Arch. Biol. Sci., Belgrade, 64 (1), 107-111, 2012 DOI:10.2298/ABS1201107P SEX EXPRESSION IN SERBIAN DENDROFLORA – A CASE STUDY OF FRAXINUS ORNUS VAR. ANGUSTIFOLIA ZORICA POPOVIĆ1, MILENA STEFANOVIĆ1, MIROSLAVA SMILJANIĆ1, RADA MATIĆ1, M. KOSTIĆ2, VERA VIDAKOVIĆ1 and S. BOJOVIĆ1 1 Institute for Biological Research “Siniša Stanković”, University of Belgrade, 11000 Belgrade, Serbia 2 Institute for Medicinal Plant Research “Josif Pančić”, 11000 Belgrade, Serbia Abstract - Out of a total number of tree species in Serbian lora, 28% are hermaphrodites, 43% are monoecious and 29% are dioecious. he dendrolora appears to have a larger proportion of unisexual than hermaphrodite species. he monoecious system is the dominant unisexual system within the tree species in Serbia. he possible sex diferentiation by RAPD markers and Correspondence Analysis was examined in a population of Fraxinus ornus var. angustifolia. On the basis of 6 selected RAPD bands which contribute to the diferentiation in frequency between male and hermaphrodite individuals, Correspondence Analysis visualized the following tendency: 20 trees of each variety were separated into two groups that mainly correspond to sexual types. Our results showed that RAPD markers assisted by Correspondence Analysis could be used in identifying male and hermaphrodite individuals in F. ornus varieties. Key words: Sexual types, trees, monoecy, dioecy, RAPD INTRODuCTION of Bojović et al. (2000) showed that RAPD markers could be used in diferentiating sexual types of Fraxinus ornus var. genuina. Interest in tree sex expression has a long and venerable history in biology. Despite the controversy that surrounds the ecology and evolution of unisexuality (monoecy and dioecy), there are only a few studies dealing with sex expression in regional loras or the ecological correlates of sex expression. Apart from the need to expand this area of research, the possibility of separating the sexes can be of great practical importance (Hormaza, 1994; Bannerjee et al., 1999). In an attempt to separate sexual types, molecular markers are increasingly used (Reamon-Buttner et al., 1999). Although less informative than codominant markers (e.g. RFLP, AFLP or SSR), RAPD markers can be useful in the irst stage, even when it comes to gender division (Khasa and Dancik 1996; Schierenbeck et al., 1997; Gallois et al., 1998; Jordano and Godoy 2000). Preliminary investigations he aims of this work were to: (1) analyze sexual expression in the dendrolora in Serbia, and (2) to evaluate how RAPD markers with statistically assisted protocol can be useful for sexuality diagnosis in F. ornus var. angustifolia and challenges for future work. MATERIALS AND METHODS Sex system information was collected for native tree species in Serbia (according to Jovanović, 2007). Introduced species were considered only if they are spontaneous in the region. All examined species (108 tree species) were classiied into one of three sex systems: hermaphroditic (all lowers on a plant bi107 108 ZORICA POPOVIĆ ET AL. Table 1. he percentage of hermaphroditic, monoecious and dioecious tree species in diferent biomes (forests). Sex strategies Our study (n=108) Tropical forest (Bawa, 1974) (n=130) Temperate forest (Curtis, 1959 ater Bawa, 1974) (n=27) Hermaphroditea 28 68 7 Monoeciousb 43 10 19 Dioeciousc 29 22 74 Note: a Hermaphrodite = all lowers on a plant hermaphroditic (bisexual), b Monoecious = male and female lowers found as separate lowers on the same individual, c Dioecious = male and female lowers found on separate individuals, n = number of examined tree species. Table 2. Attributes of the ive selected RAPD primers and 6 of 15 selected bands which difer signiicantly in frequency of fragments between the hermaphrodite (H) and male (M) individuals in F. ornus var. angustifolia. *, ** Signiicant at 0.05 and 0.01 probability levels, respectively; ns = not signiicant primer nucleotide sequence 5’ to 3’ number of polymor-phic bands band Kb showing diferences between the sex diferences between H and M χ12 OPG07 GAACCTGCGG 14 OPG11 OPG20 OPH11 TGCCCGTCGT TCTCCCTCAG CTTCCGCAGT 16 17 24 OPH15 AATGGCGCAG 12 OPG07 0.73 OPG07 0.88 OPG07 0.97 OPG07 1.01 OPG07 1.13 OPG11 0.74 OPG20 0.79 OPH11 0.64 OPH11 1.02 OPH11 1.18 OPH11 1.22 OPH11 1.40 OPH11 1.45 OPH11 1.55 