Portuguese wild hop diversity assessment by fast SNP genotyping using high-resolution melting.

A set of seven SNP markers was used to evaluate the genetic diversity of wild Portuguese hops in comparison with commercial cultivars. A collection of 110 wild genotypes and 33 cultivars was characterized by a high-resolution melting analysis of short amplicons targeting SNP loci. Most of the 143 genotypes (75%) could be differentiated. Phylogenetic analysis showed three main clusters, one included almost exclusively (98%) wild accessions, being the others constituted of both wild and commercial genotypes. The study of population genetic structure placed the accessions into three genetic units, being one exclusively of Portuguese genotypes. The study showed the great biodiversity of the Portuguese hop germplasm and the notable differences (FST = 0.163, p<0.00001) from commercial hops. Results support the usefulness of the use of these seven markers for hop discrimination, with the fast and high-throughput HRMA technique for allele calling and contribute to the affirmation of the high richness breeding potential of Portuguese wild hops.

Antimicrobial Properties of Different Hop (Humulus lupulus) Genotypes.

The antimicrobial activity of hop extracts obtained from different hop genotypes were investigated against Staphylococcus aureus and Lactobacillus acidophilus. In this study the pure xanthohumol, purified ß-acids rich fraction, as well as α-acids with ß-acids rich fraction were used to test antimicrobial activity against Staphylococcus aureus and Lactobacillus acidophilus; whereby, the antimicrobial activity of different hop extracts against Lactobacillus acidophilus was studied for the first time. Microbial susceptibility to purified hydroacetonic extracts from different hop varieties was investigated by the broth microdilution assay to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The hop hydroacetonic extracts were more effective against Staphylococcus aureus than against Lactobacillus acidophilus. Strong inverse correlations of MIC and MBC values were obtained with xanthohumol, cohumulone, n+adhumulone, colupulone and n+adlupulone contents, suggesting that the identified chemical hop compounds are directly responsible for antimicrobial effects. Moreover, the effect of the growth medium strength on the MIC values of hop extracts against Staphylococcus aureus was systematically investigated for the first time. The current study also reveals the effect of different hop extracts on Staphylococcus aureus, which responds to their presence by lag phase extension and generation time prolongation.

Metabolomic Analysis of Cannabinoid and Essential Oil Profiles in Different Hemp (Cannabis sativa L.) Phenotypes.

Hemp (Cannabis sativa L.) cannabinoids and terpenoids have therapeutic effects on human and animal health. Cannabis plants can often have a relatively high heterogeneity, which leads to different phenotypes that have different chemical profiles despite being from the same variety. Little information exists about cannabinoid and terpenoid profiles in different hemp phenotypes within the same variety. For this study, 11 phenotypes from three different varieties ("Carmagnola" selected (CS), "Tiborszallasi" (TS), and "Finola" selection (FS)) were analyzed. The components of essential oil (29) were analyzed using gas chromatography with flame ionization detection (GC/FID), and 10 different cannabinoids of each phenotype were determined using high-performance liquid chromatography (HPLC). Principal component analysis (PCA) and analysis of variance (ANOVA) showed that according to the components of essential oil, FS and TS plants were more uniform than CS plants, where there were great differences between CI and CII phenotypes. The content of cannabinoid CBD-A was the highest in all four FS phenotypes. By comparing cannabinoid profiles, FS was clearly separated from TS and CS, while these two varieties were not clearly distinguishable. Phenotypes TV and CI had the highest total content of Δ-9-THC, while all phenotypes of FS had the highest total content of CBD. The highest total content of CBG was determined in phenotype CI. Obtained results are useful for the development of new supplementary ingredients, for different pharmacy treatments, and for further breeding purposes.

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.

Cannabigerol and cannabichromene in Cannabis sativa L.

