genotype Origin Crvejne Cacak, Serbia Uebov Cacak, Serbia Moscow Selections (7) Russia OSU 1187.101 Russia (Holmskij) Seedlings (2) Russia Seedlings (3) Crimea, Ukraine Seedlings (2) Republic of Georgia (~2% of accessions show very high resistance) Very High Resistance to EFB in other Corylus species C. americana ‘Rush’, now in BC2 generation C. americana ‘Winkler’, now in BC1 generation C. heterophylla ‘Ogyoo’, now in BC2 generation Segregation ratios indicate single loci, with dominant resistance. RAPD Markers Linked to EFB Resistance from ‘Gasaway’ Robust RAPD markers are easy to score, useful in many populations. Expected ratio 1:1 (Rest:Susc) MAS because: -16 months between inoculation and canker development -desire to select for resistance in 152-800 absence of pathogen -disease phenotyping reliability (?) -expected use of ‘Gasaway’ R-gene for many years, many crosses Seedlings with one or both markers are planted in the field. Seedlings lacking both markers are discarded. 268-580 DNA Extraction using “Juice Press” high throughput needed for MAS, 200 per day easy Microsatellite Markers Corvallis: Nahla Bassil, T. Gökirmak, K. Gürcan Torino: Roberto Botta, Aziz Akkak, Paolo Bocacci SSR markers: 230 published, 10 more from internal repeats in ISSR fragments SSRs from birch and alder M.S. student Brooke Peterschmidt is developing new SSRs from RNA-Seq (expect ~200 more polymorphic markers) Transferability: About one-third of hazelnut SSRs amplify birch, alder Most Corylus avellana SSRs amplify C. americana Microsatellite Markers Amplify using PCR and fluorescent primers Size using capillary electrophoresis (size in base pairs) We have used SSR markers to : - characterize European hazelnut germplasm (270 of 500) - confirm duplicate accessions - identify parents of grower selections, cultivars - map, serve as anchor loci on a consensus map - characterize C. americana (87) and C. avellana x C. americana hybrids (68) Fusco Rubra Ruby EFB-resistant shown Albania 55 OSU 495.049 with arrow Cutleaf OSU 495.072 Gasaway UPGMA dendrogram Zimmerman Finland 187 OSU 681.078 for C. avellana OSU 408.040 Barcelonner Z. Sweden 627 from microsatellite marker data Aurea Pendula Aveline d'Angleterre Des Anglais Goc shows four main groups: Syrena Redleaf Group Red Fortrin Rote Zellernuss Central European OSU 026.072 Central European Group Black Sea Black Sea Group 1 English Black Sea Group 2 English Group 1 Spanish-Italian English Group 2 Warsaw Red Henneman #3 270 accessions incl. 72 synonyms Spanish-Italian Group Parentage indicated for 31 accs. Zeta (Gökirmak et al., 2009) 0.1 Rush x European Hybrid Group AmeIllinois#1 IL White ADF Hybrid Group1 Americana Group1 Mixed Group ADF Hybrid Group2 Americana Group2 Americana Group3 0.1 UPGMA dendrogram of 162 accessions based on 21 SSR loci shows that most hybrids were placed in one of three groups: ‘Rush’, ADF Group 1, and ADF Group 2 (with ‘Winkler’). Sathuvalli, V.R. and S.A.