Acta agriculturae Slovenica, 119/2, 1–8, Ljubljana 2023 doi:10.14720/aas.2023.119.2.13280 Original research article / izvirni znanstveni članek Characterization of nuclear DNA content and chromosome numbers of Tulipa luanica Millaku, T. kosovarica Kit Tan, Shuka & Krasniqi and T. albanica Kit Tan & Shuka Mirsade OSMANI 1, 2 , Metin TUNA 3 , Isa R. ELEZAJ 4 Received March 21, 2023; accepted May 31, 2023. Delo je prispelo 21. marca 2023, sprejeto 31. maja 2023 1 Laboratory of Biochemistry, Faculty of Food Technology, University of Mitrovica “Isa Boletini” , Mitrovicë, Republic of Kosovo 2 Corresponding author, e-mail: mirsade.osmani@umib.net 3 Department of Field Crops, Faculty of Agriculture, Namik Kemal University, Tekirdag, Turkey 4 Department of Biology, Faculty of Mathematical and Natural Sciences, University of Prishtina “Hasan Prishtina” , Prishtina, Republic of Kosovo Characterization of nuclear DNA content and chromosome numbers of Tulipa luanica Millaku, T. kosovarica Kit Tan, Shuka & Krasniqi and T. albanica Kit Tan & Shuka Abstract: The Balkan Peninsula is considered an impor- tant centre of native tulip species. Tulipa kosovarica and Tulipa luanica are new species recently discovered in Kosovo, and Tu- lipa albanica in Albania. The current study aims at the investi- gating the nuclear DNA content and chromosome number of these three tulipa species in order to provide for the first time data on their genome size and differences among these three Tulipa species. Analysis of nuclear DNA content was performed by flow cytometer (Partec CyFlow Space) in mature fresh leaves for each Tulipa species. Samples for chromosome analysis were taken from the root tip meristem of the bulbs. Results showed significantly higher amounts of nuclear DNA (2C) in T. lua- nica compared to T. kosovarica and T. albanica. The chromo- some number for these three species was 2n = 2x = 24, while the chromosome sizes of T. luanica resulted larger, compared to that of T. kosovarica and T. albanica. A correlation between the nuclear DNA content and chromosome size was found among these tulipa species. Moreover, nuclear DNA content and chromosome sizes of T. luanica, T. kosovarica and T. albanica showed clear differences among these species. Key words: tulip; DNA content; chromosome number; endemica Določitev vsebnosti jedrne DNK in kromosomskega števila treh vrst tulipanov, Tulipa luanica Millaku, T. kosovarica Kit Tan, Shuka & Krasniqi in T. albanica Kit Tan & Shuka Izvleček: Balkanski polotok je pomemben center samoni- klih vrst tulipanov.Vrsti Tulipa kosovarica in Tulipa luanica sta novi vrsti nedavno odkriti na Kosovu, in vrsta Tulipa albanica v Albaniji. Namen raziskave je bil preučiti vsebnost jedrne DNA in kromosomskega števila teh treh vrsta tulipanov in tako prvič določiti velikost njihovega genoma in razlike med temi tremi vrstami. Analiza jedrne DNA je bila narejena s pretočnim ci- tometrom (Partec CyFlow Space) v odraslih svežih listih vseh treh vrst. Vzorci za analizo kromosomov so bili vzeti z rastnih vršičkov korenin čebulic. Rezulatati so pokazali značilno večjo vsebnost jedrne DNA (2C) pri vrsti T. luanica v primerjavi z vrstama T. kosovarica in T. albanica. Kromosomsko število vseh treh vrst je bilo 2n = 2x = 24, med tem, ko je bila velikost kro- mosov vrste T. luanica večja v primerjavi z velikostjo pri vrstah T. kosovarica in T. albanica. Pri vseh treh vrstah je bila ugoto- vljena korelacija med vsebnostjo jedrne DNK in velikostjo kro- mosov. Vse tri vrste so se po vsebnosti jedrne DNK in velikosti kromosov jasno razločevale. Ključne besede: tulipan; vsebnost DNK; kromosomsko število; endemit Acta agriculturae Slovenica, 119/2 – 2023 2 M. OSMANI et al. 1 INTRODUCTION The genus Tulipa includes the most well-known, adored, and economically significant flowering plants in the world. In the World Checklist for Tulipa, 418 are named and 112 were accepted there (Govaerts 2008). Tulips species are grown in wild in North Africa, South- ern