Genetic Diversity of Loquat Accessions in Japan as Assessed by SSR Markers

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1 J. Japan. Soc. Hort. Sci. 82 (2): Available online at JSHS 2013 Genetic Diversity of Loquat Accessions in Japan as Assessed by SSR Markers Shinji Fukuda 1, Chikako Nishitani 2, Naofumi Hiehata 1, Yukiko Tominaga 1 **, Hirohisa Nesumi 1 *** and Toshiya Yamamoto 2 * 1 Agricultural and Forestry Technical Development Center, Nagasaki Prefectural Government, Omura , Japan 2 NARO Institute of Fruit Tree Science, Tsukuba , Japan The genetic variation of loquat (Eriobotrya japonica (Thunb.) Lindl.) was characterized by SSR markers developed from apple and pear, using 94 loquat accessions in Japan. Fourteen of the 24 SSR markers derived from apple could successfully produce amplified bands in loquat, whereas 10 of the 24 SSR markers derived from pear could generate amplified bands. Nine SSR markers were chosen for evaluation of the genetic diversity among 94 loquat accessions, including 61 cultivars from Japan and other countries and 33 natively grown accessions collected around Japan. A phenogram constructed using the unweighted pair-group method with arithmetic averages based on similarities between genotypes revealed two major groups. One group consisted mainly of cultivars from Japan and other countries, whereas the other group included only natively grown accessions. Some synonyms or mutants were found showing identical SSR genotypes. These results show that SSR markers can be utilized as reliable tools for genetic identification in loquat. The origins of current loquat cultivars in Japan are also discussed. Key Words: Eriobotrya japonica, genetic resource, phenogram, simple sequence repeat. Introduction Loquat (Eriobotrya japonica (Thunb.) Lindl.) belongs to the subfamily Spiraeoideae, tribe Pyreae in the family Rosaceae, along with pears (Pyrus spp.) and apples (Malus spp.). Among 20 species included in the genus Eriobotrya, the loquat (E. japonica) is the only species used commercially for fruit production (Lin, 2002). The loquat is considered to have originated in China (Ding et al., 1995), and it is indigenous to temperate and subtropical climate zones of Asia. Loquat is one of the most important fruits cultivated commercially in temperate regions. World production of loquat fruits was estimated as 310,000 t, with China (200,000 t) being a major and Japan (10,000 t) being one of the second largest producers (Caballero and Fernandez, 2002). The worldwide cultivation area has been estimated as 62,000 ha, which is mainly distributed in China, Spain, Japan, and Pakistan. The oldest description of loquat in Japan has been found in a book written in A.D. 762 by Shosoin on the Received; November 5, Accepted; January 20, * Corresponding author ( toshiya@affrc.go.jp). ** Present address: Central Nagasaki Development Bureau, Nagasaki Prefectural Government, Saikai , Japan. *** Present address: NARO Western Region Agricultural Research Center, Zentsuji , Japan. ancient history of Japan (Kikuchi, 1948). It is believed that loquat fruits have been used on a small scale in Japan since ancient times. According to Nesumi (2006), commercial cultivation started in the 19th century, after the leading cultivar Mogi was selected in Japan from seedlings introduced from China. About 150 years ago, the elite cultivar Tanaka was found in Nagasaki, Japan; it was selected and bred from seedlings that may have been introduced from China (Tanaka, 1888). Since then, many loquat cultivars have been bred in Japan by controlled pollination and bud sport selections. For example, Obusa, Suzukaze, and Yougyoku are offspring (or progeny) of Mogi or Tanaka (Iwasaki, 1967; Terai et al., 2001). Two other major cultivars, Moriowase and Morimoto, are bud sport selections of Mogi and Tanaka, respectively (Ichinose, 1983); therefore, all of the major loquat cultivars in Japan are considered to have originated from Mogi and Tanaka. However, there is very little information on the origins and characteristics of loquat cultivars in China, other foreign cultivars, and natively grown accessions in Japan. Indigenous loquat accessions are distributed across Japan (Nesumi, 2006). Although a preliminary investigation of their fruit characteristics has been conducted (Tominaga et al., 2005), the genetic relationships between cultivated and indigenous loquats have not been investigated. Therefore, it is of major interest to geneticists and breeders to explore the genetic diversity 131

