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An Affordable & Convenient Diagnostic Marker to Identify Male and Female Hop Plants – a major boost for hop breeding efficiency using PACE genotyping

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An Affordable & Convenient Diagnostic Marker to Identify Male and Female Hop Plants – a major boost for hop breeding efficiency using PACE genotyping

Discover how researchers from the National Clonal Germplasm Repository, OR and Forage Seed and Cereal Research Unit, WA of the USDA-ARS, in collaboration with the University of Minnesota and Hudson Alpha Institute for Biotechnology, have developed an accurate, low-cost, and highly scalable diagnostic marker, poised to significantly enhance efficiency in hop breeding.

Hops: A Multi-Purpose Crop for Brewing and Beyond

Hops (Humulus lupulus L.) are a crucial ingredient in beer production, belonging to the Cannabaceae family. They contribute aroma, bitterness, and preservatives to the final beer product. The US, primarily the Pacific Northwest (Washington, Idaho, and Oregon), is the largest producer of hops globally (45% in 2022), with three of the five subspecies native to North America.

Female hop plants. Image courtesy of Ninh Khuu

Hop plants are perennial, wind-pollinated, possess a diploid genome, and are dioecious – producing separate male and female plants. In commercial hop production, cones from female plants are exclusively utilised, while male plants contribute to generating novel variations in breeding programs through crossing. Currently, hop lacks a rapid and accurate diagnostic marker to determine a plant’s sex, hampering breeding programs by relying on time-consuming visual observations alone.

The absence of a diagnostic marker can extend breeding programs by 1-2 years to determine the sex of new seedlings. Understanding seedling sex earlier in the breeding cycle would facilitate more efficient breeding strategies and selections.

Previous research on sex-linked markers has been limited to specific populations or breeding programs, with hindered progress due to restricted transferability and low scalability. Recent advances and reduced costs in long-read genome sequencing and assembly now make assembling XY pairs in complex species with large genomes, such as hops, feasible expanding the possibilities for genomic study.

The study

A diverse collection of 765 male and female hop genotypes and 20,861 biallelic SNPs was assembled for GBS and GWAS studies. Association mapping revealed a highly significant SNP marker on the sex chromosome predicting sex within the study population with 96% accuracy. The SNP is in a gene predicted to encode the basic helix-loophelix transcription factor protein; a family of proteins previously implicated in male sterility in a variety of plant species.

The team developed a PACE genotyping assay for the diagnostic SNP, testing three quick extraction methodologies for a rapid, high-throughput genotyping workflow. The marker was validated in a separate population of 94 individual male and female seedlings from 15 independent crosses in the USDA-ARS hop breeding program, achieving 96% accuracy.

Hop yard growing nearly all female first-year seedlings. Image courtesy of Ninh Khuu

The marker was applied in the USDA-ARS hop breeding program in Prosser, WA in 2023 and enabled 25% increase in the ratio of female: male seedlings evaluated in the same field space as the previous year, and resulted in a 97.4% female yard.

The methodology is widely accessible to breeders, combining rapid, in-house hot-shot DNA extraction and a marker assay utilising low-cost reagents, alongside equipment and instruments commonly found in molecular laboratories. It willl allow hop breeding programs to boost efficiency (saving time, labour, money, and space) for earlier cultivar releases. It will also enhance genetic gains to counter challenges such as climate change, diseases, and addressing changing consumer trends, by liberating resources to other aspects of hop breeding programs.

Sampling hop seedlings for screening. Image courtesy of Kayla Altendorf

This new marker is diagnostic, accurate, affordable, and highly scalable, with the potential to dramatically improve efficiency in hop breeding. It is also a compelling illustration of the benefit of Marker-Assisted-Selection (MAS) techniques for hop breeding.

“The marker was applied in the USDA-ARS hop breeding program in Prosser, WA in 2023 and enabled 25% increase in the ratio of female: male seedlings evaluated in the same field space as the previous year, and resulted in a 97.4% female yard.”

References:

  • Clare, Shaun J., Ryan M. King, Anna L. Tawril, Joshua S. Havill, Gary J. Muehlbauer, Sarah B. Carey, Alex Harkess, Nahla Bassil, and Kayla R. Altendorf. “An affordable and convenient diagnostic marker to identify male and female hop plants.” G3: Genes, Genomes, Genetics (2023): jkad216.