Europe, the Middle East, and Central Asia, includ- ing China. According to Botschantzeva (1962), the Tien Shan and Pamir-Alay Mountain ranges in Central Asia are considered as primary centers while the Caucasus as a secondary gene center for Tulipa species. In the Balkan peninsula Greece, Kosovo, Bulgaria, Albania, Macedonia and Serbia have the highest number of tulip species (Go- vaerts, 2010; Millaku et al., 2018). Up to date in Kosovo there are reported five native tulipa species, T. serbica Tatić  &  Krivošej, T. australis L., T. gesneriana subsp. T. scardica L., T. luanica Millaku and T. kosovarica Kit Tan, Shuka & Krasniqi (Millaku & Elezaj, 2015; Shuka et al., 2010, 2012). While the Tulipa genus is represented by two stenoendemic species, T. kosovarica and T. serbica, which are found only in the serpentines of Kosovo. T. scardica and T. kosovarica grow in serpentine soil, well-drained and exposed to full sun but protected from high winds, while T. luanica grows in limestone soil (Osmani et al., 2018). These serpentine soils were characterized by high concentrations of metals, and the levels of metals such as Ni, Co and Cr ranged from 1500 to 1600 mg kg −1  for Ni, 130 to 140 mg kg −1 for Co, and 380 to 450 mg kg −1 for Cr (Osmani et al., 2018). This previous study reported that the enzyme δ-aminolevulinic acid dehydratase activity and concentrations of δ-aminolevulinic acid, malondial- dehyde and glutathione showed differences among these three tulipa species, especially T. kosovarica and T. al- banica (serpentine sites) in comparison with T. luanica (limestone site). Despite the existence of a large body of literature on Tulipa, taxonomy is generally considered to be difficult. Moreover, molecular analysis is a good attempt to un- derstand the relationships within Tulipa species better. Nuclear DNA content can conveniently be measured by flow cytometry using propidium iodide, a stoichiometric DNA stain that intercalates in the double helix (Zonn- eveld, 2009). It is well known that morphological charac- teristics of plants are influenced by the vegetative stage of the plant as well as by a variety of environmental condi- tions. Therefore, the identification of the genetic diversity within and among plants species based on the morpho- logical plant characterization is considered insufficient (Hunter, 2018). DNA-based markers are an advanced tool widely used to assess genetic relationships and diver- sity in plant species (Kumar, 1999; Hunter, 2018), novel molecular marker techniques have been developed in di- verse plant species (Wang et al., 2015) and the transfera- bility of molecular markers between species has been in- vestigated for many species so far (Raveendar et al. 2015; Berisha et al. 2015). The recent advances in sequencing technologies enabled the discovery of functional genes in many plant species (Abbasi et al., 2015; Chai et al., 2017). Furthermore, these initiatives have enabled the develop- ment of novel and alternative molecular markers known as gene-targeted markers (GTMs), which are based on the untranslated sections of expressed sequence tags (ESTs) (Poczai et al., 2013) or gene-targeted functional markers (GTFMs), which are gene markers implicated in phenotypic trait variation as a result of their functional gene sequences (Arnholdt-Schmitt, 2005). In addition of above mentioned techniques, flow cytometry helps in the estimation of nuclear DNA content and the ploidy level (Dolezel et al., 2021; Dolezel et al., 2004; Vlacilova et al., 2002). Flow cytometry is a method that can conveniently measure the nuclear DNA content by using propidium iodide, a stoichiometric DNA stain. A genus may con- tain numerous species with similar chromosomal num- bers but different DNA 2C-values (Ohri, 1998). Flow cytometry is a fast and practical method for elucidating systematic relationships among species within the genus. This technique was efficiently employed in ecological, physiological, molecular biology