Investigations were carried out on molecular characterization of a 1600 years old olive tree located on the Brijuni islands (Croatia) by SSR markers analysis. Measurements of fruits and leaves features were carried out on the old tree Brijunka and on the standard Istrian cultivar Buga (Buža). Measurement data on fruits and leaves
were calculated statistically.Istraživanja obuhvaćaju molekularnu karakterizaciju 1600 godina starog stabla masline Olea europea na Brijunima analizom SSR markera. Provedene su izmjere svojstava ploda i lišća starog stabla masline Brijunke i standardne istarske sorte Buga (Buža). Rezultati istraživanja obrađeni su varijacijsko-statistički

Claudio Pucci

Italo Žužić

Ivo Miljković

L. Baldoni

M. Mariotti

N.G.M. Cultrera

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I. Miljković et al.: Molecular characterization of an ancient Olea europea tree located on the Brijuni islands of (Croatia) by SSR markers analysis   3 Molecular characterization of an ancient  Olea europaea tree located on the Brijuni islands  of (Croatia) by SSR markers analysis  Molekularna karakterizacija starog stabla masline Olea europea  na Brijunima analizom SSR markera  Miljković I.1, Žužić I. 2, Pucci C., Baldoni L.3, Mariotti M.3, Cultrera N.G.M.3   SAŽETAK Istraživanja obuhvaćaju molekularnu karakterizaciju 1600 godina starog stabla masline Olea europea na Brijunima analizom SSR markera. Provedene su izmjere svojstava ploda i lišća starog stabla masline Brijunke i standardne istarske sorte Buga (Buža). Rezultati istraživanja obrađeni su varijacijsko-statistički. Ključne riječi: maslina, molekularna karakterizacija, SSR markeri  ABSTRACT Investigations were carried out on molecular characterization of a 1600 years old olive tree located on the Brijuni islands (Croatia) by SSR markers analysis. Measurements of fruits and leaves features were carried out on the old tree Brijunka and on the standard Istrian cultivar Buga (Buža). Measurement data on fruits and leaves were calculated statistically. Key words: olive tree, molecular characterisation, SSR markers  INTRODUCTION The aim of the work was to achieve the molecular characterization of an olive tree of historical interest found on the Brijuni islands, Istrian archipelago (Adriatic Sea, Croatia). From the available nature guide it is possible to deduce that the tree, the subject of this study is about 1600 years old. For the characterization and identification of the olive samples, the microsatellite markers (also called SSR, Simple sequence repeats) were used. The SSR represent, at this point, the most efficient and confident method for molecular characterization, being highly  I. Miljković et al.: Molecular characterization of an ancient Olea europea tree located on the Brijuni islands of (Croatia) by SSR markers analysis   4 polymorphic and reproducible (Baldoni et al. 2009). They allow obtaining simple profiles with one or two allelic forms for each cultivar. The SSR analysis consists of the PCR amplification of fragments containing repeated dimers (known as microsatellites) by means of primers built on their flanking regions. On the basis of the number of repeats, fragments of varying lengths are amplified, which then can be separated and observed. The obtained fingerprint is characteristic and unique to each variety and constitutes its “bar-code”. Those varieties to related each other would have similar, but not identical, fingerprints, whilst different trees of the same variety will have the equal fingerprints.  