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MEET OUR TEAM

Steve Asquith Managing Director
Steve began his career in the Genetics Division of GlaxoSmithKline, as part of the team establishing GSK’s high-throughput core genotyping laboratory. Steve joined KBioscience when it was first founded in 2002 and was a key driver in taking the company from a small start-up to a multi-national service laboratory, quickly growing the company’s revenue to over $7.5M p.a. Following the acquisition of Kbioscience by LGC in 2011, Steve was appointed Global Director of Operations for LGC Genomics, responsible for over 100 staff in Europe and N. America, successfully elevating the genotyping products and service business. Steve held a crucial leadership role until he left in 2016. In 2017 Steve joined forces with John Holme to create 3CR Bioscience, a new company with a mission to deliver outstanding, customer-focused genotyping products with innovation and affordability at its core.
Dr. John Holme Technical Director
John joined KBioscience shortly after it was founded, in 2003, and became Head of Technical Development, building the company’s genotyping and DNA extraction product portfolio and service delivery until 2011 when it was acquired by LGC. Post-acquisition, John was appointed Head of Technical Group for LGC Genomics, in charge of all Research & Development and Technical Support activities for the company. In this role John continued to build on the high-quality products and services provided to the companies growing customer base. During the 19 years John has worked in commercial R&D, he has co-invented numerous highly successful products including PACE®, ProbeSure, KASP™, KlearKall, KlearGene, KlearAmp and KlearTaq™, creating breakthrough offerings in genotyping and extraction and generating huge revenues for the companies he has worked in. In 2017, he joined forces with Steve Asquith and started 3CR Bioscience. John is dedicated to developing outstanding, innovative genotyping products and providing the very best technical support to customers globally.
Dr. Nisha JainOperations Director
Nisha has been innovating since the start of her career at Geneform Technologies developing Iso-thermal Genotyping Technologies. Nisha joined KBioscience in 2008, as Senior R&D Scientist and key account Technical Support Scientist, developing KASP and Klearkall performance and coinventing two further versions of KASP. Nisha has more than 15 years’ experience working in molecular biology and genotyping technologies, with extensive experience in the areas of R&D, Quality Assurance and Customer Technical Support. She has technically assisted many giants of the industry with their protocol development and troubleshooting and continues to deliver high-quality support and guidance. In 2018, Nisha joined 3CR Bioscience as Operations Director where she continues to develop PACE and ProbeSure for an increasing range of applications, and to grow 3CR Bioscience’s new product pipeline. Nisha is dedicated to developing outstanding, innovative genotyping products and providing the very best technical support to customers globally.
Nazma Saffin General Manager
For 20 years Nazma Saffin has worked and gained extensive expertise within the genotyping sector. Working at Kbioscience and then LGC, she has held operational leadership posts responsible for manufacturing and laboratory services. With experience of ISO 9001 implementation, production scale up and LEAN operations, Nazma has successfully led highly profitable production departments. Joining 3CR Bioscience in 2022, Nazma is committed to delivering operational excellence.
Jon Curtis Non-Executive Chair
After 8 years in The Royal Air Force, Jon moved to the Imperial Cancer Research Fund where he pioneered the use of ultra high-throughput genomic automation, capable of 46,000 PCRs per hour. In the 1990’s Jon joined GlaxoSmithKline, implementing a high-throughput genomics platform into their drug discovery pipeline. Whilst there he also developed acoustic mixing into compound management, becoming the gold standard across pharma. Jon developed the world’s first commercially viable 1536-well PCR plates, automated thermal & laser plate-sealer, plus automated liquid-handling & tip washing tools to reduce waste and costs. In 2002 Jon co-founded KBioscience with Phil Robinson, utilising ultra high-throughput PCR instrumentation & a suite of automation tools to create the company’s SNPline robotic platform, with a capacity of 250,000 PCRs/day. The business was underpinned by their ground-breaking patented genotyping chemistry, KASP™, which has over 10,000 scientific papers to date. In November 2022 Jon joined 3CR Bioscience acting as an advisor bringing his commercial and scientific experience to the company.