Phylogenetic relationships in Origanum spp. based on rDNA sequences and intra-genetic variation of Greek O. vulgare subsp. hirtum revealed by RAPD

Scientia Horticulturae 121 (2009) 103–108 Contents lists available at ScienceDirect Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti Phylogenetic relationships in Origanum spp. based on rDNA sequences and intra-genetic variation of Greek O. vulgare subsp. hirtum revealed by RAPD A. Katsiotis a,1,*, N. Nikoloudakis a,1, A. Linos a,1, A. Drossou a, T. Constantinidis b a b Department of Crop Science, Plant Breeding and Biometry Laboratory, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece Department of Biotechnology, Systematic Botany Laboratory, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece A R T I C L E I N F O A B S T R A C T Article history: Received 17 July 2008 Received in revised form 15 December 2008 Accepted 7 January 2009 Origanum species are among the most widely spread herbs in the Mediterranean basin. Eventhough they are used as a spice, evaluation of their genetic diversity and evolution has only recently drawn attention. In order to study phylogenetic relationships, 14 ITS1-5.8S-ITS2 clones belonging to the most common Origanum species were sequenced and a parsimony tree was constructed, using the approximate likelihood ratio test. All Origanum species were clearly separated from allied genera of the Mentheae tribe while a clear distinction between the Greek and the Spanish accessions was revealed. In addition the germplasm variability of the most common Greek oregano (O. vulgare subsp. hirtum) was investigated using the RAPD markers. The use of 10 random decamers resulted in 133 unambiguous and reproducible bands detected across 27 entries. Two main groups were identified by the UPGMA clustering using Jaccard’s similarity coefficient, and major genetic dissimilarities among Greek O. vulgare subsp. hirtum populations and O. onites/O. virens species were detected. Analysis of molecular variance revealed that genetic variability is distributed mainly within populations; however, significant Fst values were detected between different geographical localities, supporting noteworthy genetic differentiation among O. vulgare subsp. hirtum populations. ß 2009 Elsevier B.V. All rights reserved. Keywords: Origanum ITS RAPD Phylogenetic relationships Intra-genetic variability 1. Introduction In several countries of the Levant, there is frequently unclear distinction among aromatic plants of the Labiatae family. Amidst those is genus Origanum (tribe Mentheae), a sub-shrub that includes some of the most important culinary herbs under the vernacular name ‘oregano’. As a consequence, plants known and used as oregano belong to a number of different species. Origanum taxa have a very local distribution mostly around the Mediterranean basin and Eurasia, and are characterized by germplasm and chemical diversity (Kokkini, 1997). They can be found in habitats of different climate and soil; however, they are usually found from sea level up to 1500 m of elevation, on limestone, and are abundant in dry, sunny areas, near the coasts. Eventhough, Origanum species have been widely used in food industry for their aromatic properties, attention has been recently drawn to them due to their anti-bacterial (Aureli et al., 1992; Biondi et al., 1993), anti-fungal (Muller et al., 1995), insecticidal (Traboulsi et al., 2002), nematicidal (Oka et al., 2000) and anti- * Corresponding author. Tel.: +30 2105294634; fax: +30 2105294634. E-mail address: katsioti@aua.gr (A. Katsiotis). 1 These authors have equal contribution. 