New Set of EST-STR Markers for Discrimination of Related Papaver somniferum L. Varieties.

Papaver somniferum L. is cultivated for its edible seeds and for the production of alkaloids. A serious problem in seed trade and processing is the intentional mixing of excellent food-quality seeds with non-food-grade-quality seeds. Tracking the correct or illegitimate handling of seeds requires an efficient method for discrimination and individualization of poppy varieties. As in human and animal forensics, DNA variable regions containing short tandem repeats (STRs) located either in non-coding DNA or in gene sequences (EST-STRs) are preferred markers for discrimination between genotypes. Primers designed for 10 poppy EST-STR loci not analyzed so far were tested for their discriminatory ability on a set of 23 related P. somniferum L. genotypes. Thirty-three EST-STR alleles were identified together. Their polymorphic information content (PIC) values were in the range of 0.175-0.649. The PI value varied in the range of 0.140-0.669, and the cumulative PI was 1.2 × 10-5. PIsibs values varied between 0.436 and 0.820 and the cumulative value was lower (5.0 × 10-3). All analyzed genotypes were distinguished mutually, each with its own unique EST-STR profile. These newly developed EST-STR markers more effectively discriminated P. somniferum L. genotypes, even those genotypes whose DNA profiles were previously identical.

The Cell-Wall ß-d-Glucan in Leaves of Oat (Avena sativa L.) Affected by Fungal Pathogen Blumeria graminis f. sp. avenae.

In addition to the structural and storage functions of the (1,3; 1,4)-ß-d-glucans (ß-d-glucan), the possible protective role of this polymer under biotic stresses is still debated. The aim of this study was to contribute to this hypothesis by analyzing the ß-d-glucans content, expression of related cellulose synthase-like (Csl) Cs1F6, CslF9, CslF3 genes, content of chlorophylls, and ß-1,3-glucanase content in oat (Avena sativa L.) leaves infected with the commonly occurring oat fungal pathogen, Blumeria graminis f. sp. avenae (B. graminis). Its presence influenced all measured parameters. The content of ß-d-glucans in infected leaves decreased in all used varieties, compared to the non-infected plants, but not significantly. Oats reacted differently, with Aragon and Vaclav responding with overexpression, and Bay Yan 2, Ivory, and Racoon responding with the underexpression of these genes. Pathogens changed the relative ratios regarding the expression of CslF6, CslF9, and CslF3 genes from neutral to negative correlations. However, changes in the expression of these genes did not statistically significantly affect the content of ß-d-glucans. A very slight indication of positive correlation, but statistically insignificant, was observed between the contents of ß-d-glucans and chlorophylls. Some isoforms of ß-1,3-glucanases accumulated to a several-times higher level in the infected leaves of all varieties. New isoforms of ß-1,3-glucanases were also detected in infected leaves after fungal infection.

UPTAKE AND DISTRIBUTION OF RADIOSTRONTIUM IN TOMATO TREATED WITH ARBUSCULAR MYCORRHIZAL FUNGI.

Arbuscular mycorrhizal fungi (AMF) were inoculated into the root system of edible tomato plants to investigate their effect on the uptake and distribution of strontium radionuclide in the tissues of plants grown under laboratory conditions. The experiments were carried out in a sterile mixture of topsoil and sand, where in one series of experiments contaminated soil with 85Sr was used. Seeds were inoculated with a mixture of AMF at sowing, the second, control series was without application of AMF. Determination of radioactivity in soil and in different parts of tomato plants was carried out by gamma-spectrometric measurement. The obtained results and statistical analysis indicated a significant association between AMF inoculation of plants and the change in the distribution of 85Sr. The presence of AMF reduced the translocation of 85Sr from the root system to the aerial parts of tomato plants.

Differentiation of Apricots of Different Geographic Origin in Central and Southern Europe by Applying 87Sr/86Sr Analysis: Potential and Limitations.

