Unveiling the phenology and associated floral regulatory pathways of Humulus lupulus L. in subtropical conditions.

MAIN CONCLUSION: The hop phenological cycle was described in subtropical condition of Brazil showing that flowering can happen at any time of year and this was related to developmental molecular pathways. Hops are traditionally produced in temperate regions, as it was believed that vernalization was necessary for flowering. Nevertheless, recent studies have revealed the potential for hops to flower in tropical and subtropical climates. In this work, we observed that hops in the subtropical climate of Minas Gerais, Brazil grow and flower multiple times throughout the year, independently of the season, contrasting with what happens in temperate regions. This could be due to the photoperiod consistently being inductive, with daylight hours below the described threshold (16.5 h critical). We observed that when the plants reached 7-9 nodes, the leaves began to transition from heart-shaped to trilobed-shaped, which could be indicative of the juvenile to adult transition. This could be related to the fact that the 5th node (in plants with 10 nodes) had the highest expression of miR156, while two miR172s increased in the 20th node (in plants with 25 nodes). Hop flowers appeared later, in the 25th or 28th nodes, and the expression of HlFT3 and HlFT5 was upregulated in plants between 15 and 20 nodes, while the expression of HlTFL3 was upregulated in plants with 20 nodes. These results indicate the role of axillary meristem age in regulating this process and suggest that the florigenic signal should be maintained until the hop plants bloom. In addition, it is possible that the expression of TFL is not sufficient to inhibit flowering in these conditions and promote branching. These findings suggest that the reproductive transition in hop under inductive photoperiodic conditions could occur in plants between 15 and 20 nodes. Our study sheds light on the intricate molecular mechanisms underlying hop floral development, paving the way for potential advancements in hop production on a global scale.

Structure and genetic diversity of Canadian Maritimes wild hops.

Studies on the northeastern American native hops (Humulus lupulus ssp. lupuloides) from the Canadian Maritimes are scarce. This study aimed to evaluate the genetic structure and diversity among 25 wild-collected hops from three Canadian Maritime provinces using microsatellite (simple sequence repeat (SSR)) markers. Based on 43 SSR markers, four distinct subgroups were found, with a low molecular variance (19%) between subgroups and a high variance (81%) within subgroups. The Nei's unbiased genetic distance between clusters ranged from 0.01 to 0.08, the genetic distance between clusters 2 and 3 being the farthest and that between clusters 1 and 2 the closest. Cluster 2 captured the highest overall diversity. A total of 18 SSR markers clearly discriminated hop clones by detecting putative subspecies-specific haplotypes, differentiating clones of native-wild H. lupulus ssp. lupuloides from the naturalized old and modern hop cultivars. Seven of the 18 SSR markers also differentiated two clones from the same site from one another. The study is the first, using molecular markers, to identify SSR markers with potential for intellectual property protection in Canadian Maritimes hops. The SSR markers herein used can be prime tools for hop breeders and growers in the region.

A droplet vitrification cryopreservation protocol for conservation of hops (Humulus lupulus) genetic resources.

Hops (Humulus lupulus L.) is essentially used in the brewing industry as it contributes to flavor, and aroma of beer. However, the genetic diversity of hops is increasingly threatened by diseases, environmental changes, and urbanization. Cryopreservation has emerged as a pivotal strategy for safeguarding and maintaining the genetic diversity of hops. The present work presents a comprehensive study on the cryopreservation of hops, focusing on the development and optimization of a droplet vitrification based cryopreservation protocol. Shoot tips excised from one month old in vitro cultures were precultured on 0.3 M sucrose, dehydrated in a loading solution followed by treatment with PVS2 solution for different durations. Significant effect of PVS2 dehydration was observed on post-thaw survival and regeneration after cryoconservation with maximum 50% post-thaw regeneration observed in shoot tips dehydrated in PVS2 for 30 min. Genetic fidelity of the regenerated plants was confirmed using 30 ISSR markers. Reproducibility of the developed protocol was tested on seven other accessions and post thaw regeneration ranging from 43 to 70% was observed across the accessions. The present study reports a highly efficient protocol for conservation of hops germplasm. The results indicate that droplet vitrification can be used as a reliable and sustainable approach for hop genetic preservation, with high survival rates and minimal genetic alterations observed in cryopreserved samples. To the best of our knowledge, this is the first report on DV based cryopreservation of hops germplasm.