and genome evolution studies, as well as in plant breeding (Dolezel et al., 2021). The chromosomal karyotype parameters of Iranian Tulipa species were studied by Abedi et al. (2015) and Masoud et al. (2002). Their findings demonstrated that while the majority of Iranian species had three distinct chromosome types—m, sm, and st—and were diploid (2n = 2x = 24), their karyotype parameters varied. In the Netherlands, the genome size of a variety of Tulipa spe- cies was studied with results ranging from diploid (30 pg) to tetraploid (123 pg) (Zonneveld, 2009). Based on our previous investigation, T. kosovarica and T. albanica grow on serpentine soils while T. luanica grows in limestone soil (Osmani et al., 2018). The serpen- tine soils were characterized by higher concentrations of metals compared with limestone soils. Since these three endemic species are relatively new species discovered in recent years and they grow in different habitats, tradi- tional taxonomy based on geographic distribution and morphological characteristics will be augmented with more information on DNA content and chromosome number to clarify the relationships among these Tulipa species. The main objective of this study was to deter- mine nuclear DNA content and chromosomes number of T. kosovarica, T. luanica and T. albanica, and in line with this to have more information about differences between these species. Acta agriculturae Slovenica, 119/2 – 2023 3 Characterization of nuclear DNA content and chromosome numbers of Tulipa luanica ..., T. kosovarica ... and T. albanica 2 MATERIAL AND METHODS The plant samples were collected during flower - ing time in their natural habitats (Figure 1): Tulipa al- banica at Surroi locality in Albania (altitude 625 m a.s.l., geographical coordinates: 42º02’30” N and 20º20’15” E), Tulipa kosovarica at Mrasor locality in Kosovo (alti- tude 450 m a.s.l, geographical coordinates: 42º30’59” N and 20º34’08” E) and Tulipa luanica at Pashtrik locality in Kosovo (altitude 1100 m a.s.l, geographical coordi- nates: 42º16’17” N and 20º28’24” E). More than 30 plant samples (leaves and bulbs) from each plant species were collected. The sample collections and preparation proce- dures were carried out at a regulated temperature of 4 ºC. 2.1 FLOW CYTOMETRY ANALYSIS FCM (flow cytometry) is a great method for exam- ining the optical characteristics of small particles sus- pended in liquid, such as fluorescence and light scatter. The nuclear DNA content and polyploidy analysis of our sample set was carried out using Partec CyFlow Space flow cytometer (Munster, Germany) at analyzes the labo- ratory of genetics and plant cytogenetics at Namik Ke- mal University in Tekirdag, Turkey. Fresh mature leaves of three tulip species were collected during two seasons (2016 and 2017) during their flowering time in their natural habitats and were analyzed within the optimal time frame after their collection. The collection, storage and transport of leaves was carried out based on stand- ard procedure and under temperature control (4 ºC) with preservation of humidity. The analysis of nuclear DNA content was done with three replicates within the sam- ple (from the same leaf) and from 30 plant individuals (leaves), at least 5000 nuclei were analyzed for each type and sample. Suspension of intact nuclei was prepared us- ing commercial kits manufactured by Partec (Munster, Germany). Homogenization of the leaves (50 mg) was done together with the leaves of the standard plant in a petri dish, where 0.5 ml of extraction buffer was also added. This homogenate was filtered using 50 µm nylon filters and then transferred to standard test tubes of the apparatus where 1.5 ml of DAPI (4’,6–diamidin–2-phe- nylindole) was added; samples were left in the dark for 60 minutes at 4 ºC. (Tuna et al., 2001). DAPI was used as a fluorochrome for DNA labeling because it has more affinity for binding to the nitrogenous bases A and T. Se- cale cereale L., which has 16.55 pg / 2C