OPH15 0.48 0.00 ns 0.22 ns 0.20 ns 0.00 ns 6.67** 0.00 ns 0.00ns 13.33** 5.49* 5.05* 5.00* 1.05 ns 5.49* 1.98 ns 0.00 ns sexual), monoecious (male and female lowers found as separate lowers on the same individual) or dioecious (male and female lowers found on separate individuals). A chi-square test was used to explore diferences among diferent data sets and to compare the ratios of sex systems (Table 1). Correspondence Analysis (CA) – one of the descriptive multivariational methods capable of suggesting the structure and tendency of a set of categorical data, was carried out using the STATOSCOPE program (1997, version 1.6, Prof. M Roux, Laboratoire de Biomathématiques, Faculté des Sciences St-Jérôme, Marseille, France). he plant material (F. ornus var. angustifolia, Serbia, Košutnjak 44° 46’N, 20° 27’E) for DNA analysis consisted of 20 adult trees (10 hermaphrodite and 10 male). DNA extraction, ampliication conditions and data analysis (CA) have been described earlier for F. ornus var. genuina (Bojović et al., 2000) SEX EXPRESSION IN SERBIAN DENDROFLORA 109 Fig. 1. Correspondence Analysis of the RAPD data. Fraxinus ornus var. angustifolia. Samples identiied by their sex: H1 to H10 = hermaphrodite individuals; M1 to M10 = male individuals. RESuLTS he type of sexual system was determined for 108 tree species that are present in Serbian forests. Out of a total number of tree species, 28% are hermaphrodites, 43% are monoecious and 29% are dioecious (Table 1). Serbian dendrolora appears to have a larger proportion of unisexual than hermaphrodite species. he proportion of sexual types is diferent between diferent biomes (χ22 = from 19.08 to 45.47; P<0.05). Primers that reveal individual variation of F. ornus var. angustifolia were found (Table 2). Among 41 oligonucleotides, ive informative ones were found (OPG-07, OPG-11, OPG-20, OPH-11 and OPH-15). Each of the 6 selected bands contributed to the differentiation only partially, and none completely (Fig. 1) and the sexual types partially overlapped. he irst axis (CA) allowed the division of the trees into two separated groups that almost correspond to sexual phenotypes. 17 of 20 individuals (85%) were identiied with regard to the sexual phenotypes for F. ornus var. genuina. 110 ZORICA POPOVIĆ ET AL. DISCuSSION Serbian dendrolora appears to have a larger proportion of unisexual (monoecious and dioecious) than hermaphrodite species. he sexual systems of tree species from Serbia were compared with published data from tropical and temperate forests (Table 1). Hermaphroditic systems dominated in tropical lora, dioecy in temperate lora (deciduous forest of northeast uSA) and monoecy in Serbian lora. Dioecy prevents intra-individual self-pollination absolutely, while monoecy merely prevents intra-lower selfpollination but not intra-individual self-pollination. Monoecy is very oten studied as an evolutionary step towards dioecy (e.g. Freeman et al., 1997; Renner and Won, 2001; Dorken et al., 2002). here is considerable disagreement about the selective forces that have led to the evolution of dioecy in plants (Senarath, 2008). According to Ainsworth (2000) it is clear that there is no common underlying mechanism and that sex determination systems leading to unisexuality have originated independently many times in evolution. Despite increasing research eforts on a number of diferent plant species, there is relatively little information available on the molecular basis of sex determination and it is diicult even to estimate the numbers of genes involved. Identiication of sex is complex; there is no universal method or protocol even for related species or subspecies within the species. Although molecular approaches have not yet identiied primary sexdetermining genes in any dioecious plant species, a range of molecular markers linked to sex have been generated for agronomically important dioecious species. In a minority of dioecious plants, the males are agronomically superior to the females, being higher yielding than the females (Populus species, Tschaplinski and Tuskan, 1994; Fraxinus excelsior, Jovanović, 2007). In dioecious plants cultivated for fruit or seed it is oten diicult to identify females at an early stage of growth (Hormaza et al., 1994). 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