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. 2014 Dec 31;21(1):117–122. doi: 10.1007/s12298-014-0275-x

Initial determination of DNA polymorphism of some Primula veris L. populations from Kosovo and Austria

Naim Berisha 1,2, Fadil Millaku 1,✉, Bekim Gashi 1, Elez Krasniqi 1, Johannes Novak 2
PMCID: PMC4312321  PMID: 25650329

Abstract

Primula veris L. (Primulaceae) is a long lived perennial and well known pharmaceutical plant, widely collected for these reasons in almost all SE Europe and particularly in Kosovo. The aim of the study is to determine molecular polymorphism of cowslip (P. veris L.) populations from Kosovo. DNA extracted from leaves were  investigated in details for presence of polymorphism. RAPD analyses were conducted using 20 different short primers. Genomic DNA amplification profiles were analyzed and processed using data labelling. Comparison between cowslip populations in genetic composition revealed that samples from Bogaj were too distinct on their own. Molecular variation was observed to be more within populations (73 %) as compared to among populations (27 %). On the other hand, genetic distance of populations revealed that the highest genetic distance is between Leqinat and Maja e Madhe. Mean values of expected heterozygosity were highest in Bogaj population, while lowest in Maja e Madhe population. The obtained results indicated that Bogaj population are more polymorphic. From the obtained data it can be concluded that RAPD markers provided a useful technique to study genetic diversity in P. veris L. populations. This technology allows identification and assessment of the genetic similarities and differences among plant populations.

Keywords: Primula veris, DNA polymorphism, RAPD, Populations, Kosovo

Introduction

Primula veris (Primulaceae) is a small perennial herb, found typically on nutrient-poor grasslands, with a distribution ranging from Spain to eastern Asia (Hegi 1975). In the early spring, plants produce a rosette of several leaves and one flowering stalk (rarely two or more) that bears 5–15 yellow distylic flowers disposed in an umbel. Flowering usually begins in April and ends 3–4 weeks later in May. Seeds ripen in June–July. Flowers are mainly pollinated by Hymenoptera and Diptera (Woodell 1960). Survival rate of seedlings is generally low, though vegetative propagation through side rosettes sometimes occurs (Tamm 1972). The plant is considered to be a barochorous species, as the seeds of this species have no specieal features facilitating dispersal. Sometimes the leaves, flowers and inflorescences are damaged by insects or molluscs, while often they are destroyed by cattle grazing, affecting this way future potential performance (García and Ehrlén 2002). Primula plants have heteromorphic flowers and a sporophytically controlled, diallelic incompatibility system that prevents or reduces both self-fertilization and intramorph fertilization (Webster and Gilmartin 2006). In this paper, we consider the extent of polymorphism of cowslip (Primula veris L.) populations in Kosovo. Cowslip as a characteristic species for calcareous grasslands has shown a sharp decline not only in this part of Europe (Kosovo) but in larger parts of western Europe as well. (Kéry et al. 2000). This particularly comes as a result of overharvesting, as the plant species is widely collected for medicinal and thus commercial needs (Millaku 2010). These species are particularely at risk as their population numbers are often low and population loss is likely to result in a significant reduction in the number of their remaining populations (Glover and Abbott 1994). Biotic and abiotic processess that operate at the population level also play an important role in governing plant population dynamics (Schemske et al. 1994). While the effects of environmental and demographic stochasticity on plant population dynamics have been fairly well studied, the effects of genetic stochasticity remain to a large extent unexplored (Ouborg et al. 2006). Due to changed behaviour or isolation and/or abundance of pollinators, reduced gene flow between fragmented populations may lead to inbreeding which, in turn, may cause immediate loss of fitness (the so called inbreeding-depression; Ellstrand and Elam 1993; Young et al. 1996). In small populations, additionally the random genetic drift may erode their evolutionary potential towards adapting to changing environmental conditions (Frankham 1995; Barrett and Kohn 1991). A number of authors indeed, have shown positive relationships between genetic diversity, population size and other fitness measures, such as reproductive output (Vergeer et al. 2003; Leimu et al. 2006) suggesting that the population dynamics of a plant species may be strongly affected from decreased genetic diversity. Surprisingly though, only a few authors have integrated genetic information into studies on the demography of the plant species (Newman and Pilson 1997; Menges and Dolan 1998). In order to properly understand the relative importance of ecological and genetic factors in determining population viability, this kind of research is crucial (Reed et al. 2002; Oostermeijer et al. 2003). In Kosovo, Primula veris is mainly distributed in Albanian Alps of Kosovo, though it is found also in other places in lower number of present populations (Millaku 1999, 2005, 2010; Rexhepi, 2003). The Albanian Alps are divided into three parts by a boundary between Kosovo, Albania, and Montenegro. The part of these mountains that is under the administration of Kosovo has a North–East position and is influenced by the continental climate. By collecting plant samples from 3 different cowslip populations in Albanian Alps, detailed information was gathered about their life cycle, plant associations, habitat types and soil composition. Having needed knowledge about genetic varieties of cowslip populations in Kosovo would be important for having sustainable plant sources in the future. We have aimed to find out, in this research, how many existing morphs are present within and between analyzed cowslip populations, which of the observed populations are more closely related to each other and what are the chances of effective breeding and domestification of the species. Furthermore, by getting to know more about the genetic constitution of cowslip populations, we will enable ourselves to deduce quota for harvesting on wild populations and ensuring sustainable collection.

