Hop Breeding Using Molecular Marker Technology - IBD Asia Pacific Convention...

Hop Breeding Using Molecular Marker

Technology

Russell Falconer MBrew FIBD

Managing Director – Steiner Hops Ltd

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

Hopsteiner Breeding Locations

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USA-

Greenhouses, Nursery,

Laboratories, Growing

Molecular Screening

Germany-

Greenhouse, Nursery,

Application of

Molecular Screening,

Laboratory

Taxonomy of Humulus lupulus

4

Order: Rosales

Family: Cannabaceae

Species: Humulus lupulus

Humulus japonicus

Humulus yunnanesis

Hop Genome: 2.8 Gb

Ploidy: 2n=20

Seeds

x

Hop breeding must be integrated with production

• Chemical traits

• Stability

• Unique aromas

• Hop Acid composition / Co-Humulone

• Story

• Agronomics

• Maturity

• Growth habit

• Less pesticides

• Climate Change

• Lower carbon footprint

• Diseases

• Regulations

EnvironmentGrower

ProcessorCustomer

6

Main focus of our Breeding Goals using

advanced lines to date…

Focus Areas:

1. Chemistry

2. Agronomy

3. Aroma and Flavor

7

Focus on Quality: advanced line profiles

Delta

Calypso

Bravo

Apollo

Super Galena

Lemondrop

Eureka!

Denali

04190

07270

09238

09326

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Varieties Chemical Profiles Agronomic Traits

Low CoHumulone

Novel Aroma

High Oil Content

Select Polyphenols

Storage Stability

Durable Downy

Mildew Resistance

and Powdery Mildew

Resistance

Vigor

Virus Tolerance

Pickability

Compact Cones

Focus: Bitter acids and yield

Variety GalenaSuper

Galena07270 Zeus Apollo Bravo

Alpha acids

% w/w10.0 - 13.5 13.0 - 16.0 18-20 12.0 - 16.5 15.0 - 19.0 14.0 - 17.0

Beta acids %

w/w7.0 - 9.0 8.0 - 10.0 4.5-6.0 4.0 - 6.0 5.5 - 8.0 3.0 - 5.0

CoH % w/w

of α-acids35 - 40 35 - 40 27-29 27 - 35 24 - 28 29 - 34

Total Oil

ml/100g0.9 - 1.2 1.5 - 2.5 3.0 1.0 - 2.0 1.5 - 2.5 1.6 - 2.4

Stability 75 - 80% 75 - 80% 85% 50 - 60% 80 - 90% 60 - 70%

Powdery

Mildew*Susceptible “Resistant” Resistant Susceptible “Resistant” “Resistant”

Yield lbs/acre1,600 -

2,220

2,500 -

2,800 2,500-3,400

2,400 -

3,000

2,600 -

3,000

2,700 -

3,100

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Focus: Aroma and flavor

Variety Cascade Calypso Centennial Lemondrop Denali

Alpha acids

% w/w4.5-7.0 13.0 - 16.0 9.5-11.5 4.5-6.5 15-18

Beta acids %

w/w4.5-7.0 8.0 - 10.0 3.5-4.5 4.0-6.0 4.0-5.0

CoH % w/w

of α-acids33-40 35 - 40 29-30 30-33 22-26

Total Oil

ml/100g0.8-1.5 1.5 - 2.5 1.5-2.3 1.5-2.0 4.0

Stability 48% 75 - 80% 45-55% 65% 80%

Powdery

Mildew*Tolerant Susceptible Tolerant Tolerant Tolerant

Yield lbs/acre 1600-2200 2,500 - 2,800 1,700-2,000 2,000 – 2,800 2,600 - 3,200

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Denali

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Experimental Variety 06277

20142nd Most Popular

Experimental Hop

Alpha: 15-17% Beta: 4-5%

CoH: 22-25% Total Oil: 4.0-6.0

Av. Yield: 3500 lbs/acre

Susceptible to PM in greenhouse

Emerald 5-acre; Roza MHN: 80-hills

(voted 1st favorite in 2013)

Content





HTS Phenometrics

HTS Chemometrics

HTS Metabolomics

HTS Genomics


Cone development and variation

13

Content





HTS Phenometrics

HTS Chemometrics

HTS Metabolomics

HTS Genomics


Prototypical trichome gland metabolites are extracellular

secretory cells are specialized

extraction of subcuticular space is

easier than leaf epidermis cells

.16

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:

