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Until now in Europe has not been cultivated any hybrid cultivar of oilseed rape based on the Shaan 2A cytoplasmic male sterility (CMS), a widely used CMS system in China. The aim of Czech breeders now is to produce new, improved cultivars of rapeseed based on this CMS system. Sterile Shaan 2A CMS line (S; rf/rf), its corresponding maintainers (N; rf/rf) and fertility restorers (S; Rf/Rf) were analyzed on molecular level for the presence of functional CMS cytoplasm. Two new primer pairs covering CMS-associated gene (so called orf224-1) present in Shaan 2A CMS line were developed and selection capability of the developed primers was successfully evaluated. These primers can be used for early selection of plants with functional Shaan 2A CMS system in breeding programmes.
† Background and Aims A detailed knowledge of cytotype distribution can provide important insights into the evolutionary history of polyploid systems. This study aims to explore the spatial distribution of different cytotypes in Pilosella echioides at various spatial scales (from the whole distributional range to the population level) and to outline possible evolutionary scenarios for the observed geographic pattern.
† Methods DNA-ploidy levels were estimated using DAPI flow cytometry in 4410 individuals of P. echioides from 46 populations spread over the entire distribution range in central Europe. Special attention was paid to the cytotype structure in the most ploidy-diverse population in south-west Moravia.
†Key Results Five different cytotypes (2x, 3x, 4x, 5x and 6x) were found, the last being recorded for the first time. Although ploidy-uniform (di- or tetraploid) sites clearly prevailed, nearly one-quarter of the populations investigated harboured more (up to all five) cytotypes. Whereas penta- and hexaploids constituted only a minority of the samples, a striking predominance of the triploid cytotype was observed in several populations.
†Conclusions The representative sampling confirmed previous data on cytotype distribution, i.e. the spatial aggregation of mixed-ploidy populations in south-west Moravia and Lower Austria and the predominance of ploidy-uniform populations in other parts of the area investigated. Recurrent origin of polyploids from diploid progenitors via unreduced gametes and their successful establishment are considered the key factors promoting intrapopulational ploidy mixture (‘primary hybrid zones’). As an alternative to the generally accepted theory of cytotype co-existence based on the development of different means of inter-ploidy reproductive isolation, it is suggested that a long-term ploidy mixture can also be maintained in free-mating populations provided that the polyploids originate with a sufficient frequency. In addition, the prevalence (or subdominance) of the triploid cytotype in several mixed-ploidy populations represents the first evidence of such a phenomenon in plant systems with exclusively sexual reproduction.
Two methods recommended for analysis of denatured proteins, automated Experion chip electrophoresis and SDS-PAGE, are compared. The Experion method is a novel technique based on combination of the LabChip microfluidic separation technique (Caliper Life Sciences) and sensitive fluorescent detection. Both methods were compared in molecular weight (MW) determination of a protein standard mixture, resolution of protein pairs of near molecular weights and estimation of the abundance of a target protein in the mixture to be purified. Both the methods are appropriate for MW determination and in purification of proteins. The accuracy of the methods is approximately the same (ca. 8 %), but the Experion method shows better reproducibility (ca. 1.48 %) than SDS-PAGE (ca. 2.17 %).
In the year 2007, there were one hundred and seventy-eight potato varieties enlisted in the Czech list of registered potato varieties. The classical morphometric approach to characterization is not effective for such a number of varieties especially for identification at the level of tubers. The needfulness of variety identification at the level of tubers is important mainly for trade aspect. The Czech law no.110/1997 Sb. about the food-stuff and tobacco products and the consequential ordinance (MZe č. 332 / 1997 Sb.) require guarantee of variety declaration in commercial relation for table potato. In this study we analyzed twenty potato varieties (Solanum tuberosum L.) cultivated in the Czech Republic. Every variety was represented by four independent replicates. This set of samples was analyzed by methods of PCR-SSR (Simple Sequence Repeats) and PCR-ISSR (Inter Simple Sequence Repeats). We discovered that both of tested methods afford sufficient polymorphism for variety identification, but the method of PCR-ISSR is not utilizable, because we observed the variability within variety. For outright identification of the whole set of potato varieties cultivated in the Czech Republic we recommend to use SSR, AFLP and retrotransposene-based markers as well as morphological markers.
Premise : Studies of hybridizing species are facilitated by the availability of species-specifi c molecular markers for identifying early- and later-generation hybrids. Cattails are a dominant feature of wetland communities, and a better understanding of the prevalence of hybrids is needed to assess the ecological and evolutionary effects of hybridization. Hybridization between Typha angustifolia and T. latifolia produce long-lived clones, known as Typha × glauca , which are considered to be invasive. Although morphological variation in cattails makes it diffi cult to recognize early- and later-generation hybrids, several dominant, species-specifi c RAPD markers are available. Our goal was to fi nd codominant, species-specifi c markers with greater polymorphism than RAPDs, to identify later-generation hybrids more effi ciently.