Materials and methods

Plant material

This research was conducted in 2010, being split in two main parts. In early spring we have collected plant samples, while the laboratory analyses were conducted during the time period October–December. Plant samples of cowslip (Primula veris L.) were collected from a random sample of individuals, growing in populations located in Albanian Alps of Kosovo (Table 1.). From three surveyed populations we took approximately 20 individuals each, sampled from large populations at Bogaj, Maja e Madhe and Leqinat, which contain several thousand individuals. For comparison reasons, in our study we have also included one set of Austrian cowslip samples, collected in suburbs of Vienna (Pötzeleinsdorfer Park). The plant samples were naturally dried at room temperature and voucher specimens of the populations are kept at the Faculty of Natural Sciences herbarium.

Table 1.

List of Primula veris populations from Kosovo and the Austrian samples

No. Locality name Population code
1 Maja e Madhe L01-XK
2 Bogaj L02-XK
3 Liqenat L03-XK
4 Pötzeleinsdorfer Park L04-AT
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Genomc DNA purification

Genomic DNA was extracted from dried leaves of Primula veris using a modified cetyltrimethyl ammonium bromide (CTAB) extraction method (Doyle and Doyle 1990). Leaves were ground into a fine powder in liquid nitrogen with pestle and a mortar, resuspending it in 25 ml CTAB extraction buffer. For 20 min the suspension was incubated at 65 °C before the addition of 10 ml chloroform:isoamyl alcohol and gentle mixing for 20 min at room temperature. DNA quality was tested on 1.4 % agarose gel and its concentration was measured with fluorometer. DNA quantitation data were generated using reagents and protocol contained in the Total DNA Quantitation Kit (Briggs and Panfili 1991). This method is based on the action of a compound known as Hoechst and TEN—commercially known buffer.

RAPD PCR

Amplification reactions were performed in a 25 μl volume containing 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 2.25 mM MgCl2, 0.001 % gelatin, 0.2 mM each dNTP and 1.75 units Taq (Super-Taq, HT Biochemicals). Each reaction contained 5–50 ng of genomic DNA as template. The PCR cycling started with an initial phase of 15 min (for the Taq HOT FIREPol® polymerase) at 95 °C, then 40 cycles of 10 s at 95 °C, 20 s at 60 °C and a 20 s elongation step at 72 °C. High resolution melting was carried out immediately following PCR from 70 °C to 90 °C at steps of 0.05 °C, each step with a 1 s hold. PCR reaction were mixed in 4 μl loading dye and run on 2 % agarose gel. In order to enable measuring of DNA band lengths, in the left and the right side of the agarose gel we have put 1 μl of standard DNA (100 bp ADN-Ladder, 0,5 mg/ml, Peqlab Biotechnologie GmbH, Erlagen, Deutschland). After this, gel was stained with  ethidium bromide solution in order to enable DNA bands be visualized by UV fluorescence. Only clear, distinct and reproducible bands were included in the diversity analysis. Bands of the same size were scored as identical. The reproducible bands were converted into binary data (presence = 1, absence = 0), and the resulting data set was analyzed.