DNA, was used as a standard. The results were processed with the FloMax analysis software program and expressed in pg 2C DNA (picograms of diploid DNA) (Figure 2). The amount of nuclear DNA was calculated based on the following for- mula: 2C DNA = [(average G1 peak of sample / average G1 peak of standard)] × amount of 2C DNA standard (pg DNA) 2.2 KARYOTYPE ANALYSIS Samples for chromosome (karyotype) analysis were taken from the root tip meristem of the bulbs. First, the bulbs were collected from their natural habitats, and then they were grown in a vegetative room. After germination, approximately 2 mm root tips were cut and treated with 0.05 % colchicine for 5 hours, then fixed with acetic acid/ ethanol in a ratio of 1:3 for 24-48 hours. Hydrolysis of Figure 1: Tulipa species during flowering time in their natural habibitats: A) T. kosovarica, B) T. luanica and C) T. albanica Acta agriculturae Slovenica, 119/2 – 2023 4 M. OSMANI et al. the tip of the roots was done using 1N HCl for 12 min- utes at a temperature of 60 °C, while staining was done by using acetocarmine. Karyotyping, determination of cen- tromere position and chromosome type was performed in the metaphase according to the nomenclature of Le- van et al. (1964). 2.3 DATA ANALYSES Statistical analysis of the results was carried out with Sigma stat 32 programs 2004 STAT Software. The data presented in the paper represent the average of at least four independent experiments with ± S.E. Each continu- ous variable, a distribution form was determined, and the significant differences between means were checked by Student’s t test.  3 RESULTS AND DISCUSSION Seven different taxa of Tulipa are found in Kosovo, three of which are stenoendemic (T. luanica, T. kosovari- ca, and T. serbica), one of which is a local endemic (T. scardica), while the other three taxa, T. gesneriana and T. sylvestris which is represented by two subspecies (T. syl- vestris subsp. australis and T. sylvestris subsp. sylvestris), have a wider distribution (Millaku et al., 2018; Millaku & Elezaj, 2015; Shuka et al., 2010, 2012). The presence of a high number of Tulipa species/taxa in Kosovo and their sympatric area in serpentine substrate in the Deva local- ity, in the south of Kosovo, close to the border with Alba- nia, makes it an important regional and global habitat of native species of the genus Tulipa. According to Millaku et al. (2018), considering tulip species high variability, the application of molecular analyses is of crucial impor- tance to accurately classify Tulipa species of Kosovo and of the Balkans and in their taxonomic differentiation. 3.1 NUCLEAR DNA CONTENT The results indicate a significantly higher amount (p < 0.001) of nuclear DNA (2C) in T. luanica (47.49 pg) compared to T. kosovarica (45.71 pg) and T. albanica (43.86 pg) (Table 1). Based on differences in the amount of nuclear DNA expressed as a percentage, we can pre- sume that T. luanica and T. kosovarica differ by 3.75 %, T. luanica and T. albanica differ by 7.64 %, while T. kosovarica and T. albanica differ by 4.05 %. Our results regarding the genome size (2C DNA) of T. luanica, T. ko- sovarica and T. albanica are in accordance with those re- ported by other authors; Zonneveld (2009) reported that the genome size of plants of the genus Tulipa sp. with dip- loid number (2n) of chromosomes varied from 32-69 pg 2C DNA. Our genome size results (2C DNA) showed a range of 43.86 – 47.49 pg in the three tulip species, which are within the values reported for tulips with diploid chromosome number by Zonneveld (2009). In our study the 2C DNA content of the T. albanica resulted 43.86 pg, while in the same speciesthe 2C DNA content was pre- viously reported to be 54.15 pg (Shuka et al. 2010). The Figure 2: Flow Cytometer histogram for the amount of nuclear DNA (2C) in: 1) standard plant and 2) Tulipa luanica Acta agriculturae Slovenica, 119/2 – 2023 5 Characterization of nuclear DNA content and chromosome numbers of Tulipa luanica ..., T. kosovarica ... and T. albanica observed differences in the genome size in the same tu- lipa species can be due to the fact that, in our analysis, we used adult leaves collected at the time of blooming from naturally grown plants, while according to Shuka et al. (2010), the plant material used for the amount of DNA estimation was taken both by the germinated seeds and the adult leaves. Fresh leaves that have nearly completed growth are often preferred; very young leaves might not be as suited because they contain more inhibitors than older leaves (Dolezel et al., 2007). The genome size (2C DNA) reported for other tulips that grow in the Balkans (Albania and Kosovo) was 61.5 pg in T. schrenkii Regel and 69 pg in T. scardica Bornm. (Zonneveld, 2009). If we compare species of the genus Tulipa that extend from east to west, from North Pakistan to the Balkans, a grad- ual increase in the amount of nuclear DNA is observed, from 32 pg to 69 pg (Zonneveld, 2009). The transfer of DNA sequences from the nucleus into mitochondria and chloroplasts may have been one of the causes for smaller genome sizes (Karimzadeh et al., 2010). The results of the number of base pairs calculated using a value of 978 mega base pairs (Mbp) for one pi- cogram, showed that T. luanica had a greater number of mega base pairs compared to the T. kosovarica and T. al- banica (Table 1). Based on these results, T. luanica has about 1740 Mbp more than T. kosovarica and about 3550 Mbp more than T. albanica, while T. kosovarica has about 1809 Mbp more than T. albanica. Genome size results were also given with flow cytometer histograms for each tulip species. According to previous studieson genome size, 1pg is equal to several thousand genes or about 978 Mbp (mega base pairs) (Zonneveld, 2009; Dolezel et al., 2003). According to our results, T. luanica has a larger ge- nome, a larger number of genes and base pairs, compared to other tulip species under study, T. kosovarica and T. albanica, the latest are grown on serpentine soils. Ac- cording to Knight et al. (2005) and Temsch et al. (2010), large genomes are a burden for plant organisms and limit their adaptation. On this regards, plant species that grow under stressful habitats face greater risk; therefore, we presume that the two plant species T. kosovarica and T. albanica have adapted in terms of genome size to live in serpentine environments, which are considered poten- tially more stressful, while T. luanica in limestone envi- ronments has a larger genome. 3.2 CHROMOSOME NUMBER AND KARYOTYPE CHARACTERISTICS Analysis of the results for chromosome number and karyotype characteristics for T. luanica, T. kosovarica and T. albanica are presented in table 2 and figure 4. Based on these results we can conclude that the number of chro- mosomes in the three species under study, T. luanica, T. kosovarica and T. albanica, is 2n = 2x = 24 (Figure 3). The size of the chromosomes in T. luanica ranged from 5 - 12 µm, where two pairs of chromosomes are metacentric (I and VIII), two pairs are submetacentric (X and XII) and the other eight are subtelocentric (II, III, IV, V, VI, VII, IX and XI). In T. kosovarica the size of the chromosomes ranged from 5 - 10 µm, where two pairs of chromosomes are metacentric (X and XII), five are submetacentric (II, IV , V , VI and XI) and the other five subtelocentric (I, III, VII, VIII and IX). In T. albanica the size of the chromo- somes ranged from 5 - 8 µm, where two pairs are meta- centric (V and VIII), three pairs submetacentric (VI, X and XII) and the other seven chromosomes are subtelo- centric (I, II, III, VI, VII, IX and XI). Our results for chromosome number and karyotype characteristics of T. albanica and T. luanica were in ac- cordance with those reported by other authors (Shuka et al., 2010; Millaku & Elezaj, 2015). According to Zonn- eveld (2009), most of the species of Tulipa have the same basic chromosome number, 2n = 2x = 24. In addition, many species in this genus have