Genetic structure of the Greek olive germplasm revealed by RAPD, ISSR and SSR markers
Introduction
Olive (Olea europaea L.) is a universal symbol of peace and, without a doubt, the most characteristic plant of the Mediterranean Basin. Since antiquity, it has a significant trading and economic impact among the oil producing countries. The olive domestication most likely occurred in the Middle East about 6000 years ago (Zohary and Spiegel-Roy, 1975), and spread westwards through commercial shipping and land migration across the Mediterranean Basin (Damania, 1995, Lavee, 2013). Eventough Greece was considered as a secondary centre of domestication; still, recent scenarios for olive domestication based on DNA markers reveal Greece as a primary centre of domestication (Breton et al., 2008, Breton et al., 2012)
The advancement of molecular techniques facilitated studies that focused primarily on introgressive hybridization and it has been concluded that several multilocal domestication events have occurred (Claros et al., 2000, Besnard et al., 2001). This assumption is also fortified by two major facts: firstly, the immense variety of clonally propagated cultivars in the established olive-producing countries (Rallo, 2005) and secondly, the spread out of the wild olive (Olea europaea subsp. europaea var. sylvestris), the ancestor of the cultivated olive, as indigenous vegetation throughout the Mediterranean countries (Díez et al., 2011).
So far, a large number of olive varieties have been described, the majority of which have been established via the empirical selection of the growers in different localities (Besnard et al., 2001). The richness of the cultivated olive germplasm is an extraordinary case among horticultural crops, due to the tree's longevity and the lack of turnover with new breeding genotypes (Belaj et al., 2012). Currently, according to the literature, more than 1200 varieties are cultivated in orchards, and roughly 4200 genotypes have been recorded in 79 international and national collections situated in 24 countries (Bartolini et al., 2005). Throughout the extensive history of olive cultivation, some cultivars have been given the same name without being genetically similar (homonymy), while others were named differently although they were genetically identical or closely related (synonymy), among and within olive-growing countries (Besnard et al., 2001). Thus the number of the existing true cultivars is uncertain and the extent of olive germplasm needs to be clarified.
As a rule, efforts have been focused on the collection and conservation of olive genetic resources which lead to the establishment of ex situ germplasm banks around the world. The primary role of a germplasm bank is to obtain, preserve and make accessible representative plant genetic resources of the crop of concern (Belaj et al., 2012). Till now, this vast genetic variability of olive germplasm has been investigated by different molecular markers, such AFLPs (Rao et al., 2009), DARTS (Dominguez-Garcia et al., 2012), ISSRs (Hess et al., 2000, Vargas and Kadereit, 2001), RAPD (Nikoloudakis et al., 2003, Hagidimitriou et al., 2005, Durgac et al., 2010), RFLPs (Besnard and Berville, 2002, De Caraffa et al., 2002), SCARs (Shu-Biao et al., 2004), SNPs (Reale et al., 2006, Rekik et al., 2010) and SSRs (Diaz et al., 2006, Sarri et al., 2006, Noormohammadi et al., 2007, Bracci et al., 2009, Erre et al., 2010, Díez et al., 2011). All of the above mentioned work targeted cultivar identification and diversity studies. However, the gap between the available diversity and its effective use remains unevaluated (Belaj et al., 2011).
Nowadays, olive tree cultivation is experiencing a shift conversion from traditional to modern grove, planted with only a few varieties, featuring appealing agronomic and adaptive traits. Furthermore, super high density systems of orchards have further reduced the varieties being used, to a fraction of its potential. For instance, the ‘Picual’ and ‘Arbequina’ varieties have been widely planted over the last two decades in Spain, replacing traditional varieties (Belaj et al., 2010). An alike trend is expanding in Portugal, with the main cultivar ‘Galega’ grown in about 80% of the olive groves (Gemas et al., 2004), and in Morocco where ‘Picholine Marocaine’ is the major variety all over the country (Khadari et al., 2008). Hence, in contrast to the profound diversity, modern orchards are largely relying on a fraction of the available germplasm, leading to gradual genetic erosion.
Still, the existing genetic diversity may possibly be a significant pool for selection and breeding of modern olivoculture. In fact, the modern olive oil industry necessitates new and more productive cultivars with specific traits in order to respond to the market's demands. Almost in all olive-producing countries, breeding programmes aim to establish new olive varieties that are more suitable for the modern olive oil industry (Lavee, 1990). Straightforward breeding techniques, such as hybridization and introgression, clonal selection, and induced mutagenesis, are still extensively implemented in order to enhance the olive germplasm pool (Panelli et al., 1990, Lavee and Avidan, 2000). However, less common cultivars (mainly located in remote regions and occasionally threatened) could support olive growing, particularly in relation to the effects of global change (D’Imperio et al., 2011).
The goal of the present study was to uniquely fingerprint and explore the genetic relationships among 101 Greek cultivars using RAPD, ISSR and SSR molecular markers. The genetic structure of this germplasm collection was investigated using a model-based Bayesian clustering method to allocate genotypes into defined gene pools. This study also provides crucial baseline information for the exploitation of Greece's less common and locally preserved cultivars for the first time in literature. This is an essential first step towards optimizing preservation of the Greek olive genetic resources and consequently for genetic association studies to identify QTLs of interesting agronomic traits based on the most comprehensive Greek olive collection.
Section snippets
Plant material and DNA extraction
A total of 101 O. europaea entries, collected throughout Greece, along with O. chrysophylla Lam. and Olea europaea subsp. cuspidata (Wall. ex G. Don) Cif., as outgroups, were included in the present study (Table 1), all kindly provided by Kostelenos nurseries. DNA was extracted from healthy young leaves using the Invisorb Spin Plant Mini Kit (Invitek). DNA quality was tested in a 1% Agarose gel electrophoresis and its concentration was determined spectrophotometrically.
RAPD reactions
From the 30 RAPD primers
Genetic diversity
The combined analysis proved to be highly effective for discriminating the 103 genotypes included. The total amount of amplified loci was 165 for RAPD, 49 for ISSRs and 40 for SSRs, summing up to a total of 254 loci. In general, the majority of bands were polymorphic (more than 90%) amongst genotypes (Table 3). On average ISSR markers detected more polymorphic bands per assay; however RAPD markers were proven to be more informative. Nonetheless, all techniques were highly informative, since all
Discussion
In recent times, the organization of olive germplasm collections and diversification of olive varieties, have advanced genetic implement at the molecular level in most of the olive-growing countries (Baldoni and Belaj, 2010). Thus, considerable efforts have been made in order to characterize and utilize the Olea spp. genetic structure of collections. Unfortunately, even though Greece is the third largest producer globally (FAOSTAT), producing more than 10% of total crop, Greek cultivars are
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Cited by (0)
- 1
Current address: Vegetative Propagation Material Control Station, Hellenic Ministry of Rural Development and Food, Greece.
- 2
Current address: Department of Agricultural Science, Biotechnology and Food Science, Cyprus University of Technology, Limassol, Cyprus.