0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.01.015 oxidant (Gouladis et al., 2003; Tepe et al., 2004; Nakiboglu et al., 2007; Ozkan et al., 2007; Gortzi et al., 2007) properties of their essential oils. Out of a total of 49 species, it appears that 46 Origanum taxa have a very local distribution within the Mediterranean basin and most of them are local endemics (Kokkini, 1997). In Greece nine restricted Origanum spp. can be found (six of them are local endemics). Among them, the most economically important species is O. vulgare, widely known as ‘Greek oregano’, which is generally accepted as one of the species giving the best quality oregano (Calpouzos, 1954; Fleisher and Sneer, 1982; Fleisher and Fleisher, 1988). A taxonomic study of wild populations in Greece has shown that three subspecies are present, subsp. vulgare, subsp. viridulum (Martin-Donos) Nyman, and subsp. hirtum (Link) Ietswaart (Kokkini et al., 1991). Subspecies hirtum is a typical East Mediterranean taxon, found mainly in the southern part of the Balkan Peninsula and in Turkey (Vokou et al., 1993). In Greece it can be found at the islands of the Aegean Sea and at the southern mainland (Kokkini et al., 1991). Despite the importance of O. vulgare, little is known about its genetic affinities and evolution. Genetic diversity studies have mostly been based on morphological traits (Bosabalidis and Tsekos, 1984; Bosabalidis and Kokkini, 1997; Gündüz and Özörgücü, 1999; Novak et al., 2002) and comparative studies of 104 A. Katsiotis et al. / Scientia Horticulturae 121 (2009) 103–108 their essential oils (Kokkini and Vokou, 1993; Kokkini et al., 1994). However, most of those traits are influenced by the environment. Nucleotide data and DNA-based fingerprinting among Origanum species are limited (Kaufmann and Wink, 1994; Gounaris et al., 2002; Ayanoglu et al., 2006). As a result, considerable confusion exists regarding the identity of many accessions (Ayanoglu et al., 2006) and the estimation of genetic variation within the genus is essential. The purpose of the present work is to utilize both nucleotide data of ITS1-5.8S-ITS2 sequences and genetic RAPD marker data, in an attempt to evaluate phylogenetic relationships among Origanum spp. and to reveal the extent and distribution of intragenetic diversity of the Greek O. vulgare subsp. hirtum germplasm reservoir, respectively, as a first step for initiating breeding programs. 2. Materials and methods 2.1. Plant material Thirty-four different entries belonging to seven Origanum species were included in the present study. Their name, origin and collection sites are listed in Table 1. Sampled entries had a wide morphological and biogeographical diversity. 2.2. DNA isolation—PCR amplification Total genomic DNA was extracted from young leaves using the Invisorb Spin Plant Mini Kit (Invitek). The purity and quantity of genomic DNA was determined spectrophotometrically and confirmed using 0.8% agarose gel electrophoresis. For the amplification of the ITS1-5.8S-ITS2 fragments, the universal plant-specific primers 50 -ACG AAT TCA TGG TCC GGT GAA GTG TTC G-30 (18S) and 50 -TAG AAT TCC CCG GTT CGC TCG CCG TTA C-30 (26S) were used. Each 50-mL reaction contained 20 ng of total DNA, 1.5 mM MgCl2, 200 mM dNTPs, 15 pmol of each primer and 1 U Taq polymerase (Promega). The cycling profile consisted of an initial denaturation step of 4 min, followed by 30 cycles of 1 min at 94 8C, 30 s at 50 8C, and 2 min at 72 8C and a final elongation step of 8 min at 72 8C before cooling to 10 8C. For the RAPD amplification, the 25-mL reaction contained 50 ng of total DNA, 1.5 mM MgCl2, 200 mM dNTPs, 100 pmol of a decamer primer and 1 U Taq polymerase (Promega). The cycling profile consisted of an initial denaturation step for 6 min, followed by 40 cycles of 30 s at 94 8C, 30 s at 39 8C, and 60 s at 72 8C, with a final elongation step of 8 min at 72 8C, before cooling to 10 8C. Amplification products were analyzed by electrophoresis in 1.6% agarose gels containing 0.004% (w/v) ethidium bromide in 0.5 TBE buffer. Bands were visualized under UV and digitally photographed. 2.3. Cloning and sequencing Cloning of the ITS1-5.8S-ITS2 PCR products was carried out using the USER Friendly Cloning Kit (NEB) according to manufacturer’s instructions. Clones carrying inserts were identified using blue/white colony selection. Sequencing was performed at the Foundation for Research and Technology (Iraklion, Greece) with the universal M13 forward and reverse primers using an automated sequencer. 