An affordable and convenient diagnostic marker to identify male and female hop plants

Authors: Clare, Shaun; King, Ryan; Tawril, Anna; HAVILL, JOSHUA - University Of Minnesota; MUEHLBAUER, GARY - University Of Minnesota; CAREY, SARAH - Hudsonalpha Institute For Biotechnology; HARKESS, ALEX - Hudsonalpha Institute For Biotechnology; Bassil, Nahla; Altendorf, Kayla

Submitted to: G3, Genes/Genomes/Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/8/2023
Publication Date: 11/14/2023
Citation: Clare, S.J., King, R., Tawril, A., Havill, J.S., Muehlbauer, G.J., Carey, S., Harkess, A., Bassil, N.V., Altendorf, K.R. 2023. An affordable and convenient diagnostic marker to identify male and female hop plants. G3, Genes/Genomes/Genetics. 14(1). Article jkad216. https://doi.org/10.1093/g3journal/jkad216.
DOI: https://doi.org/10.1093/g3journal/jkad216

Interpretive Summary: Hop is a dioecious species meaning it has both male and female plants. Hop cones, which are the end-product used in brewing beer, are produced exclusively by female hop plants. Plant breeders seeking to identify new and improved varieties of hop are primarily selecting among female plants. Prior to flowering male and female plants are visually indistinguishable and breeders typically wait 1-2 years for plants to mature to determine plant sex by observing floral structures. A molecular marker, or a known DNA sequence associated with a specific plant trait, in this case sex, can aid breeders in making selections prior to observing the trait visually. Previous work to develop a sex-linked marker in hop have suffered from low-scalability and limited transferability across populations. We used a large collection of 855 hop lines with known sex, genotyping by sequencing data, and association mapping and identified a highly significant marker associated with plant sex that was predictive 96.2% of the time. We optimized a protocol for utilizing the marker in the lab using a rapid DNA extraction technique. The marker, when tested using DNA from a new population with known sex phenotypes, was 96% accurate. The DNA marker is in a gene that is predicted to encode a transcription factor protein, which has been previously implicated in male sterility in a variety of plant species. The marker is diagnostic, accurate, affordable, and highly scalable and has the potential to improve efficiency in hop breeding.

Technical Abstract: Hop production utilizes exclusively female plants, whereas male plants only serve to generate novel variation within breeding programs through crossing. Currently, hop lacks a rapid and accurate diagnostic marker to determine whether plants are male or female. Without a diagnostic marker, breeding programs may spend one to two years determining the sex of new seedlings. Previous research on sex-linked markers has been limited to specific populations or breeding programs and therefore had limited transferability or suffered from low scalability. A large collection of 855 hop genotypes with known sex phenotypes, genotyping-by-sequencing, and genome wide association mapping revealed a highly significant marker on the sex chromosome (LOD score = 208.7) that predicted sex within our population with 96.2% accuracy. We developed a PACE assay for the diagnostic SNP and tested three quick DNA extraction methodologies for rapid, high-throughput genotyping. Additionally, the marker was validated in a separate population of 94 individuals from fifteen families from the USDA-ARS hop breeding program in Prosser, WA with 96% accuracy. This diagnostic marker is situated in a gene predicted to encode basic helix-loop-helix transcription factor protein, a family of proteins that have been previously implicated in male sterility in a variety of plant species, which may indicate a role in determining hop sex. The marker is diagnostic, accurate, affordable, and highly scalable and has the potential to improve efficiency in hop breeding.