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Preliminary Report of Cytometric Evaluation of Genotypes and Morphometric Data of Selected Taxa Prunus

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Preliminary Report of Cytometric Evaluation of Genotypes and Morphometric Data of Selected Taxa Prunus PRELIMINARY REPORT OF CYTOMETRIC EVALUATION OF GENOTYPES AND MORPHOMETRIC DATA OF SELECTED TAXA PRUNUS Adela Kišacová, Ľuba Ďurišová, Ľudmila Galuščáková, Tibor Baranec Department of Botany, Faculty of Agrobiology and Food Resources, Slovak University of Agriculture in Nitra, Slovakia Corresponding author: [email protected] Abstract Representatives of the genus Prunus are among widespread taxa in the communities of wild bushes lining the lowlands. In our research we focused on monitoring and the analysis of the genome size of Prunus x fruticans, Prunus × dominii and Prunus × fetchneri from the sites Dolné Lefantovce, Bádice and Podhorany. We determined the genome size of the monitored species using flow cytometry. By comparing the reference samples and standards we set the absolute amount of DNA in nucleus. The results were recorded using a histogram. For the measurement of plant material by flow cytometry, it was necessary to release the nuclei of cells from plant tissues. In preparing the samples, we used the fluorescent colouring agent Propidium Iodide, which is able to absorb light and emit radiation of different wavelengths. Colouring agents used in the analysis of the nuclear contents had to specifically bind to DNA. The relative size of the nuclear genome was determined by comparing the arithmetic mean of the top of the sample and the standard in the histogram. The highest average genome size was observed at 1.91 ± 0.11 pg in P. × fetchneri and the smallest 1.36 ± 0 pg at P. × dominii. Morphological evaluation of selected taxa showed the smallest range in the average stone length of 9.00 ± 0.38 mm for P. × fruticans and 13.46 ± 0.29 mm for P. × fetchneri. The greatest weight of the stones showed P.fetchneri 0.49 ± 0.10 mm and the smallest 0.17 ± 0.015 mm P. × dominii. The length of kernels, width, thickness and weight of the kernels showed considerable variability of studied taxa. Key words: Prunus variability, flow cytometry, genome size, kernels 1 Introduction Family Rosaceae is a very large group, which ranks among the genus Prunus. Representatives of this family, combined with other types form wild forest belts corridors. Kalkman (2004) wrote that, Rosaceae are a moderately large family, comprising an estimated 85 genera and approximately 2000 sexual species. The family Rosaceae distribution is cosmopolitan to sub-cosmopolitan, but is diversified, particularly in the Northern hemisphere. The herbaceous species grow in temperate forests as understory plants, in salt or freshwater marshes, in arctic tundra, in old fields, and along roadsides. Woody members are pioneer species, and are prominent in the early stages of forest succession. Rosaceous trees may also be minor components of mature mixed deciduous forests (Judd et al., 1999). In Slovakia there are abundant indigenous scrub communities (series Prunetaila spinosae), in which the dominant species belong to the genus Prunus, Crataegus and Rosa. Among the main components of these shrubs belong Prunus spinosa and P. × fruticans. Baranec et al. (2010) in the evaluation of local populations noted that autochthonous hybrids are P. × fruticans, P. × fetchnerii and P. × dominii. The dominant taxon of phytocoenoses becomes P. × fruticans. We have no data about the presence of this hybrids in Slovakia. Domin stated the populations near Štúrovo, which can be considered a hybrid between P. × fruticans and P. spinosa subsp. dasyphylla (Bertová et al., 1992). Fruits of studied specimens P. × fruticans from the site Čechynce mature in September (Rybnikárová et al., 2009). The aim of the work was the analysis of genome size and evaluation of variability of selected morphometric characteristics of stones. Flow cytometry for cytometric analysis was used. 47 2 Materials and Methods The subject of the observation were the following taxa: cherry plum - Prunus × fruticans, Prunus × dominii, and Prunus × fetchneri. Taxa occurr as part of unmaintained shrub strips (bio-corridors) on the edges of agricultural areas. Observed sites were: Dolné Lefantovce, Bádice and Podhorany which are in the land of the city Nitra. 2.1 Characteristics of the observed sites The areas of interest lie in the northwestern foothills of Tribeč on the alluvial cones of left sided of the river Nitra. According to the average monthly and annual amounts of solar emission 2517 kWh.m2 reaching the upper limit of the atmosphere, we include the territory of Nitra in warm areas. Average monthly and annual sums of global emission (1961 - 1990), represent for this territory 1237 kWh.m2 a year (Šiška, et al., 2005). Biological material was collected from 5 of the marked bushes of selected bio-corridors. For flow cytometeric measurements we used drupes, from which were isolated nuclei with extraction buffer. 