the same chromosome number, differences in DNA 2C value, when present, have proven to be very effective in delimiting infrageneric 2C ADN pg Difference in % Equivalent in Mbp T. luanica 47.49 ± 0.53 TL : TK = 3.75 % 46445.22 T. kosovarica 45.71 ± 0.29 TL : TA = 7.64 % 44704.38 T. albanica 43.86 ± 0.59 TK : TA = 4.05 % 42895.08 Significance TL : TK < 0.001 TL : TA < 0.001 TK : TA < 0.001 Table 1: The amount of nuclear DNA in picograms (pg) in the leaves of T. luanica, T. kosovarica and T. albanica, the difference be- ing expressed as a percentage (%) between these species and the approximate number of mega base pairs (Mbp) TL – T. luanica; TK – T. kosovarica; TA – T. albanica. The results are expressed as mean and standard error (±). Significant differences were found between different species for p < 0.001 Acta agriculturae Slovenica, 119/2 – 2023 6 M. OSMANI et al. divisions in a number of taxa (Ohri, 1998). Genome size has been demonstrated to differ between taxa that share identical chromosome numbers. Moreover, Greilhuber (1998, 2005) has clearly shown that intraspecific varia- tion of genome size is much less than assumed. In this case, results show that the size of the genome (2C DNA) and chromosomes of T. luanica are larger compared to T. kosovarica and T. albanica. A correlation was also found between the 2C DNA content and chromosome size; the larger the genome, the larger are the chromosomes in these three types of tulips. In addition, morphological differences between these species reported from Millaku and Elezaj (2015) show that capsule and seed size of T. luanica are bigger in comparison with T. kosovarica and T. albanica. These findings are consistent with a consid- erable amount of evidences that suggest that the size of reproductive organs might be related to genome size and that the variations in genome size, both increases and Species Chromosome number Chromosome size Karyotype characteristics Metacentric Submetacentric Subtelocentric T. luanica 24 5-12 µm I, VIII X, XII II, III, IV , V , VI, VII, IX, XI T. kosovarica 24 5-10 µm X, XII II, IV , V , VI, XI I, III, VII, VIII, IX T. albanica 24 5-8 µm V , VIII VI, X, XII I, II, III, VI, VII, IX, XI Table 2: Number of chromosomes and karyotypes of T. luanica, T. kosovarica and T. albanica Figure 3: Karyotyping of chromosomes in metaphase for T. luanica (TL), T. kosovarica (TK) and T. albanica (TA) Figure 4: Karyogram and presentation of chromosomes in metaphase Acta agriculturae Slovenica, 119/2 – 2023 7 Characterization of nuclear DNA content and chromosome numbers of Tulipa luanica ..., T. kosovarica ... and T. albanica decreases, might have contributed to the evolution and diversification of the genus, even within closely related species (Seijo & Fernandez, 2003). 4 CONCLUSIONS The combination of analysis of nuclear DNA con- tent carried out in flow cytometry and the number of chromosomes resulted very useful to determine the re- lationship status among three Tulipa species. Moreover, nuclear DNA content and chromosome sizes of T. luani- ca, T. kosovarica and T. albanica showed clear differenc- es among these species. The nuclear DNA content and chromosome size of T. luanica resulted larger, compared to that of T. kosovarica and T. albanica. Furthermore, we presume that the two plant species T. kosovarica and T. albanica have adapted in terms of genome size to live in serpentine environments, which are considered poten- tially more stressful, while T. luanica which is grown in a less stressful limestone environment resulted in a larger genome size. 5 ACKNOWLEDGEMENTS This study was carried out in collaboration between the Department of Biology, University of Pristina “Hasan Prishtina, Republic of Kosovo (promoter Prof. Dr. Isa Elezaj) and Faculty of Agriculture, Namik Kemal Univer- sity, Tekirdag, Turkey (promoter Prof. Dr. Metin Tuna), and the results of this paper are part of a doctoral dis- sertation.. 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