2.4. Data analysis The obtained sequences were checked for homology using the NCBI BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/). Table 1 Origanum entries analyzed, NCBI accession numbers for the sequenced ITS1-5.8SITS2 clones and collection sites of taxa included in the present study. Species NCBI accession no. Origin Collection site 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. O. O. O. O. O. O. O. O. O. O. Greece Greece Greece Greece Greece Greece Greece Greece Greece Spain Crete Attica Attica Crete Crete Ikaria Vrachos Othris Crete Madrid 11. O. vulgare Spain Madrid 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. EU252137 EU252134 EU252135 EU252136 EU252132 EU252133 EU252130 EU252131 EU252138 EU252126 EU252127 EU252128 EU252129 EU252139 Greece Greece Greece Greece Greece Greece Greece Greece Greece Greece Greece Greece Greece Greece Greece Greece Greece Greece Greece Greece Greece Greece Spain Vrachos Vrachos Vrachos Vrinena Vrinena Agrafa Agrafa Agrafa Agrafa Agrafa Agrafa Agrafa Agrafa Agrafa Agrafa Agrafa Karditsa Karditsa Vrachos Sourpi Sourpi Sourpi Madrid dictamnus majorana (microphylla) majorana (macrophylla) microphylum onites onites scabrum scabrum scabrum virens vulgare vulgare vulgare vulgare vulgare vulgare vulgare vulgare vulgare vulgare vulgare vulgare vulgare vulgare vulgare vulgare vulgare vulgare vulgare vulgare vulgare vulgare vulgare subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. subsp. hirtum-B1 hirtum-B2 hirtum-B4 hirtum-BR1 hirtum-BR2 hirtum-K1 hirtum-K2 hirtum-K3 hirtum-K4 hirtum-K5 hirtum-K6 hirtum-K7 hirtum-K8 hirtum-K10 hirtum-K12 hirtum-K13 hirtum-KAR1 hirtum-KAR2 hirtum-N2 hirtum-S5 hirtum-SR hirtum-SR1 hirtum-SP Nucleotide multiple alignments were done with ClustalW algorithm (Thompson et al., 1997) using the default parameters. For phylogenetic analysis the maximum likelihood (ML) method, as implemented in the phyML software (Guindon and Gascuel, 2003), was used. An approximate Likelihood-Ratio Test (aLRT) was performed using the SH-like parameter. Trees were depicted with the TreeView software (Page, 1996). Patterns of nucleotide substitutions and the invertion/transvertion ratio for the ITS1-5.8S-ITS2 fragment were estimated using the DAMBE software (Xia and Xie, 2001). Reproducible RAPD fragments were scored as present (1)/absent (0) for each reaction and were assembled in a binary data matrix table. Genetic similarities between taxa were calculated using the Jaccard (1908) similarity coefficient with the SIMQUAL command and an UPGMA dendrogram was constructed. Mantel test was used to compute the cophenetic correlation, i.e., to test the goodness of fit of the cluster analysis to the similarity matrix. All of the above analyses were performed using the NTSYS-PC 2.01 software (Rohlf, 1998). Genotypic variations were assessed across various populations by means of analysis of molecular variance (AMOVA) using GenALEx 6 (Peakall and Smouse, 2006). This analysis not only allows the partition of the total variation into within-group and among-group variation components, but also provides a measure of inter-group genetic distances as the proportion of the total variation residing between accessions (Phi statistics) (Excoffier et al., 1992). The significance of the resulting variance components and inter-population genetic distances was tested using 999 random permutations. A. Katsiotis et al. / Scientia Horticulturae 121 (2009) 103–108 3. Results and discussion 3.1. Size and nucleotide frequencies of the ITS1-5.8S-ITS2 region From the 14 Origanum spp. clones that were sequenced and aligned, a consensus sequence for the ITS1-5.8S-ITS2 rDNA fragment was obtained, having in total 626 bp (235 bp for ITS1, 155 bp for 5.8S and 236 bp for ITS2). The GC content was 57.78%. The ITS1-5.8S-ITS2 region was found to be composed of a mosaic of conserved and polymorphic (75 nucleotide sites) regions, 105 with major differences detected between species. As expected, the highest substitution rates were found in the transcribed spacers (ITS1 and ITS2), while the 5.8S coding region was more conserved. A 1.29:1 transition/transvertion ratio was calculated for the ITS1-5.8S-ITS2 DNA fragment. The most frequent single type nucleotide substitution was the G $ A transition followed by the C $ T transition, even though transvertions are expected to occur twice as frequently as transitions, assuming equal probabilities for all nucleotide positions (Soltis and Soltis, 1998). Fig. 1. Phylogenetic aspects of the Mentheae tribe. Accessions in bold represent clones of accessions sequenced for the present study. High affinity of Origanum spp. sequences as well as segregation from allied genera is supported by high branch support values. 