• Methods : We screened nine SSR (simple sequence repeat) loci that were described from populations in Ukraine, and we surveyed 31 cattail populations from the upper Midwest and eastern USA.
• Key results : Seven SSR loci distinguished the parent taxa and were consistent with known species-specifi c RAPD markers, allowing easier detection of backcrossing. We used linear discriminant analysis to show that F 1 hybrid phenotypes were intermediate between the parent taxa, while those of backcrossed plants overlapped with the hybrids and their parents. Log(leaf length/leaf width), spike gap length, spike length, and stem diameter explained much of the variation among groups.
• Conclusions : We provide the fi rst documentation of backcrossed plants in hybridizing cattail populations in Michigan. The diagnostic SSR loci we identifi ed should be extremely useful for examining the evolutionary and ecology interactions of hybridizing cattails in North America.
1. By increasing vigour and broadening ecological tolerances, hybridization between native and introduced species may serve as a primary driver of invasiveness.
2. Cattails (Typha, Typhaceae) are clonal wetland graminoids that are known to hybridize where anthropogenic influences have resulted in distributional overlap.
3. In order to gauge the relative performance of hybrid vs. pure Typha, we characterized hybridization and clonal growth where native Typha latifolia and introduced Typha angustifolia occur together in the Western Great Lakes Region of North America.
4. Based on microsatellite markers, we documented F1 hybrids as the most common class at five intensively sampled sites, constituting up to 90% of the genets and 99% of the ramets. Backcrosses to one or the other parent constituted 5–38% of the genets. Pure T. latifolia was rare and never constituted more than 12% of the genets.
5. F1 hybrid genets achieved the highest mean ramet numbers at three sites, and were second in size only to T. angustifolia at two sites; however, these differences were not significant based on sitespecific one-way anovas.
6. F1 hybrids exhibited little height advantage over other Typha classes, although there was a general tendency for hybrids in relatively mixed stands to be among the tallest genets in shallow water, but among the shortest genets in deeper water.
7. Native T. latifolia was found growing at the shallowest water depths at the only site where it was sufficiently abundant to be included in statistical comparisons.
8. Synthesis. The role of hybridization in plant invasions can be difficult to confirm in the absence of molecular data, particularly for clonal species where the boundaries separating individuals are otherwise difficult to discern. Here, we used molecular markers to document the prevalence and performance of hybrid genets in five invasive Typha stands covering a broad area of the Western Great Lakes Region. We found an extremely high prevalence of F1 hybrids within mixed Typha stands. This, coupled with the typically larger sizes of hybrid genets, suggests that hybrids are capable of outperforming other Typha spp. and that hybridization has played an influential role in the North American cattail invasion.
There is mounting evidence that the clonal dynamics of foundational plant species, including exotic invaders such as hybrid Typha x glauca, have a profound effect on wetland function. Here, we report on the clonal structure of five intensively sampled Typha stands from the Upper Midwest region where invasions have been especially disruptive. Each of these stands consisted of a large proportion of F1 hybrids between T. latifolia and T. angustifolia, although backcrosses to both parents were also observed, and provided a means of determining relative age of invasion. We found clonal richness, measured as the proportion of ramets representing distinct genets, to vary positively with age of invasion over a range from 0.20 to 0.45, whereas Simpson’s Evenness was relatively consistent among sites due to a pattern of dominance by a few large clones accompanied by many smaller clones. Ramets were significantly clumped within genets over a range of approximately 20 m, although many clones included ramets separated by as much as 60 to 90 m, suggesting some degree of clone fragmentation over time. Related genets were significantly clumped over approximately 10 m, suggesting that seedling cohorts may frequently recruit in close proximity to one another.
We analyzed a set of twenty most grown potato (Solanum tuberosum L.) varieties listed in the Czech Variety List using the PCR-IRAP (Inter-Retrotransposon Amplified Polymorphism) method in order to distinguish fast and unambiguously the varieties. In total, 62 polymorphic alleles were amplified using the three primers P-Tst-1, P-Tst-3 and P-Tst-6. The recorded pattern of markers was stable and reproducible. The analyses were repeated three times and identical results were always obtained. The level of polymorphism varied from 11% to 79% depending on the respective primer. All analysed varieties could be reliably distinguished after multivariate statistics have been applied to the data obtained by the PCO and UPGMA analyses. The best resolution of individual varieties was obtained if all three primers were evaluated as a complex. The use of retrotransposonbased markers appears to be suitable for the differentiation of large sets of potato samples and should be an eligible complement to other molecular markers used in potato variety identification such as Simple Sequence Repeats (SSR) and Amplified Fragment Length Polymorphisms (AFLP).
