Jernej Jakse

Characterization of Verticillium albo-atrum Field Isolates Using Pathogenicity Data and AFLP Analysis

Since 1997, hop wilt induced by a virulent pathotype of Verticillium albo-atrum has caused considerable economic losses in hop fields in Slovenia. In all, 20 isolates of V. albo-atrum, including 12 from plants affected with the lethal form (PG2) of hop wilt, 6 from plants with the mild form (PG1), 1 from cucumber, and 1 from petunia, as well as 1 isolate of V. dahliae each from hop and green pepper, were analyzed by amplified fragment length polymorphism (AFLP). Differences in the virulence of hop isolates were confirmed by pathogenicity tests on hop cultivars. The AFLP method was optimized for analysis of these fungi and 7 of 39 primer combinations tested were used for the analysis of polymorphism among isolates. Cluster analysis of AFLP data divided the isolates into two, well-separated V. albo-atrum and V. dahliae clusters, confirming that the two species are genetically distinct. Within the V. albo-atrum cluster, isolates were further separated into two distinct groups: the A1 group contained PG1 hop pathotype and cucumber and petunia isolates, and the A2 group all hop isolates of the PG2 pathotype. Minor genetic variation was detected within pathotype-associated AFLP groups, but the clear separation of V. albo-atrum hop isolates according to their level of virulence shows genetic differentiation among hop V. albo-atrum pathotypes.

Origins of Allium ampeloprasum horticultural groups and a molecular phylogeny of the section Allium (Allium: Alliaceae)

The subgenus Allium section Allium includes economically important species, such as garlic and leek, as well as other polyploid minor crops. Phylogenetic studies within this section, with a focus on horticultural groups within A. ampeloprasum, were performed on 31 accessions of 17 species using the nuclear ribosomal DNA internal transcribed spacer (ITS) region and the chloroplast trnL-F and trnD-T regions. The results confirmed the monophyly of section Allium. Four main clades were identified on all ITS analyses but the relationships among those and the remaining species studied within section Allium remained unresolved. Trees based on cpDNA recovered two major clades and a topology only partly congruent with that of the ITS tree. Intra-individual polymorphism of the ITS region proved useful in tracking putative parent species of polyploid taxa. The allopolyploid origin of great headed garlic (GHG), A. iranicum and A. polyanthum was confirmed. No signs of hybridization in leek or kurrat were detected but possible introgression events were identified in pearl onion and bulbous leek. Although GHG is often used as a garlic substitute, molecular analysis revealed only a distant relationship with garlic. We also clarified the previous incorrect classification of cultivated forms within A. ampeloprasum, by showing that leek, kurrat, pearl onion, and bulbous leek should be considered separately from GHG.

High throughput isolation of microsatellites in hop (Humulus lupulus L.)

Procedures to generate usable microsatellite marker sequences should be optimized for cost-effectiveness in each species. For hop, we have used a cocktail of several restriction enzymes to digest the genomic DNA. This is followed by capture of microsatellite-containing sequences with long microsatellite probes attached to a membrane. The enrichment level for GA and GT libraries was 37% and 35%, respectively, and 100% of the clones contained microsatellite sequences. Libraries can be generated from genomic DNA in approximately 10 d.

High-throughput genotyping of hop ( Humulus lupulus L.) utilising diversity arrays technology (DArT)

Implementation of molecular methods in hop (Humulus lupulus L.) breeding is dependent on the availability of sizeable numbers of polymorphic markers and a comprehensive understanding of genetic variation. However, use of molecular marker technology is limited due to expense, time inefficiency, laborious methodology and dependence on DNA sequence information. Diversity arrays technology (DArT) is a high-throughput cost-effective method for the discovery of large numbers of quality polymorphic markers without reliance on DNA sequence information. This study is the first to utilise DArT for hop genotyping, identifying 730 polymorphic markers from 92 hop accessions. The marker quality was high and similar to the quality of DArT markers previously generated for other species; although percentage polymorphism and polymorphism information content (PIC) were lower than in previous studies deploying other marker systems in hop. Genetic relationships in hop illustrated by DArT in this study coincide with knowledge generated using alternate methods. Several statistical analyses separated the hop accessions into genetically differentiated North American and European groupings, with hybrids between the two groups clearly distinguishable. Levels of genetic diversity were similar in the North American and European groups, but higher in the hybrid group. The markers produced from this time and cost-efficient genotyping tool will be a valuable resource for numerous applications in hop breeding and genetics studies, such as mapping, marker-assisted selection, genetic identity testing, guidance in the maintenance of genetic diversity and the directed breeding of superior cultivars.