106 A. Katsiotis et al. / Scientia Horticulturae 121 (2009) 103–108 3.2. Phylogenetic evaluation of the ITS1-5.8S-ITS2 sequences Sequences from species belonging to the Mentheae tribe along with the above-mentioned Origanum sequences were aligned and a maximum likelihood dendrogram was constructed (Fig. 1). All Origanum clones were well separated from those of the outgroup species. Within Origanum, two clades were formed according to the country of origin. The first cluster (A) was composed of an O. vulgare subsp. hirtum clone, two O. onites clones, two O. majorana clones, an O. dictamnus clone, an O. microphylum clone and three O. scabrum clones, all of Greek origin. The second cluster (B) included two O. vulgare and two O. virens clones, all of Spanish origin, while an O. vulgare clone (AY506647) from Canary Islands was related to this group. Origanum species were well separated from the related genus Thymus, supporting the probable monophyletic nature of Origanum. However, parallel lineages of oregano cannot be ruled out uncritically. The fact that O. vulgare clones (Spanish origin) grouped separately from O. vulgare subsp. hirtum (Greek origin) sequences indicates that polyphyletic/paraphyletic events may have contributed to the evolution of Origanum spp. Furthermore, there are reports demonstrating that allied species of the Nepetoideae, e.g. Lepechinia (Hart, 1983), Satureja (Cantino et al., 1992), and Salvia (Walker and Sytsma, 2007) are nonmonophyletic, modifying the nomenclature at the genic level for the Mentheae tribe. 3.3. RAPD profiles and genetic relationships From the 15 RAPD primers tested, 10 showed polymorphism and were used (Table 2). A total of 133 unambiguous and reproducible bands were detected across a set of 27 entries, ranging from 150 bp to 1920 bp, with 126 of them (94.7%) being polymorphic (Table 2). Primers OPR-13 and OPB-1 yielded a total of 20 and 13 polymorphic bands, respectively, both having a discriminating power (Tessier et al., 1999) of 0.97. Primers OPA-9, OPAH-1 and OPA-20 each yielded only nine polymorphic bands with a discriminating power of 0.86, 0.92 and 0.95, respectively. The highest genetic similarity was found among O. vulgare subsp. hirtum entries K5 and K7 (0.849), followed by K2 and K10 (0.841), while the lowest was found between the O. virens and O. majorana species (0.271). Genetic distances among O. vulgare subsp. hirtum entries in the present study were equivalent to those estimated for Turkish oregano (O. onites) germplasm by means of AFLP analysis (Ayanoglu et al., 2006). Pairwise similarities between the O. onites entries ranged from 0.396 to 0.725, implying that genetic heterozygosity within the Origanum species is extremely high. According to the UPGMA dendrogram, that had a high degree of fit (cophenetic correlation coefficient, r = 0.93), entries were clustered into two main groups (Fig. 2). Group A included all Greek O. vulgare subsp. hirtum entries (22) having a similarity coefficient higher than 0.45. Within this group, four subgroups were formed. Subgroup A1 included entries K2, K10 and K13, originating from the area of Agrafa. Another five genotypes, also from Agrafa formed subgroup A2, while subgroup A3 was largest including 11 genotypes having mixed geographical origins. Within this subgroup the two entries from Vrinena (BR1 and BR2) had the highest genetic value (0.80), while entries from Karditsa had a 0.70 genetic similarity. Subgroup A4 consisted of three entries originating from Sourpi. Group B contained three Origanum species, O. vulgare subsp. hirtum and O. virens, both of Spanish origin, and O. onites from the island of Ikaria (Greece). In a previous study using RAPD (Gounaris et al., 2002), eight primers were found sufficient to discriminate O. onites and O. vulgare species and were able to position hybrids between their parents. In the present study, group B entries showed intermediate genetic affinity to each other and low similarity to those of group A. The remaining two O. majorana entries, that according to RAPD analysis were found genetically identical, formed an outer group. Fig. 2. UPGMA cluster analysis of the 27 Origanum genotypes using Jaccard coefficient of similarity. Codes correspond to origin as shown in Table 1. 