Purple loosestrife (Lythrum salicaria), a native Eurasian species, belongs among highly invasive angiosperms in North American wetlands. Ploidy levels of 1860 progenies arising from nearly 600 parental plants sampled in both primary and secondary areas of distribution were estimated using flow cytometry in order to study potential differences in cytotype population structure. Considerable cytotype variation (2x, 3x, 4x and 6x) was found across the native range (76 populations covering 14 European and Middle East countries). Triploid and hexaploid cytotypes were detected for the first time. Tetraploids clearly prevailed in the area studied, while diploids and hexaploids were recorded only from Israel and Turkey, respectively. Triploid progenies occurred in one population from Hungary (together with 4x). Sympatric occurrence of tetraploid and hexaploid individuals was also encountered in progenies from Turkey. In contrast to a large variation in primary area, cytotype uniformity is a typical feature of the non-native American material (68 populations covering 13 states or provinces of the USA and Canada) where only tetraploids were revealed.
The effect of the type of leaf tissue selected for the study of green fluorescent protein (GFP) fluorescence intensity was investigated here using the T1 generation of transgenic tobacco expressing the m-gfp5- ER gene. The fluorescence of GFP was detected by fluorescence binocular microscope coupled with the CCD camera and quantified by means of image analyses using the Lucia1 software. Mean brightness values from various leaf tissues were compared. First, an original data revealing the significant differences in the fluorescence intensity between the abaxial and adaxial surfaces are given. Stronger signal was detected on the abaxial side. Subsequently, the effect of the tissue location within the leaf surface was investigated and higher fluorescence was detected on the samples detached from leaf tips. Finally, the effect of the physiological age of leaves was studied using the in vitro clonally propagated plants. Leaves from the analogous positions within the plant body of three clones were investigated. The decrease in the fluorescence towards the plant top (youngest leaves) was observed in all studied plants. Surprisingly, the variability of the fluorescence within the clones of studied genotype was high enough to conclude, that the fluorescence of each individual is unique and affected by particular genotype and environment. Our study showed that the origin of leaf tissue selected for the GFP quantification is crucial and that the fluctuations in the fluorescence intensity should be taken into account when comparing the GFP fluorescence patterns of different plants. Moreover, the degree of fluorescence variability seems to be individually affected.
Constitutive promoters are the most common promoters used to drive the expression of various genes in monocots and dicots. Therefore, it is of intense interest to ascertain their expression patterns in various plant species, organs and during their ontogenic development. In this study, the activity of the CaMV 35S promoter in transgenic tobacco plants was assessed. In contrast to other studies, performed rather on the primary transformants (T0 generation), here, individuals of T1 and T2 generations were used. The expression profiles of the CaMV 35S promoter were tracked within various plant organs and tissues using the GFP marker. Special attention was given to floral tissues for which the original data regarding the CaMV 35S expression were obtained. As expected, distinct developmental and organ/tissue specific expression patterns in a plant body were observed. CaMV 35S activity was detected in most of the plant tissues and during different developmental stages. The GFP signal was not visible in dry seeds only, but it became clearly apparent within 24–48 h after sowing onto the medium, what, among other things, enables the discrimination of transgenic and non-transgenic seeds/seedlings. Afterwards, the most pronounced GFP fluorescence intensity was usually visible in various vascular tissues of both, T1 and T2 plants, indicating the high promoter activity. A stable manifestation of the promoter was retained in the next T2 generation without any evident changes or losses of activity, showing the expression stability of the CaMV 35S.
Aim This study aimed to document precisely the patterns of DNA ploidy variation in the native and secondary ranges of Lythrum salicaria distribution. The hypothesis that species invasiveness had been induced by a switch in ploidy level was addressed.
Location Europe, Middle East, North America.
Methods DNA ploidy levels of 1884 progenies of 578+ plants collected at 124 localities were determined by DAPI flow cytometry.
Results Large cytotype variation (2x, 3x, 4x and 6x) was found across the native area of distribution (64 populations covering 12 European and two Middle Eastern countries). DNA hexaploids were detected for the first time, and rare DNA triploids were reliably confirmed. DNA tetraploids largely prevailed across the native range studied, while DNA diploids and DNA hexaploids were recorded only in Israel and Turkey, respectively. DNA triploid progenies occurred in one population from Hungary (together with DNA tetraploids). Sympatric growth of DNA tetraploids and DNA hexaploids was repeatedly encountered in Turkey. In contrast, cytotype uniformity was a typical feature of the invasive North American plants. Sixty populations, covering 13 states of the USA and provinces of Canada, were characterized by the presence of only DNA tetraploids.
Main conclusions Several L. salicaria cytotypes (2x, 3x, 4x, 6x) occur in the native range of distribution, with much variation concentrated in the Middle Eastern countries, whereas only DNA tetraploids appeared to occur in North America. Our data show that the invasive spread of North American populations was not triggered by differences in ploidy level. Alternative explanations should be sought.