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Aldehydes

Lactones

Norcarotenoids

Thiols

Esters

Flavanol glucosides

Many, many others

Classic Separation

Reverse phase, C18

Isocratic (MeOH, H3PO4)

Long separation time

ASBC HPLC of Bitter Acids

18

19

Rocket UPLC

325 nm

370 nm

Alpha Beta

XN

2.5 minutes

Polyphenols

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21

Time5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00 85.00

%

4

Procyanidin B1

Catechin

Epicatechin

Quercetin-3-O-glucoside

Rutin

Kaempferol-3-O-rutinoside

Astragalin

Quercetin

LC-qToF-MS TIC

of 80% MeOH

hop extract

Phenylalanine

Terpenoids Two biosynthetic origins

Many cyclases

Variable oxidative decorations

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Content





HTS Phenometrics

HTS Chemometrics

HTS Metabolomics

HTS Genomics


Molecular phenotyping and genotyping

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Genome

Metabolome

DNA

Sugar FlavonoidesAmino

acidsLipids

LC-qTOF

Orbitrap

(IPK/IPB)

Illumnia Hi Seq

(Buckler lab, Cornell)

Understand the genetic regulation of

resistance and metabolism

Phenotype / Function

mGWAS

Understand interrelation between

metabolome and phenotype

e.g.

Metabolomics for disease resistance

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Genome Phenome

Metabolome

GWAS

Trait dissection

Content





HTS Phenometrics

HTS Chemometrics

HTS Metabolomics

HTS Genomics


Evolution of breeding systems technology

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Single CrossSelect

Quantitative

Genetics

Molecular

Quantitative

Genetics

Time-line of molecular marker development

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600 Diversity Array Technology

(DArT) markers

Microarray hybridization to

detect presence vs. absence

of individual fragments

2010

1.000 Simple Sequence

Repeat (SSR) markers

short, non-coding

repeats in the genome;

mostly detected at the

same loci

2011

300.000 Single Nucleotide

Polymorphisms (SNP)

Variation of a single base pairs

in the genome

Next Generation Sequencing

20132012

Motivation for molecular marker

development

What are markers good for?

• Trueness-to-type determination

• Parent determination

• Variety rights protection

• Marker-assisted selection (MAS)

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Figure 2. DNA is extracted from a group of plants

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.

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.

Genome Wide Association Studies

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1. Making a Reference Genome

DNA SequencingHop Genome: 2.8 Gb

Diploid: 2n=20

Genome Current Condition

Total number of scaffolds = 340.401

Sum (bp) = 1.798.099.671

Max scaffold size (bp) = 684.849


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2. Generating GBS Markers

Phenotype Extract DNA

Cut and

Sequence

Genomic DNA


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3. Genome Based Selection

R = resistant S = susceptible

Mapping Reads to Reference

Q-Q plot of Mixed Linear Modelling

associations

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Manhattan Plot of Mixed Linear Modelling

associations

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Time-line of variety development too slow

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Year 0 - 1

• Selection of parents

• Seedling Screening

• Diseases

• Marker screening

Year 2 – 3Single Hill Evaluation

• Disease resistance

• Chemical traits

• Maturity

Year 4 – 6Multi Hill Evaluation

• Agronomic traits

• Chemical traits

• Different environments

• 1st brewing trials

Year 7 – 10Semi Commercial

• Confirmation on agronomic and chemical traits

• Extensive brewing tests

2 varieties100 plants, 20 plants

≈7,000 plants50 crosses,

≈25,000 seedlings

Greenhouse

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Nursery

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Commercial field

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80% of breeding efforts are

breeding for disease resistance

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• Plant microbial diseases

Powder mildew (Podosphaera macularis)

Downy mildew (Pseudoperonospora humuli)

Viruses and viroids (stunt viroid)

Powdery mildew resistance breeding

41

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?

Normalized breeding values showing

stackedness for two traits: R1,R3 and R2

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GEBV_R1R3norm

GEBV_R2norm

High GEBV for both sets of markers

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

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With thanks to Paul D. Matthews Ph.D.

and Alex Feiner Msc

Thank you for your attention.

Hop Breeding Using Molecular Marker Technology - IBD Asia Pacific Convention...

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