MATERIAL AND METHODS Investigations were carried out on a 1600 years old olive tree located on the Brijuni islands (Croatia). The Brijuni are the climatic part of north Mediterranean with mild Mediterranean climate, lots of sun and warm weather, with plenty of humidity creating rich vegetation. The average air temperature in winter is 6.3°C, in spring 12.2°C, in summer 22.2°C and in autumn 14.8°C, The average annual precipitation is 817 millimetres with relatively high percent age of moisture in the air (76%). The soil is terra rossa, typical, luvic, anthropogenized, and medium deep. Ecological conditions for olive culture are very favourable (Miljković 1976; Miljković and Žužić 1987). Two samples collected from the foliage and from the trunk respectively of the olive tree investigated were analyzed. The DNA was extracted from the leaves using the kit GenElute (Sigma). Each sample was subjected to fingerprinting using the microsatellite markers. Sixteen highly polymorphic SSR loci, as resulting from previous investigations (Baldoni et al., 2009), were analyzed. It has to be taken into account that the use of only three loci (DCA9, DCA 16 and DCA17) is reported to be sufficient to discriminate between more than 100 varieties (Sarrie et al., 2006).  Name of locus Authors DCA3, DCA4, DCA5, DCA7, DCA9, DCA13, DCA15, DCA16, DCA17, DCA18 Sefc et al., 2000 GAPU45, GAPU71B, GAPU103A, GAPU101 Carriero et al., 2002 UDO43 Cipriani et al., 2002 EMO-90 de La Rosa et al., 2002  I. Miljković et al.: Molecular characterization of an ancient Olea europea tree located on the Brijuni islands of (Croatia) by SSR markers analysis   5 In order to verify possible and to examine the similarity level between the studied genotype and those present in neighboring areas or those of other ancient olive trees, the data were compared with those already available at the CNR Institute of Plant Genetics, Perugia. in particular the comparison was made with 28 samples from Albania and more than 80 varieties from Italy and other Mediterranean Countries.   Figure 1.  Old olive tree on the Brijuni  RESULTS Results of investigation of elaiographic features of fruits and leaves of the old olive tree Brijunka and the standard Istrian cultivar Buga (Buža) are presented in tables 1, 2 and 3 and illustrated in Fig. 2, 3, 4, 5, 6 and 7.   I. Miljković et al.: Molecular characterization of an ancient Olea europea tree located on the Brijuni islands of (Croatia) by SSR markers analysis   6 Table 1.  Biometric characteristics of fruit of old olive tree Brijunka Feature _ x (mm)  Range of variation (mm) St. dev. s Variance s2 cv Min. Max. Length of fruit 15,80 14 17 0,913 0,833 5,78 Width of fruit 12,28 11 14 0,614 0,377 5,00 L/W 1,29 1,17 1,42 0,068 0,005 5,27 Weight of fruit 1,147 0,868 1,411 0,124 0,015 10,8 Weight of stone 0,322 0,219 0,399 0,039 0,002 12,1 Weight of flesh 0,825 0,649 1,018 0,089 0,008 10,8 Length of stone 12 11 13 0,640 0,410 5,33 Width of stone 8 7 9 0,5 0,25 6,25 L/W 1,51 1,33 1,71 0,087 0,008 5,76  Table 2. Biometric characteristics of fruit of old Istran olive cultivar Buga (Buža) Feature _ x (mm)  Range of variation (mm) St. dev. s Variance s2 cv Min. Max. Length of fruit 22,64 21 25 0,952 0,906 4,21 Width of fruit 20,60 20 22 0,614 0,037 2,81 L/W of fruit 1,10 1,05 1,20 0,041 0,002 3,73 Weight of fruit 4,897 4,261 5,794 0,408 0,167 8,33 Weight of stone 0,523 0,420 0,640 0,053 0,003 10,13 Weight of flesh 4,374 3,774 5,158 0,371 0,138 8,48 Length of stone 14,36 13 17 1,113 1,240 7,75 Width of stone 9,60 9 10 0,5 0,25 5,20 L/W of stone 1,50 1,30 1,78 0,129 0,17 8,60  Table 3. Biometric characteristics of leaves Cultivar Feature _ x (mm)  Range of variation (mm) St. dev. s Variance s2 cv Min. Max. Old olive tree Brijunka Length of leaf 50,76 46 56 3,004 9,023 5,92 Width of leaf 13,4 12 16 1,190 1,417 8,88 Istrian cultivar Buga (Buža) Length of leaf 57,20 48 76 5,923 35,083 10,35 Width of leaf 13,9 9 21 3,807 14,490 27,35  I. Miljković et al.: Molecular characterization of an ancient Olea europea tree located on the Brijuni islands of (Croatia) by SSR markers analysis   7  Figure 2 Old olive tree Brijunka   Figure 3  Fruit and the stone of Brijunka   Figure 4  Leaves of Brijunka I. Miljković et al.: Molecular characterization of an ancient Olea europea tree located on the Brijuni islands of (Croatia) by SSR markers analysis   8  Figure 5  Cultivar Buga (Buža)   Figure 6  Fruit and stone of Buga (Buža)   Figure 7  Leaves of Buga (Buža) I. Miljković et al.: Molecular characterization of an ancient Olea europea tree located on the Brijuni islands of (Croatia) by SSR markers analysis   9 Greater variability properties of fruits were found in an old olive Brijunka than in the standard Istrian cultivar Buga (Buža). Buga