Populations of Phragmites australis (CAV.) TRIN. ex STEUD. were studied in the littoral zones of two man-made lakes located in the Trˇebonˇ Basin (South Bohemia, the Czech Republic): (1) Opatovicky´ fishpond, a shallow artificial lake constructed in 1510–1514 by damming a shallow valley and used since for carp production, and (2) Hala´mky sand pit, a new lake formed by sand extraction in 1970–1994. Phenotypic variability was assessed on the basis of shoot morphological and growth characteristics, measured at the time of seasonal maximum aboveground biomass. Genotypic variability was detected using RAPDs, which demonstrated a high clonal diversity in both habitats. The clonal diversity would be strongly underestimated if it were based only on morphological differences. Higher genotypic variability was found in the fishpond reed, not corresponding with low variability in its phenotype performance. Based on analysis of 160 samples, four patterns of genotypic variation were detected: (1) Some stands were genetically uniform and were therefore considered to be monoclonal in both populations studied. (2) Some stands consisted of several clones at the Hala´mky sand pit. However, these clones showed more similarity within the particular stands than with clones of adjacent stands. (3) In the Opatovicky´ fishpond population, multiclonal stands consisted of clones with a low degree of similarity. (4) Identical clones were detected in several neighbouring stands separated by gaps in the Opatovicky´ fishpond population. The findings support a model of colonization postulating that populations initiated by seeds are initially genetically diverse and over time become dominated by a few clones as a result of competition and selection. These processes then decrease both genetic and morphological variability.
At present, protease inhibitors are one of the most studied natural substances. They represent a very heterogeneous group for which uniform rules for their classification have not yet been established. Protease inhibitors occurring in potato tubers can be classified in two ways: according to general classification of protease inhibitors or specific classification for protease inhibitors from potato tubers. Attention is devoted to protease inhibitors especially for their potential use in plant protection and medicine. Potato-tuber protease inhibitors are active against some insect pests such as larvae of genus Diabrotica. In medicine, they can be active in treatment of cancer, dermatitis and obesity.
Wild potato species (genus Solanum, section Petota) represent a tremendously diverse gene pool which is traditionally utilized as a source of diverse traits for potato breeding. Abiotic and biotic stress tolerance and resistance belong to the most frequently utilized traits of wild species in potato breeding programs. This review provides an introduction to the taxonomy, centre of diversity, genetic characteristics, evolution and important tolerance and resistance traits of wild potatoes and their use for potato breeding. The review has been written for readers who are interested in the problems of finding and utilization of new resistance genes from the wild genetic resources.
Populations of common reed (Phragmites australis [Cav.] Trin. Ex Steud.) were studied in the littoral zones of two human-made lakes in the Trˇebonˇ Basin (South Bohemia, Czech Republic): (1) Opatovicky´ fishpond, a 500-year-old fishpond, and (2) Hala´mky sand pit, a new lake formed by sand extraction in the 1970–1990s. Clones were identified using the RAPD method within morphologically different stands. The clones were propagated vegetatively and cultivated under comparable conditions in a common garden experiment for one vegetation season. Morphological and growth characteristics of the cultivated plants were recorded and compared with those found in the original reed stands. The younger (30-year old) population was genotypically more diverse than the older (500-year old) population. In addition, the younger population demonstrated correspondence of genotypic and phenotypic variation, while the older did not. The results indicate that the genetically determined morphological features (especially shoot length) are manifested more fully in the phenotype of the plants of the younger (Hala´mky) population, whereas environmental factors affect the phenotype to a greater extent in the older (Opatovicky´) population.
This short review is focused on antifungal plant proteins, their classification and characterization. There are 11 groups of the proteins: PR-1, PR-2, PR-3, PR-4, PR-5 proteins, defensins, thionins, CLPs, RIPs, LTPs, and protease inhibitors. The mechanisms of action of these proteins are as different as their sources and include degradation of fungal cell wall polymers, formation of membrane channels or damage of cellular ribosomes. The mode of action of many proteins remains unknown. The range of fungi that are inhibited by these proteins is very broad, including pathogens of many plants. The genes encoding antifungal proteins can be used to create transgenic plants with increased fungal field resistance. Some antifungal proteins (e.g. zeamatin) are tested for therapeutical use.
Contemporary cultivation of field crops utilizes new findings of biotechnologies, specifically recombinant DNA and transgenous techniques to an ever-increasing extent. Transgenic plants enriched in various new genes already became common practice in agriculture of a number of developed but also developing countries. The research in this field intensively grows, also entirely new directions, in addition to already proved gene manipulations, are the subject of interest. The contribution deals with classification and function of some protease inhibitors utilizable in transgenosis of plants. It concentrates also on the aspects associated with possible use of their recombined genes in enhancement of resistance of plants to insect pests and some pathogens. Some examples are given of important transgenic plants which already express genes for most important protease inhibitors.