In addition to delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), other phytocannabinoids, such as cannabigerol (CBG) and cannabichromene (CBC), also have beneficial effects on human health. A high content of CBG is found in plants with the B0 genotype, whereas CBC is independent of the allelic chemotype locus B. In basic research models such as mice or rats, CBG has demonstrated anticancer properties, particularly against breast cancer. CBG has shown anti-inflammatory effects on murine colitis and on inflammatory bowel disease as well as stimulatory effects on the feeding behaviors of mice. It has also exhibited inhibition of aldose reductase, which is known to cause an accumulation of sorbitol and increase glucose levels in the blood, which may lead to diabetes. Cannabinoid CBC has also shown anti-inflammatory effects and reduced hypermobility in the gut and has displayed potential in vitro effect on adult neural stem progenitor cells. CBC also exerts modest analgesic properties in rodents, as well as anti-fungal, anti-bacterial, pro-apoptotic, and anti-proliferative effects in tumor cells.

New Male Specific Markers for Hop and Application in Breeding Program.

Male specific DNA sequences were selected from a Diversity Arrays Technology (DArT) mapping study to evaluate their suitability for determination of the sex phenotype among young seedlings in a hop (Humulus lupulus L.) breeding program. Ten male specific DArT markers showed complete linkage with male sex phenotype in three crossing families. Following optimization, four were successfully converted into PCR markers and a multiplex PCR approach for their use was developed. Among 197 plants (97 from the world collection; 100 from three segregating families), 94-100% positive correlation with sex phenotypic data was achieved for the single PCR amplification, whereas the multiplex approach showed 100% correlation. To develop a fast and low-cost method, crude sample multiplex PCR was evaluated in 253 progenies from 14 segregating populations without losing accuracy. The study describes, for the first time, the routine application of molecular markers linked to male sex in an intensive Slovenian hop breeding program. The methods described could be employed for screening of sex at the seedling stage in other hop programs worldwide, thereby saving resources for desirable female plants.

Hop (Humulus lupulus L.) response mechanisms in drought stress: Proteomic analysis with physiology.

Drought is one of the major environmental devastating stressors that impair the growth and productivity of crop plants. Despite the relevance of drought stress, changes in physiology and resistance mechanisms are not completely understood for certain crops, including hop (Humulus lupulus L.). In this research the drought response of hop was studied using a conventional physiological approach (gas exchange techniques, fluorescence, relative water content measurements) and proteomic analysis (2D-DIGE). Plants of two cultivars (Aurora and Savinjski golding) were exposed to progressive drought in a pot experiment and analysed at different stress stages (mild, moderate and severe). Measurements of relative water content revealed a hydrostable water balance of hop. Photosynthesis was decreased due to stomatal and non-stomatal limitation to the same extent in both cultivars. Of 28 identified differentially abundant proteins, the majority were down regulated and included in photosynthetic (41%) and sugar metabolism (33%). Fifteen % of identified proteins were classified into the nitrogen metabolism, 4% were related to a ROS related pathway and 7% to other functions.

Antioxidant and Antimicrobial Properties of Veronica spicata L. (Plantaginaceae).

Tea made from Veronica spicata L. (syn. Pseudolysimachion spicatum (L.) Opiz, family Plantaginaceae) herb is used in traditional medicine as expectorant for cough and throat rinsing. To get insight into chemical compounds of V. spicata, the essential oil content (analysed by GC-FID and GC-MS), the content of phenolic compounds (analysed by HPLC), the content of macroelements and trace elements (analysed by ICP-AES), quantity of total phenols and total flavonoids (analysed by UV/Vis spectrophotometer), and antioxidant and antimicrobial properties were investigated. The main compounds of the essential oil were phytol (21.13%), heptacosane (10.22%) and pentacosane (8.91%). The most abundant investigated macroelement was K (8261 mg/kg) while Fe was the most represented element (32.49 mg/kg) among investigated micronutrients. Ten phenolic compounds (chrysin, rutin, quercitrin, quercitrin, and cichoric, ferulic, protocatehuic, rosmarinic, syringic and tannic acid) were identified and quantified. Additionally, V. spicata extract demonstrated notable radical-scavenging and chelating properties. The bacterial and fungal strains used in study were found to be susceptible toward methanolic and ethyl-acetate extracts with MIC values between 1.25 and 5.00 mg/mL using microdilution method. Aquose extracts were found to be antimicrobial inactive.

Antimicrobial and Antioxidant Properties of Satureja Montana L. and S. Subspicata Vis. (Lamiaceae).

