An improved assembly of the "Cascade" hop (Humulus lupulus) genome uncovers signatures of molecular evolution and refines time of divergence estimates for the Cannabaceae family.

We present a chromosome-level assembly of the Cascade hop (Humulus lupulus L. var. lupulus) genome. The hop genome is large (2.8 Gb) and complex, and early attempts at assembly were fragmented. Recent advances have made assembly of the hop genome more tractable, transforming the extent of investigation that can occur. The chromosome-level assembly of Cascade was developed by scaffolding the previously reported Cascade assembly generated with PacBio long-read sequencing and polishing with Illumina short-read DNA sequencing. We developed gene models and repeat annotations and used a controlled bi-parental mapping population to identify significant sex-associated markers. We assessed molecular evolution in gene sequences, gene family expansion and contraction, and time of divergence from Cannabis sativa and other closely related plant species using Bayesian inference. We identified the putative sex chromosome in the female genome based on significant sex-associated markers from the bi-parental mapping population. While the estimate of repeat content (~64%) is similar to the estimate for the hemp genome, syntenic blocks in hop contain a greater percentage of LTRs. Hop is enriched for disease resistance-associated genes in syntenic gene blocks and expanded gene families. The Cascade chromosome-level assembly will inform cultivation strategies and serve to deepen our understanding of the hop genomic landscape, benefiting hop researchers and the Cannabaceae genomics community.

Downy mildew resistance is genetically mediated by prophylactic production of phenylpropanoids in hop.

Downy mildew in hop (Humulus lupulus L.) is caused by Pseudoperonospora humuli and generates significant losses in quality and yield. To identify the biochemical processes that confer natural downy mildew resistance (DMR), a metabolome- and genome-wide association study was performed. Inoculation of a high density genotyped F1 hop population (n = 192) with the obligate biotrophic oomycete P. humuli led to variation in both the levels of thousands of specialized metabolites and DMR. We observed that metabolites of almost all major phytochemical classes were induced 48 hr after inoculation. But only a small number of metabolites were found to be correlated with DMR and these were enriched with phenylpropanoids. These metabolites were also correlated with DMR when measured from the non-infected control set. A genome-wide association study revealed co-localization of the major DMR loci and the phenylpropanoid pathway markers indicating that the major contribution to resistance is mediated by these metabolites in a heritable manner. The application of three putative prophylactic phenylpropanoids led to a reduced degree of leaf infection in susceptible genotypes, confirming their protective activity either directly or as precursors of active compounds.

Identification of tandem repeat families from long-read sequences of Humulus lupulus.

Hop (Humulus lupulus L.) is known for its use as a bittering agent in beer and has a rich history of cultivation, beginning in Europe and now spanning the globe. There are five wild varieties worldwide, which may have been introgressed with cultivated varieties. As a dioecious species, its obligate outcrossing, non-Mendelian inheritance, and genomic structural variability have confounded directed breeding efforts. Consequently, understanding the hop genome represents a considerable challenge, requiring additional resources. In order to facilitate investigations into the transmission genetics of hop, we report here a tandem repeat discovery pipeline developed using k-mer filtering and dot plot analysis of PacBio long-read sequences from the hop cultivar Apollo. From this we identified 17 new and distinct tandem repeat sequence families, which represent candidates for FISH probe development. For two of these candidates, HuluTR120 and HuluTR225, we produced oligonucleotide FISH probes from conserved regions of and demonstrated their utility by staining meiotic chromosomes from wild hop, var. neomexicanus to address, for example, questions about hop transmission genetics. Collectively, these tandem repeat sequence families represent new resources suitable for development of additional cytogenomic tools for hop research.

3D Molecular Cytology of Hop (Humulus lupulus) Meiotic Chromosomes Reveals Non-disomic Pairing and Segregation, Aneuploidy, and Genomic Structural Variation.

