Prunus (subfamily Prunoideae of the family Rosaceae) is a large and diverse genus comprising ≈400 species of trees and shrubs. This genus originated chiefly in the Northern hemisphere and is widely represented in Europe (Dosba et al., 1994). The basic chromosome number of Prunus is x = 8. Almond [P. dulcis D.A. Webb (formerly P. amygdalus Batsch)], peach [P. persica (L.) Batsch], apricot (P. armeniaca L.), and sweet cherry (P. avium L.) are diploids with 2n = 2x = 16. Tetraploid species include sloe (P. spinosa L.), sour cherry (P. cerasus L.), and P. fruticosa Pall. (2n = 4x = 32) and hexaploid species are represented by European plums [P. domestica L. and P. insititia L. (2n = 6x = 48)]. Prunus is economically important because of its diversified uses as fruit [e.g., European plums, japanese plum (P. salicina Lindl.), peach, apricot, sour cherry, sweet cherry, and almond], ornamentals, forestry, or for industrial purposes (Webster, 2005). The presence of a large number of Prunus species increases the possibilities of interspecific hybridization and makes the establishment of botanical classification quite complicated (Layne, 1987). Traditional taxonomy of Prunus has used morphology to estimate relationships among the species in question and to provide taxonomy for such genus. Systematic classification in Prunus is still controversial. De Tournefort (1700) recognized six distinct genera within Prunus “sensu lato” based on fruit morphology: Amygdalus L., Armeniaca Mill., Cerasus Mill., Laurocerasus Duhamel, Persica Mill., and Prunus “sensu stricto”. Bentham and Hooker (1865) were the first to unite the six genera of De Tournefort (1700) into a single genus Prunus, which was subdivided into seven sections: Amygdalopsis, Amygdalus, Armeniaca, Cerasoides, Cerasus, Laurocerasus, and Prunus. Koehne (1893) initially divided the genus Prunus into seven subgenera, but later (Koehne, 1911) recognized only four: Amygdalus, Cerasus, Padus, and Prunophora (= Prunus). Rehder (1940) reviewed the previous treatments and divided the genus into five subgenera: Prunus [= Prunophora (Neck.)] Focke, Amygdalus (L.) Benth. Hook., Cerasus (Adans.) Focke, Padus (Moench) Focke, and Laurocerasus (Ser.) Rehd. The subgenus Cerasus, as defined by Rehder, was composed of a large and diverse group of species and was later divided by Ingram (1948) into subgenera Cerasus and Lithocerasus Ingram. At present, biochemical (isoenzyme) and molecular (DNA analysis) approaches allow a more accurate estimate of genetic affinities and evolutionary relatedness among Prunus taxa that permits a comparison with traditional classifications established by taxonomists. Several investigations were carried out to assess genetic variation among Prunus species using isozyme markers (Arulsekar et al., 1986; Mowrey and Werner, 1990). Molecular markers such RFLPs, RAPDs, and SSRs were also used for phylogenetic and genetic diversity studies among Prunus species (Casas et al., 1999; Kaneko et al., 1986; Rohrer et al., 2004).
Noncoding regions of cpDNA have been explored under the assumption that these regions should be under less functional constraint than coding regions and should hence provide greater levels of variation for phylogenetic analyses (Gielly and Taberlet, 1994). Universal primers (Demesure et al., 1995; Dumolin-Lapegue et al., 1997; Taberlet et al., 1991) have been used to amplify fragments of noncoding regions of cpDNA followed by digestion with restriction enzymes. Fragment length polymorphism was consequently revealed within the amplified fragments (Demesure et al., 1996; El Mousadek and Petit, 1996; King and Ferris, 1998). This PCR-RFLP method, also known as cleaved amplified polymorphic sequences (CAPS) (Konieczny and Ausubel, 1993), is a readily accessible laboratory technique that can be used to evaluate large portions of the chloroplast genome in numerous individuals in a short time and at a lower cost. This method has been used to analyze phylogenetic relationships of cultivated Prunus species (Badenes and Parfitt, 1995; Panda et al., 2003) and to determine the phylogeography and the population diversity in P. spinosa and P. avium (Mohanty et al., 2000, 2001, 2002).
Current trends in breeding stone fruit rootstocks are based on the production of interspecific hybrids, aiming at putting together favorable traits that appear in different species (Moreno, 2004). Commercial Prunus rootstocks that are a result of uncontrolled interspecific pollinations are available on the market. Moreover, the pedigree of most of the clones is unknown due to the lack of parental control, and this can be a major constraint for their use in breeding programs. It is subsequently desirable to appropriately characterize the parentage of the rootstock material as a means to evaluate the success of various crosses.
In this study, a characterization of Prunus species and rootstocks (interspecific hybrids) on the basis of cpDNA screening was carried out with the following objectives: 1) to analyze the genetic diversity of interspecific hybrids and species belonging to the Prunus genus; 2) to test the usefulness of the method to confirm the maternal origin of Prunus interspecific hybrids; 3) to investigate genetic relationships among Prunus species. The characterization of this plant material and the verification of its pedigree before it is introduced into the breeding programs of the Experimental Station of Aula Dei at Zaragoza, Spain, will be useful to increase the genetic background in these programs.
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