The hops (Humulus lupulus) genome contains a mid-sized terpene synthase family that shows wide functional and allelic diversity.

BACKGROUND: Hops (Humulus lupulus L.) are a dioecious climbing perennial, with the dried mature "cones" (strobili) of the pistillate/female inflorescences being widely used as both a bittering agent and to enhance the flavour of beer. The glandular trichomes of the bract and bracteole flowering structures of the cones produce an abundance of secondary metabolites, such as terpenoids, bitter acids and prenylated phenolics depending on plant genetics, developmental stage and environment. More knowledge is required on the functional and allelic diversity of terpene synthase (TPS) genes responsible for the biosynthesis of volatile terpenes to assist in flavour-directed hop breeding. RESULTS: Major volatile terpene compounds were identified using gas chromatography-mass spectrometry (GC-MS) in the ripe cones of twenty-one hop cultivars grown in New Zealand. All cultivars produced the monoterpene ß-myrcene and the sesquiterpenes α-humulene and ß-caryophyllene, but the quantities varied broadly. Other terpenes were found in large quantities in only a smaller subset of cultivars, e.g. ß-farnesene (in seven cultivars) and α-pinene (in four). In four contrasting cultivars (Wakatu™, Wai-iti™, Nelson Sauvin™, and 'Nugget'), terpene production during cone development was investigated in detail, with concentrations of some of the major terpenes increasing up to 1000-fold during development and reaching maximal levels from 50-60 days after flowering. Utilising the published H. lupulus genome, 87 putative full-length and partial terpene synthase genes were identified. Alleles corresponding to seven TPS genes were amplified from ripe cone cDNA from multiple cultivars and subsequently functionally characterised by transient expression in planta. Alleles of the previously characterised HlSTS1 produced humulene/caryophyllene as the major terpenes. HlRLS alleles produced (R)-(-)-linalool, whilst alleles of two sesquiterpene synthase genes, HlAFS1 and HlAFS2 produced α-farnesene. Alleles of HlMTS1, HlMTS2 and HlTPS1 were inactive in all the hop cultivars studied. CONCLUSIONS: Alleles of four TPS genes were identified and shown to produce key aroma volatiles in ripe hop cones. Multiple expressed but inactive TPS alleles were also identified, suggesting that extensive loss-of-function has occurred during domestication and breeding of hops. Our results can be used to develop hop cultivars with novel/improved terpene profiles using marker-assisted breeding strategies to select for, or against, specific TPS alleles.

Identification of quantitative trait loci associated with R1-mediated resistance to powdery mildew and sex determination in hop (Humulus lupulus L.).

KEY MESSAGE: Two QTL were identified using linkage mapping approaches, one on hop linkage group 3 (qHl_Chr3.PMR1) associated with powdery mildew resistance and a second on linkage group 10 (cqHl_ChrX.SDR1) associated with sex determination. Hop (Humulus lupulus L.) is a dioecious species cultivated for use in beer. Hop powdery mildew, caused by Podosphaera macularis, is a constraint in many growing regions. Thus, identifying markers associated with powdery mildew resistance and sex provides the opportunity to pyramid R-genes and select female plants as seedlings, respectively. Our objectives were to characterize the genetic basis of R1-mediated resistance in the cultivar Zenith which provides resistance to pathogen races in the US, identify quantitative trait loci (QTL) associated with R1 and sex, and develop markers for molecular breeding-based approaches. Phenotypic evaluation of the population indicated that R1-based resistance and sex are inherited monogenically. We constructed a genetic map using 1339 single nucleotide polymorphisms (SNPs) based upon genotype-by-sequencing of 128 F1 progeny derived from a Zenith × USDA 21058M biparental population. SNPs were assigned to 10 linkage groups comprising a map length of 1204.97 cM with an average density of 0.94 cM/marker. Quantitative trait locus mapping identified qHl_Chr3.PMR1, associated with R1 on linkage group 3 (LOD = 23.57, R2 = 57.2%), and cqHl_ChrX.SDR1, associated with sex on linkage group 10 (LOD = 5.42, R2 = 25.0%). Kompetitive allele-specific PCR (KASP) assays were developed for both QTL and assessed against diverse germplasm. Our results indicate that KASP markers associated with R1 may be limited to materials that are pedigree-related to Zenith, whereas markers associated with sex may be transferable across populations. The high-density map, QTL, and associated KASP markers will enable selecting for sex and R1-mediated resistance in hop.