2.2 Characteristics of the measuring equipment - flow cytometer FCM Flow cytometry (FCM) is a fast and effective way of simultaneously analysing several optical properties (fluorescence, light scatter) of single particles in suspension as they move in a narrow liquid stream through a powerful beam of light (Shapiro, 2004). The analysis of samples was performed by flow cytometry CyFlow ® ML (Partec, Germany) using a solid laser emitting green light of wavelength 532 nm. Measurement of each sample was performed in triplicate and measured values were processed in the form of a histogram of fluorescence intensity of measured particles. Flow cytometry, a fast and accurate method for the estimation of DNA content, has become the predominant technique for establishing plant genome size. The most common procedure of sample preparation involves chopping plant organs/tissues (mainly leaves) in a nuclei- isolation buffer and measuring the fluorescence of a fluorochrome intercalated into the DNA double helix (Jedrzejczyk, Sliwinska, 2010). 2.3 Determination of genome size of plant material Biological material was collected from 5 of the marked bushes of given bio-corridors. For flow cytometeric measurements we used kernels, from which were isolated nuclei with extraction buffer. The Rosaceae family is economically very important because many of the species are cultivated for their fruits (e.g., Malus, Pyrus, Prunus, Fragaria, and Rubus) or have ornamental value (Rosa). It has been well studied, although the systematic position and evolution of many taxa are still not clear. Knowledge of the genome size would be helpful in their classification. Within the Rosaceae, polyploidy series from diploid to 12 – ploid or higher occur. Since the chromosomes are small and often numerous, ploidy estimation by chromosome counts is difficult. In addition, microscopic chromosome counting is time- consuming and limited to a few tissues. Therefore, flow cytometry (FCM) is a more convenient alternative for establishing the ploidy/genome size of Rosaceae species (Jedrzejczyk, Sliwinská, 2010). Biological material was collected from 5 of the marked bushes of given bio-corridors. For flow cytometeric measurements we used kernels, from which were isolated nuclei with extraction buffer. To determine the genome size of assessed taxa we used as internal standard soya beans (Glycine max cv. Polanka) with the genome size (2C = 2.50 pg). For staining we used the solution of 50 ml propidium iodide (PI) and 60 ml of RNAse solution. 48 1. Preparation of nuclear suspensions For the analysis of genome size using the flow cytometry, we used seed segments of assessed taxa (key leaves and embryonic axis). Segments of the seeds were stored in a refrigerator at 4 °C for 4 months. Before analyzing the seeds were freed of sclerogenic endocarp and seed coat. From the internal standard (soya bean – Glycine max cv. Polanka), we used fresh young leaves (0.5 cm2). Using the methodology Doležel - Bartoš (2005) - Two-step OTO I and II we did our preparation of samples. Segment seeds were cut with a razor blade in a Petri dish (about 40 seconds) in 500μl extraction solution. The incubation period in the extraction solution lasted 30-90 seconds and then the sample was filtered into a tube. As a filter we used nylon filter (pore size 42 μm). To the filtrate was added a 1% solution polyvinylpyrolidone (PVP) (Jedrzejczyk, Sliwinska, 2010), with the staining solution propidium iodide (PI 50 ml) and RNAse (60 ml). Subsequently, the sample was incubated (60-90 min) at 4 °C in a refrigerator. The incubation period of internal standard (soybean) lasted 15 minutes. Before analyzing the samples in a flow cytometry, the sample was with a suspension of the internal standard in a 1:1 ratio. 2. The analysis using the flow cytometer Compared to ploidy measurement by conventional chromosome counting. FCM offers a valuable, rapid, simple, accurate and fairly cheap alternative. For this reason, it has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty, determination of reproductive pathways, and estimation of absolute DNA amount or genome size (Doležel and Bartoš 2005). Unlike microspectrophotometry and image cytometry, flow cytometry analyses microscopic particles in suspension, which are constrained to flow in single the within a fluid stream throught the focus of intense light. Pulses of scattered light and fluorescence are collected and converted to electric current pulses by optical sensors and classified. Because the particles are analysed individually and at high speed, large populations can be measured in a short time and the presence of subpopulations may be detected (Shapiro, 2003). The quality of nuclear suspension is best judged by analysing a histogram of relative nuclear DNA content. The histogram should contain minimal amounts of background debris, G1 (G2) peaks should be symmetrical and the variation should be low. The variation is usually expresed as the coefficient variation (CV) = standard deviation/peak mean x 100 %. Unlike the standard deviation, CV does not depend on peak mean and hence the precision of measurements with peaks at different positions may be directly compared. As shown by Doležel and Göhde (1995), histograms with peak CVs lower than 1 % may be obtained under specific conditions.
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