By Dr. Alexander Feiner, Hopsteiner, Germany

Food security, the production of renewable raw materials and combating climate change, as well as issues of sustainability and the preservation of the cultural landscape, are among the most important tasks facing our society (1). As a result, innovations in plant breeding have become enormously important.

Since the discovery of the laws of genetics by Gregory Mendel in 1866, plant breeders have continually incorporated the latest innovations in plant biology into their daily work to develop improved crops.

Requirements for hop breeding

The greatest challenge in hop cultivation in recent years and in the future is to ensure stable yields in hop production. Extreme climatic conditions with exceptionally long dry periods and high temperatures have had a major influence on widely fluctuating yields and quality in the past years.

Climate change is an essential component in plant breeding. According to climate researchers, temperatures are expected to rise, precipitation will be distributed differently, and it will become increasingly difficult to secure the water supply needed for agriculture (2). That is why, in addition to new solutions for water supply, plants that can adapt to these changes and perform with the available resources are needed.

Therefore, desirable characteristics of hop varieties are:

• Yield potential and yield security with improved resource utilization
• Resistance to pests and diseases and higher tolerance to climatic influences
• Ingredient composition regarding aroma and increased content of bitter substances or processing properties

From the grower’s perspective, high yields together with great disease resistance are desirable.

The fungal pathogens Pseudoperonospora humuli and Podosphaera macularis are the cause of downy and powdery mildew respectively. These two diseases could potentially result in a loss of significant tonnage per year and require multiple fungicide sprays. Naturally, for ‘bitter’ varieties the alpha acid content is the key driver with values > 20% w/w now being realized.

The time of maturation is also important. Having the plants mature at different times over a four to six week window allows for more efficient use of farmers labor, picking equipment, cleaning and kilning facilities. When cleaning the hops from the residual leaf and strig material, derived from the bines upon which they grow, pick-ability is important. Large, dense, compact cones which separate easily from the leaf and stem ‘waste’ material without losing valuable cones and bracts are a desired trait, too.

The stability of alpha acid post-harvest is also a factor that needs to be considered, as some alpha varieties can lose up to 25% of their alpha acid content in just a few weeks from harvest, if not processed into more stable products. Certain varieties are also bred which are easier to pelletize and extract due to their better solubility in extraction solvents such as liquid CO2.

Finally, the needs of the brewer drive the market, with significant attention being given lately to breeding unique hop aromas and flavor. Hop alpha acid composition, such as low co-humulone content is still requested by some brewers.

The breeding timeline

Hop, Humulus lupulus L., is a dioecious perennial member of the Cannabaceae family (3). Its female flowers are mainly used in beer brewing as a flavoring as well as bittering agent because of the high abundance of secondary metabolites, including bitter acids, terpenes and polyphenols (4).

The basis of classical hop breeding is the targeted cross-breeding of female (Figure 1A) and male (Figure 1B) hops and the selection of progeny with desirable traits.

Figure 1: Female (A) and male (B) inflorescence of Humulus lupulus.

Plants with desirable trait composition are vegetatively propagated and reach the market as a new variety after various growing trials as well as brewing trials. The traditional timeline for developing a new hop variety is up to 10 years. The following describes a typical breeding cycle:

• Year 1: female and male parents are selected based on performance as well as breeding history and crossed.
• Year 2: resulting seedlings are screened for disease resistance using marker-assisted-selection.
• Years 2-3: seedlings are planted out on single hill plots and evaluated for disease resistance, maturity, and chemical traits and aroma.
• Years 4-6: promising genotypes are propagated into multi hill plots at multiple locations and evaluation continues for agronomic and chemical traits, but also in different environments. First brewing trials are executed.
• Years 7-10: advanced genotypes are move to semi commercial production. Agronomic and chemical traits have to be confirmed. Brewing trials are extensive.
• Year 10: In case of very promising results, a variety approval is targeted.

Breeding technology

Biotechnology plays a significant role in maximizing the probability of success in hop breeding. Using DNA markers to support plant breeding can significantly increase efficiency and precision.

The use of DNA markers in plant breeding is called marker-assisted selection (MAS). In MAS, molecular biology techniques are used to screen cross progeny for genetic composition and compare them to the parent plants. By creating such a “genetic fingerprint”, it is possible to determine already in young plants whether they carry the desired traits or not. The breeding effort and selection time are thus reduced.

By using Genome Wide Association Studies (GWAS) with the use of Single Nucleotide Polymorphism (SNP) molecular markers, hop breeding is now looking to significantly reduce the numbers of seedlings required in year one and condense the timeline down to six years by reducing the front-end evaluation (5). Genotyping by Sequencing (GBS) is used to develop molecular markers for applications of MAS. GBS is a next-generation sequencing based method that targets a large, but manageable number of SNP sites by restriction digest-based reduced representation.