Optimization and Validation of Singleplex and Multiplex RT-qPCR for Detection of Citrus bark cracking viroid (CBCVd), Hop latent viroid (HLVd), and Hop stunt viroid (HSVd) in Hops (Humulus lupulus).

Direct crop losses due to plant diseases and the measures used to control them have significant agricultural and economic impacts. The shift from diverse small-scale to large-scale genetically uniform monoculture production, along with agricultural intensification and climate change, has led to several known epidemics in man-made agroecosystems that have been rendered more vulnerable to pathogens. One such example is hop growing, which is threatened by highly aggressive hop viroids. Since 2007, almost one-third (about 500 ha) of Slovenian hop gardens have been affected by severe hop stunt disease caused by Citrus bark cracking viroid (CBCVd), which continues to spread despite strict prevention measures. We have developed and validated a multiplex RT-qPCR (mRT-qPCR) for the sensitive detection of CBCVd, Hop latent viroid (HLVd), and Hop stunt viroid (HSVd). Singleplex RT-qPCR assays were designed individually and subsequently combined in a one-step mRT-qPCR assay. Hop-specific mRNA170 and mRNA1192 internal controls were also developed to detect possible PCR inhibition. Analytical specificity was tested on 35 samples from different hosts, geographic regions, and combinations of viroids. Method validation showed that mRT-qPCR had lower sensitivity than singleplex RT-qPCR, while specificity, selectivity, repeatability, and reproducibility remained unchanged. The newly developed assays were found to be robust, reliable, and suitable for large-scale screening of hop viroids.

"Pathomorphogenic" Changes Caused by Citrus Bark Cracking Viroid and Transcription Factor TFIIIA-7ZF Variants Support Viroid Propagation in Tobacco.

Viroids are small, non-coding, pathogenic RNAs with the ability to disturb plant developmental processes. This dysregulation redirects the morphogenesis of plant organs, significantly impairing their functionality. Citrus bark cracking viroid (CBCVd) causes detrimental developmental distortions in infected hops (Humulus lupulus) and causes significant economic losses. CBCVd can infect cells and tissues of the model plant tobacco (Nicotiana tabacum), provided it is delivered via transgenesis. The levels of CBCVd in tobacco were enhanced in plant hybrids expressing CBCVd cDNAs and either the tobacco or hop variant of TFIIIA-7ZF, a viroid-mediated splicing derivative of transcription factor IIIA, which is important for viroid replication by DNA-dependent RNA polymerase II. The TFIIIA-7ZF variants can change the tobacco morphogenesis if expressed in leaves and shoots. In addition to the splitting of shoots, the "pathomorphogenic" network in hybrid plants expressing CBCVd and HlTFIIIA-7ZF induced leaf fusions and malformations. Moreover, CBCVd can dramatically change another morphogenesis into teratomic and petal-like tissues if propagated above some limit in young transgenic tobacco microspores and anthers. By comparative RNA profiling of transgenic tobacco shoots bearing TFIIIA-7ZFs and CBCVd-transformed/infected anthers, we found a differential expression of many genes at p < 0.05. As the main common factor showing the differential up-regulation in shoot and anther tissues, a LITTLE ZIPPER 2-like transcription factor was found. We propose that this factor, which can interact as a competitive inhibitor of the also dysregulated homeobox-leucin zipper family protein (HD-ZIPIII) in apical meristem, is essential for a network responsible for some morphological changes and modifications of plant degradome within shoot meristem regulation and secondary xylem differentiation.