2 132 S. Fukuda, C. Nishitani, N. Hiehata, Y. Tominaga, H. Nesumi and T. Yamamoto within E. Japonica, including cultivars present in Japan, cultivars present in China, and indigenous accessions. Morphological investigations (Nagato et al., 1996), isozyme analyses (Degani and Blumenfeld, 1986; Nagato et al., 1997), and the use of RAPD markers (Fukuda et al., 2002; Vilanova et al., 2001) have been the major methods used to differentiate loquat cultivars and to assess the genetic variability within E. japonica; however, morphological differences among cultivars in Japan are slight and indistinct, leading to errors in the identification of propagated materials. The true genetic variations and relatedness among loquat cultivars therefore remain unclear. Recently, simple sequence repeat (SSR) markers have been used to identify genetic diversity and genetic relationships in apple and pear (Bao et al., 2007; Hokanson et al., 2001; Katayama et al., 2007; Kimura et al., 2002; Oraguzie et al., 2005). SSR markers have several advantages owing to their codominant inheritance, large number of alleles per locus, and abundance in genomes (Weber and May, 1989). Recent reports indicated that SSR markers could be applicable across genera within the sub-family Spiraeoideae, tribe Pyreae, which includes apple, pear, loquat, and quince (Fukuda et al., 2007; Liebhard et al., 2002; Soriano et al., 2005; Yamamoto et al., 2001, 2004). In loquat, SSR markers originating from apple have been used to analyze cultivars present in Europe (Soriano et al., 2005), cultivars present in China (He et al., 2011), and polyploid cultivars present in Japan (Watanabe et al., 2008); however, there have been few reports on the genetic diversity of East Asian loquats. We performed SSR analysis to investigate the genetic variations and relationships among loquat cultivars in East Asia, cultivars in Japan, and wild-type accessions. Genetic relationships among loquat cultivars in China and Japan are discussed. Our findings will provide important information for loquat breeding programs. Materials and Methods Plant materials and DNA isolation Ninety-four loquat accessions were used in this study (Table 1), including 30 cultivars in Japan, 13 cultivars derived from China; and 18 cultivars derived from Israel, USA, Vietnam, Mexico, and Greece. Thirty-three indigenous accessions were also collected from Nagasaki, Fukui, Niigata, Iwate, Yamaguchi, and Oita Prefectures, Japan. Seventeen cultivars ( Fusahikari, Mizuho, Murotowase, Nagaowase, Nagasakiwase, Biwa Nagasaki 2, Biwa Nagasaki 3, Biwa Nagasaki 5, Nojyuwase, Obusa, Otatsu, Reigetsu, Suzukaze, Togoshi, Tomifusa, Tsukumo, and Yougyoku ) were bred by crossbreeding. Three cultivars ( Moriowase, Morimoto, and Taisho ) were derived from bud sport mutation. Seven accessions collected from Fukui Prefecture were obtained from the National Center for Seeds and Seedlings (Kanaya Station, Shizuoka, Japan). The other loquat accessions were maintained at the Fruit Tree Research Division, Agricultural and Forestry Technical Development Center, Nagasaki Prefectural Government (Nagasaki, Japan). Genomic DNA was isolated from fresh leaves of each individual using the CTAB (cetyltrimethylammonium bromide) method (Doyle and Doyle, 1987). The extracted genomic DNAs were evaluated using a Mini Fluorometer (Hoefer, Holliston, MA, USA) and diluted to 10 mg L 1 for SSR-PCR analysis. SSR analysis In our preliminary experiments, some SSR markers originating from apple and pear that have already been applied to loquat (He et al., 2011; Soriano et al., 2005; Watanabe et al., 2008) were evaluated for loquat materials, including indigenous accessions, in this study. It was found that these SSR markers sometimes had disadvantages, for example, unstable amplification, difficulty in scoring and a small number of SSR alleles, maybe due to different plant materials and analysis systems; therefore, we screened additional SSR markers suitable for this study. Forty-eight SSR markers, including 24 SSR markers derived from apple and 24 SSR markers from pear (Liebhard et al., 2002; Yamamoto et al., 2002a, b), were tested for cross-genera amplification (Table 2). Sixteen accessions were used for SSR amplification. SSR markers transferable to loquat were used for further analysis of the genetic diversity of the 94 loquat accessions. PCR amplification was performed in a 20 μl solution of 10 mm Tris-HCl (ph 8.3), 50 mm