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

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.

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.

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. The CV with 3 % are considered fully acceptable (Marie and Brown, 1993; Galbraith et al., 1998).

2.4 The size and weight of the stones of studied taxa Kernels weight were determined by laboratory scales KERN 440-45N. The length and width of kernels were determined by caliper (we weighed and measured 20 kernels from each individual). Fruits of studied taxa, which are bare stone fruit, ripen in late summer. Ripe fruits of P. spinosa and P. × fruticans are round, black, blue oinovated as the Bertová states (1992).

Fig. 1 Morphometric characterization kernels of observed taxa

Fig. 2 Measurement length, width and thickness of kernel Prunus × fruticans from locality Dolné Lefantovce

3 Results and discussion 3.1 The size of the genome of studied taxa The 2C – values in the Rosaceae ranges from 0,20 to 7,30 pg. Out of four subfamilies, a relatively large genome size occurs in the Spiraeoideae, which supports the polyploidy origin of this subfamily. Although crosspollination between individuals with different ploidy levels is possible in the Rosaceae, no differences in ploidy were observed here between leaves and seeds originating from the same tree/shrub. Nevertheless, in seeds of species where there is a polyploidy series it is possible that hybridization between plants of different ploidies has occurred, and that the DNA content in the embryo may not correspond to that of the mother plant. In such cases, the leaves of the parent should be analyzed as a ploidy control (Jedrzejczyk, Sliwinská, 2010). Our results with genome size of plum taxa range from 1,36 to 1,91 pg. Jedrzejczyk and Sliwinska (2010) indicate that 2C DNA content of seeds of Prunus padus was 1,15 ± 0,02 pg. The size of the genome studied taxa was evaluated by cytometric analysis. The genome sizes of cherry plums was 1,53 ± 0,20 pg from the site Dolné Lefantovce while the size of the genome of the same genotype from the site Podhorany showed 1,48 ± 0,02 pg. The amount of the DNA of genotypes P. × dominii varied from 1,36 ± 0 pg to 1,39 ± 0,20 in the site Dolné Lefantovce. The size of the genome of P. × fetchneri had values at 1,91 ± 0,11 pg in site Bádice. Cytometric analysis showed a significant difference in genome size in P. × fetchneri from other studied genotypes. By comparing the measurements with seeds genome size of cherry plum with genotypes results Muráňová et al. (2011) in P. × fruticans from the site Čechynce, which reached values between 1,42 ± 0,01 pg to 1,55 ± 0,01 pg.

Fig. 3 Flow cytometric profile of Prunus × fruticans with internal standard nuclei (Glycine max) in average 1,53 ± 0,20 pg of locality Dolné Lefantovce.

Fig. 4 Flow cytometric profile of Prunus × fruticans with internal standard nuclei (Glycine max) in average 1,48 ± 0,02 pg of locality Podhorany.

