The Genetic Variability of Sicilian Lemon Germplasm Revealed by Molecular Marker Fingerprints
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JOBNAME: jashs 133#2 2008 PAGE: 1 OUTPUT: February 19 06:42:40 2008 tsp/jashs/158968/01231 J. AMER.SOC.HORT.SCI. 133(2):242–248. 2008. The Genetic Variability of Sicilian Lemon Germplasm Revealed by Molecular Marker Fingerprints Mirko Siragusa1, Fabio De Pasquale, Loredana Abbate, Letizia Martorana, and Nicasio Tusa Institute of Plant Genetics—CNR, Research Division Palermo, Corso Calatafimi 414, I-90129 Palermo, Italy ADDITIONAL INDEX WORDS. Citrus limon, genetic diversity, ‘Femminello’, ‘Monachello’, ‘Lunario’, ISSR, RAPD ABSTRACT. There is a high level of diversity among lemons [Citrus limon (L.) Burm. f. (2n = 2x = 18)] in Sicily, where each growing area has a wide range of landraces mostly derived from bud mutation. Because this variability represents an important resource for future breeding programs and genetic improvement, the relationships among the principal 36 accessions of Sicilian lemon, belonging to three different cultivars (Femminello, Monachello, and Lunario), were examined by intersimple sequence repeat and random amplified polymorphic DNA markers. Three ‘Femminello’ accessions from nearby Italian regions were also examined to study the genetic flow from the continent. The disputed case of the accession ‘Eureka Messina lemon’ was also examined, using ‘Frost Eureka’ as a control. Our results confirmed the extreme polymorphic nature of the three principal Sicilian cultivars and the presence of a wide range of different genotypes. Twenty-two Sicilian genotypes were recognized as unique accessions, reflecting the richness of the lemon germplasm present in Sicily. Each growing area showed the presence of several genetically different landraces, probably preserved by genetic isolation, whereas the continental accessions appeared extremely similar to the island genotypes, showing an exchange of germplasm from the island to the continent. Lemon is an economically important crop widely grown in about their genetic variability. Until now, Sicilian lemon southern Italy and especially in Sicily, the first Italian region for characterization has been done only through phenotypic anal- lemon production (Cottone et al., 1996; Forte, 1999). While in ysis (Crescimanno et al., 1992). This characterization requires a the other principal producing countries one or few cultivars large set of data, which is often difficult to access, and in some prevail over the others (‘Eureka’ and ‘Lisbon’ in the United cases, dependent upon environmental conditions. States, ‘Genoa’ and ‘Limoneira Lisbon’ in Argentina, ‘Galego’ Polymerase chain reaction (PCR)-based DNA marker tech- in Brazil and ‘Berna’ in Spain; Russo and Spina, 1985; Saunt, nology has already become a useful tool in characterizing 2000), in Italy, each lemon-cultivating area (Palermo, Catania, lemon cultivars. In particular, PCR methods using arbitrary Siracusa, Messina, Reggio Calabria, Salerno, and Naples) has a primers have become very popular, as they do not require any wide range of landraces mostly derived from bud mutation from information about DNA sequences in addition to being cost- the cultivar Femminello (Russo and Spina, 1985). and time-effective (Fang and Roose, 1997). As the most domesticated fruit crop species over the past Fang and Roose (1997) used intersimple sequence repeat century, the Italian lemon has also suffered a dramatic (ISSR) to discriminate between five genotypes of the cultivars reduction in the gene pool due to the best characteristics (high Eureka and Lisbon, whereas Gulsen and Roose (2001a) used yields, good fruit size, high juice content, low seededness) of ISSR, microsatellites, and isozymes to assess diversity and some grown cultivars and the presence of ‘‘mal secco’’ disease phylogenetic relationships in several lemon accessions and re- {citrus wilt [Phoma tracheiphila (Petri) L.A. Kantachveli and lated taxa. Recently, 10 autochthonous lemon cultivars of the Gikachvili]}, which allowed only tolerant cultivars (such as Italian region Campania were identified through ISSR (Cappar- Monachello) to survive (Crescimanno et al., 1992). Therefore, elli et al., 2004) and random amplified polymorphic DNA some landraces with interesting breeding characteristics are at (RAPD) techniques (Mariniello et al., 2004). risk of disappearance. Phylogenetic relationships between lemon and other Citrus In recent years, the collection and the characterization of L. species and Citrus-related genera have been also investi- fruit crop germplasm has become a common concern among gated by SSR (Barkley et al., 2006), RAPD, and restriction geneticists and breeders to identify and preserve the genetic fragment length polymorphism (Federici et al., 1998; Nicolosi diversity of species and for