KCl, 1.5 mm MgCl 2, 0.01% gelatin, 0.2 mm of each dntp, 10 pmol of each forward primer labeled with a fluorescent chemical (Fam, Tet, or Hex) and unlabeled reverse primer, 10 ng genomic DNA, and 0.5 units of Taq polymerase (Invitrogen, Grand Island, NY, USA). Amplification was conducted under the following conditions: initial denaturation at 94 C for 1 min, followed by 35 cycles at 94 C for 1 min, 55 C for 2 min, and 72 C for 2 min, for denaturation, annealing, and primer extension, respectively. The PCR products were separated and detected with a PRISM 3100 DNA sequencer (Applied Biosystems, Foster City, CA, USA). The size of the amplified bands was calculated against that of internal standard DNA (GeneScan-350 TAMRA; Applied Biosystems) using GeneScan software (Applied Biosystems). Statistical analysis The observed heterozygosity (H O ) and expected heterozygosity (H E ) were assessed for microsatellite loci using CERVUS version 2.0 software (Marshall et al., 1998) and MarkerToolKit version 1.0 (Fujii et al., 2008). H O was calculated as a ratio of the heterozygous genotypes scored at each locus. H E was calculated using an unbiased formula from allele frequencies: 1 Σpi 2

3 J. Japan. Soc. Hort. Sci. 82 (2): Table 1. Loquat accessions used in this study. Code Accession name Origin z A1 Amakusagokuwase Japan A2 Amakusawase Japan A3 Amamishiro Japan A4 Biwa Nagasaki 2 bred by AFTDC, Japan A5 Biwa Nagasaki 3 bred by AFTDC, Japan A6 Biwa Nagasaki 5 bred by AFTDC, Japan A7 Fukujyuin Japan A8 Fusahikari bred by SPHI, Japan A9 Ikeda Japan A10 Kusunoki Japan A11 Mizuho bred by NIFTS, Japan A12 Mogi Japan A13 Morimoto Japan A14 Moriowase Japan A15 Murotowase Japan A16 Nagaowase Japan A17 Nagasakiwase bred by AFTDC, Japan A18 Nojyuwase Japan A19 Obusa bred by NIFTS, Japan A20 Otatsu Japan A21 Reigetsu bred by AFTDC, Japan A22 Shiromogi bred by AFTDC, Japan A23 Suzukaze bred by AFTDC, Japan A24 Taisho Japan A25 Tanaka Japan A26 Togoshi bred by NIFTS, Japan A27 Tomifusa bred by SPHI, Japan A28 Tsukumo bred by NIFTS, Japan A29 Wasedai Japan A30 Yougyoku bred by AFTDC, Japan B1 Baisha China B2 Banhong China B3 Changhong3hao China B4 Dahongpao China B5 Guangdong China B6 Hongganben China B7 Jiajiao China B8 Jiefangzhong China B9 Shanghaipipa China B10 Suzhoubai China B11 Wanhong China B12 Xiolou China B13 Zaohuang China C1 Akko1 Israel C2 Akko13 Israel C3 Heads Mamuth Israel C4 Success Israel Code Accession name Origin z C5 Yehuda Israel C6 Zrifin8 Israel D1 Advance USA D2 Champagne USA D3 Gold Nugett USA E1 Vietnam loquat Col. No Vietnam E2 Vietnam loquat Col. No Vietnam E3 Vietnam loquat Col. No Vietnam E4 Vietnam loquat Col. No Vietnam F1 Mexican loquat No. 1 Mexico F2 Mexican loquat No. 2 Mexico F3 Mexican loquat No. 3 Mexico G1 Greece loquat Col. No Greece G2 Greece loquat Col. No Greece H1-1 Kawatana mamebiwa 1 collected at Nagasaki, Japan H1-2 Kawatana mamebiwa 2 collected at Nagasaki, Japan H1-3 Kawatana mamebiwa 3 collected at Nagasaki, Japan H1-4 Kawatana mamebiwa 4 collected at Nagasaki, Japan H1-5 Kawatana mamebiwa 5 collected at Nagasaki, Japan H1-6 Mamebiwa collected at Nagasaki, Japan H1-7 Tsushima 12 collected at Nagasaki, Japan H1-8 Tsushima 15 collected at Nagasaki, Japan H2-1 Fukui 1 collected at Fukui, Japan H2-2 Fukui 2 collected at Fukui, Japan H2-3 Fukui 3 collected at Fukui, Japan H2-4 Fukui 4 collected at Fukui, Japan H2-5 Fukui 5 collected at Fukui, Japan H2-6 Fukui 6 collected at Fukui, Japan H2-7 Fukui 10 collected at Fukui, Japan H2-8 Fukui 24 collected at Fukui, Japan H2-9 Fukui 54 collected at Fukui, Japan H2-10 Fukui 116 collected at Fukui, Japan H2-11 Fukui 197 collected at Fukui, Japan H2-12 Fukui 208 collected at Fukui, Japan H3-1 Sado 8 collected at Niigata, Japan H3-2 Sado 164 collected at Niigata, Japan H3-3 Sado 199 collected at Niigata, Japan H3-4 Sado 245 collected at Niigata, Japan H3-5 Sado 256 collected at Niigata, Japan H3-6 Sado 259 collected at Niigata, Japan H4-1 Oita 16 collected at Oita, Japan H4-2 Oita 71 collected at Oita, Japan H4-3 Oita 81 collected at Oita, Japan H4-4 Oita 85 collected at Oita, Japan H4-5 Oita 235 collected at Oita, Japan H5-1 Iwate 16 collected at Iwate, Japan H6-1 Yamaguchi 7S-176 collected at Yamaguchi, Japan z AFTDC: Agricultural and Forestry Technical Development Center, Nagasaki Prefectural Government. SPHI: Southern Prefectural Horticulture Institute, Chiba Prefectural Agriculture and Forestry Research Center. NIFTS: NARO Institute of Fruit Tree Science.