Identification and characterization of long non-coding RNA and their response against citrus bark cracking viroid infection in Humulus lupulus.

Long non-coding RNAs (lncRNAs) are a highly heterogeneous class of non-protein-encoding transcripts that play an essential regulatory role in diverse biological processes, including stress responses. The severe stunting disease caused by Citrus bark cracking viroid (CBCVd) poses a major threat to the production of Humulus lupulus (hop) plants. In this study, we systematically investigate the characteristics of the lncRNAs in hop and their role in CBCVd-infection using RNA-sequencing data. Following a stringent filtration criterion, a total of 3598 putative lncRNAs were identified with a high degree of certainty, of which 19% (684) of the lncRNAs were significantly differentially expressed (DE) in CBCVd-infected hop, which were predicted to be mainly involved in plant-pathogen interactions, kinase cascades, secondary metabolism and phytohormone signal transduction. Besides, several lncRNAs and CBCVd-responsive lncRNAs were identified as the precursor of microRNAs and predicted as endogenous target mimics (eTMs) for hop microRNAs involved in CBCVd-infection.

Tracing foreign sequences in plant transcriptomes and genomes using OCT4, a POU domain protein.

Contaminations in sequencing data, especially in reference genomes, lead to inevitable errors in downstream analyses. Similarly, presence of contaminants in transcriptomes, misrepresents the molecular basis of various interactions. In this study, we report the presence of a large number of plant transcriptomes contaminated with RNAs encoding POU domain proteins; a family of proteins that has not been reported in plants and fungi. Besides, our findings illustrated that there are four POU domain protein-coding sequences in the reference genome of Rhodamnia argentea. It turned out that the existing foreign fragments are related to arthropods that are considered as plant pests. We also identified two contaminated draft genomes, Humulus lupulus and Cannabis sativa that contained complete rDNA sequences originating from Tetranychus species. As a result, careful screening of sequencing data before releasing them in public databases or checking existing genomes for possible contaminations is recommended.

Plant genera Cannabis and Humulus share the same pair of well-differentiated sex chromosomes.

We recently described, in Cannabis sativa, the oldest sex chromosome system documented so far in plants (12-28 Myr old). Based on the estimated age, we predicted that it should be shared by its sister genus Humulus, which is known also to possess XY chromosomes. Here, we used transcriptome sequencing of an F1 family of H. lupulus to identify and study the sex chromosomes in this species using the probabilistic method SEX-DETector. We identified 265 sex-linked genes in H. lupulus, which preferentially mapped to the C. sativa X chromosome. Using phylogenies of sex-linked genes, we showed that a region of the sex chromosomes had already stopped recombining in an ancestor of both species. Furthermore, as in C. sativa, Y-linked gene expression reduction is correlated to the position on the X chromosome, and highly Y degenerated genes showed dosage compensation. We report, for the first time in Angiosperms, a sex chromosome system that is shared by two different genera. Thus, recombination suppression started at least 21-25 Myr ago, and then (either gradually or step-wise) spread to a large part of the sex chromosomes (c. 70%), leading to a degenerated Y chromosome.

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.

Noncatalytic chalcone isomerase-fold proteins in Humulus lupulus are auxiliary components in prenylated flavonoid biosynthesis.