HRM and data processing

PCR and HRM were performed on a RotorGene 6500 (Corbett Research Pty Ltd, Sydney, Australia) with a HRM-module, the results were analyzed using the RotorGene 6000 series software, Version 1.7.65. In order to obtain interrun comparability standard, samples were used in every run. Analyzed genetic parameters—Principal Coordinate Analyses (PCA), Percentage of Molecular Variance, Genetic Distance, Mean and SE for each population, were calculated using a software program ‘GenAleX 6’ (Peakall and Smouse 2006).

Results

Using high resolution melting curve analysis (HRM) as a technique for measuring exactly the decreasing fluorescence of intercalating dye in the process of dissociation of double stranded DNA, we tried to find appropriate molecular markers suitable for detecting  polymorphism between analysed Primula veris populations of Kosovo. For this reason 20 molecular markers were used. Despite of the fact that HRM is a very specific, time-and labour-saving method for identifying DNA sequence variations and is ideally suitable for routine PCR analysis (Schmiderer et al. 2010), in our study none of the used markers showed any polymorphism, (Fig. 1) where all the melting curves are the same. After this, we tested different microsatellite primers, that were previously developed but yet not tested in Primula veris samples, with the aim to verify if any genetic polymorphism between and within the populations from Kosovo can be found. Again, out of 20 different microsatellite markers that were used, no one was polymorphic. These HRM graphs did not indicate any ploymorphism, so all these used molecular markers failed (Fig. 1). After this, we switched the rest part of the work to RAPD (Random Amplification of Polymorphic DNA), that is a PCR reaction type but with the difference that the amplified DNA segments are random. Also, the use of RAPD markers is favourable over other methods (e.g., AFLP, nuclear (SSRs), or allozymes) for their technical simplicity and for providing genome-wide information from many loci (Daskalova et al. 2012). Using RAPD we were able to find and use good primers and detect and evaluate the genetic constitution of Primula veris populations from Kosovo.

Fig. 1.

Fig. 1

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Overwiew of all curve forms appearing from four Primula veris population sample

We have successfully tested 15 RAPD primers. As shown in Fig. 2, we have formation of DNA bands in different lengths. Afterwards, we continued with data processing. We observed that samples from Bogaj (L02-XK) are too distinct within their own, while the Austrian set of samples (L04-AT) are not so far from the rest of Kosovarian set of Primula veris samples (Fig. 3). We have had variations between analyzed populations, either though the real genetic distance between populations at site was not so high (with exception of Austrian samples). While analyzing the percentage of molecular variation (Fig. 4 & Table 2) of populations, we have observed that the variation among populations is lower (27 %) when compared with expressed variation within populations, which is surprisingly higher (73 %). Within our L04-AT samples we have had also some hybrids of Primula veris x vulgaris, which surely have increased values of variance inside populations versus those among populations. The analysis of genetic distance (Table 3) between populations varies between 0.028 (L02-XK and L04-AT) and 0.088 (L01-XK and L03-XK, having so the largest genetic distance). Also, we have variations between populations when it comes to pairwise matrix of genetic identity (Table 4). Pairwaise distances between populations vary from 0.972 between L02-XK and L04-AT to 0.916 between L01-XK and L03-XK.

Fig. 2.

Fig. 2

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Example of PCR-RAPD amplification profile, generated from genomic DNA of Primula veris populations, resolved on 2.0 % gel agarose

Fig. 3.

Fig. 3

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Principal Coordinate Analysis (PCA), showing the difference of genetic composition between analysed populations

Fig. 4.

Fig. 4

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Scatter plot for relationship in terms of molecular variation within and between Primula veris populations

Table 2.

Summary of AMOVA—molecular variation in %

Source df SS MS Est. Var. %
Among Pops 3 16.250 5.417 0.427 27 %
Within Pops 36 41.400 1.150 1.150 73 %
Total 39 57.650 1.577 100 %
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Table 3.

Matrix of populations genetic distance according to Nei

Primula veris Populations
L02-XK L01-XK L03-XK L04-AT
0.000 L02-XK
0.032 0.000 L01-XK
0.060 0.088 0.000 L03-XK
0.028 0.040 0.049 0.000 L04-AT
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Table 4.