4 134 S. Fukuda, C. Nishitani, N. Hiehata, Y. Tominaga, H. Nesumi and T. Yamamoto Table 2. SSR markers used in this study. SSR marker z,y Origin Citation CH01c06, CH01d08, CH01d09, CH01e01, CH01e12, CH01f02, CH01f07a, CH01f09, CH01g05, CH01g12, CH01h01, CH01h02, CH01h10, CH02b03b, CH02b07, CH02b10, CH02c02b, CH02c06, CH02c09, CH02c11, CH02d08, CH02d11, CH02d12, CH02f06 apple Liebhard et al., 2002 z y BGT23b, BGT24, HGA8b, HGT6, KA4b, NB102a, NB111a, NB113a, NH004a, NH005b, NH008b, NH013a, NH014a, NH015a, NH020a, NH022a, NH023a, NH024b, NH025a, NH026a, NH030a, NH036b, NH039a, RLG Underlined SSR markers showed discrete polymorphic amplified bands in all samples. SSR markers in italics did not show amplification. pear Yamamoto et al., 2002a, b (i ranges from 1 to m), where m is the number of alleles at the target locus and pi is the allele frequency of the ith allele at the target locus. A phenogram of the 94 accessions was constructed using the unweighted pairgroup method using arithmetic averages (UPGMA) based on Nei s genetic identity (Nei, 1972). The program NTSYS-pc version 2.01 was used to construct the phenogram (Rohlf, 1998). Results Amplification of SSR markers in loquat A total of 48 SSR markers developed from apple and pear were evaluated and tested for amplification in loquat. Of the 24 SSR markers derived from apple, amplified fragments were observed for 16 SSR markers (ca. 67%), whereas the other 8 SSR markers did not show amplification (Table 2). Among the 16 SSR markers showing amplification, 11 (CH01e01, CH01f07a, CH01h01, CH01h02, CH01h10, CH02b03b, CH02b10, CH02c06, CH02c09, CH02d12, and CH02f06) produced clear polymorphic amplified fragments for all tested samples. The remaining five SSR markers were unstable or monomorphic. Seven SSR markers (CH01e01, CH01h01, CH01h02, CH02b03b, CH02b10, CH02c06, and CH02d12) were chosen for further analysis because of their reproducibility, clear shape for easy scoring, and degree of polymorphism. Ten of the 24 SSR markers derived from pear produced amplified fragments (Table 2). Only three SSR markers (NH026a, NH036b, and NH039a) showed clear polymorphic amplified fragments for all tested samples. Two SSR markers (NH036b and NH039a) were chosen for further analysis because of their reproducibility, clear shape for easy scoring, and degree of polymorphism. Genetic diversity of loquat The nine apple and pear SSR markers generated 39 putative alleles for the 94 loquat accessions, with an average value of 4.3 per marker (Table 3). The number of alleles per locus ranged from three (CH02b03b and NH036b) to six (CH02c06). The H O values ranged from at CH02c06 to at CH01e01, with an average value of (Table 3). CH02b03b and CH02d12 also showed rather high H O values: and 0.538, respectively. The H E values ranged from at CH02b03b to at CH02c06, with an average value Table 3. SSR locus Characteristics of nine SSR loci in loquats. Size range (bp) No. of alleles CH01e CH01h CH01h CH02b03b CH02b CH02c CH02d NH036b NH039a Average of Five of the nine SSR markers had high H E values of more than Genetic relatedness A phenogram constructed for the 94 loquat accessions on the basis of the SSR analysis revealed two major groups (Fig. 1). Group I, which consisted of 69 mainly cultivated accessions, could be divided into three subgroups, I-1, I-2, and I-3. Cultivated loquats derived from Japan and China were in subgroups I-1 and I-2, whereas nine cultivars introduced from Vietnam, Mexico, and Greece were included in subgroup I-3. Group II included 25 indigenous accessions from Japan. Subgroup I-1 consisted of 31 accessions, including many cultivars presumably derived from Mogi (A12 in Fig. 1). Twenty-eight accessions were included in subgroup I-2, in which some cultivars were presumably derived from Tanaka (A25 in Fig. 1). The phenogram suggested that the ancestral cultivars Mogi and Tanaka have rather different genetic backgrounds. Loquat cultivars derived from Vietnam, Mexico, and Greece formed subgroup I-3 and had different genetic backgrounds from those of cultivars in Japan and China and Japanese indigenous accessions. Because the origins of cultivars from Vietnam, Mexico, and Greece remain unclear, it was very difficult to identify their transmission and introduction. Cultivars from USA and Israel were positioned mainly in subgroup I-2, whereas one USA cultivar (D1: Advance ) and one Israeli cultivar (C3: Heads Mamuth ) were included in subgroups I-1 and I-3, respectively. H O H E