Transformation of plants is a popular tool for modifying various desirable traits. Marker genes, like those encoding for bacterial b-glucuronidase (GUS), firefly luciferase (LUC) or jellyfish green fluorescent protein (GFP) have been shown to be very useful for establishing of efficient transformation protocols. Due to favourable properties such as no need of exogenous substrates and easy visualization, GFP has been found to be superior in to other markers in many cases. However, the use of GFP fluorescence is associated with some obstacles, mostly related to the diminishing of green fluorescence in older tissues, variation in fluorescence levels among different tissues and organs, and occasional interference with other fluorescing compounds in plants. This paper briefly summarizes basic GFP properties and applications, and describes in more detail the contribution of GFP to the establishment, evaluation and improvement of transformation procedures for plants. Moreover, features and possible obstacles associated with monitoring GFP fluorescence are discussed.
The terrestrial orchid species Neotinea ustulata has recently been split into two subspecies, differing remarkably in their flowering time, but only slightly in morphological characteristics, which makes their taxonomic status uncertain. We have analyzed morphometric and genetic differences between the early- and late-flowering populations in Central Europe. Our results on morphology are ambiguous. Indirect gradient analysis has not shown a distinct separation of early- and late-flowering individuals in the ordination space. However, according to MANOVA, populations of early- and late-flowering plants can be distinguished by plant height, leaf length, numbers of basal (rosette) and stem leaves and even better by certain ratios of these numbers. All genetic analyses, on the other hand, are definite and consistently distinguish two groups. Random amplified polymorphic DNA (RAPD) markers have shown that the early- and late-flowering populations differ significantly from one another. Principal coordinate analysis (PCoA) based on presence/absence matrix of RAPD bands separated the two groups, implying that the difference in flowering phenology could form an effective barrier to gene exchange. Partitioning of genetic diversity in analysis of molecular variance (AMOVA) has shown that the genetic divergence between the two groups, early- and late-flowering populations, is somewhat greater (33%) than the genetic variability among populations within particular group (23%). Using the Mantel test, we found that genetic differentiation coefficients between populations closely correspond to their geographic distribution. After elimination of the effect of sample origin from the model, direct gradient analysis (RDA) has shown that the early- and late-flowering groups differ significantly in their RAPD spectra. To conclude, our results indicate the presence of two genetically and phenologically distinct taxa, but the weak morphological differentiation supports the taxonomic rank of variety rather than subspecies.
Hyphomycete Paecilomyces fumosoroseus that is well known as saprophytic and entomopatogenic fungus was investigated for its mycoparasitism on the cucumber powdery mildew pathogen. Mycoparasitism was documented by using standard bioassay and SEM. Effects of mycoparasitism were evaluated in three types of experiments. Paecilomyces fumosoroseus was applied in the form of graded suspensions into a colony of powdery mildew on a leaf segment. Interaction between both fungi was observed as the percentage of colonized area vs. experimental time. In the second experiment, young cucumber plants were sprayed with a suspension of Paecilomyces fumosoroseus 24 h before inoculation of Sphaerotheca fuliginea. Pre-treatment with P. fumosoroseus reduced development and spreading of powdery mildew infection significantly 15 days post-inoculation in contrast to pre-treatments with sulfur fungicide and distilled water. The development of pure culture powdery mildew under determined experimental conditions was observed and compared with treated variants. In the third experiment, mildewed plants were treated with a suspension of P. fumosoroseus. The control treatments with sulfur fungicide and distilled water were tested. Effects of P. fumosoroseus on the dispersion of powdery mildew during a 21-day period were observed. P. fumosoroseus suppressed the development and spread of cucumber powdery mildew significantly during the time of the experiment. The mechanical and physical damages and disruptions of vegetative and fruiting structures of powdery mildew were recorded under light microscopy and S.E.M. Results were concluded in pursuance to differences between the natural behaviour and development of S. fuliginea on cucumber plants treated with P. fumosoroseus and non-treated plants.
The effects of environmental conditions (year, growing site) on protein compounds in buckwheat (Fagopyrum esculentum Moench) were studied. Small-plot experiments with four buckwheat cultivars (tetraploid Emka and diploid Kora, Pyra, and Krupinka) were established in two growing sites in 1999 and 2000. Crude and pure protein content, proportion of protein solubility fractions, SDS-PAGE of protein fractions, and immunoassay of gluten-like proteins were determined in buckwheat flour. The growing site had the most significant effect on variability of crude protein content. On the contrary, pure protein content was most of all affected by year. Apparently a higher occurrence of later developed buckwheat seeds in 1999 harvest (as a consequence of weather abnormalities) could generally cause lower content of pure protein in this year (7.32 %) than in 2000 (9.10 %). Alcohol-soluble protein fraction (prolamins like proteins, PLP), considered as inconvenient for gluten-free diet, was generally found at low level, but at the average of experiment, significant differences (P < 0.05) between years 1999 and 2000 were recorded, 2.12 and 5.37 % of crude protein, respectively. On the other hand, specific determination of harmful gluten-like proteins (GLP) by immunochemical assay (ELISA) revealed, that in the all tested samples their contents were below the detection limit of the method 12.5 mg . kg-1. Obtained SDS-PAGE subunit profile of AGLP was divided into four groups with range of molecular weight 5 – 51 kDa. PLP profile was only represented by a double-band with 17 kDa and by a band with molecular weight of about 8 kDa.