setting up new breeding programs et al., 2000). aimed at genetic improvement. In the present study, 41 phenotypically diverse lemon Currently in Sicily, there are several lemon landraces with accessions, mostly collected from different parts of Sicily, interesting agronomic traits, but little information is available were analyzed by ISSR and RAPD methods to detect genetic polymorphisms useful for setting up a molecular reference system that would allow precise identification. Data obtained from this study have been also used to provide much informa- Received for publication 13 Sept. 2007. Accepted for publication 15 Jan. 2008. tion about genetic relationships among the accessions exam- This research was supported by the research grant ‘‘Scrigno Project’’ (Develop and Characterization of Genetic Resources Native in Market Gardening) from ined. This information, with morphological and phenological Ministry of University and Research (MIUR). descriptors, could be useful in assessing the basis of breeding 1Corresponding author. E-mail: [email protected]. programs aimed at the genetic improvement of lemons. 242 J. AMER.SOC.HORT.SCI. 133(2):242–248. 2008. JOBNAME: jashs 133#2 2008 PAGE: 2 OUTPUT: February 19 06:42:42 2008 tsp/jashs/158968/01231 Materials and Methods then visualized with a ultraviolet transilluminator at 300 nm. To confirm the reproducibility of the banding patterns, the PCR PLANT MATERIALS AND DNA EXTRACTION. A total of 41 plants experiments were repeated three times. of C. limon, including 34 ‘Femminello’, 3 ‘Monachello’, 2 RAPD ANALYSIS. Twenty-two arbitrary decamer primers ‘Lunario’, and 2 ‘Eureka’ accessions were used in the inves- i.e., OPA01, OPAT 04, OPAT14, OPH04, OPH15, OPK04, tigation. The sampling sites, the codes, and the more remark- OPM04, OPM06, OPN07, OPN14, OPO14, OPQ11, and P130 able quality traits are listed in Table 1. The terms ‘Femminello’, (reported by Coletta Filho et al., 1998) and UBC219, UBC234, ‘Monachello’, and ‘Lunario’ commonly are used to identify UBC237, UBC239, UBC247, UBC251, UBC264, UBC266, different cultivars, each constituted from a phenotypically and UBC272 (reported by Wang et al., 1999) were used for the diverse, wide range of plants. Thirty-one ‘Femminello’, three amplification of DNA sequences. The primers were purchased ‘Monachello’, and two ‘Lunario’ accessions differing in mor- from MWG-Biotech AG. phological and physiological traits were collected from several DNA amplification reactions were performed in a volume of sites located in the major Citrus diffusion areas of Sicily. The 25 mL with 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2, localization of the sampling sites of Sicilian plants is shown 800 mM dNTP, 0.4 mM of each primer, 1 U of Platinum Taq in Fig. 1. Two ‘Femminello’ plants, ‘Femminello a foglia larga’ polymerase (Life Technologies, Grand Island, NY), and 30 ng (FW) and ‘Femminello Favazzina’ (FV), were collected from of template DNA. The amplification was performed in a Calabria region, the closest peninsular Italian region to Sicily, 98-Well GeneAmp PCR System 9700 thermocycler (Applied whereas a ‘Femminello common’ (FC7) was collected from Biosystems) equipped with a Hot Bonnet under the following Salerno (Campania region). Among the two ‘Eureka’ acces- cycle program: initial denaturation step for 90 s at 94 °C, sions, only ‘Frost Eureka’ (EF) was certified, and it was used followed by 40 cycles at 94 °C for 1min (denaturation), 35 °C as external control. The ‘Eureka Messina lemon’ (EM) was a for 2 min (annealing), and 72 °C for 2 min (extension), followed putative ‘Eureka’ plant collected from a grower in the Siracusa by a final extension step at 72 °C for 10 min. PCR-amplified area. All the plants were introduced in the germplasm collection DNA fragments were visualized as described above. To con- in the Lascari field station (lat. 38°N, long. 14°E). firm the reproducibility of the banding patterns, all analyses Genomic DNA was extracted from leaves as described were repeated three times. by Doyle and Doyle (1987). The leaves were collected from DATA ANALYSIS. Amplified bands from each primer were mature trees present in the germplasm collection and were scored as present (1) or absent (0) for all cultivars studied. Only carefully washed (as advised to avoid insects and fungal con- those bands showing consistent amplification were considered; tamination; Fang and Roose, 1997), frozen in liquid nitrogen, smeared and weak bands were excluded from the analysis. and stored at –80 °C. The samples were ground in a mortar Dice’s (1945) coefficient of similarity (Dij) was determined with liquid nitrogen. DNA was quantified by measuring OD260 between each pair of accessions. Dice’s coefficient has been as described by Sambrook et al. (1989). recommended for the evaluation of genetic similarities when ISSR ANALYSIS. A total