Hop (Humulus lupulus L.) is an important crop worldwide, known as the main flavoring ingredient in beer. The diversifying brewing industry demands variation in flavors, superior process properties, and sustainable agronomics, which are the focus of advanced molecular breeding efforts in hops. Hop breeders have been limited in their ability to create strains with desirable traits, however, because of the unusual and unpredictable inheritance patterns and associated non-Mendelian genetic marker segregation. Cytogenetic analysis of meiotic chromosome behavior has also revealed conspicuous and prevalent occurrences of multiple, atypical, non-disomic chromosome complexes, including those involving autosomes in late prophase. To explore the role of meiosis in segregation distortion, we undertook 3D cytogenetic analysis of hop pollen mother cells stained with DAPI and FISH. We used telomere FISH to demonstrate that hop exhibits a normal telomere clustering bouquet. We also identified and characterized a new sub-terminal 180 bp satellite DNA tandem repeat family called HSR0, located proximal to telomeres. Highly variable 5S rDNA FISH patterns within and between plants, together with the detection of anaphase chromosome bridges, reflect extensive departures from normal disomic signal composition and distribution. Subsequent FACS analysis revealed variable DNA content in a cultivated pedigree. Together, these findings implicate multiple phenomena, including aneuploidy, segmental aneuploidy, or chromosome rearrangements, as contributing factors to segregation distortion in hop.

Non-Mendelian Single-Nucleotide Polymorphism Inheritance and Atypical Meiotic Configurations are Prevalent in Hop.

Hop ( L.) breeding programs seek to exploit genetic resources for bitter flavor, aroma, and disease resistance. However, these efforts have been thwarted by segregation distortion including female-biased sex ratios. To better understand the transmission genetics of hop, we genotyped 4512 worldwide accessions of hop, including cultivars, landraces, and over 100 wild accessions using a genotyping-by-sequencing (GBS) approach. From the resulting ∼1.2 million single-nucleotide polymorphisms (SNPs), prequalified GBS markers were validated by inferences in population structures and phylogeny. Analysis of pseudo-testcross (Pt) mapping data from F families revealed mixed patterns of Mendelian and non-Mendelian segregation. Three-dimensional (3D) cytogenetic analysis of late meiotic prophase nuclei from two wild and two cultivated hop revealed conspicuous and prevalent occurrences of multiple, atypical, nondisomic chromosome complexes including autosomes. We used genome-wide association studies (GWAS) and fixation index (F) analysis to demonstrate selection mapping of genetic loci for key traits including sex, bitter acids, and drought tolerance. Among the possible mechanisms underlying the observed segregation distortion from the genomic data analysis, the cytogenetic analysis points to meiotic chromosome behavior as one of the contributing factors. The findings shed light on long-standing questions on the unusual transmission genetics and phenotypic variation in hop, with major implications for breeding, cultivation, and the natural history of .

Increase in cone biomass and terpenophenolics in hops ( Humulus lupulus L.) by treatment with prohexadione-calcium.

Humulus lupulus L. (hop), a specialty crop bred for flavor characteristics of the inflorescence, is an essential ingredient in beer. Hop inflorescences, commonly known as hop cones, contain terpenophenolic compounds, which are important for beer flavoring and of interest in biomedical research. Hop breeders focus their efforts on increasing cone biomass and terpenophenolic content. As an alternative to traditional breeding, hops were treated with prohexadione-calcium (Pro-Ca), a growth inhibitor previously shown to have positive agronomic effects in several crops. Application of Pro-Ca to hop plants during cone maturation induced increases in cone biomass production by 1.5-19.6% and increased terpenophenolic content by 9.1-87.3%; however, some treatments also induced significant decreases in terpenophenolic content. Induced changes in cone biomass production and terpenophenolic accumulation were most dependent on cultivar and the developmental stage at which plants were treated.

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 photodiode array detection, and light microscopy techniques.

Hop (Humulus lupulus L.) inflorescences, commonly known as "hop cones", are prized for their terpenophenolic contents, used in beer production and, more recently, in biomedical applications. In this study we investigated morphological and phytochemical characteristics of hop cones over five developmental stages, using liquid chromatography coupled to time-of-flight mass spectrometry (LC-TOF-MS), and ultrahigh performance liquid chromatography photodiode array detection (UHPLC-PDA) methods to quantitate 21 polyphenolics and seven terpenophenolics. Additionally, we used light microscopy to correlate phytochemical quantities with changes in the morphology of the cones. Significant increases in terpenophenolics, concomitant with glandular trichome development and associated gross morphological changes, were mapped over development to fluctuations in contents of polyphenolic constituents and their metabolic precursor compounds. The methods reported here can be used for targeted metabolic profiling of flavonoids, phenolic acids, and terpenophenolics in hops, and are applicable to quantitation in other crops.