5 J. Japan. Soc. Hort. Sci. 82 (2): Fig. 1. Phenogram of the 94 loquat accessions evaluated in this study. The phenogram was produced using the UPGMA method. The 33 indigenous accessions collected from six regions of Japan are underlined. Most indigenous loquat accessions collected from six areas in Japan formed group II (Fig. 1), which was distinctly separated from cultivated loquats in Japan. Indigenous loquat accessions collected from the same areas did not cluster, but were distributed across group II. These results suggest that indigenous loquats in Japan are genetically different from cultivated loquats in Japan. However, eight of the 33 indigenous accessions (Kawatana mamebiwa 2 [H1-2 in Fig. 1], Kawatana mamebiwa 3 [H1-3], Kawatana mamebiwa 4 [H1-4], Tsushima 12 [H1-7], Tsushima 15 [H1-8], Fukui 2 [H2-2], Oita 81 [H4-3], and Yamaguchi 7S-176 [H6-1]), were included in group I with the cultivated loquats, suggesting that these accessions might have been

6 136 S. Fukuda, C. Nishitani, N. Hiehata, Y. Tominaga, H. Nesumi and T. Yamamoto genetically influenced by cultivated loquats. Synonyms and bud sport cultivars Among the 94 accessions evaluated, some cultivars had identical SSR genotypes and could not be differentiated using the nine SSR markers. The cultivars Mogi and Taisho had the same SSR genotype and similar morphological characteristics, suggesting that Taisho may be a synonym of Mogi or have originated from a bud sport mutant of Mogi. Tanaka, Morimoto, and Nojyuwase could not be differentiated by SSR analysis, suggesting that these were bud sport mutants or synonyms. Indeed, Morimoto was a bud sport of Tanaka (Ichinose, 1983). Three cultivars Amakusagokuwase, Amakusawase, and Moriowase had the same SSR genotype, suggesting that these are bud sport or synonymous cultivars. Moriowase which is thought to be a bud sport of Mogi (Ichinose, 1983), produced different amplified bands for several SSR markers (CH01h01, CH02b03b, CH02b10, NH036b, and NH039a) from those of the original cultivar Mogi, and was located in a different position on the phenogram. These results indicate that Moriowase is not a bud sport of Mogi. Discussion According to Yamamoto et al. (2001), SSR markers developed from apple could be applied across genera within the subfamily Spiraeoideae, tribe Pyreae and used in pears (Pyrus spp.), and nucleotide sequences of SSR fragments are highly conserved between apple and pear. Liebhard et al. (2002) reported that all the tested SSR markers derived from apple could amplify fragments in other genera (e.g., Amelanchier, Cotoneaster, Pyrus, Cydonia). In loquat, SSR markers from apple could be used to analyze the genetic diversity of cultivars present in Europe (Soriano et al., 2005) and cultivars present in China (He et al., 2011). Three SSR markers (CH01h02, CH02c06, and CH02d12) applicable to loquat accessions in this study were used and applied to loquat cultivars by Soriano et al. (2005) and He et al. (2011) and were in good accordance. On the other hand, some SSR markers (CH01h01, CH02b03b, and CH02b10) successfully used in this study failed to reveal a polymorphism or amplified undetermined products (Soriano et al., 2005). These inconsistencies might have derived from different plant materials and analysis systems. Watanabe et al. (2008) reported that about half of the SSR markers derived from apple and pear were applicable to loquat and about 30% showed polymorphisms; the rate of SSR markers applicable to loquat were similar between pearand apple-derived markers. Here, however, we found that 67% of apple SSR markers (16/24) and 42% of pear SSR markers (10/24) could be used for loquat. Previous studies have examined the expected heterozygosity of