We recorded the thickness, width, length and weight of the kernels by morphometric characteristics. The length of the kernel of sampled specimens of P. × fruticans was at 9.00 ± 0.38 mm, of the genotypes of P. × dominii 9.35 ± 0.53 mm and 10.25 ± 0.49 mm from the site Dolné Lefantovce. Prunus × fetchneri showed the length 13.46 ± 0.29 mm from the site Bádice and P. × fruticans 9.79 ± 0.06 mm from the site Podhorany. Muráňová et al. (2011) states that the length of the stone of fruits of sampled specimens from the site Čechynce CH19 and CH39 P. × fruticans was 9 ± 0.79 mm, the genotype CH49 at about 9.6 ± 0.54 mm and shrub CH29 was the longest kernels (mean 12.2 ± 0.82 mm). Rybnikárová, et al. (2010) states, that the average length of kernels P. × fruticans ranging from 8.56 to 11.06 mm. The coefficient of variation in this character varies in the range 4.85 to 6.39 %. The significant differences were not confirmed. According to our calculations, coefficient of variation of the length kernels varied in the range from 0.64 to 11.11 %. The width of kernels no. 1 was 7.46 ± 0.36 mm and thickness 5.60 ± 0.21 mm, at no. 2 7.03 ± 0.25 mm and thickness 4.66 ± 0.35 mm and at an individual no. 3 the width 8.13 ± 0.22 mm and a thickness 5.40 ± 0.21 mm from the site Dolné Lefantovce. The kernels width was 9.87 ± 0.69 mm and a thickness 6.90 ± 0.74 mm from locality Bádice. The shrub from the location Podhorany showed the greatest width of the kernel of taxa P. × fruticans 8.59 ± 0.42 mm and a thickness 6.34 ± 0.35 mm. Muráňová et al. (2011), states that the greatest width of the stones was seen in the individual CH19 P. × Fruticans 8.1 ± 0.31 mm from the site Čechynce. Rybnikárová, et al. (2010) followed the same taxon from the same site, whereby the average width of stones varied ranging from 8.09 to 9.26 mm. We noted the weight of kernels of studied taxa from 0.17 to 0.49 g. The lowest weight was recorded for P. × dominii 0.17 g and 0.49 g of the largest P. × fetchneri. Observed morphometric characters showed statistically highly significant differences between the studied subjects. The results confirmed the observations of Rybnikárová et al. (2010), which

states that the average weight of stones varied from 0.29 to 0.61 g. The coefficient of variation in this character varies in the range 9.89 to 20.57 %. According to our measurement of coefficient of variation of the same character varies between 4.16 to 20.40 %. Results of the analysis of genome size of observed taxa show a diversity of studied taxa, which consist only of pure hybrids, but also transitional forms. The results of the morphological characters of measurement of length, width and weight of the stones didn´t show established relationships compared with the size of the studied genome taxa. The increased size of the genome did not ultimately affect the elevated levels of morphometric data. High variability was observed in the coefficient of variation in the length of the stones ranging from 0.64 to 11.11 %, which indicates a significant variability in the observed

indicators.

Morphometric analysis of kernels 16 thickness width length weight 14

1.Prunus x fruticans 2.Prunus x dominii 3.Prunus x dominii 4.Prunus x fetchneri 5.Prunus x fruticans

Fig. 5 Morphometric analysis are looked width, length and weight all taxa of choosing localities. The biggest different were in length and smallest were of kernels weight.

Morphometric analysis of length kernels in mm

8 9 9,35 10,25 9,79 6 13,46

0 1 2 3 4 5

Fig. 6 Average length of kernels from 9 to 13,46 mm. The longest kernel was from taxon Prunus × fetchneri and smallest stone was of taxon Prunus × fruticans

The differences obtained in our analysis may have been caused by significant agricultural activities on adjacent managed areas, giving bigger assumes for increasing hybridization with non-originating cultural species. Morphometric data demonstrate the considerable variability among the bushes of the selected sites. Discovered significant differences of observed taxa in genome size and morphometric characters suggest that there is a constant biocorridor hybridization. The results can be used to revitalize indigenous scrubland communities.

4 Conclusions Within the family Rosaceae, which is characterized by its great variability and frequency, hybridization often takes place. Wild species of the genus Prunus are often found along the biocenoses, which are exposed to adverse biotic and abiotic conditions. Under these conditions, their mutual intersections often happen. We conducted an analysis of variability between the genotypes taxa P. × fruticans, P. x dominii and P. × fetchneri comparing the genome size and morphometric evaluation of characters of stones. Analysis of variability and genome size is important for determination of taxa and their diacritical characteristics. The biological material was collected during the month of October 2011, from marked wild bushes. The objective of our research was focused on analyzing the variability in width, length and weight of the stone. In determining the absolute size of the genome taxa assessed by flow cytometry, we used seeds freed from seed coat. As the internal standard was used soya bean (Glycine max cv. Polanka). The size of the genome of individual genotypes listed taxa reached the measured values ranging from 1.36 to 1.91 pg. The morphometric characteristics of the stones showed the greatest length of Prunus × fetchneri. High variability was observed in the long stones and normal variability were measured of the width and weight of the stones.

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