17 1. Introduction The subgenus Cerasus of the genus Prunus includes more than 50 species, most of which are distributed in temperate areas in the Northern Hemisphere, especially in China, where 33 wild species occur (Yu and Li, 1986). In Japan, nine native species are recorded: P. jamasakura Sieb. ex Koidz., P. sargentii Rehder, P. verecunda (Koidz,) Koehne, P. incisa Thumb. ex Murray, P. nipponica Mat- sum., P. apetala (Sieb. et Zucc.) Fr. et Sav., P. lannesiana (Carr.) Wilson var. speciosa (Koidz.) Makino, and P. pen- dula f. ascendens (Makino) Ohwi. In addition, three wild species, P. pseudo-cerasus Lindl., P. cerasoides D. Don and P. campanulata Maxim., have been popularly cultivat- ed since their introduction from China, Taiwan, and Nepal, respectively (Kawasaki, 1991). Several classifications based on morphological obser- vations have been proposed for Japanese flowering cher- ries (Kawasaki, 1991; Kobayashi, 1992; Ohba, 1992), and they have been classified into five sections: Apetalae (P. apetala), Incisae (P. incisa and P. nipponica), Sargentiella (P. jamasakura, P. sargentii, P. verecunda, and P. lannesi- ana), Phyllomahaleb (P. maximowiczii), and Microcalym- ma (P. pendula). The phylogenetic relationships among these taxa have been investigated using restriction fragment length polymorphism (RFLP) analysis of chloroplast DNA (Kaneko et al., 1986), randomly amplified polymorphic DNA (RAPD) analysis (Shimada et al., 2001), and analyses of rDNA ITS sequences (Lee and Wen, 2001), SSR markers for nuclear DNA (Ohta et al., 2005), and plastid subtype identity (PSID) sequences (Ohta et al., 2006). More than 250 cultivars of flowering cherries, includ- ing Prunus × yedoensis Matsum. ‘Somei-yoshino’ (Ik- etani et al., 2006), have been created through repeated natural and artificial hybridizations among wild Cerasus species (Kawasaki, 1993). ‘Somei-yoshino’ was first pro- posed to have arisen as a hybrid between P. lannesiana var. speciosa and P. pendula f. ascendens (Wilson, 1916). Takenaka (1962, 1965) produced hybrids between the two species and noted that they had similar morphologi- cal characters to ‘Somei-yoshino.’ However, these hybrid plants, such as ‘Amagi-yoshino’ and ‘Izu-yoshino,’ were taller and produced many more flowers with white pet- als than ‘Somei-yoshino.’ Based on SSR marker analysis, Iketani et al. (2007) pointed out clonal status of ‘Somei- yoshino,’ which has been propagated by grafting. Origin of Prunus × yedoensis ‘Somei-yoshino’ based on sequence analysis of PolA1 gene Nakamura I.(1)*, Takahashi H.(1), Ohta S.(2), Moriizumi T.(3), Hanashiro Y.(4), Sato Y.-I.(5), Mii M.(1) (1) Graduate School of Horticulture, Chiba University, Matsudo, Matsudo 271-8510, Japan. (2) Department of Agriculture, Shizuoka University, Ohya, Shizuoka 422-8529, Japan. (3) Bex Co. Ltd., Itabashi, Tokyo 173-0004, Japan. (4) Ocean Exposition Commemorative National Government Park Management Foundation, Kunigami, Okinawa 905-0206, Japan. (5) Future Center, Kyoto Sangyo University, Kamigamo, Kita-ku, Kyoto 603-8555, Japan. Key words: Flowering cherry, phylogenetic relationships, PolA1 gene, RNA polymerase I largest subunit. Abstract: Prunus × yedoensis ‘Somei-yoshino’ is the most popular cultivar of flowering cherry in Japan. Although the origin of this cultivar has been considered hybrid between P. pendula f. ascendens and P. lannesiana var. speciosa, the paternity of P. lannesiana has not been clearly proven by molecular analysis. To reveal the origin of ‘Somei-yoshino,’ we analyzed sequences of intron 19 and exon 20 of PolA1, a single-copy nuclear gene encoding the largest subunit of RNA polymerase I. One of two exon 20 sequences found in ‘Somei-yoshino’ was the same as that of P. pendula, whereas the other sequence was shared with several taxa in seven wild species, including P. jamasakura and P. lannesiana. ‘Somei- yoshino’ contained two different haplotypes of the intron 19 sequences; one was the same as that of P. lannesiana, which is endemic to the Izu and Boso Peninsula in Japan. While another haplotype of ‘Somei-yoshino’ was different from that of P. pendula by two SNPs but identical to one of two haplotypes of P. pendula ‘Komatsu-otome,’ which is a cultivar found in the Ueno Park, Tokyo. These results indicated that ‘Somei-yoshino’ probably originated by the hybridization of cultivars derived from P. pendula and P. lannesiana. Adv. Hort. Sci., 2015 29(1): 17-23 * Corresponding author: inakamur@faculty.chiba-u.jp Received for publication 14 November 2014 Accepted for publication 13 February 2015 18 Adv. Hort. Sci., 2015 29(1): 17-23 Previously, Kaneko et al. (1986) showed that P. pen- dula might be the maternal parent of ‘Somei-yoshino’ based on RFLP patterns of chloroplast DNA. A recent analysis of PSID sequences provided additional support that ‘Somei-yoshino’ had the 10A-T-4A haplotype spe- cific to P. pendula, but not the 14A haplotype for P. lan- nesiana and P. jamasakura (Ohta et al., 2006). Recently, Roh et al. (2007) proposed that variations for nuclear ISSR markers and two plastid DNA sequences of the P. yedoen- sis population on Korean Jeju Island overlapped those of ‘Somei-yoshino.’ Their data, however, did not prove the paternal origin of ‘Somei-yoshino.’ Utilizing the associa- tions of nuclear DNA markers dispersed over the genome, it is especially difficult to identify the paternal parent in Cerasus species. Because the Cerasus species have com- plete self-incompatibility, those DNA markers recombine every generation. To resolve the paternity of ‘Somei-yoshino,’ we decid- ed to compare a relatively short sequence within a single- copy gene, because a short DNA sequence is thought to be a block consisting of many closely linked DNA markers, for which recombination is difficult. Sang (2002) stated that the sequences of single- or low-copy nuclear genes are particularly helpful for understanding the inter- and in- traspecific relationships of various plant groups. Recently, we were interested in PolA1 as a candidate single-copy gene, which encoded the largest subunit of the RNA poly- merase I complex. The DNA sequences of intron 19 of the PolA1 gene were highly polymorphic whereas the exon 20 sequences showed species-specific variations in the gen- era Petunia (Zhang et al., 2008), Oryza (Takahashi et al., 2009), and Triticum (Takahashi et al., 2010). The present study was initiated to reveal the origin of ‘Somei-yoshino’ through the analysis of intron 19 and exon 20 sequences in PolA1 gene. 2. Materials and Methods Plant material Most of the DNA samples used in this study were pro- vided from the Faculty of Agriculture, Shizuoka Univer- sity, and some DNA samples were extracted from leaves of the clonally propagated plants that were maintained in the Tama Forest Science Garden, Tokyo, Japan. A to- tal of 42 individuals of nine wild species native to Japan (Table 1) were analyzed; P. apetala (three individuals), P. incisa (four), P. nipponica (four), P. jamasakura (eight), P. sargentii (two), P. verecunda (four), P. lannesiana (three), P. maximowiczii (three), and P. pendula (six), and three alien wild species, P. campanulata (two), P. cerasoides (two), and P. pseudo-cerasus (one) (Table 1). Two culti- vars, ‘Somei-yoshino,’ P. pendula f. ascendens ‘Komastu- otome’ Hayashi & Nshida (Hayashi, 1989), and five Edo- higan trees were collected in Ueno Park, Tokyo, Japan. One individual of apricot (Prunus armeniaca L.) was also analyzed as out-group material. Genomic DNA isolation and PCR amplification Genomic DNA was extracted from ca. 50 mg of young leaves using a modified CTAB method (Doyle and Doyle, 1987). The forward primer designated as 19ex5P (5’- CTC- GCTGGACGGGGTGAGATGAATG-3’) and the reverse primer designated as 21ex3P (5’-ATTTACTGGCAATC- CAAGACAGAT-3’) were designed based on PolA1 gene (GenBank accession No. NM_125397) of Arabidopsis thaliana and EST (GenBank accession No. BQ641151) of almond (Prunus dulcis Mill.), respectively. DNA frag- ments containing intron 19 and exon 20 sequences of PolA1 gene were amplified by PCR using a pair of 19ex5P and 21ex3P primers (Fig. 1). The reaction mixture of 25 µl contained 10-50 ng of genomic DNA, 1 unit of Ex Taq DNA polymerase (TaKaRa Co., Japan), 2.5 µl of 10× buf- fer (100 mM Tris-Cl, 500 mM KCl, and 15 mM MgCl 2 , pH8.0), 2 µl of 2.5mM dNTPs, 1 µl of 2.0 µM each primer (19ex5P and 