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

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 take 1-2 years to determine the sex of new seedlings. Previous research on sex-linked markers was restricted to specific populations or breeding programs and therefore had limited transferability or suffered from low scalability. A large collection of 765 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. In this study, we developed a PCR allele competitive extension (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 15 families from the USDA-ARS hop breeding program in Prosser, WA with 96% accuracy. This diagnostic marker is located in a gene predicted to encode the 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.

Keywords: Humulus lupulus; association mapping; diagnostic marker; hops; sex determination.

Fig. 1.

a) Manhattan plot of the association models for hop sex using GLM and BLINK algorithms. Chromosomes and marker density are indicated on the x-axis, where marker densities are plotted as the number of markers per 10 Mb window, with low and high marker density as represented by heat map on the right. The most significant marker used for marker development is highlighted with a vertical bar. The LOD score is on the y-axis with Bonferroni thresholds indicated by the solid (α-level 0.05) and dashed (α-level 0.01) lines. b) Quantile–quantile plot for the GLM and BLINK association models for hop sex with the expected LOD score on the x-axis and observed LOD scores on the y-axis. The solid line indicates whether all data points did not deviate from the expected LOD score.

Fig. 2.

Jitter plot of allelic distribution of markers surrounding the significant marker from the association mapping. Two flanking markers upstream (UM2 and UM1) and 2 markers downstream (DM1 and DM2) of the most SM1 are shown in physical order from the association mapping. Each panel is separated into female (left) and male (right) and allele class of homozygous reference (A), heterozygous (H), and homozygous alternative (B).

Fig. 3.

Scatter plot of DNA assay for design assay design and cycling conditions. Fluorescence from the FAM fluorophore is on the x-axis and HEX fluorophore is on the y-axis, with the sense design on the top facet and antisense design on the bottom facet. Number of standard PCR cycles posttouchdown ranging from 26× to 36× cycles are shown in 2× increments for both sense and antisense designs. NTCs, males, females, monoecious plants and unknown samples are shown. Clusters are outlined as NTCs, male, and female.

Fig. 4.

Scatter plot of DNA assay using different DNA extraction methods. Fluorescence from the FAM fluorophore is on the x-axis and HEX fluorophore is on the y-axis. Crude DNA extraction methods include HotSHOT, modified HotSHOT, and QuickExtract extraction protocols along with “clean” DNA from Gentra Puregene (30× sense panel of Fig. 3) are shown for comparison. NTCs, males, females, monoecious, and unknown samples are shown. Clusters are outlined as NTC, male, and female.

Fig. 5.

Scatter plot of the DNA assay using the validation plate. Fluorescence from the FAM fluorophore is on the x-axis and HEX fluorophore on the y-axis utilizing the modified HotSHOT and optimized assay design. NTCs, males, females, erroneous samples, failed to amplify (FTA) male and female, respectively, are shown. Clusters are outlined as NTC, male, female, FTA: male, and FTA: female.

Fig. 6.

Scatter plot of DNA assay in comparison with a recently developed assay (Havill et al. 2023). Fluorescence from the FAM fluorophore on the x-axis and HEX fluorophore on the y-axis with the previously developed assay (FM014) on the top facet and the DNA assay from this study on the bottom facet using 4 validation plates from the University of Minnesota (UMN). NTCs, males, females, erroneous, and undetermined samples are shown.