Consumers prefer food commodities of certain origins over the same products of other provenances and are willing to pay higher prices for them. Thus, it is possible to increase profit simply by giving an incorrect geographic origin to a product. To effectively control the declared geographic origin of food, the product itself has to be investigated to discover whether it actually originates from the declared origin, or if it has been mislabeled. Conventionally, control of a geographic origin is conducted by stable isotope analysis of the main elements, which has proven to be successful in numerous cases, but often reference data have to be produced anew for every harvest to control, resulting in additional costs and delays. Applying entirely geogenic parameters for the control of provenance requires reference data to be produced only once. As they do not vary between years and harvests, they can often be used for different (food) commodities. Here, we investigate whether the geographic origin of apricot samples can be controlled by their 87Sr/86Sr ratios measured by TIMS. The results show that Slovak and Hungarian apricots can be differentiated from the Wachau apricots, a well-known regional Austrian brand, and those from other regions in Austria, even though the differentiation from the latter is only partial. 87Sr/86Sr investigations can be a very potent tool; however, its success depends on the exact question that needs to be answered.

Formation of Potential Heterotic Groups of Oat Using Variation at Microsatellite Loci.

An evaluation of polymorphism at the microsatellite loci was applied in distinguishing 85 oat (Avena sativa L.) genotypes selected from the collection of genetic resources. The set of genotypes included oats with white, yellow, and brown seeds as well as a subgroup of naked oat (Avena sativa var. nuda Koern). Variation at these loci was used to form potential heterotic groups potentially used in the oat breeding program. Seven from 20 analyzed microsatellite loci revealed polymorphism. Altogether, 35 microsatellite alleles were detected (2-10 per locus). Polymorphic patterns completely differentiated all genotypes within the subgroups of white, brown, and naked oats, respectively. Only within the greatest subgroup of yellow genotypes, four pairs of genotypes remained unseparated. Genetic differentiation between the oat subgroups allowed the formation of seven potential heterotic groups using the STRUCTURE analysis. The overall value of the fixation index (Fst) suggested a high genetic differentiation between the subgroups and validated a heterotic grouping. This approach can be implemented as a simple predictor of heterosis in parental crosses prior to extensive field testing or development and implementation of more accurate genomic selection.

The impact of sewage sludge on the fungal communities in the rhizosphere and roots of barley and on barley yield.

Current problems with sewage sludge (SS) disposal could be solved by application to agricultural land considering its fertilizer properties and ability to improve soil condition. However, SS may contain heavy metals as well as pathogenic microorganisms. In this study, molecular analysis of partial 18S rRNA gene was used to study the impact of SS application into the soil on the genetic diversity of fungal communities, especially arbuscular mycorrhizal fungi in the rhizosphere and roots of barley. These samples were collected on three dates from the control soil without SS and from the soil with the addition of SS at the concentrations of 5 and 15 t ha-1. Fungal alpha diversity in the rhizosphere of barley was affected by SS differently than in barley roots. In addition, principal component analysis and cluster analysis revealed that fungal communities were strongly influenced by the SS addition into the soil, sample type, and the sampling date. This approach was complemented by an evaluation of the basic parameters of barley production and the response of these parameters to the presence of SS in the soil. The plant height increased with increasing SS concentration and the thousand seed weight significantly increased at the concentration of 5 t ha-1 SS but significantly decreased in 15 t ha-1.

Superabsorbent Polymer Seed Coating Reduces Leaching of Fungicide but Does Not Alter Their Effectiveness in Suppressing Pathogen Infestation.

Superabsorbent polymers (SAPs) applied to soil have been recognized as water reservoirs that allow plants to cope with periods of drought. Their application as a seed coat makes water available directly to the seeds during their germination and early growth phase, but on the other hand, it can affect the efficiency of plant protection substances used in seed dressing. In our experiments, we evaluated the effect of seed coating with SAP on fungicide leaching and changes in their effectiveness in suppressing Fusarium culmorum infestation. Leaching of fungicide from wheat seeds coated with SAP after fungicide dressing, as measured by the inhibition test of mycelium growth under in vitro conditions, was reduced by 14.2-15.8% compared to seeds without SAP coating. Germination of maize seeds and growth of juvenile plants in artificially infected soil did not differ significantly between seeds dressed with fungicide alone and seeds treated with SAP and fungicide. In addition, plants from the seeds coated with SAP alone grew significantly better compared to untreated seeds. Real-time PCR also confirmed this trend by measuring the amount of pathogen DNA in plant tissue. Winter wheat was less tolerant to F. culmorum infection and without fungicide dressing, the seeds were unable to germinate under strong pathogen attack. In the case of milder infection, similar results were observed as in the case of maize seeds.

Responses of Rhizosphere Fungal Communities to the Sewage Sludge Application into the Soil.

