HORTSCIENCE 41(1):84–89. 2006. In particular, PCR methods using arbitrary primers have become very popular among researchers since 1990 (Williams et al., 1990), Identifi cation of Sour Orange as they do not require any information about DNA sequences. Of these techniques, randomly Accessions and Evaluation of Their amplifi ed polymorphic DNA (RAPD), using 10 base pairs random primers, is the most Genetic Variability by Molecular widely applied (Lanham et al., 1995; Mailer et al., 1994; Stiles et al., 1993), principally due to the easiness of the procedure and the very Marker Analyses low amount of DNA required for analysis. Mirko Siragusa, Fabio De Pasquale, Loredana Abbate, and In Citrus, RAPD markers have been used Nicasio Tusa1 previously for studies about genetic diversity (Coletta Filho et al., 1998), hybrid and mutant Institute of Plants Genetic (Section Palermo), National Council of Researches, identifi cation (Deng et al., 1995; Elisiàrio et al., Corso Calatafi mi 414, I-90129 Palermo, Italy 1999), mapping (Cai et al., 1994), and linkage analysis (Cheng and Roose, 1995; Gmitter et Additional index words. Citrus aurantium, Citrus rootstock, ISSR, RAPD, genetic al., 1996; Ling et al., 2000). variability, Dice’s coeffi cient, UPGMA Inter-simple sequence repeat (ISSR) tech- Abstract. A collection of 18 accessions of sour orange (Citrus aurantium L.) coming from nique also uses arbitrary primers composed of Sicily and other countries was investigated by two polymerase chain reaction (PCR)-based a microsatellite sequence repeated in tandem DNA marker technologies. Ten inter-simple sequence repeat (ISSR) primers and fi fteen (core) and a fl anking sequence of two to four de- randomly amplifi ed polymorphic DNA (RAPD) primers were used to identify and to evalu- generate nucleotides (anchor). Because ISSR ate the genetic variability and relationship of accessions. A total of 111 ISSR and 145 RAPD primers require high-annealing temperature, amplifi ed fragments were used to estimate the Dice’s coeffi cient of similarity for cluster this technique guarantees high reliability and analysis using a unweighted pair-group method using an arithmetic averaging (UPGMA) repeatability, in addition to cost- and time-ef- algorithm. The genetic relationships identifi ed using ISSR and RAPD markers were highly fectiveness (Fang and Roose, 1997), especially concordant, such that the correlation between ISSR and RAPD genetic distance (GD) if compared with RAPD. A few genetic studies estimates was r = 0.93. The ISSR and RAPD analysis of 18 sour orange accessions found a using ISSR markers have been carried out high grade of genetic diversity in foreign accessions, while a low variability was detected on Citrus and Citrus-related species mainly in local accessions. Sicilian accessions could be grouped in two distinct clusters, including to characterize closely related commercial indistinctly plants from three origin regions. Some markers could be linked to the different varieties (Fang et al., 1997; Fang and Roose, growing areas. The ISSR and RAPD molecular reference system seems to be suitable for a 1997), experimental somatic hybrids (Scarano fi ne identifi cation of tightly related plants and the obtained results can form the basis for et al., 2002), and to discriminate zygotic and future setting up of Citrus rootstock genetic improvement projects. nucellar plants (Lambardi et al., 2004). In the present study, phenotypically diverse sour orange accessions collected from different Sour orange (Citrus aurantium L., 2n = 2x therefore uniformity is almost complete. parts of Sicily and some foreign countries were = 18) is one of the most widely used Citrus Off-type seedlings are generally eliminated analysed by RAPD and ISSR methods to detect rootstocks in the world. Despite its decreasing by nurserymen, but zygotic seedlings are genetic polymorphisms useful for setting up a cultivation because of spreading citrus tristeza sometimes diffi cult to identify and they could molecular reference system that would allow a virus (CTV), sour orange is still cultivated in develop plants with very different agronomic precise identifi cation. This information, with many countries, in particular in the Mediter- traits. Currently, in southern Italy, there are morphological and phenological descriptors, ranean Sea coasts. Its importance is due mainly several sour orange populations with interest- could be useful for assessing the basis of to its capability of growing in calcareous and ing agronomical traits, but no information is breeding programmes aimed at the genetic saline soil and being tolerant to several seri- available about their variability, and no type improvement of sour orange. Data obtained ous Citrus diseases (such as phytophthora, characterization