Xanthohumol (XN) and demethylxanthohumol (DMX) are specialized prenylated chalconoids with multiple pharmaceutical applications that accumulate to high levels in the glandular trichomes of hops (Humulus lupulus L.). Although all structural enzymes in the XN pathway have been functionally identified, biochemical mechanisms underlying highly efficient production of XN have not been fully resolved. In this study, we characterized two noncatalytic chalcone isomerase (CHI)-like proteins (designated as HlCHIL1 and HlCHIL2) using engineered yeast harboring all genes required for DMX production. HlCHIL2 increased DMX production by 2.3-fold, whereas HlCHIL1 significantly decreased DMX production by 30%. We show that CHIL2 is part of an active DMX biosynthetic metabolon in hop glandular trichomes that encompasses a chalcone synthase (CHS) and a membrane-bound prenyltransferase, and that type IV CHI-fold proteins of representative land plants contain conserved function to bind with CHS and enhance its activity. Binding assays and structural docking uncover a function of HlCHIL1 to bind DMX and naringenin chalcone to stabilize the ring-open configuration of these chalconoids. This study reveals the role of two HlCHILs in DMX biosynthesis in hops, and provides insight into their evolutionary development from the ancestral fatty acid-binding CHI-fold proteins to specialized auxiliary proteins supporting flavonoid biosynthesis in plants.

Core RNA Interference Genes Involved in miRNA and Ta-siRNA Biogenesis in Hops and Their Expression Analysis after Challenging with Verticillium nonalfalfae.

RNA interference is an evolutionary conserved mechanism by which organisms regulate the expression of genes in a sequence-specific manner to modulate defense responses against various abiotic or biotic stresses. Hops are grown for their use in brewing and, in recent years, for the pharmaceutical industry. Hop production is threatened by many phytopathogens, of which Verticillium, the causal agent of Verticillium wilt, is a major contributor to yield losses. In the present study, we performed identification, characterization, phylogenetic, and expression analyses of three Argonaute, two Dicer-like, and two RNA-dependent RNA polymerase genes in the susceptible hop cultivar Celeia and the resistant cultivar Wye Target after infection with Verticillium nonalfalfae. Phylogeny results showed clustering of hop RNAi proteins with their orthologues from the closely related species Cannabis sativa, Morus notabilis and Ziziphus jujuba which form a common cluster with species of the Rosaceae family. Expression analysis revealed downregulation of argonaute 2 in both cultivars on the third day post-inoculation, which may result in reduced AGO2-siRNA-mediated posttranscriptional gene silencing. Both cultivars may also repress ta-siRNA biogenesis at different dpi, as we observed downregulation of argonaute 7 in the susceptible cultivar on day 1 and downregulation of RDR6 in the resistant cultivar on day 3 after inoculation.

Mapping the Gene Expression Spectrum of Mediator Subunits in Response to Viroid Infection in Plants.

The mediator (MED) represents a large, conserved, multi-subunit protein complex that regulates gene expression through interactions with RNA polymerase II and enhancer-bound transcription factors. Expanding research accomplishments suggest the predominant role of plant MED subunits in the regulation of various physiological and developmental processes, including the biotic stress response against bacterial and fungal pathogens. However, the involvement of MED subunits in virus/viroid pathogenesis remains elusive. In this study, we investigated for the first time the gene expression modulation of selected MED subunits in response to five viroid species (Apple fruit crinkle viroid (AFCVd), Citrus bark cracking viroid (CBCVd), Hop latent viroid (HLVd), Hop stunt viroid (HSVd), and Potato spindle tuber viroid (PSTVd)) in two model plant species (Nicotiana tabacum and N. benthamiana) and a commercially important hop (Humulus lupulus) cultivar. Our results showed a differential expression pattern of MED subunits in response to a viroid infection. The individual plant MED subunits displayed a differential and tailored expression pattern in response to different viroid species, suggesting that the MED expression is viroid- and plant species-dependent. The explicit evidence obtained from our results warrants further investigation into the association of the MED subunit with symptom development. Together, we provide a comprehensive portrait of MED subunit expression in response to viroid infection and a plausible involvement of MED subunits in fine-tuning transcriptional reprogramming in response to viroid infection, suggesting them as a potential candidate for rewiring the defense response network in plants against pathogens.

Evaluation of Disease Severity and Global Transcriptome Response Induced by Citrus bark cracking viroid, Hop latent viroid, and Their Co-Infection in Hop (Humulus lupulus L.).