This short review is focused on potato tuber proteins - their classification, importance, and potential application in practice. A new approach to classification of tuber proteins by molecular weight is mentioned. The main and most important component of tuber proteins is patatin proteins. These are a heterogeneous group of protease inhibitors and other proteins. Patatin proteins are a family of immunologically identical glycoproteins with monomer molecular weights of ca. 40−43 kDa. Multiple enzymatic activities were found in patatin proteins, but their lipid acyl hydrolase activity is predominant. Enzymatic and other biochemical properties of patatin proteins determine their future practical application in food industry. In general, potato tuber proteins are high-quality plant proteins with an excellent nutritional and biological value.
The different molecular analysis for specification of white clover (Trifolium repens L.) populations was studied between 2002 and 2003. RAPD, SSR (microsatellites), rDNA and PCR-RFLP markers were used for this study. The high genetic variation was detected among the cultivars but also within the cultivars by RAPD markers. For this reason RAPD markers were not found as a suitable marker system for determination of white clover cultivars. The distribution of low genetic variation of rDNA and PCR-RFLP markers was not able to differentiate cultivars. SSR and rDNA markers did not show variability of patterns within one cultivar. The different sizes of PCR fragments were obtained after amplification with microsatellite primers. SSR markers are therefore suggested as the suitable markers for the identification of different T. repens cultivars.
Autoinkompatibilita u odrůd, donorů kvality a autoinkompatibilních linií Brassica napus byla analyzována použitím identifikace S lokusu. U několika odrůd B. napus jeden S lokus genu SLG byl detekován jako dominantní a druhý S lokus jako recesivní. Amplifikací SLG genu třídy II byl odhalen recesivní gen ve všech analyzovaných vzorcích (autokompatibilních i autoinkomatibilních). DNA fragment recesivního genu sekvenčně souhlasil s SLG genem W, který byl objeven u odrůdy Westar. S haplotypy byly analyzovány metodou PCR-RFLP. Různé odrůdy B. napus měly identická elektroforetická spektra, která odpovídala nefunkční A10 alele v B. campestris. V B. napus A10 alela je lokalizována v genomu A, který pochází z B. campestris. Funkční recesivní alela SLG genu je pravděpodobně lokalizována v genomu C. Byl navržen model segregace. Autokompatibilní a autoinkompatibilní rostliny segregovaly v F2 generaci v poměru přibližně 3:1. To potvrzuje recesivní monogenní založení autoinkomaptibility v experimentálních populacích.
Three different types of molecular markers, RAPD, SSR and fluorescence-based AFLP, were evaluated and compared for their ability to identify oilseed rape cultivars. The direct comparison of RAPD, SSR and AFLP approaches in cultivar identification showed that the AFLP methodology detected polymorphisms more efficiently than either RAPD or SSR methods. For the characterisation of six oilseed rape cultivars, 60 RAPD primers were tested and only eight of them (14%) detected sufficient levels of polymorphism. Five microsatellites out of fifteen tested were polymorphic, but in all loci, except one, only two different alleles were detected. This result indicated the limited degree of polymorphism found in Brassica napus. Each of the six tested AFLP combinations detected polymorphisms, the best combination (M-CAA/E-ACT) had 26% polymorphic peaks from a total of 90 peaks and could distinguish the analysed cultivars and 4 out of 5 core lines of cultivars. The results presented show that florescence-based AFLP is, for the purposes of oilseed rape cultivar fingerprinting, a more suitable approach than either RAPD or SSR.
Biochemical variability between thirteen European and five Czech potato (Solanum tuberosum L.)cultivars grown in the Czech Republic was studied by soluble protein, isoesterase, and isoperoxidase electrophoretic patterns. It was confirmed that cultivar differences in protein polymorphism can be revealed by applied electrophoreotis patterns. It was shown that the different character of protein and isozyme profiles required different approaches to their evaluation. For complex patterns such as electrophoreotic soluble protein spectra, it is more conventient to use the evaluation of their absorbance profiles and for simpler profiles of isozymes the evaluation based on the presence or absence af a band in a definite position (simple matching) should be used. In spite of the complexity of tetraploid disposition of analysed cultivars, the results suggested higher similarity of profiles between relative cultivars and the also indicated the existence of higher similarity between cultivars from the same breeding firm.
Four protein fractions: 1 – albumins and globulins, 2 – gliadins, 3 – glutenins (extracted in NaOH), 4 – glutenins (extracted in SDS) separated by SDS-PAGE were used as biochemical markers for evaluation of polymorphism level in three spelt wheat cultivars – Hercule, Altgold and Rouquin, three new–breeders’ spelt lines – H92.27, H92.28 and M92.20 (originated from hybridisation between spelt and common wheat) and reference common wheat cultivar Brea. Electrophoretic phenotypes and zymograms were evaluated by means of digital image analysis and Nei and Li coefficient of similarity was used to evaluate the relation of analysed genotypes. Entire evaluation of all four-marker systems showed differences between common wheat cultivar Brea and spelt cultivars and spelt breeders’ lines. Also significant differences between old spelt cultivars (Hercule, Altgold and Rouquin) and new spelt breeders’ lines were found. The reality of the mutual passing of protein fractions (gliadins and glutenins), based on Osborne extraction was confirmed. In this sense it is necessary to see both fractions as dynamic overlapping structures.