21ex3P), and 17.5 µl of distilled water. PCR was performed with a condition of 35 cycles of 94°C for 1 min denaturation, 58°C for 1 min annealing, and 72°C for 2 min elongation in PTC200 Thermocycler (MJ Research Co., USA). Direct sequencing of PCR products containing the intron 19 and exon 20 The amplified PCR products were subjected to 1.2% agarose gel electrophoresis, purified using QIAquick PCR Purification Kit (Qiagen Co., USA), and directly se- quenced with 19ex5P or 21ex3P primer used for the PCR- amplification by ABI3100 Automated DNA Sequencer with a BigDye Terminator Cycle Sequencing Kit (Life Technologies Co., USA). Either 20ex3P (5’-TTGAAGAT- GTTCAGGTATGGGGAG-3’) or 20ex5P (5’-ATAAGTT- GAAGAAAATCAC TGTGG-3’) primer were also used as an internal sequencing primer. The two internal primers were designed based on the partially determined sequenc- es of PolA1 exon 20 of Cerasus in this study. The determined sequences of the intron 19 and exon 20 of PolA1 gene were analyzed using a NCBI web-based Blast server (Altschul et al., 1990), and aligned using web server of Mafft ver 6.0 (Kato and Toh, 2008), and then the aligned sequences were subjected to phylogenetic analy- sis using UPGMA software, with bootstrap analysis using 1,000 replicates, in the Mega 4.0 (Tamura et al., 2007). Fig. 1 - DNA fragments containing intron 19 (PI19) and exon 20 (PE20) of PolA1 gene were amplified using a pair of 19ex5P (PE19) and 21ex3P (PE21) primers. The sequences were determined by direct sequencing using primers for the initial amplification and internal sequence primers, 20ex5P and 20ex3P. 19 Nakamura et al., Origin of Prunus × yedoensis ‘Somei-yoshino’ based on sequence analysis of PolA1 gene Table 1 - Samples used in this study Species Name (Z) Locality PI19 (y) PE20 (y) Prunus apetala (Sieb. et Zucc.) Fr. et Sav. TJ063 Kawaguchiko, Yamanashi 507 nd. TJ093 Chino, Nagano 507 847 TJ164 Hachioji, Tokyo (TFSG) 507 847 P. incisa Thumb. MM048 Gotenba, Shizoka 507 847 MM131 Fujimi, Nagano 507 847 MM160 Amatsukominato, Chiba (TFSG) 507 847   MM165 Fujiyoshida, Yamanashi (TFSG) 507 nd. P. nipponica Matsum. TK077 Shizuoka, Shizuoka 507 nd. TK113 Ashiyasu, Yamanashi 507 nd. TK140 Fujimi, Nagano 507 847 TK188 Kusatsu, Gunma 507 847 P. jamazakura Sieb. ex Koidz. YM001 Morimachi, Shizuoka 507 nd. YM011 Ishikawa Forest Exper. Station 485,507 nd. YM038 Amagiugashima, Shizuoka 485 823 YM154 Hachioji, Tokyo (TFSG) 507 nd. YM245 Kushikino, Kagoshima 507 847 YM256 Izumi, Kumamoto 507 847 YM272 Kinkai, Nagazaki 507 nd.   YM277 Yayoi, Notsu, Oita 485,507 nd. P. sargentii Rehder OY024 Ishikawa Forest Exper. Station 507 nd. OY162 Mamurogawa, Yamagata (TFSG) 507 847 P. verecunda (Koidz.) Koehne KS016 Ishikawa Forest Exper. Station 507 847 KS136 Fujimi, Nagano 507 nd. KS183 Yahiko, Niigata 507 nd. KS211 Nishiki, Yamaguchi 507 847 P. lannesiana (Carr.) Wilson var. speciosa (Koidz.) Makino OS017 Ishikawa Forest Exper. Station 507 847 OS166 Miyake, Tokyo (TFSG) 507 847 OSMTD Matsudo, Chiba 507 nd. P. maximowiczii Rupr. MY076 Shizuoka, Shizuoka 507 823 MY139 Fujimi, Nagano 498 nd.   MY158 Chichibu, Saitama (TFRG) 507 823 P. pendula Maxim. f. ascendens (Makino) Ohwi. EH015 Ishikawa Forest Exper. Station 506 nd. EH149 Ochiai, Okayama (TFRG) 506 nd. EH150 Takekawa, Yamanashi (TFRG) 506 823 EH155 Oya, Hyogo (TFRG) 506 nd. EH163 Oguchi, Kagoshima (TFRG) 506 823 EHFSG a mountain behind TFRG 506 nd. P. pseudo-cerasus Lindl. Shinami SN009 Ishikawa Forest Exper. Station 507 823 P. campanulata Maxim. KN014 Ishikawa Forest Exper. Station 507 823 KN101 Tsukubo Botanical Garden 507 nd. P. cerasoides D. Don. HM001 Katomandu, Nepal (Shizuoka U.) 507 823 HM002 Katomandu, Nepal (Shizuoka U.) 507 nd. P. armeniaca L. ANZ Chiba University 510 823 P. × yedoensis Matsum. ‘Somei-yoshino’   Chiba University 505, 507 823, 847 P. pendula Maxim. ‘Komatsu-otome’   Ueno Park, Tokyo 505, 506 823 (Z) Nos. are according to Ohta et al. (2006). (y) length (bp). Nd= not determined. 