107 A. Katsiotis et al. / Scientia Horticulturae 121 (2009) 103–108 Table 2 Primers selected and degree of polymorphism obtained. Primer name Primer sequence OPB-8 OPB-1 OPH-18 OPR-13 OPA-20 OPAH-9 OPA-1 OPA-9 OPAH-1 OPN-20 50 -GTCCACACGG-30 50 -GTTTCGCTCC-30 50 -GGGCTAGTCA-30 50 -GGACGACAAG-30 50 -GTTGCGATCC-30 50 -AGAACCGAGG-30 50 -CAGGCCCTTC-30 50 -GGGTAACGCC-30 50 -TCCGCAACCA-30 50 -GGTGCTCCGT-30 Total bands Total 12 13 18 20 9 12 11 9 9 13 133 Discriminating power (Dj) of the jth assay unit (Tessier et al., 1999): D j ¼ 1  number of pattern generated by the jth assay unit. i¼1 Table 3 AMOVA for partitioning the RAPD generated variation in Origanum vulgare subsp. hirtum accessions between and within populations. Source of variation d.f. Sum of squares %Total variance Fst p-Value Among populations Within populations 4 17 103.250 267.114 15 85 0.146 0.011 Table 4 Population differentiation based on RAPD analysis (upper diagonal matrix, Fst values) and level of confidence (lower diagonal matrix, p values). Vrachos 0.188 0.082 0.187 0.149 Vrinena Agrafa Karditsa Sourpi 0.062 0.047 0.047 0.079 0.329 0.028 0.024 0.206 0.020 0.104 0.149 0.377 0.291 0.152 0.132 0.351 Discrimination power (Dj) 0.91 0.97 0.96 0.97 0.95 0.94 0.94 0.86 0.92 0.91 126 PI Analysis of molecular variance for the O. vulgare subsp. hirtum entries revealed that a high proportion (85%) of the total genetic diversity is present within the five populations (Table 3). The highest variability was recorded for the Agrafa group (SS = 172.364), followed by Vrachos (SS = 49.750) and Sourpi (SS = 26.000). Moreover, AMOVA also revealed that the genetic distance between populations was significant (Table 3, Fst = 0.146, p = 0.011). Fst values suggest the presence of differentiation between populations. Karditsa population is clearly distinguished from Vrinena (Fst = 0.329, p = 0.377) and Sourpi populations (Fst = 0.104, p = 0.351). The closer proximity between Origanum spp. was found among the Sourpi and Agrafa (Fst = 0.020, p = 0.132), Agrafa and Karditsa (Fst = 0.028, p = 0.152) and Sourpi and Vrachos populations (Fst = 0.024, p = 0.149) which are in geographical proximity (Table 4). Low similarity coefficients among O. vulgare subsp. hirtum entries reported in the present study are in agreement with the results reported by Ayanoglu et al. (2006). This low affinity and heterogeneity among entries is probably related to the genus pollination system. For instance, O. onites is reported as both selfand cross-pollinated species. Flower structure and frequent pollinator’s visits are good indicators of a highly cross-pollinating mating system. In addition, genetic constrains hindering crosspollination are absent (Ayanoglu et al., 2006), and the presence of identical chromosome numbers (2n = 30) within Origanum species (Ietswaart, 1980) might facilitate crossabilities among species. As a result, naturally derived interspecific hybrids have been reported such as Origanum  intercedens, a hybrid between O. onites and O. vulgare subsp. hirtum (Kokkini and Vokou, 1993). Finally, gynodioecy in O. vulgare has been recorded (Jain, 1968), with Vrachos Vrinena Agrafa Karditsa Sourpi Polymorphic bands 12 13 18 20 9 14 14 10 9 14 pi ðN pi  1Þ=N  1, where pi is the frequency of the ith pattern; N, sample size; I, total the presence of female (male-sterile) plants next to hermaphrodite individuals in various European populations, at a rate as high as 50%. The evolutionary significance of such a mating system, promoting outbreeding (with the proportion of females regulating the degree of outbreeding) and supporting genetic variability, is essential (Crowe, 1964; Baker, 1966), and could explain the high within population heterogeneity in Origamun. Acknowledgments We would like to thank Jose Iriondo for providing plant material from Spain. 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