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Genetic Relatedness Among Cultivars of the Greek Plum Germplasm

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Genetic Relatedness Among Cultivars of the Greek Plum Germplasm Available online at www.notulaebotanicae.ro Print ISSN 0255-965X; Electronic 1842-4309 Not Bot Horti Agrobo, 2013, 41(2):491-498 Notulae Botanicae Horti Agrobotanici Cluj-Napoca Genetic Relatedness among Cultivars of the Greek Plum Germplasm Ioannis ATHANASIADIS1, Nikolaos NIKOLOUDAKIS2, Marianna HAGIDIMITRIOU1* 1 Agricultural University of Athens, Department of Crop Science, Pomology Laboratory, Iera Odos 75, 118 55 Athens, Greece; [email protected] (*corresponding author) 2 Hellenic Ministry of Rural Development and Food, Vegetative Propagation Material Control Station, Greece; [email protected] Abstract Genetic diversity of the Greek plum germplasm collection was investigated using a combined RAPD and ISSR molecular markers approach. Twenty-six genotypes held at NAGREF-Naoussa were analyzed, producing in total 150 loci, of which 116 were polymorphic. Both techniques were highly informative and had a discrimination power greater than 0.9. RAPD and ISSR dendrograms were fairly correlated. The accessions were clustered according to ploidy and species. All Prunus domestica genotypes were grouped together and showed greater similarity to P. insititia and P. cerasifera genotypes compared to P. salicina, which was found genetically diverged. Bayesian structural analysis revealed significant admixture among genotypes. Greek varietiesP. domestica ‘Goulina’ and ‘Asvestochoriou’ exhibited a distinctive genetic background, differentiating them from foreign varieties. This feature could make them attractive for breeding programs, since they can increase genetic diversity. Keywords: AMOVA, ISSR, Prunus spp., RAPD, STRUCTURE Introduction Prunus salicina, a diploid (2n = 2x = 16) species do- mesticated in China from ancient times (its wild forms Plum species belong to genus Prunus L. (Rosaceae), are believed to thrive in the regions of Shensi and Kansu), and are naturally distributed in the temperate regions of was introduced in Japan 200-400 years ago (Ramming the Northern Hemisphere (Mabberley, 2008). This taxa and Cociu, 1990). These plums were initially improved is a diverse group of plants with various botanical species in Japan and later, to a much greater extent, in the Unit- that have been cultivated for 2000-4000 years (Banegal, ed States (Okie and Ramming, 1999) where subsequent 1954). The most economically important plum species are breeding resulted in larger fruit cultivars (latter half of the generally classified into two groups: the European (Pru- 19th century). nus domestica L.) and the Japanese (Prunus salicina Lindl.) Traditionally, classification within the genus Prunus plum. was mostly based on fruit morphology and thus being de- It has been proposed that P. domestica originated in batable (Aradhya et al., 2004). Furthermore, phenotype Southern Europe or Western Asia around the Caucasus is influenced by environmental factors, mainly as a result Mountains and the Caspian Sea (Cullinan, 1937). How- of the long generation time and large size of the trees. ever, it is also widespread in the Balkans and the Medi- Therefore, precise characterization for Prunus spp. and terranean countries, including Greece. Prunus domestica germplasm evaluation is a prerequisite, in order to develop has been cultivated in Europe for at least 2000 years, but effective conservation and breeding strategies. In order to and up to now no distinctly wild form is documented indisputably explore the genetic diversity of these numer- (Westwood, 1993). Thus the evolution of the European ous plum varieties and their interrelationships, molecular plum remains a controversial matter. Crane and Lawrence marker technologies can prove valuable. (1952) suggested that P. domestica, a hexaploid (2n = 6x In the present study, two different molecular marker = 48), originated through an interspecific cross between approaches (RAPD and ISSR) were employed in order to the diploid (2n = 2x = 16) P. cerasifera Ehrh. (Myrobalan evaluate the degree of genetic diversity of the Greek Na- plum) and the tetraploid (2n = 4x = 32) P. spinosa L., ei- tional Plum Collection held at NAGREF-Naoussa, and ther through chromosome doubling of the hybrid triploid to further analyze the genetic structure of three related or through the action of unreduced parental gametes. On plum species: P. domestica, P. salicina and P. insititia (L.) the contrary, Zohary (1992) proposed that P. domestica C.K. Schneid. Additionally, the elucidation capability and was an autopolyploid derived from P. cerasifera – rather effectiveness of RAPD and ISSR markers on genetic rela- than an allopolyploid, while Eryomine (1991) suggested tionships among plum genotypes was examined. that a number of