Viroids are small non-capsidated, single-stranded, covalently-closed circular noncoding RNA replicons of 239-401 nucleotides that exploit host factors for their replication, and some cause disease in several economically important crop plants, while others appear to be benign. The proposed mechanisms of viroid pathogenesis include direct interaction of the genomic viroid RNA with host factors and post-transcriptional or transcriptional gene silencing via viroid-derived small RNAs (vd-sRNAs) generated by the host defensive machinery. Humulus lupulus (hop) plants are hosts to several viroids among which Hop latent viroid (HLVd) and Citrus bark cracking viroid (CBCVd) are attractive model systems for the study of viroid-host interactions due to the symptomless infection of the former and severe symptoms induced by the latter in this indicator host. To better understand their interactions with hop plant, a comparative transcriptomic analysis based on RNA sequencing (RNA-seq) was performed to reveal the transcriptional alterations induced as a result of single HLVd and CBCVd infection in hop. Additionally, the effect of HLVd on the aggressiveness of CBCVd that underlies severe stunting in hop in a mixed infection was studied by transcriptomic analysis. Our analysis revealed that CBCVd infection resulted in dynamic changes in the activity of genes as compared to single HLVd infection and their mixed infection. The differentially expressed genes that are involved in defense, phytohormone signaling, photosynthesis and chloroplasts, RNA regulation, processing and binding; protein metabolism and modification; and other mechanisms were more modulated in the CBCVd infection of hop. Nevertheless, Gene Ontology (GO) classification and pathway enrichment analysis showed that the expression of genes involved in the proteolysis mechanism is more active in a mixed infection as compared to a single one, suggesting co-infecting viroids may result in interference with host factors more prominently. Collectively, our results provide a deep transcriptome of hop and insight into complex single HLVd, CBCVd, and their coinfection in hop-plant interactions.

Genome-wide transcriptome profiling of transgenic hop (Humulus lupulus L.) constitutively overexpressing HlWRKY1 and HlWDR1 transcription factors.

BACKGROUND: The hop plant (Humulus lupulus L.) is a valuable source of several secondary metabolites, such as flavonoids, bitter acids, and essential oils. These compounds are widely implicated in the beer brewing industry and are having potential biomedical applications. Several independent breeding programs around the world have been initiated to develop new cultivars with enriched lupulin and secondary metabolite contents but met with limited success due to several constraints. In the present work, a pioneering attempt has been made to overexpress master regulator binary transcription factor complex formed by HlWRKY1 and HlWDR1 using a plant expression vector to enhance the level of prenylflavonoid and bitter acid content in the hop. Subsequently, we performed transcriptional profiling using high-throughput RNA-Seq technology in leaves of resultant transformants and wild-type hop to gain in-depth information about the genome-wide functional changes induced by HlWRKY1 and HlWDR1 overexpression. RESULTS: The transgenic WW-lines exhibited an elevated expression of structural and regulatory genes involved in prenylflavonoid and bitter acid biosynthesis pathways. In addition, the comparative transcriptome analysis revealed a total of 522 transcripts involved in 30 pathways, including lipids and amino acids biosynthesis, primary carbon metabolism, phytohormone signaling and stress responses were differentially expressed in WW-transformants. It was apparent from the whole transcriptome sequencing that modulation of primary carbon metabolism and other pathways by HlWRKY1 and HlWDR1 overexpression resulted in enhanced substrate flux towards secondary metabolites pathway. The detailed analyses suggested that none of the pathways or genes, which have a detrimental effect on physiology, growth and development processes, were induced on a genome-wide scale in WW-transgenic lines. CONCLUSIONS: Taken together, our results suggest that HlWRKY1 and HlWDR1 simultaneous overexpression positively regulates the prenylflavonoid and bitter acid biosynthesis pathways in the hop and thus these transgenes are presented as prospective candidates for achieving enhanced secondary metabolite content in the hop.

Diversity and evolution of the repetitive genomic content in Cannabis sativa.

