Genetic analysis of the genus Diospyros ssp. using RAPD and i-PBS methods

RADDOVA, J., PTACKOVA, H. , CECHOVA, J., ONDRASEK, I. : Genetic analysis of the genus Diospyros ssp. using RAPD and i-PBS methods. Acta univ. agric. et silvic. Mendel. Brun. , 2012, LX, No. 8, pp. 205–216 Molecular techniques (RAPD and i-PBS) were used to study genetic diversity within persimmon collection at Horticulture Faculty of Mendel University in Lednice. The aim of the work was to distinguish 14 known and 6 of unknown origin persimmon cultivars. The basic screening of 20 OPT primers was applied to 4 cultivars diff erring in the place of origin. Within the group of screened primers there were chosen those, which gave polymorphic repeatable strong and middle strong bands. Selected primers were used for the RAPD reactions within the whole persimmon collection. Three OPA primers previously described in the literature were also used for the RAPD reactions within the whole persimmon collection. Additional 16 i-PBS primers previously described in the literature were also used for i-PBS analysis of the whole group of cultivars. Amplifi cation was successful with 12 i-PBS primers. The FreeTree so ware package was used to gen erate a similarity matrix and then to produce a dendrogram using UPGMA analyses. The similarity dendrograms of all persimmon cultivars were created based on both approaches and also on combination of both analyses by program Tree View. All the dendrograms clearly separated the assessed cultivars into 4 clusters. There are cluster of American persimmons – Meader’ (1), ’Garretson’ (2) and ’Early Golden’ (3). They are representatives of D. virginiana. Further part of dendrogram includes single D. lotus (5), which is also clearly separated from other cultivars of the genus Diospyros. The third cluster includes interspecifi c hybrids ’Rossiyanka’ (10) and ’Nikitskaiya Bordovaiya’ (13), which arised from crosses of D. virginiana and D. kaki. The last cluster is formed by cultivars of Japanese persimmon – ’Mikatani Gosho’, ’Zenjimaru’, ’Tone Wase’, ’Hiratanenashi’, ’Fuyu’, Chinese cultivar – ’Sansi’ and two Italian cultivars ’Vaniglia’ and ’Tipo’. They are clustered without signifi cant distinction. The similarities and the diff erences revealed among incorporation of cultivars into groups were compared with the literature fi ndings. persimmon, RAPD, i-PBS, primer, dendrogram Persimmon belongs to important fruit cultures. Persimmon is mainly cultivated in the subtropical areas nevertheless some very early ripenig cultivars and interspecifi c cultivars can be grown under climate conditions in the Czech Republic (Ondrasek and Krska, 2009). The largest distributions are in the countries of its origin such as China, Japan and Korea. China is the biggest world producer of persimmon and Italy is major European producer of persimmon (Faostat, 2009). There are three the most important species of Diospyros, namely, Diospyros kaki Thunb. (Japanese persimmon), Diospyros virginiana L. (American persimmon) and botanical species Diospyros lotus L. Diospyros kaki is the most important species from commercial point of view (Yonemori et al., 2000; Giordani, 2002). Japanese persimmon can be classifi ed into four types based on the relationship among astringency in the fruit at harvest, presence of seed, and fl esh colour, i.e., pollination constant and non-astringent (PCNA), pollination variant and non-astringent (PVNA), pollination variant and astringent (PVA), pollination constant and astringent (PCA) (Sugiura, 2005). 206 J. Raddova, H. Ptackova, J. Cechova, I. Ondrasek More than 900 cultivars are known nowadays. Identifi cation of persimmon cultivars is one of the main problems in the present due to misleading transliterations, local names, synonyms and homonyms. Cases of synonymies and homonyms do exist accessions collected in diff erent sites, and possibly the names of a given genotype do not correspond to the denomination of the true to type accession held by reference germplasm collections of the countries of origin (namely Japan). At the European level 37% of accessions are positively identifi ed, while the status of identifi cation of the remaining 63% is probable (55%) and totally uncertain (8%) (Bellini and Giordani, 2003). For characterization of germplasm it is essential to identify individual genotypes. Therefore one of the important aspects of fruit growing is the reliable determination of grown cultivars (Giordani, 2002; Sugiura, 2005; Yakushiji and Nakatsuka, 2007). Authenticating the identity of germplasm resources of persimmon would be a great value for breeding (Badenes et al., 2003). There are various methods for cultivar identifi cation and distinctness. First, there are methods based on morphological traits, and biochemical markers as analysis of isozymes (Mondini et al., 2009). Limiting factors of these techniques for cultivar identifi cation led to the development of methods based on polymerase chain reaction (PCR) (Akbulut et al., 2008). Therefore to identify diff erent varieties of persimmon there are nowadays applied techniques based on restriction fragment length polymorphisms (RFLP) (Nakamura and Kobayashi, 1994), random amplifi cation of polymorphic DNA (RAPD) (Thaipong et al., 2003; Yamagishi et al., 2005; Yýldýz, et al., 2007; Akbulut et al., 2008; Zahng et al., 2009), amplifi ed fragment length polymorphism (AFLP) (Yonemori et al., 2008) or single sequence repeat (SSR) (Guo and Luo, 2011). RAPD markers have been used successfully for estimating genetic relationships in several fruit crops such as apple (Koller et al., 1993), peach (Badenes et al., 1998), almond (Bartolozzi et al., 1998), plum (Boonprakob et al., 2001), guava (Prakash et al., 2002), grapevine (Ulanovsky et al., 2002), apricot (Ercisli et al., 2009), citrus fruit (Ji et al., 2011) and also persimmon (Thaipong et al., 2003; Bellini et al., 2003). The RAPD method seems to be a very eff ective indicator of diff erent persimmon cultivars (Nakamura and Kobayashi, 1994). One recently described retrotransposon-based molecular marker technique was used to identify the varieties (Mondini et al., 2009). There are several retrotransposon-based molecular methods which require sequence information to design elementspecifi c primers (Kalendar, 2011; Kalendar et al., 2011). Retrotransposons are kind of elements which can be found in the genome of many eukaryotic organisms (Kumar and Bennetzen, 1999). One retrotransposon-based molecular method does not require previous sequence knowledge, it’s i-PBS (inter-primer binding site). I-PBS primers were designed according the highly conservative sequences called Primer Binding Site (PBS) to which tRNA binds, acting as a primer for reverse transcriptase during the replication cycle of retroviruses and LTR retrotransposons. I-PBS is a universal and effi cient method from identifi cation of polymorphism (Kalendar et al, 2010; Kalendar, 2011; Kalendar et al., 2011). The aim of this paper was to diff erentiate persimmon collection by means of RAPD and i-PBS methods, study genetic diversity within persimmon collection and to incorporate a unknown persimmon cultivars in the dendrogram. MATERIAL AND METHODS