Pairwise population matrix of Nei genetic identity

Primula veris Populations
L02-XK L01-XK L03-XK L04-AT
1.000 L02-XK
0.968 1.000 L01-XK
0.942 0.916 1.000 L03-XK
0.972 0.961 0.952 1.000 L04-AT
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The expected level of Heterozigosity (He) was also measured among other values in studied populations, (Table 5). He is higher in L02-XK when compared with three other populations, while lowest He values were recorded in L01-XK. Expected level of heterozygosity is an important value when it comes to genetic variation of natural populations. Higher He means higher genetic variability and vice versa.

Table 5.

Mean values of population genetic variation for four  Primula veris populations

Mean and SE over Loci for each Population
Pop N Na Ne I He UHe
L02-XK Mean 10.000 1.000 1.241 0.234 0.151 0.159
SE 0.000 0.277 0.087 0.072 0.049 0.051
L01-XK Mean 10.000 0.929 1.102 0.141 0.080 0.084
SE 0.000 0.267 0.038 0.049 0.029 0.030
L03-XK Mean 10.000 1.000 1.143 0.176 0.103 0.109
SE 0.000 0.277 0.055 0.055 0.035 0.037
L04-AT Mean 10.000 0.714 1.218 0.194 0.130 0.136
SE 0.000 0.266 0.091 0.075 0.051 0.054
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SE Standard error; N Number of individuals; Na Number of alleles; Ne Number of effective alleles; I Shannon Index (or Diversity Index); He Expected heterozigosity, Uhe Expected neutral heterozigosity

Discussion

This study is the first one to use molecular markers such as DNA sequencing, to determine the polymorphism of Primula veris populations in Kosovo. The levels of genetic variation in plant populations are highly variable (Hamrick and Godt, 1990). In this work, the very low level of genetic polymorphism was found in natural populations of Primula veris in Kosovo which confirms the similar results reported by other authors (Antrobus and Lack 1993; Young et al. 1996; Morozowska and Krzakowa 2003). Plant species exhibit great morphological and functional variation, much of which is thought to be adaptive. In order to understand the process of adaption, it is required to synthesize several disparate levels of information. These levels include information from knowledge about measured genetic diversity and exploring the relationship between genetic variants and the environment. (Wright and Gaut 2005).

Analysed Primula veris populations showed that the genetic distance between them is not so high. Our results do indicate that the Primula veris samples from L02-XK are with higher genetic polymorphism, being so much more suitable and far more resistant to environmental  changes. This is an important factor for sustainability of natural Primula veris populations and harvesting of these plants in wild. Additionally, we have observed that the variation between populations is lower when compared to molecular variance within populations, what makes us believe that the plant populations are composed also of hybrids, most probably of P. veris × P. vulgaris, as both species do not differ significantly also in sexual organ positions (Keller et al. 2012). Especially at some populations, we have found mixed populations of Primula veris x Primula vulgaris, an occurrence phenomenon also reported by Brys and Jacquemyn (2009).

The results show that the Primula veris populations in this part of Kosovo are not so genetically diverse, though observed diversity suggests that populations are dynamic and it is possible to increase population size with adequate management (Sørensen et al. 2014). This should encourage medicinal plant cultivators to expand Primula veris propagation with the aim of conserving natural populations. Monitoring of reproduction of Primula veris populations may therefore provide important indicators of future population trends.

Even though Primula veris is not listed as a rare or endangered species in Kosovo (Millaku et al. 2013), its occurrence in Kosovo was formerly more common than it is today. In many areas of its original distribution, the abundance and occurrence of Primula veris declined mainly as a result of changed land-use practices, ongoing habitat fragmentation, harvesting as a medicinal plant, etc. Combining intense grazing, in order to allow seedling establishment and relatively relaxed grazing, allowing seed production, might be the optimal management strategy to maintain viable Primula veris populations in long-term scale (Brys et al. 2005; Brys and Jacquemyn 2009). This sort of management regime could be achieved through applying a year-round variation in grazing combination.

Acknowledgments

This work has been carried out with financial support from the German phytoneering company Bionorica SE, which is gratefully acknowledged.

Abbreviations

RAPD

Random amplification of polymorphic DNA

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