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Inter-simple sequence repeat (ISSR) markers were used for cultivar identification and for determination of the phenetic relationships among 24 pear cultivars (Pyrus communis L.). The ability of several molecular marker systems including randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP), inter-simple sequence repeats (ISSR), simple sequence repeats (SSR), and selective amplification of microsatellite polymorphic loci (SAMPL) to detect variation among clones of the most significant Portuguese cultivar, Rocha, was also investigated. Each of the eight ISSR primers tested was able to distinguish the 24 pear cultivars. The ISSR primers generated 337 markers, 79.5% of which were polymorphic. The cultivar dendrogram obtained with the ISSR marker data was very similar to that obtained with previous RAPD+AFLP analysis, confirming the genetic divergence of ‘Pérola’, ‘Carvalhal’ and ‘Lawson’ from the other cultivars. Eight out of 15 apple [Malus sylvestris (L.) Mill. var domestica (Borkh.) Mansf.] SSR primers tested also amplified microsatellites in pear. None of the five molecular marker systems analyzed (with a total of 1082 markers) detected reproducible polymorphisms among the nine ‘Rocha’ clones, in spite of the presence of clear phenotypic differences. informativeness, SSR markers have become a tool for establishment of unique genetic identities or fingerprints in many different plant species for example in soybean [Glycinea max (L.) Merrill] (Rongwen et al., 1995). Development of microsatellite markers involves laborintensive procedures including cloning, sequencing, and design of locus-specific primers. However, flanking regions are sometimes highly conserved in related species. The primer pairs designed for one species can be useful for amplification of the same DNA region in related genomes (Moore et al., 1991). SSR markers have not yet been developed for pears, but a number of SSR primers have been described for apples (Malus sylvestris var. domestica) (Gianfranceschi et al., 1998; Guilford et al., 1997; Hokanson et al., 1998). Inter-simple sequence repeat (ISSR) amplification (Zietkiewicz et al., 1994) is another microsatellite-based technique useful for genome studies. The primers used in ISSR amplifications are based on SSR sequences (di-, tri-, tetra-, or penta-nucleotide repeats), anchored to genomic sequences flanking each side of the targeted simple sequence repeats (SSRs) (5' or 3') by using two to four arbitrary, and often degenerate, nucleotides. Unlike SSRs, this technique does not require prior sequence information and generates a high number of polymorphisms. ISSR markers are thought to be particularly useful for study of closely related individuals which exhibit low levels of polymorphism (Zietkiewicz et al., 1994) and have been applied as a very useful alternative to fingerprinting and genetic analysis in fruit crops including citrus (Citrus L. sp.) (Fang and Roose, 1997; Fang et al., 1998), grape (Vitis vinifera L.) (Moreno et al., 1998), gooseberry (Ribes L.) (Lanham and Brennan, 1999) and plum (Prunus L. sp.) (Goulão et al., 2001). Another PCR-based DNA technique, selective amplification of microsatellite polymorphic loci (SAMPL), developed by Morgante and Vogel (1994) is a high multiplex ratio marker system which combines the advantages of microsatellites and AFLP markers, and has been used recently in the analysis of conifer genomes (Paglia and Morgante, 1998). Development of techniques for discrimination of clones, sports, and seedlings obtained by hybridization or selfing is very useful for selection and certification of plant material. Studies related to discrimination of very closely related material have been performed. While Cipriani et al. (1994), Loureiro et al. (1998), Vignani et al. (1996), and Ye et al. (1998) failed to obtain differences among clones of grape using RAPD and SSR markers, Cervera et al. (1998), Moreno et al. (1998), and Sensi et al. (1996) reported discrimination Received for publication 28 Dec. 2000. Accepted for publication 29 May 2001. This work was supported by Fundação para a Ciência e Tecnologia (FCT), project PRAXIS XXI 3/3.2/HORT/2163/95. The authors thank Rui de Sousa (ENFVN) for providing plant material from ‘Rocha’ clones, Amado Silva (DRARO), Fernanda Lampreia, and António Moacho (DGPC) for supplying plant material from the rest of pear cultivars, as well as António Monteiro (ISA) for critical reading of the manuscript. