Hop Breeding Using Molecular Marker Technology

Transcription

1 Hop Breeding Using Molecular Marker Technology Russell Falconer MBrew FIBD Managing Director Steiner Hops Ltd

2 Content What do brewers want in a new variety? Variety development goals What s needed for Good Breeding Practice Vertically Integrated Variety Development HTS Phenometrics HTS Chemometrics HTS Metabolomics HTS Genomics Systems approach (Genome-Wide Association Studies) 2

3 3 Hopsteiner Breeding Locations USA - Greenhouses, Nursery, Laboratories, Growing Molecular Screening Germany - Greenhouse, Nursery, Application of Molecular Screening, Laboratory

4 Taxonomy of Humulus lupulus x Order: Rosales Family: Cannabaceae Species: Humulus lupulus Humulus japonicus Seeds Hop Genome: 2.8 Gb Ploidy: 2n=20 Humulus yunnanesis 4

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6 6 Hop breeding must be integrated with production Climate Change Lower carbon footprint Diseases Regulations Agronomics Maturity Growth habit Less pesticides Environment Grower Customer Processor Unique aromas Hop Acid composition / Co-Humulone Story Chemical traits Stability

7 7 Main focus of our Breeding Goals using advanced lines to date Focus Areas: 1. Chemistry 2. Agronomy 3. Aroma and Flavor

8 8 Focus on Quality: advanced line profiles Varieties Chemical Profiles Agronomic Traits Delta Calypso Bravo Apollo Super Galena Lemondrop Eureka! Denali Low CoHumulone Novel Aroma High Oil Content Select Polyphenols Durable Downy Mildew Resistance and Powdery Mildew Resistance Vigor Virus Tolerance Storage Stability Pickability Compact Cones

9 9 Focus: Bitter acids and yield Variety Alpha acids % w/w Beta acids % w/w CoH % w/w of α-acids Total Oil ml/100g Galena Super Galena Zeus Apollo Bravo Stability 75-80% 75-80% 85% 50-60% 80-90% 60-70% Powdery Mildew* Yield lbs/acre Susceptible Resistant Resistant Susceptible Resistant Resistant 1,600-2,220 2,500-2,800 2,500-3,400 2,400-3,000 2,600-3,000 2,700-3,100

10 10 Focus: Aroma and flavor Variety Cascade Calypso Centennial Lemondrop Denali Alpha acids % w/w Beta acids % w/w CoH % w/w of α-acids Total Oil ml/100g Stability 48% 75-80% 45-55% 65% 80% Powdery Mildew* Tolerant Susceptible Tolerant Tolerant Tolerant Yield lbs/acre ,500-2,800 1,700-2,000 2,000 2,800 2,600-3,200

11 11 Denali Experimental Variety (voted 1 st favorite in 2013) nd Most Popular Experimental Hop Alpha: 15-17% Beta: 4-5% CoH: 22-25% Total Oil: Av. Yield: 3500 lbs/acre Susceptible to PM in greenhouse Emerald 5-acre; Roza MHN: 80-hills

12 Content What do brewers want in a new variety? Variety development goals What s needed for Good Breeding Practice Vertically Integrated Variety Development HTS Phenometrics HTS Chemometrics HTS Metabolomics HTS Genomics Systems approach (Genome-Wide Association Studies) 12

13 Cone development and variation 13

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15 Content What do brewers want in a new variety? Variety development goals What s needed for Good Breeding Practice Vertically Integrated Variety Development HTS Phenometrics HTS Chemometrics HTS Metabolomics HTS Genomics Systems approach (Genome-Wide Association Studies) 15

16 Prototypical trichome gland metabolites are extracellular secretory cells are specialized extraction of subcuticular space is easier than leaf epidermis cells. 16

17 17 Chemical Diversity: Unique Flavors in Perpetuity The bitter acids of hops are terpenophenolics Diverse polyphenols contribute to haze, foam stability and bitterness Volatile terpenes are aromas Many flavors come from: Aldehydes Lactones Norcarotenoids Thiols Esters Flavanol glucosides Many, many others

18 18 ASBC HPLC of Bitter Acids Classic Separation Reverse phase, C18 Isocratic (MeOH, H3PO4) Long separation time

19 19 Rocket UPLC 325 nm Alpha Beta 2.5 minutes 370 nm XN

20 Polyphenols 20

21 LC-qToF-MS TIC of 80% MeOH hop extract Rutin Quercetin-3-O-glucoside Phenylalanine Quercetin % Procyanidin B1 Kaempferol-3-O-rutinoside Epicatechin Astragalin Catechin Time 21

22 22 Terpenoids Two biosynthetic origins Many cyclases Variable oxidative decorations