The objective of this study was an improvement on oat identification procedure for laboratory applications, and the comparison of albumin-globulin and avenin protein patterns in five hulled and naked oat cultivars: Abel (CZ) and Izák (CZ) – naked oats, Auron (CZ), Edmund (D) and Expander (D) – hulled oats. The last object of this study was the authenticity verification of standardly prepared meal samples with various proportions of admixture. It was confirmed that avenins, characterised under SDS-PAGE conditions, are reliable implements for the identification of oat cultivars. It was found that oat grain contains, on the basis of Osborne fractionation, another significant protein fraction – glutelins. The question of the protein fraction analysis that was used for the admixture identification stays still open. In sufficiently different cultivars, the certainty of the admixture detection in meal samples may be high. Nevertheless, in other cases (higher cultivar similarity) it will be necessary to use some other, more sensitive techniques.
The genetic diversity of re-established population of endangered species Allium angulosum L. was tested as a one part of rescue program. Founder individuals were picked in Chropyne - Zarici area (North Moravia, Czech Republic) and new population was set in Protected Landscape Area Litovelske Pomoravi (North Moravia, Czech Republic). The task was whether the newly founded population was made by representative individuals to cover (include) the genetic variability of source (mother) population. Items were tested with variability assay of six isozyme systems (G-6- PDH, AAT, PGM, EST, ACP, PGI) using discontinuous polyacrylamide gel electrophoresis (PAGE). The method stated relatively sufficient level of variability on condition that new population would be raised to prevent genetic changes. Application of more tests checking the genetic diversity within population could be useful during reintroduction and management of endangered plant species.
Entomopathogenic and mycoparasitic fungi were characterised by RAPD technique, with special attention to evaluate the genetic stability of strains that are used as active ingredients in commercial biopesticides. Strain-specific fingerprints were constructed for Paecilomyces fumosoroseus – strain PFR 97 Apopka, Gliocladium virens – strain GL 21 and Verticillium lecanii – strain MYCOTAL. Genetic stability and homogeneity was confirmed among re-isolates that were obtained from commercial batches of bio-insecticide PFR 97TM 20%WDG and bio-fungicide SoilGardTM12G that had been produced in 1995–1999. RAPD analysis indicated the genetic identity of V. lecanii strains re-isolated from the two different bio-insecticides MYCOTAL® and VERTALEC®. The usefulness of RAPD technique was demonstrated when P. fumosoroseus strain PFR 97 Apopka was reliably identified after having passed through adults of the spruce bark beetle Ips typographus, and by analysis of the relationship between fungi of the genus Gliocladium.
Nový typ molekulárního markeru AFLP, založeného na fluorescenci, byl používán pro svou schopnost identifikovat odrůdy a homogenitu DH linií řepky olejné. Každá ze šesti testovaných AFLP kombinací detekovala polymorfismy, nejlepší kombinace (M-CAA/E-ACT) měla 26% polymorfních píků z celkového počtu 90 a bylo možné rozlišit analyzované odrůdy a 4 z 5 DH linií. Genetická podobnost byla vypočítána použitím klastrové analýzy (metody UPGMA). Ukázalo se, že genetická podobnost mezi DH liniemi je nízká, i když tyto linie byly získány mikrosporogenezí. PCO analýza odrůd ukázala dva uzavřené klastery. Pouze u odrůdy Arabela byla zaznamenána větší odlišnost.
Seven cultivars of common buckwheat were tested in field trials under two levels of nitrogen fertilisation on two experimental sites during 1998–2000. The aim of the experiments was to evaluate the influence of cultivar, nutrition and year on main technological quality parameters (thousand achenes weight, volume weight, proportion of fractions on sieves 4.5 and 4 mm, proportion of husks and yield of groats). The differences were observed between buckwheat cultivars in all observed parameters of technological value. Nitrogen fertilisation before sowing (50 kg.ha–1) did not influence any parameter. On the contrary, buckwheat technological value was influenced by sequence weather (particularly rainfalls) during flowering and achenes formation periods (July). The influence of year was manifested especially on development of endosperm and husks of achenes. Better growing conditions on experimental site caused lower values of volume weight, lower proportion of pericarp (husks) and considerably higher proportion of fraction over 4.5 mm.
