Branka Javornik

Evaluation of genetic variability among common buckwheat (Fagopyrum esculentum Moench) populations by RAPD markers

Genetic variability among 80 accessions of common buckwheat was evaluated using random decamer primers in polymerase chain reaction (PCR) amplification of bulked DNA samples. Thirteen primers, previously chosen on the basis of the number and intensity of the fragments they produced, were used for amplification and yielded a total of 141 bands, of which 77 were polymorphic. An NTSYS-pc computer program was further employed for data analysis using a simple matching coefficient of similarity as a base for dendrogram construction via the UPGMA method. A moderate level of polymorphism and relatively close relationships among the studied common buckwheat populations were observed. The dendrogram separated accessions into European and Asian groups, with a number of additional clusters and some inconsistant populations from both regions. Clusters corresponded reasonably well to the geographical origins of examined populations, with some unexpected but interesting exceptions. The effectiveness of RAPD analysis of bulked DNA samples for the study of genetic variability in common buckwheat was demonstrated, and this approach seems very useful for identification and phylogenetic studies.

Chemical composition and protein quality of buckwheat (Fagopyrum esculentum Moench)

The chemical composition and protein quality of two varieties of buckwheat were measured and compared with the values of wheat. The protein quality was based on amino acid composition and true protein digestibility, biological value, net protein utilization, and utilizable protein obtained in N-balance experiments with rats. The protein content in buckwheat was approximately 12% and thus very much the same as in wheat. The fat content in buckwheat was close to 3% whereas the crude fiber concentration was very high (12.7 and 17.8%, respectively, for two varieties). The high fiber content caused a low concentration of soluble carbohydrates with the lowest value of 48.7%. Both buckwheat varieties had a high tannin content (1.76 and 1.54%, respectively). The protein quality was very high, with biological values above 90%. This can be explained by a high concentration of most essential amino acids, especially lysine, threonine, tryptophan, and the sulphur-containing amino acids. However, due to the high contents of crude fiber and tannin, the true protein digestibility was slightly below 80%.

Studies of gynogenesis in onion (Allium cepa L.): induction procedures and genetic analysis of regenerants

Gynogenesis of four onion cultivars was induced, starting from ovules and ovaries, using a 2-step culture procedure. The induction frequency was much higher from ovaries and was strongly affected by genotype. Thidiazuron (2 mg/l) had a positive effect on regeneration and subsequent in vitro culture of regenerants. Esterase isozyme analysis showed that 59% of regenerants were homozygous. Chromosome counting of root tip cells and flow cytometry of leaf tissue showed haploid, doubled haploid and mixoploid structures. Within cloned plants, some showed segregation of the ploidy level with a tendency towards endoreduplication. Analysis of random amplified polymorphic DNA (RAPD) profiles of homozygous regenerants revealed several missing bands compared with donor plants, but micropropagated clones of different ploidy level were identical among each other. Gametoclonal variation was detected by the appearance of a novel RAPD band, present in two out of 12 regenerants and not in the donor plants.

Ectomycorrhizae of Lactarius lignyotus on Norway spruce, characterized by anatomical and molecular tools

The ectomycorrhizae of Lactarius lignyotus on Norway spruce are comprehensively described by morphological and anatomical characteristics. Identification of ectomycorrhizae was performed by tracing mycelia to the fruitbodies and also by molecular tools, using polymerase chain reaction (PCR) amplification of the fungal DNA. The newly described ectomycorrhiza is compared to ectomycorrhiza of the related Lactarius picinus. The amplified DNA products of the two fungi and their ectomycorrhizae could be distinguished by characteristic fragments after digestion with Hinf1.

Genetic variability of economically important Asparagus species as revealed by genome size analysis and rDNA ITS polymorphisms

Abstract Ten species (13 accessions) of the genus Asparagus were analysed to reveal their genome size structure and genetic similarity. The choice of species was based on their possible application in crossbreeding with Asparagus officinalis L. and their agricultural value . Flow cytometric measurement of genome size revealed two distinctive groups, based on 1C values, the European dioecious species having on average twice the genome size of the South African hermaphrodite species. Variability among species was relatively high both as absolute values (from 1.43 to 7.87 pg per nucleus) or expressed as 1C values (0.67–1.71 pg/1C). Additionally, Asparagus maritimus was determined as hexaploid in contrast to previous reports of it being tetraploid. Based on restriction fragment analysis of internal transcribed spacers (ITSs) of nuclear ribosomal DNA, the same two, distinctly distant groups of species were formed, which clearly separate species by their gender and geographic origin. The clustering obtained by the UPGMA method is in agreement with some botanical classifications. The genetic similarity and ploidy level results explain the successful interspecific hybridisation reported for A. officinalis and Asparagus tenuifolius and the failure of hybridisation between A. officinalis and some other Asparagus species.

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.

Genetic structure and differentiation in hop (Humulus lupulus L.) as inferred from microsatellites

A set of 67 wild and cultivated hop accessions, representative of hop diversity, was genotyped with 29 SSR markers in order to investigate the population structure and genetic diversity among hop genotypes. A total of 314 alleles was detected, with an average of 10.8 alleles per locus and an average PIC content of 0.607. Model-based clustering placed the accessions into five germplasm groups. A distance-based tree showed good agreement with five germplasm groups, and additionally assigned accessions omitted from model-based analysis into two additional germplasm groups. The 67 hop accessions were thus subdivided in seven germplasm groups, with three corresponding to major breeding groups and four to wild hops. This finding is in accordance with two biogeographically separated hop germplasms (European and North American origin) and with the known history of the accessions. North American hop germplasm was partitioned into native and cultivated germplasm groups. European germplasm was divided into two groups of hop cultivars representing distinguishable European germplasms and three new groups of native hops, which were differentiated for the first time by this analysis. Admixture analysis showed shares of various ancestries in hop cultivars, mostly congruent with pedigree data, and the introgression of various ancestries in some native hops. The above results have so far given the most detailed insight to date into the population structure of hop diversity, which is important for its effective use in hop breeding.

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.

Gynogenic lines of onion (Allium cepa L.): evidence of their homozygosity.

Haploid induction via gynogenesis offers the possibility of using doubled haploid (DH) inbred lines in onion breeding. A first DH line that originated from the open-pollinated (OP) cultivar ‘Dorata di Parma’ was obtained after overcoming difficulties associated with the haploidy of the regenerants. Spontaneous chromosome doubling occurs seldom in onion. The first DH line obtained was cloned and selfed to produce sufficient seeds for genetic studies. The homozygosity of the DH gynogenic line was revealed on the basis of the low standard deviations of the bulb traits polar diameter, shape index and weight with respect to those of the S1 line or the OP cultivar. In the DH line, moreover, segregation of RAPD and alpha esterase markers was not noted. Out of four primers revealing polymorphism at 16 ge-netic loci in the OP cultivar ‘Dorata di Parma’, none produced polymorphism in the DH gynogenic line. The Est-1 locus, homozygous in 22 plants (Est-11/1 in 3 and Est-12/2 in 19) and heterozygous (Est-11/2) in 11 plants of the OP cultivar, always carried the same alleles in the DH line. We also tested genetic stability during micropropagation of a second halpoid line obtained via gynogenesis from var. ‘Senshyu Yellow’. Seventeen plants of this line were tested to detect changes occurring during the tissue culture process. Again no polymorphism was observed. The high genetic homogeneity observed in the two gynogenic lines of onion could be related to the absence of the callus phase during the gynogenic process.

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.