species, such as P. microcarpa C.A. Mey., P. salicina, P. armeniaca L., and P. persica L., could have participated in the lineage of P. domestica. Athanasiadis I. et al. / Not Bot Horti Agrobo, 2013, 41(2):491-498 492 Materials and methods using standard λ-phage molecular weights. All samples were diluted at a concentration of 5 ng/μL by adding T.E., Plant material and DNA extraction for use in PCR reactions, and stored at -20oC. Twenty-two prune varieties (P. domestica and P. sali- nica), three P. insititia and one P. cerasifera accessions held RAPD and ISSR reactions at NAGREF-Naoussa, were included in the present study More than 30 RAPD primers (Invitrogen) were initial- (Tab. 1). ly tested in preliminary experiments of which ten resulted in unambiguous polymorphic products among genotypes. Tab. 1. Plant material utilized for RAPD and ISSR analyses, For the ISSR analysis ten primers were tested and five were including the number assigned, cultivar or common name, selected (Tab. 2). species and assignment of each variety to clusters based on Bayesian simulations (K = 2, K = 5) Tab. 2. List of the primers used in RAPD and ISSR analyses Assignment Sequence Cultivar or Primer Sequence (5-3) Primer No Species to clusters (5-3) common name RAPD ISSR K = 2 K = 5 OPB-1 GTAGACCCGT 818 (CA)8G 1 ‘Goulina’ Prunus domestica 1 1 OPB-11 GTTTCGCTCC 825 (AC)8Τ 2 ‘Asvestochoriou’ Prunus domestica 1 1 OPH-13 GACGCCACAC 844 (CT)8RC 3 ‘Bluefree’ Prunus domestica 1 1 OPH-18 GAATCGGCCA 861 (ACC)6 4 ‘Black Beauty’ Prunus salicina 2 Admixed OPAH-17 CAGTGGGGAG 889 DBD(AC)7 5 ‘Friar’ Prunus salicina 2 2 OPBD-7 GAGCTGGTCC 6 ‘Calita’ Prunus salicina Admixed Admixed OPA-9 GGGTAACGCC 7 ‘Stanley’ Prunus domestica 1 1 RAPD-3 AGAACCGAGG 8 ‘Angeleno’ Prunus salinica 2 Admixed RAPD-5 TCCAACGGCT ‘Anna Späth’ 9 Prunus domestica 1 Admixed RAPD-20 TCCGGGTTTG Oradea 10 ‘Anna Späth’ Pitesti Prunus domestica 1 1 11 ‘President’ Prunus domestica 1 1 12 ‘Tuleu gras’ bistro Prunus domestica 1 1 The RAPD and ISSR protocols followed Despotaki et 13 ‘Kisnanai’ Prunus domestica 1 Admixed al. (2011). The amplified products were resolved on 1.5% 14 ‘Tuleu dulce’ Prunus domestica 1 Admixed and 2% agarose gels respectively, buffered with 1x TAE ‘Koromilo and stained with EtBr. 15 Prunus insititia 1 3 roumanias’ 16 ‘Feher besztercei’ Prunus domestica 1 1 Data analysis 17 ‘Kesley’ Prunus salicina Admixed Admixed The RAPD/ ISSR banding patterns were visualized 18 ‘11/11’ Prunus insititia 1 3 and photographed using a Canon A630 and a UV table 19 ‘Scoldus’ SS Prunus domestica 1 1 (Serva). Reproducible fragments were scored as present ‘Mirabelle de (1)/absent (0) for each reaction and were assembled in a 20 Prunus domestica 1 Admixed Nancy’ binary data matrix table. Comparison of the discriminating capacity, level of polymorphism and informativeness were 21 ‘Gilej’ Prunus domestica 1 Admixed estimated following the procedure described by Belaj et al. (2003). Genetic similarities between taxa were calculated 22 ‘Daw dean’ Prunus insititia 1 Admixed using the Dices coefficient and an UPGMA dendrogram 23 ‘Myrobalanos’ Prunus cerasifera Admixed Admixed was constructed. Mantel test was used to compute the co- 24 ‘Santa Rosa’ Prunus salicina 2 Admixed phenetic correlation, i.e., to test the goodness of fit of the 25 ‘Black Diamond’ Prunus salicina 2 Admixed cluster analysis to the similarity matrix and the goodness 26 ‘Black Gold’ Prunus salicina Admixed Admixed of fit of cluster analysis to the similarity matrices (999 per- mutations). All of the above analyses were performed using Young leaves were collected, cleaned with moist paper the NTSYS-PC 2.01 software (Rohlf, 2000). Bootstrap towels and flash-frozen in liquid nitrogen. The tissue was analysis was performed using the FreeTree program (Pav- stored at -80 °C. Genomic DNA was extracted as previ- licek et al., 1999) and displayed with TreeView (http:// ously described by Doyle and Doyle (1987). DNA con- taxonomy.zoology.gla.ac.uk/rod/treeview.html). centration and quality was calculated spectrophotometri- Genotypic variations were assessed across various pop- cally (Unicam Helios; OD260nm/OD280nm ratios were ulations by means of analysis of molecular variance (AM- above 1.8) and confirmed with 1% agarose electrophoresis OVA) using GenALEx 6 (Peakall and Smouse, 2006). Athanasiadis I. et al. / Not Bot Horti Agrobo, 2013, 41(2):491-498 493 The significance of the resulting variance components and primer was 889 that amplified 11 loci and the least were inter-population genetic distances were tested using 999 818, 825 and 861, producing eight loci. Strikingly, all loci random permutations. Bayesian model-based clustering amplified by primers 818 and 825 were polymorphic. On approach to identify the genetic structure in the plum ger- the contrary, primer 889 revealed the lowest polymorphic mplasm was performed using
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