has a much higher variability of length and width of the leaf than the old olive Brijunka as is evident from the values of s, s2 and cv. The microsatellite alleles obtained from the SSR analysis are reported in Table 4. The profiles of the two parts of the plant were identical, showing that the plant was propagated using shoots or cuttings, and not by grafting (the rootstock would be different from the scion). This method of propagation brings the studied tree close to the ones from central/Northern Italy, that are usually never grafted, whilst those from Southern Italy and from the Islands are commonly grafted onto wild olive trees (Baldoni et al., 2006).  Table 4. Fingerprints (allelic length of the 16 SSR markers) of the two samples from the ancient olive tree located on the Brijuni (Croatia)  DCA3 DCA4 DCA5 DCA7 DCA9 DCA13 DCA15 DCA16 Samples from crown 244-244 167-189 204-206 149-149 175-187 118-134 242-242 144-150 Samples from suckers 244-244 167-189 204-206 149-149 175-187 118-134 242-242 144-150  DCA17 DCA18 GAPU45 GAPU71b GA101 GA103 UDO43 EMO-90 Samples from crown 111-125 176-178 181-195 121-127 192-198 175-175 176-178 195-195 Samples from suckers 111-125 176-178 181-195 121-127 192-198 175-175 176-178 195-195  The comparison of the SSR profiles of the analyzed plant with those of the above indicated samples, has demonstrated that this genotype does not correspond with any of them. On the other hand, the sample has shown a significant similarity with the following sample: - cv Dobrovnica (Slovenia) - cv Tendellone (Italy, minor variety of Umbria) - some ancient trees from Albania  Nevertheless, for some alleles it is possible to find differences in these samples as well, therefore it is possible to exclude identity with these cultivars and confirm only a significant relationship with them. These results prove the uniqueness of the genotype analyzed and advise recovering and propagation of the old tree, aiming its conservation. I. Miljković et al.: Molecular characterization of an ancient Olea europea tree located on the Brijuni islands of (Croatia) by SSR markers analysis   10 REFERENCES BALDONI L., CULTRERA N.G.M., MARIOTTI R., RICCIOLINI C., ARCIONI S., VENDRAMIN G.G., BUONAMICI A., PORCEDDU A., SARRI V., OJEDA M.A., TRUJILLO I., RALLO L., BELAJ A., PERRI E., SALIMONTI A., MUZZALUPO I., CASAGRANDE A., LAIN O., MESSINA R., TESTOLIN R., 2009. 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SARRI V., BALDONI L., PORCEDDU A., CULTRERA N.G.M., CONTENTO A., FREDIANI M., BELAJ A., TRUJILLO I., CIONINI P.G., 2006. Microsatellite markers are powerful tools for discriminating among olive cultivars and assigning them to geographically defined populations. Genome, 49(12):1606-1615. MILJKOVIĆ I., 1976: Globalna valorizacija ekoloških uvjeta za uzugoj i zaštitu masline na našem kršu na osnovi meteoroloških ekvivalenata., međuakademski odbor JAZU (HAZU) “ Ekološka va.lorizacoja primorskog krša” str. 143- 145. HAZU , Zagreb. MILJKOVIĆ I., ŽUŽIĆ I., 1987: Iskustva s intenzivnim uzgojem maslina u plantažama “Agrolaghuna” Poreč ., Agrnoski glasnik 4, str. 32-42. I. Miljković et al.: Molecular characterization of an ancient Olea europea tree located on the Brijuni islands of (Croatia) by SSR markers analysis   11SEFC K.M., LOPES M.S., MENDONÇA D., RODRIGUES DOS SANTOS M., LAIMER DA CAMARA MACHADO M., DA CAMARA MACHADO A., 2000. Identification of microsatellite loci in olive (Olea europaea) and their characterization in Italian and Iberian olive trees. Mol. Ecol. 9:1171-1173.  Author’s address: 1Ivo Miljković, 10000 Zagreb, Čazmanska 2, Croatia 2Italo Žužić, 52465 Poreč, Pod brajdom 3, Croatia Claudio Pucci, Universitá degli Studi della Tuscia, Viterbo, Italy 3Baldoni L., Mariotti M., Cultrera N.G.M, CNR - Institute of Plant Genetics, Via Madonna Alta 130, 06128 Perugia, Italy I. Miljković et al.: Molecular characterization of an ancient Olea europea tree located on the Brijuni islands of (Croatia) by SSR markers analysis   12  

Molekularna karakterizacija starog stabla masline Olea europea na Brijunima analizom SSR markera

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