Due to the increasing sewage sludge production in the world and problems with its disposal, an application of sludge to the soil appears to be a suitable solution considering its fertilizer properties and ability to improve the soil physical conditions. On the other hand, the sludge may also contain undesirable and toxic substances. Since soil microorganisms are sensitive to environmental changes, they can be used as indicators of soil quality. In this study, we used sewage sludge (SS) from two municipal wastewater treatment plants (SS-A and SS-B) in the dose of 5 t/ha and 15 t/ha in order to determine possible changes in the fungal community diversity, especially arbuscular mycorrhizal fungi (AMF), in the rhizosphere of Arundo donax L. Rhizosphere samples were collected in summer and autumn for two consecutive years and the fungal diversity was examined using terminal restriction fragment length polymorphism and 18S rDNA sequencing. Fungal alpha diversity was more affected by SS-A than SS-B probably due to the higher heavy metal content. However, based on principal component analysis and ANOSIM, significant changes in overall fungal diversity were not observed. Simultaneously, 18S rDNA sequencing showed that more various fungal taxa were detected in the sample with sewage sludge than in the control. Glomus sp. as a representative of AMF was the most represented. Moreover, Funneliformis in both samples and Rhizophagus in control with Septoglomus in the sludge sample were other representatives of AMF. Our results indicate that the short-term sewage sludge application into the soil does not cause a shift in the fungal community composition.

Progress in the genetic engineering of cereals to produce essential polyunsaturated fatty acids.

Essential polyunsaturated fatty acids with more than two double bonds and length of carbon chain 18-22 must be taken in the diet to prevent diseases and imbalances caused by their deficiency. Terrestrial sources of polyunsaturated fatty acids are limited to only a few plant species whose large-scale cultivation is not possible and the production of their seeds and oil is ineffective. The complete biosynthetic pathway of fatty acids is known in organisms, including plants. After the first gene encoding the enzyme catalysing the initial steps of PUFA biosynthesis (ω-3 desaturase, Δ6-desaturase) were isolated, isolation of other genes encoding relevant enzymes of the PUFA pathway from different donor organisms followed. Genetic transformations of model plants by the desaturase- and elongase-encoding genes opened the way for the genetic engineering of oilseed crop species. Some of the developed transgenic plants produced PUFAs, including eicosapentaenoic and docosahexaenoic acids. Seed oils extracted from them were similar to fish oil. Tools of the synthetic biology can be applied in modifications of the PUFA pathway and also in overcoming of limitations when the gene and its expression product are absent in the pathway. Such progress in cereals (barley, wheat, maize) has been made only recently, when the first successful modifications of the ω-3 and ω-6 PUFA pathways succeeded. This review focuses on genetic modifications of the PUFA biosynthetic pathway in cereals in relation to the status reached in model plants and oilseed crops.

Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene.

The artificial gene D6D encoding the enzyme ∆6desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T0 and T1 generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%-0.32% (v/v) of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T1 generation as well as in immature and mature grains of the T2 generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%-1.40% (v/v) and 0%-1.53% (v/v) from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat.

Genetic structure of Pyrenophora teres net and spot populations as revealed by microsatellite analysis.

The population structure of the fungal pathogen Pyrenophora teres, collected mainly from different regions of the Czech and Slovak Republics, was examined using a microsatellite analyses (SSR). Among 305 P. teres f. teres (PTT) and 82 P. teres f. maculata (PTM) isolates that were collected, the overall gene diversity was similar (h = 0.12 and h = 0.13, respectively). A high level of genetic differentiation (FST = 0.46; P < 0.001) indicated the existence of population structure. Nine clusters that were found using a Bayesian approach represent the genetic structure of the studied P. teres populations. Two clusters consisted of PTM populations; PTT populations formed another seven clusters. An exact test of population differentiation confirmed the results that were generated by Structure. There was no difference between naturally infected populations over time, and genetic distance did not correlate with geographical distance. The facts that all individuals had unique multilocus genotypes and that the hypothesis of random mating could not be rejected in several populations or subpopulations serve as evidence that a mixed mating system plays a role in the P. teres life cycle. Despite the fact that the genetic differentiation value between PTT and PTM (FST = 0.30; P < 0.001) is lower than it is between the populations within each form (FST = 0.40 (PTT); FST = 0.35 (PTM); P < 0.001) and that individuals with mixed PTT and PTM genomes were found, the two forms of P. teres form genetically separate populations. Therefore, it can be assumed that these populations have most likely undergone speciation.