Satureja montanaL. and S. subspicata Vis. (Lamiaceae) are used for centuries in traditional medicine of Balcanic people in the healing of the lymphatic nodule and respiratory system inflammation. In this paper the amount of total phenols and flavonoids (analyzed by UV/Vis spectrophotometry), phenolic compounds profile (analyzed by HPLC), antimicrobial and antioxidant activities were studied in samples collected in seven per species populations of S. montanaand S. subspicatain Croatia. Eight phenolic compounds (rutin, quercetin, caffeic, p-coumaric, ellagic, protocatehuic, rosmarinic, and syringic acid) were identified and quantified using HPLC in methanolic and ethanolic extracts. Results showed that both species contained polyphenolics and other antioxidant compounds with chelating and radical-scavenging properties. The extracts prepared from both species showed broad spectrum of antimicrobial activity on in vitrotested microbial species (Staphylococcus aureus, Escherichia coli, Candida albicans, C. dubliniensis, C. krusei, C. glabrata, C. parapsilosis, and Microsporum gypseum).

Influence of two hop (Humulus lupulus L.) varieties on in vitro dry matter and crude protein degradability and digestibility in ruminants.

BACKGROUND: Hop cones contain several antimicrobial substances. The aim of the study was to determine the effects of two hop varieties, Aurora and Dana, on substrate (diet for a dairy cow, producing 30 kg milk daily) in vitro dry matter (DM) and crude protein (CP) degradability and digestibility. RESULTS: In the in vitro trial freshly ground hops were added to the buffered rumen fluid in concentrations simulating the cow's daily intake of 50, 100 and 200 g of hops. Increasing the concentration of hops decreased (P < 0.05) both the average in vitro DM degradabilities of substrate from 725 to 592, 553 and 481 g kg(-1), respectively, and in vitro CP degradabilities of substrate from 752 to 566, 561 and 478 g kg(-1), respectively. The reduction of in vitro DM and CP degradability is counterbalanced by the (invariable) in vitro DM and CP digestibility. The difference between CP digestibility and degradability represents an estimate of the amount of rumen 'bypass' protein which increased with increasing concentration of hops from 172 to 454 g kg(-1). CONCLUSIONS: Decreased DM and CP degradability and increased amount of rumen 'bypass' protein could lower the amounts of protein required by high-producing ruminant animals. However, this supposition needs a validation with in vivo trials.

Quantitative trait loci in hop (Humulus lupulus L.) reveal complex genetic architecture underlying variation in sex, yield and cone chemistry.

BACKGROUND: Hop (Humulus lupulus L.) is cultivated for its cones, the secondary metabolites of which contribute bitterness, flavour and aroma to beer. Molecular breeding methods, such as marker assisted selection (MAS), have great potential for improving the efficiency of hop breeding. The success of MAS is reliant on the identification of reliable marker-trait associations. This study used quantitative trait loci (QTL) analysis to identify marker-trait associations for hop, focusing on traits related to expediting plant sex identification, increasing yield capacity and improving bittering, flavour and aroma chemistry. RESULTS: QTL analysis was performed on two new linkage maps incorporating transferable Diversity Arrays Technology (DArT) markers. Sixty-three QTL were identified, influencing 36 of the 50 traits examined. A putative sex-linked marker was validated in a different pedigree, confirming the potential of this marker as a screening tool in hop breeding programs. An ontogenetically stable QTL was identified for the yield trait dry cone weight; and a QTL was identified for essential oil content, which verified the genetic basis for variation in secondary metabolite accumulation in hop cones. A total of 60 QTL were identified for 33 secondary metabolite traits. Of these, 51 were pleiotropic/linked, affecting a substantial number of secondary metabolites; nine were specific to individual secondary metabolites. CONCLUSIONS: Pleiotropy and linkage, found for the first time to influence multiple hop secondary metabolites, have important implications for molecular selection methods. The selection of particular secondary metabolite profiles using pleiotropic/linked QTL will be challenging because of the difficulty of selecting for specific traits without adversely changing others. QTL specific to individual secondary metabolites, however, offer unequalled value to selection programs. In addition to their potential for selection, the QTL identified in this study advance our understanding of the genetic control of traits of current economic and breeding significance in hop and demonstrate the complex genetic architecture underlying variation in these traits. The linkage information obtained in this study, based on transferable markers, can be used to facilitate the validation of QTL, crucial to the success of MAS.