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. Graduate student. Associate professor and corresponding author; e-mail: crismoniz@isa.utl.pt. The common pear (Pyrus communis) is one of the most widely cultivated temperate fruit crops in the world. More than 5000 cultivars exist today, although only a small percentage of them are cultivated commercially (Bell et al., 1996). The existence of cultivar stability in European pear production is an exceptional phenomenon in tree fruits. The main commercial cultivars of pear were derived by selection among open-pollinated seedlings, especially in France and Belgium, during the golden age of pear improvement from 1750 to 1850 (Bell et al., 1996). The need for pear germplasm collection and preservation has lead to establishment of national and international programs for conservation of genetic resources. The preservation of pear germplasm in Portugal has been performed by Estação Nacional de Fruticultura Vieira de Natividade, Alcobaça. This collection includes some European and Asian cultivars, many traditional Portuguese cultivars with unknown pedigrees, and several clones of the main Portuguese cultivar, Rocha. To be used more efficiently, germplasm collections should be evaluated and well characterized. Molecular markers have been used for studying genetic diversity, relationships, and origins of the cultivars, as well as for cultivar discrimination and fingerprinting of several fruit crops (e.g., Cervera et al., 1998; Dirlewanger et al., 1998; Fang and Roose, 1997; Gianfranceschi et al., 1998; Hokanson et al., 1998; Koller et al., 1993). Polymerase chain reaction (PCR)-based molecular markers such as randomly amplified polymorphic DNA (RAPD) (Williams et al., 1990) and amplified fragment length polymorphisms (AFLPs) (Vos et al., 1995) have already been used for cultivar identification and taxonomic relationship studies in pears (Botta et al., 1998; Oliveira et al., 1999; Monte-Corvo et al., 2000). Microsatellites, also known as simple sequence repeats (SSR), are composed of tandemly repeated 2 to 5 base pair (bp) DNA sequences. Due to their great of grape clones using AFLP or ISSR assays. In other fruit crops like apple (Gianfranceschi et al, 1998; Goulão et al., 2001) or almond [Prunus dulcis (Mill.) D.A. Webb]. (Bartolozzi et al., 1998) distinction was also not achieved. However, in citrus (Fang and Roose, 1997) and peach [Prunus persica (L.) Batsch (Peach Group)] (Dirlewanger et al., 1998), cultivars obtained from somatic mutations could be distinguished with ISSR and AFLP, respectively. ‘Rocha’ is the most economically important Portuguese cultivar, accounting for 75% of pear production in Portugal. ‘ Rocha’ originated from a chance seedling on a farm located in Sintra, Portugal, between 1830 and 1840 (Matta, 1928). Although this cultivar is not yet well known outside Portugal, it has some very interesting characteristics, such as long storage life and resistance to injury during transport. However, there have not been any breeding efforts to improve ‘Rocha’. ‘Rocha’ is characterized by high withincultivar phenotypic variability which is probably the result of accumulation of natural mutations (sports). The following research was conducted with two objectives. The first objective was to use ISSR primers to study phenetic variation among pear cultivars and for cultivar identification, and to compare these results with RAPD and AFLP data, reported previously (Monte-Corvo et al., 2000) and the second objective was to investigate the potential of RAPD, AFLP, ISSR, SSR and SAMPL markers for detecting polymorphisms in mutant clones of the cultivar Rocha. Materials and Methods PLANT MATERIAL. The same 24 Pyrus communis cultivars studied by Monte-Corvo et al. (2000) were used for ISSR analysis. The cultivars and their immediate parents where known were ‘Conference’, ‘William’s’, ‘Passe Crassane’, ‘Beurré Hardy’, ‘Doyenné du Comice’, ‘General Leclerc’, ‘Beurré Bosc’, ‘Beurré Clairgeau’, ‘Souvenir du Congré’, ‘Alexandrine Douillard’, ‘Doctor Jules Guyot’, ‘Delbard Première’ (‘Akca’ x ‘D. Jules Guyot’), ‘Lawson’, ‘Clapp’s Favourite’ (‘Flemish Beauty’ x ‘William’s’), ‘Highland’ (‘William’s’ x ‘Doyenné du Comice’), ‘D. Joaquina’, ‘Sete Cotovelos’, ‘Lambe-os-dedos’, ‘Carvalhal’, ‘Pérola’, ‘Carapinheira Parda’, ‘Rocha’, ‘Packham’s Triumph’ (‘Uvedale St. Germain’ x ‘William’s’) and ‘Butirra Precoce Morettini’ (‘Ercolini’ x ‘William’s’). For clone discrimination, nine ‘Rocha’ clones with clear differences in flower morphology, fruit set, yield, fruit color or fruit size (Sousa et al., 1993) obtained from the germplasm collection of the Estação Nacional de Fruticultura Vieira de Natividade were analyzed. DNA EXTRACTION. DNA from the different cultivars and clones was extracted from fresh young, expanded leaves using a hexadecyltrimethylammonium bromide (CTAB) protocol described previously by Oliveira et al. (1999), with additional purification according to Monte-Corvo et al. (2000). Two replicate extractions per tree were performed. For ‘Rocha’ clone discrimination, material from two trees, vegetatively propagated from the same source tree, was extracted for each clone. ISSR ANALYSIS OF CULTIVARS. PCR amplifications were performed in 20 μL of reaction mixtures containing the following reagents: 30 ng of template DNA,

ISSR Analysis of Cultivars of Pear and Suitability of Molecular Markers for Clone Discrimination