In Brassica species, self-incompatibility has been mapped genetically to a single chromosomal location. In this region several closely linked genes have been identified. One of them, S-locus receptor kinase (SRK), determines Shaplotype specificity of the stigma and it’s the key protein for SI reaction. The role of the Slocus glycoprotein (SLG) gene remains unclear. In the last decade approximately 15 additional genes linked to S-locus have been found. Recently, a gene has been identified (SCR) that encodes a small cysteine-rich protein which is a candidate for the pollen ligand. In addition to Slocus linked genes there are unlinked SLR genes (S-locus related genes). In this review, we discuss the role of these genes and the current view on the self-incompatibility mechanism in Brassica.
Extracts of cotyledons of Brassica napus plants (seed progenies of doubled haploid plants) were separated by electrophoresis on polyacrylamide gels and stained for acid phosphatase (ACP - E.C. 22.214.171.124.) and leucine aminopeptidase (LAP - E.C. 126.96.36.199.) enzymes to investigate the possibility of utilising isozymes as markers of homogeneity (purity) of plant populations. One zone of activity for acid phosphatase and two zones of activity for leucine aminopeptidase were identified on gels, some variation in isozyme patterns occurred in several androgenetic lines. This method is appropriate and lines-progenies of doubled haploid (D.H.) plants.
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Cílem metodiky je podat přehled o v praktickém šlechtění použitelných postupech pro selekci cílových rostlin - postupech založených na technikách molekulární biologie a přístupech molekulárního markerování.
Matice podobnosti pro určení genetické vdálenosti mezi odrůdami řepky olejky na základě použití tří molekulárních markerů: SSR, ISSR a AFLP.
Řepka (brukev řepka olejka, Brassica napus L.) patří mezi nejvýznamnější olejniny a v současné době se ve světovém měřítku řadí na druhé místo hned za sóju. V evropě je z hlediska výměry nejvýznamější olejninou, je pěstována na 60% (6,5mil ha) celkové plochy celkové plochy této skupiny (10,8 mil. ha.). Nejinak je tomu i v České republice, kde sklizňové plochy řepky olejky kolísají v rozmezí 354-400 tis.ha. řepka olejka je tak v ČR co do výměry třetí nejvýznamnější plodinou (po pšenici a jarním ječmenu). Řepkový olej je velmi kvalitní a téměř srovnatelný s vysoce ceněným olejem olivovým. V průběhu šlechtění došlo k významným posunům ve skladbě mastných kyselin. V současné době existuje také řada šlechtitelských programů cíleně zaměřených na získání odrůd se změněnou skladbou mastných kyselin dle specifického využití dané odrůdy.
V současné době je šlechtění řepky zaměřeno na tvorbu F1 hybridů. Při produkci hybridního osiva je nutné zamezit samosprášení. U řepky je možné využít autoinkompatibility, přirozené se vyskytujícího systému zabraňujícího opylení vlastním pylem u některých planých druhů rodu Brassica i u některých linií B.napus. U brukvovitých rostlin jde o sporofytický typ AI, kde pylový determinant je produktem buněk tapeta. K hybridnímu šlechtění jsou vhodné pouze AI linie s vysokou účinností AI reakce, aby byl podíl hybridního osiva maximální i polních podmínkách. Podíl hybridního osiva je možno stanovit prakticky pouze na základě vhodného genetického markeru.
Kulturní brambor, Solanum tuberosum L., je jednou ze čtyř nejvýznamnějších plodin světa spolu s pšenicí, rýží a kukuřicí a využívá se jak ve výživě lidí, tak i ve výživé hospodářských zvířat. Významné jsou i jeho využití pro účely průmyslového zpracování. Geneticky modifikované organismy (GMO) jsou žijící organismy, jejichž geentická informace byla změněna pomocí technik genové manipulace, tzv. technik rekombinantí DNA. Touto genetickou manipulací je obvykle vnesení sekvence cizorodé DNA (insertu) do recipientního modifikovaného organismu. Tento proces se nazývá transformace.
Technické řešení se týká bílkovinného koncentrátu, který se připravuje z hlízové vody brambor, a ve kterém je zachována rozpustnost izolovaných bílkovin pro použití tohot bílkovinného koncentrátu v potravinářském průmyslu a v biotechnologických aplikacích.
Rok 2008 je z pohledu bramborářství oproti ostatním rokům obzvláště významný, neboť OSN jej vyhlásila Mezinárodním rokem brambor. Heslem se stalo "HIDDEN TREASURE", neboli SKRYTÝ POKLAD, či spíše SKRYTÉ BOHATSTVÍ. Brambory jsou celosvětově významnou plodinou, řadí se do tetrády nejvýznamnějších plodin. V současné době se pěstují na téměř 20 mil.ha a celosvětově je registrováné přes 4200 odrůd, které jsou pěstovány ve více než 100 zemích světa. V České republice je ve Státní odrůdové odrůdové knize zapsáno 161 odrůd - stav z roku 2008. Platný zákon č. 110/1997 Sb. o potravinách a tabákových tabákových výrobcích a vyhláška na něj navazující (Vahláška MZe č. 332/1997 Sb.) vyžadují u konzumních brambor garanci odrůdové deklarace při obchodním styku.