23 Content What do brewers want in a new variety? Variety development goals What s needed for Good Breeding Practice Vertically Integrated Variety Development HTS Phenometrics HTS Chemometrics HTS Metabolomics HTS Genomics Systems approach (Genome-Wide Association Studies) 23

24 24 Molecular phenotyping and genotyping Genome DNA Illumnia Hi Seq (Buckler lab, Cornell) Metabolome e.g. Sugar Flavonoides Amino acids Lipids LC-qTOF Orbitrap (IPK/IPB) Phenotype / Function mgwas Understand the genetic regulation of resistance and metabolism Understand interrelation between metabolome and phenotype

25 25 Metabolomics for disease resistance Genome GWAS Phenome Trait dissection Metabolome

26 Content What do brewers want in a new variety? Variety development goals What s needed for Good Breeding Practice Vertically Integrated Variety Development HTS Phenometrics HTS Chemometrics HTS Metabolomics HTS Genomics Systems approach (Genome-Wide Association Studies) 26

27 27 Evolution of breeding systems technology Single Cross Select Quantitative Genetics Molecular Quantitative Genetics

28 28 Time-line of molecular marker development 600 Diversity Array Technology (DArT) markers Microarray hybridization to detect presence vs. absence of individual fragments Single Nucleotide Polymorphisms (SNP) Variation of a single base pairs in the genome Next Generation Sequencing Simple Sequence Repeat (SSR) markers short, non-coding repeats in the genome; mostly detected at the same loci

29 29 Motivation for molecular marker development What are markers good for? Trueness-to-type determination Parent determination Variety rights protection Marker-assisted selection (MAS)

30 Genome Wide Association Studies Figure 2. DNA is extracted from a group of plants Figure 1. DNA is extracted from Apollo hop variety and is run on Next Generation Sequencing machines. The raw reads produced are then groomed and trimmed. The cleaned reads are then assembled using massive computing into a reference genome. selected to optimize the power of the experiment. This example shows three plants, but a typical population size for an association study is ~200. Using an enzyme the DNA is then cut at specific sites. These fragments are then sequenced. 30

31 31 Genome Wide Association Studies 1. Making a Reference Genome DNA Sequencing Hop Genome: 2.8 Gb Diploid: 2n=20 Genome Current Condition Total number of scaffolds = Sum (bp) = Max scaffold size (bp) =

32 32 Genome Wide Association Studies 2. Generating GBS Markers Phenotype Extract DNA Cut and Sequence Genomic DNA

33 Genome Wide Association Studies 3. Genome Based Selection Mapping Reads to Reference R = resistant S = susceptible 33

34 34 Q-Q plot of Mixed Linear Modelling associations

35 35 Manhattan Plot of Mixed Linear Modelling associations

36 Time-line of variety development too slow Year 0-1 Selection of parents Seedling Screening Diseases Marker screening Year 2 3 Single Hill Evaluation Disease resistance Chemical traits Maturity Year 4 6 Multi Hill Evaluation Agronomic traits Chemical traits Different environments 1 st brewing trials Year 7 10 Semi Commercial Confirmation on agronomic and chemical traits Extensive brewing tests 50 crosses, 25,000 seedlings 7,000 plants 100 plants, 20 plants 2 varieties 36

37 Greenhouse 37

38 Nursery 38

39 Commercial field 39

40 40 80% of breeding efforts are breeding for disease resistance Plant microbial diseases Powder mildew (Podosphaera macularis) Downy mildew (Pseudoperonospora humuli) Viruses and viroids (stunt viroid)

41 41 Powdery mildew resistance breeding Powdery mildew resistance genes against evolving fungal virulence strains Gene Source Status in USA R1, R3, Rb Zenith Tolerance R2 Wye Target Resistance R4 Early Choice Tolerance R5 Cascade Tolerance R6 Nugget Broken 19058mR6 Broken Kazak 2000R Kazak 2000 Resistant, HSR Might stacked resistance genes confer durable resistance/tolerance?

42 Normalized breeding values showing stackedness for two traits: R1,R3 and R2 High GEBV for both sets of markers GEBV_R1R3norm GEBV_R2norm

43 43 Summary Hops exhibit great morphological, genetic and chemical diversity Hop breeding must take advantage of diversity with available technologies Chemo-analytic methods have improved allowing deep chemical profiles Next generation DNA sequencing has revolutionized marker development New markers have been successfully applied to selection of disease resistance Complicated traits, such as aroma and flavor, are now feasible breeding targets using metabolomics

44 With thanks to Paul D. Matthews Ph.D. and Alex Feiner Msc Thank you for your attention.