The GBS method allows high throughput genotyping of large numbers of individuals at tens of thousands of polymorphic markers, providing cost effectiveness (6).

Using metabolomics to understand downy mildew resistance

Metabolomics is the science concerned with the unique range of metabolic products that different organisms produce, and how from this they might be ‘fingerprinted’. Secondary metabolites, for example, sugars, flavonoids, amino acids, or lipids associated with a particular phenotype are studied to understand the genetic regulation of this metabolite and trait expression.

As written above, Pseudoperonospora humuli, the causal organism of hop downy mildew, is an obligate biotrophic oomycete pathogen and has been a serious threat in recent years. To identify the molecular processes of natural resistance as well as genetic and metabolic markers for hop breeding, a metabolome-genome-wide association study was carried out on 192 genotypes.

The researchers found out that some hop metabolites, especially phenylpropanoids, are correlated with resistance to downy mildew. In an independent validation experiment, a mixture of three putative prophylactic phenylpropanoids was applied on susceptible genotypes together with fungal spores.

This external application of the substances, which are overrepresented in resistant genotypes, resulted in a reduced leaf infection (Figure 2). This confirmed their protective activity either directly or as precursors of active compounds (7).

Figure 2: Different degrees of downy mildew seven days after infection. A) resistant, B) tolerant, C) slightly susceptible, D) susceptible, E) highly susceptible.

The metabolic and genetic markers obtained through this study provide a better understanding of the underlying resistance to downy mildew. In the future, this will allow a more precise selection of breeding partners and progeny in hop breeding.

Conclusion

Only a mix of new adaptable varieties and smart farming methods will be able to ensure the economic sustainability of agriculture and thus of hop growing. In order to be able to drive forward the necessary changes, it will be important to take new directions at all levels, from cultivation to the usage of hops. New varieties will enrich the market by providing good yields, improvements in cultivation and the full characteristics of beer.

References

1. Anderson, Robyn, Philipp E Bayer, and David Edwards. 2020. “Climate Change and the Need for Agricultural Adaptation.” Current Opinion in Plant Biology 56 (August): 197–202. https://doi.org/10.1016/j.pbi.2019.12.006.

2. Deutscher Wetterdienst. 2021. “German Climate Atlas.” 2021. https://www.dwd.de/EN/climate_environment/climateatlas/climateatlas_node.html.

3. Neve, R. 1991. Hops. Dordrecht: Springer Netherlands. https://doi.org/10.1007/978-94-011-3106-3.

4. Cleemput, M., K. Cattoor, K. Bosscher, G. Haegeman, D. Keukeleire, and A. Heyerick. 2009. “Hop (Humulus Lupulus)-Derived Bitter Acids as Multipotent Bioactive Compounds.” Journal of Natural Products 72: 1220–30. https://doi.org/10.3399/bjgp12X616418.

Kavalier, A., A. Litt, C. Ma, N. Pitra, M. Coles, E. Kennelly, and P. Matthews. 2011. “Phytochemical and Morphological Characterization of Hop (Humulus Lupulus L.) Cones over Five Developmental Stages Using High Performance Liquid Chromatography Coupled to Time-of-Flight Mass Spectrometry, Ultrahigh Performance Liquid Chromatography Photod.” Journal of Agricultural and Food Chemistry 59 (9): 4783–93. https://doi.org/10.1021/jf1049084.

Keukeleire, J. De, G. Ooms, A. Heyerick, I. Roldan-Ruiz, E. Van Bockstaele, and D. De Keukeleire. 2003. “Formation and Accumulation of α-Acids, β-Acids, Desmethylxanthohumol, and Xanthohumol during Flowering of Hops (Humulus Lupulus L.).” Journal of Agricultural and Food Chemistry 51 (15): 4436–41. https://doi.org/10.1021/jf034263z.

5. Matthews, P., M. Coles, and N. Pitra. 2013. “Next Generation Sequencing for a Plant of Great Tradition: Application of NGS to SNP Detection and Validation in Hops (Humulus Lupulus L .).” BrewingScience 66 (December): 185–91.

6. Glaubitz, J., T. Casstevens, F. Lu, J. Harriman, R. Elshire, Q. Sun, and E. Buckler. 2014. “TASSEL-GBS: A High Capacity Genotyping by Sequencing Analysis Pipeline.” PLoS ONE 9 (2). https://doi.org/10.1371/journal.pone.0090346.

7. Feiner, Alexander, Nicholi Pitra, Paul Matthews, Klaus Pillen, Ludger A. Wessjohann, and David Riewe. 2020. “Downy Mildew Resistance Is Genetically Mediated by Prophylactic Production of Phenylpropanoids in Hop.” Plant, Cell & Environment, no. September: 1–16. https://doi.org/10.1111/pce.13906.