Investigation of chemical compounds, antioxidant and antimicrobial properties of teucrium arduini L. (lamiaceae).

In this paper chemical composition of the essential oil (analysed by GC and GC-MS), the content of phenolic compounds (analysed by HPLC), quantity of total phenols and total flavonoids (analysed by UV/Vis spectrophotometer), antioxidant and antimicrobial activities of ethanolic extracts were investigated in endemic Teucrium arduini L. in population of Mt Biokovo (Croatia). The oil was characterized by a high concentration of sesquiterpene hydrocarbons (70.4%) of which ß-caryophyllene (35.2%) and germacrene D (18.7%) being the major compounds. Three phenolic compounds (quercetin, ferulic acid and rosmarinic acid) were identified and quantified in ethanolic extract of T. arduini using HPLC. The results also showed that T. arduini is a source of polyphenolic and other antioxidants with radical-scavenging and chelating properties. The ethanol extracts prepared from the leaf of T. arduini showed broad spectrum of antimicrobial activity on Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Aspergillus brasiliensis, which are susceptible on concentration below or equal to 4.00 mg/mL, whilst Microsporum gypseum was resistant at investigated concentrations.

Identification of quantitative trait loci for resistance to Verticillium wilt and yield parameters in hop (Humulus lupulus L.).

Verticillium wilt (VW) can cause substantial yield loss in hop particularly with the outbreaks of the lethal strain of Verticillium albo-atrum. To elucidate genetic control of VW resistance in hop, an F1 mapping population derived from a cross of cultivar Wye Target, with the predicted genetic basis of resistance, and susceptible male breeding line BL2/1 was developed to assess wilting symptoms and to perform QTL mapping. The genetic linkage map, constructed with 203 markers of various types using a pseudo-testcross strategy, formed ten major linkage groups (LG) of the maternal and paternal maps, covering 552.98 and 441.1 cM, respectively. A significant QTL for VW resistance was detected at LOD 7 on a single chromosomal region on LG03 of both parental maps, accounting for 24.2-26.0 % of the phenotypic variance. QTL analysis for alpha-acid content and yield parameters was also performed on this map. QTLs for these traits were also detected and confirmed our previously detected QTLs in a different pedigree and environment. The work provides the basis for exploration of QTL flanking markers for possible use in marker-assisted selection.

Genetic mapping of hop (Humulus lupulus L.) applied to the detection of QTLs for alpha-acid content.

The map locations and effects of quantitative trait loci (QTLs) were estimated for alpha-acid content in hop (Humulus lupulus L.) using amplified fragment length polymorphism (AFLP) and microsatellite marker (simple sequence repeat (SSR)) genetic linkage maps constructed from a double pseudotestcross. The mapping population consisted of 111 progeny from a cross between the German hop cultivar 'Magnum', which exhibits high levels of alpha-acids, and a wild Slovene male hop, 2/1. The progeny segregated quantitatively for alpha-acid content determined in 2002, 2003, and 2004. The maternal map consisted of 96 markers mapped on 14 linkage groups defining 661.90 cM of total map distance. The paternal map included 70 markers assigned to 12 linkage groups covering 445.90 cM of hop genome. QTL analysis indicated 4 putative QTLs (alpha1, alpha2, alpha3, and alpha4) on linkage groups (LGs) 03, 01, 09, and 03 of the female map, respectively. QTLs explained 11.9%-24.8% of the phenotypic variance. The most promising QTL to be used in marker-assisted selection is alpha2, the peak of which colocated exactly with the AFLP marker. Three chalcone synthase-like genes (chs2, chs3, and chs4) involved in hop bitter acid synthesis mapped together on LG04 of the female map. Saturation of the maps, particularly the putative QTL regions, will be carried out using SSR markers, and the stability of the QTLs will be tested in the coming years.