BACKGROUND: The repetitive content of the genome, once considered to be "junk DNA", is in fact an essential component of genomic architecture and evolution. In this study, we used the genomes of three varieties of Cannabis sativa, three varieties of Humulus lupulus and one genotype of Morus notabilis to explore their repetitive content using a graph-based clustering method, designed to explore and compare repeat content in genomes that have not been fully assembled. RESULTS: The repetitive content in the C. sativa genome is mainly composed of the retrotransposons LTR/Copia and LTR/Gypsy (14% and 14.8%, respectively), ribosomal DNA (2%), and low-complexity sequences (29%). We observed a recent copy number expansion in some transposable element families. Simple repeats and low complexity regions of the genome show higher intra and inter species variation. CONCLUSIONS: As with other sequenced genomes, the repetitive content of C. sativa's genome exhibits a wide range of evolutionary patterns. Some repeat types have patterns of diversity consistent with expansions followed by losses in copy number, while others may have expanded more slowly and reached a steady state. Still, other repetitive sequences, particularly ribosomal DNA (rDNA), show signs of concerted evolution playing a major role in homogenizing sequence variation.

The Use of Metabolomics to Elucidate Resistance Markers against Damson-Hop Aphid.

Phorodon humuli (Damson-hop aphid) is one of the major pests of hops in the northern hemisphere. It causes significant yield losses and reduces hop quality and economic value. Damson-hop aphid is currently controlled with insecticides, but the number of approved pesticides is steadily decreasing. In addition, the use of insecticides almost inevitably results in the development of resistant aphid genotypes. An integrated approach to pest management in hop cultivation is therefore badly needed in order to break this cycle and to prevent the selection of strains resistant to the few remaining registered insecticides. The backbone of such an integrated strategy is the breeding of hop cultivars that are resistant to Damson-hop aphid. However, up to date mechanisms of hops resistance towards Damson-hop aphids have not yet been unraveled. In the experiments presented here, we used metabolite profiling followed by multivariate analysis and show that metabolites responsible for hop aroma and flavor (sesquiterpenes) in the cones can also be found in the leaves, long before the hop cones develop, and may play a role in resistance against aphids. In addition, aphid feeding induced a change in the metabolome of all hop genotypes particularly an increase in a number of oxidized compounds, which suggests this may be part of a resistance mechanism.

Hop stunt viroid: Effect on Host (Humulus lupulus) Transcriptome and Its Interactions With Hop Powdery Mildew (Podospheara macularis).

Viroids are the smallest known plant pathogens that exploit host systems for their replication and cause diseases in many hosts. In this study, the host response of hop plants to Hop stunt viroid (HSVd) infection was studied through transcriptome analysis. RNA sequence analysis of hop leaves infected with HSVd revealed dynamic changes in hop gene expression. Defense-related genes and genes involved in lipid and terpenoid metabolism are the major categories that showed differential expression due to HSVd infection. Additionally, the effect of HSVd on development of hop powdery mildew (Podospheara macularis) (HPM) was studied. Transcriptome analysis followed by quantitative reverse transcription-polymerase chain reaction analysis showed that transcript levels of pathogenesis-related (PR) genes such as PR protein 1, chitinase, and thaumatin-like protein genes are induced in leaves infected with HPM alone. The response in these genes to HPM is significantly down-regulated in leaves with HSVd-HPM mixed infection. These results confirm that HSVd alters host metabolism, physiology, and plant defense responses. Nevertheless, in detached leaf assays, HPM consistently expanded faster on HSVd-negative leaves relative to HSVd-positive leaves. Although HSVd infection suppresses elements associated with the host immunity response, infection by HSVd is antagonistic to HPM infection of hops.

Identification of male-specific AFLP and SCAR markers in the dioecious plant Humulus scandens.

In this study, 17 male-specific amplified fragment length polymorphism (AFLP) markers were identified between male and female Humulus scandens plants. BLAST analysis revealed that 7 of the 17 sex-linked sequences were highly similar to retrotransposons. Two stable male-specific sequence-characterized amplified regions (SCAR) markers were developed. These AFLP and SCAR markers are novel molecular probes that can be used efficiently to identify the genetic gender of H. scandens and may provide a basis for further investigations on the evolution of sex chromosomes.