Genetic variability and virulence among Verticillium albo-atrum isolates from hop

Verticillium wilt, caused by Verticillium albo-atrum or V. dahliae, is an important disease of many worldwide crop species. In Europe, V. albo-atrum isolates infecting hop express different levels of virulence, inducing mild or lethal disease syndromes, and it is therefore an attractive model for studying the virulence of this pathogen. In this work, eleven amplified fragment length polymorphism (AFLP) primer combinations were used to analyze genetic variability among 55 V. albo-atrum hop isolates from four European hop growing regions, as well as isolates from other hosts and V. dahliae isolates. Cluster analysis divided V. albo-atrum and V. dahliae isolates into two well-separated groups. Within the V. dahliae cluster, isolates were separated without host specific grouping, although no host adapted isolates were included. In V. albo-atrum, the alfalfa isolates were distinct from isolates of other hosts, where a high association with virulence was observed in hop and tomato isolates. All lethal hop isolates were genetically different from mild hop isolates. The lethal hop isolates from England and Slovenia expressed the same virulence phenotype, although they showed a different AFLP pattern. The mild hop isolates formed two subgroups, to which isolates clustered irrespective of geographical location. These data suggest multiple origins of V. albo-atrum hop isolates, and the possible appearance of new virulent isolates in the future in other hop growing regions.

Trinucleotide microsatellite repeat is tightly linked to male sex in hop (Humulus lupulus L.)

Hop is a dioecious perennial, the female plants of which are cultivated for production of resin, used mainly in the brewing industry. Sex determination of hop plants is phenotypically determined in the second year of growth, so early sex determination at seedling stage, before planting, is important for breeding and cultivation purposes. We analyzed a microsatellite locus to determine sex specific differences in hop. Alleles of the locus showed tight linkage to male character, since no cross-over event was detected in two analyzed families with 181 progenies. The complete heterozygosity in 50 analyzed diverse males indicates a potential use of this marker for any desired parental pair. The marker is amplified in homologous chromosomes, which rules out incorrect determination of non-amplified samples, as can be the case with the dominant (presence/absence) type of markers. The described microsatellite locus is also highly variable, with 35 sized alleles, the distribution of which in different hop germplasms and their sequence variability are discussed. The sex specific marker determined in our study can contribute to studies of sex determination mechanisms and can be readily used in hop breeding and cultivation.

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

BackgroundHop (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.ResultsQTL 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.ConclusionsPleiotropy 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.

Development of Pathotype-Specific SCAR Markers for Detection of Verticillium albo-atrum Isolates from Hop

Rapid polymerase chain reaction (PCR) assays were developed for the identification and detection of Verticillium albo-atrum hop pathotypes PG1 and PG2 from Slovenia. Of 17 pathotype-linked amplified fragment length polymorphism (AFLP) markers, 11 were cloned successfully and sequenced. To convert polymorphic AFLP markers into pathotype-specific sequence-characterized amplified region (SCAR) markers, 22 PG2- and 10 PG1-specific primer pairs were designed from 16 sequences. When primer specificity was tested on a wide range of Verticillium isolates, 10 PG2- and 6 PG1-specific primer pairs retained amplification specificity for V. albo-atrum Slovene hop isolates, but also amplified sequences in V. albo-atrum and V. dahliae hop isolates from different hop production areas in Europe, as well as in some isolates from other hosts. Primer combinations obtained from the AFLP-9-1 marker were specific only for V. albo-atrum PG2 isolates. The highly specific primers were used in multiplex PCR and a nested PCR to detect the V. albo-atrum PG2 pathotype in xylem tissue of hop plants. These new SCAR markers provide a valuable tool for rapid identification of V. albo-atrum PG1 and PG2 hop pathotypes.

Broad taxonomic characterization of Verticillium wilt resistance genes reveals an ancient origin of the tomato Ve1 immune receptor

Plant-pathogenic microbes secrete effector molecules to establish themselves on their hosts, whereas plants use immune receptors to try and intercept such effectors in order to prevent pathogen colonization. The tomato cell surface-localized receptor Ve1 confers race-specific resistance against race 1 strains of the soil-borne vascular wilt fungus Verticillium dahliae which secrete the Ave1 effector. Here, we describe the cloning and characterization of Ve1 homologues from tobacco (Nicotiana glutinosa), potato (Solanum tuberosum), wild eggplant (Solanum torvum) and hop (Humulus lupulus), and demonstrate that particular Ve1 homologues govern resistance against V. dahliae race 1 strains through the recognition of the Ave1 effector. Phylogenetic analysis shows that Ve1 homologues are widely distributed in land plants. Thus, our study suggests an ancient origin of the Ve1 immune receptor in the plant kingdom.

New polymorphic dinucleotide and trinucleotide microsatellite loci for hop Humulus lupulus L.

One hundred and thirty‐five microsatellite markers were developed for hop Humulus lupulus L. from di‐ and trinucleotide‐enriched libraries. Seventy‐eight primers showed amplification in two tested genotypes. Twenty‐four loci were further characterized on a population of 34 hop samples and the number of alleles per locus, observed heterozygosity and expected heterozygosity ranged from two to 20 (9.7 on average), from 0.0294 to 0.9412 (0.6234 on average) and from 0.0294 to 0.9170 (0.6720 on average), respectively. These microsatellite markers will be further used for studying population structures and relationships and for identifying important qualitative and quantitative loci of hop.