has been done yet. Because from this study may also be used to provide exocortis, and xyloporosis viroid and blight). the rootstock can greatly affect performance useful information about genetic relationships Furthermore, sour orange guarantees consistent of trees budded onto them (Roose and Traugh, among the accessions examined. yields, good fruit quality, and ability to import 1988), an improved understanding of sour cold hardiness to the scion (Castle, 1987). In orange genetic diversity is desirable to select Materials and Methods nurseries, sour orange cultivation is represented lines suitable for increasing the productivity by local populations seed-propagated and of Citrus cultivations. It also would permit to Plant materials and DNA extraction. A total maintained over generations by selection based isolate, within the best local selections, indi- of 18 accessions of Citrus aurantium L. includ- on morphological traits (Herrero et al., 1996). viduals amenable for setting up new breeding ing 15 Sicilian accessions and three foreign As with other polyembryonic Citrus species, programmes for genetic improvement. These clones were used in the investigation. Plants seedling populations of sour orange are geneti- breeding programmes aim to constitute mainly different for morphological and physiological cally quite uniform because they arise from new CTV-resistant hybrid rootstocks having the traits (Tusa et al., 1979) were collected from apomictic seed through nucellar embryony same good qualities of sour orange (Grosser several sites located in the three major Citrus (Castle, 1987; Esan, 1973; Xiang and Roose, et al., 2004). diffusion areas of Sicily: northwest (province of 1988), allowing clonal propagation. However, Polymerase chain reaction (PCR)-based Palermo), northeast (province of Messina), and sour orange produces seeds that contain both DNA marker technologies have represented southeast (province of Siracusa-Ragusa). They nucellar and zygotic (sexual) embryos, and a useful tool to characterize closely related were introduced in the germplasm collection plants (Barcaccia et al., 2003; Imazio et al., in the Lascari fi eld station (38ºN, 14ºE). The Received for publication 7 June 2005. Accepted for 2002) and to evaluate the genetic homogene- Sicilian accessions were ‘Barcellona 1’ (BC1), publication 26 Sept. 2005. This research was sup- ity and purity, respectively, of inbreeds and ported by research grant of Scrigno Project (Develop ‘Barcellona 2’ (BC2), ‘Barcellona 3’ (BC3), and Characterization of Genetic Resources Native hybrids (Bellamy et al., 1996; Scarano et al., ‘Palermo 1’ (PA1), ‘Palermo 2’ (PA2), ‘Palermo in Market Gardening) from Ministry of University 2002). They were also used to investigate 3’ (PA3), ‘Palermo 4’ (PA4), ‘Siracusa’ (SR), and Research (MIUR). the phylogenetic relationship between plants ‘Noto 1’ (NT1), ‘Noto 2’ (NT2), ‘Rosolini’ 1Corresponding author; e-mail nicasio.tusa@igv. and populations belonging to the same genus (RSL), ‘Ispica 1’ (IS1), ‘Ispica 2’ (IS2), ‘Sco- cnr.it. (Burstin et al., 2001; Rossetto et al., 2002). glitti’ (SCO) and ‘Campofelice’ (CM), according 84 HORTSCIENCE VOL. 41(1) FEBRUARY 2006 FFebruaryBookebruaryBook 1 8844 112/14/052/14/05 110:51:260:51:26 AAMM and 30 ng of template DNA. The amplifi cation was performed in a MJ Research thermocy- cler (Genenco) equipped with a Hot Bonnet under the following cycle program: initial denaturation step for 90 s at 94 °C, followed by 40 cycles at 94 °C for 1min (denaturation), 35 °C for 2 min (annealing) and 72 °C for 2 min (extension), followed by a fi nal exten- sion step at 72 °C for 10 min. PCR-amplifi ed DNA fragments were visualized as described above. To confi rm the reproducibility of the banding patterns, all analyses were repeated three times. Data analysis. Amplifi ed bands from each primer were scored as present (1) or absent (0) for all the accessions studied. Only those bands showing consistent amplifi cation were consid- ered; smeared and weak bands were excluded from the analysis. Dice’s (1945) coeffi cient of similarity (Dij) was determined between each pair of accessions. Dice’s coeffi cient has been recommended for the evaluation of genetic similarities when using RAPD data (Lamboy, 1994). The genetic distance (GD) between two samples was calculated as: GD = 1 – Dij. Fig. 1. Localisation of the 15 Sicilian sour orange accessions used in this study. Most of the plants are The degree of concordance between ISSR and concentred in three growing areas. Northwest (province of Palermo): ‘Palermo 1’ (PA1); ‘Palermo 2’ RAPD markers was determined by visual ap- (PA2); ‘Palermo 3’ (PA3); ‘Palermo 4’ (PA4). Northeast (province of Messina): ‘Barcellona 1’ (BC1); praisal of graphic depictions generated from the ‘Barcellona