loquat, with values for mostly cultivars in Europe ranging from 0.27 to 0.64, with an average value of 0.46 (Soriano et al., 2005), and those of cultivars in China ranging from 0.11 to 0.76, with an average value of 0.50 (He et al., 2011). These H E values are similar to the values obtained in this study. According to Soriano et al. (2005), the average value of putative alleles per marker was 2.4 in cultivars in Europe. He et al. (2011) reported this value as 3.4 in cultivars in China; this is smaller than the average value of 4.3 for loquat cultivars collected mainly from Japan and China and for the indigenous accessions in this study. The relatively small number of alleles for loquat cultivars in Europe might be because most cultivars in Europe were derived from cultivars introduced from Japan after 1784 (Lin et al., 1999). Watanabe et al. (2008) noted that the phenogram of 15 diploid loquat cultivars showed no distinctive separation of commercial cultivars in Japan from cultivars introduced from China, in good accordance with the phenogram in this study. Both phenograms showed that the ancestral cultivars Mogi and Tanaka had rather different genetic backgrounds, although some minor disagreements were found. Of 40 loquat accessions in Soriano et al. (2005), only three cultivars were used in this study, and eight out of 54 accessions in He et al. (2011) overlapped in this study. Since different SSR marker sets were applied for different loquat accessions in these reports, it is difficult to compare genetic relationships. It will be necessary to establish a standard SSR marker set for loquat and to combine data sets from different research groups in order to understand comprehensively the genetic diversity in loquat accessions distributed worldwide. Our findings indicate that indigenous loquat accessions are genetically separated from cultivated loquats in Japan. Some indigenous loquats showed unique characteristics of small fruits with thin flesh, unlike cultivated loquats (Tominaga et al., 2005). Although the indigenous accessions Kawatana mamebiwa 3 and Oita 81 had small fruits, the two accessions were placed in group I and had SSR genotypes similar to that of Mogi or Tanaka. These results suggest that SSR analysis might be a more accurate method than morphological comparison for the classification of indigenous or cultivated loquats. In a study based on RAPD markers, Fukuda et al. (2002) reported that the genetic diversity in cultivated loquats was very small and that no distinct groups were found within cultivated loquats. Nagato et al. (1997) noted that isozyme analysis could not differentiate cultivars. In this study, SSR analysis could identify the genetic backgrounds of the accessions and differentiated them into three major groups based on indigenous/ cultivated nature and the country where they were collected. Thus the analysis of SSR markers is a very informative and reliable DNA fingerprinting technique that is applicable to loquat, which has relatively little genetic diversity. The information obtained here can be

7 J. Japan. Soc. Hort. Sci. 82 (2): used for cultivar identification, DNA profiling, genetic research, and loquat breeding programs. Acknowledgments We thank Dr. Hiroyuki Iketani (NARO Institute of Fruit Tree Science) for valuable discussion and suggestions on the experiments and the manuscript. Literature Cited Bao, L., K. Chen, D. Zhang, Y. Cao, T. Yamamoto and Y. Teng Genetic diversity and similarity of pear (Pyrus L.) cultivars native to East Asia revealed by SSR (simple sequence repeat) markers. Genet. Resour. Crop. Evol. 54: Caballero, P. and M. A. Fernandez Loquat, production and market. First International Symposium on Loquat: Degani, C. and A. Blumenfeld The use of isozyme analysis for differentiation between loquat cultivars. HortScience 21: Ding, C., Q. Chen, T. Sun and Q. Xia Germplasm resources and breeding of Eriobotrya japonica Lindl. in China. Acta Hort. 403: Doyle, J. J. and J. L. Doyle A rapid DNA isolation procedure for small quantities of fresh leaf tissue. 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