20 Adv. Hort. Sci., 2015 29(1): 17-23 3. Results Polymorphisms of the PolA1 intron 19 sequences Using total DNA extracted from 43 individuals in ten species as template, ca. 2.2-kb-long DNA fragments containing intron 19 and exon 20 of the PolA1 gene were clearly amplified by PCR (Fig. 2). The PolA1 intron 19 sequences of most Cerasus species were 507 bp in length (Table 1). All six individuals of P. pendula contained 506 bp because of a one-base insertion at position 49 and a two-base deletion at position 349-350. One individual (YM038) of P. jamasakura and one individual (MY139) of P. maximowiczii had shorter intron 19 lengths of 485 and 498 bp, respectively. Two individuals (YM011 and YM277) of P. jamasakura had two intron 19 sequences of different lengths (485 and 507 bp), although these se- quences could not be confirmed. The DNA sequences described in this paper have been deposited in DDBJ DNA database (accession nos. LC010372- LC010416). Polymorphisms of the PolA1 exon 20 sequences Prunus pendula, P. maximowiczii, P. pseudo-cerasus, P. campanulata, P. cerasoides, and P. armeniaca (out- group) contained an 823-bp exon 20 (Table 1), whereas, seven species (P. apetala, P. incisa, P. nipponica, P. ja- masakura, P. sargentii, P. verecunda, and P. lannesiana) showed a long 847-bp-long exon 20 with a 24-bp inser- tion. One individual (YM038) of P. jamasakura had the short exon 20 (823 bp), and two individuals (YM011 and YM277) possessed both long and short exons 20 (Table 1). The DNA sequences described in this paper have been deposited in DDBJ DNA database (accession nos. LC010540 - LC010565). Phylogenetic tree of the PolA1 intron 19 and exon 20 se- quences The 41 sequences determined for intron 19 and the 23 sequences for exon 20 were aligned using Mafft and sub- jected to phylogenetic analysis using the UPGMA method in MEGA 4.0 with 1,000 bootstap replicates. In the phy- logenetic tree for exon 20, the nine Japanese wild species were classified into two groups, Jamasakura and Pendula (Fig. 3). Prunus campanula belonged to a distantly-related clade. In the Jamasakura group, P. jamasakura, P. nippon- ica, P. incisa, P. lannesiana, and P. apetala shared a long exon 20 and formed a closely-related clade, and ten indi- viduals of these five species contained the same sequence for the exon 20. One individual (KS016) of P. verecunda and one individual (OY162) of P. sargentii also shared an identical exon 20. One individual (YM038) possessed the short exon 20 and was distantly related to the other indi- viduals in the Jamasakura group. The remaining two spe- cies, P. pendula and P. maximowiczii, formed the Pendula Fig. 2 - PCR products (arrow) of DNA fragments containing intron 19 and exon 20 of PolA1 gene in the Cerasus species, 1: Prunus lannesiana var. speciosa, 2: P. pendula f. ascedens, M: marker (λDNA/HindIII plus øx144 DNA/HaeIII). Fig. 3 - Phylogenetic UPGMA tree of the exon 20 sequences in the PolA1 genes from 22 individuals in nine Cerasus species. Indi- viduals used are listed in Table 1. PCR amplification 21 Nakamura et al., Origin of Prunus × yedoensis ‘Somei-yoshino’ based on sequence analysis of PolA1 gene group together with P. pseudo-cerasus and P. cerasoides. Unlike the species in the Jamasakura group, the four spe- cies in the Pendula group were clearly differentiated from one another (Fig. 3). In the phylogenetic tree for intron 19, P. pendula was positioned as the most distantly-related clade (Fig. 4) be- cause this species contained a unique insertion (position 49) and a unique deletion (positions 349-350) (Fig. 5). Ex- cept for P. pendula, the individuals in the Jamasakura and Pendula groups formed two independent clades. Although the species in the Pendula group were clearly differenti- ated from one another, concurring with the results for exon 20, most individuals of the six species in the Jamasakura group, except for YM038, shared similar sequences at in- tron 19, and 14 individuals of the six species possessed the same intron 19 sequence. Three individuals of P. lannesi- ana had the same sequence and belonged to an indepen- dent sub-clade (Fig. 4). Analysis of the origin of P. × yedoensis ‘Somei-yoshino’ When the two allelic sequences of exon 20 of ‘Somei- yoshino’ were determined, one was identical to that of P. pendula, and the other was the same as that shared by five species in the Jamasakura group. For the intron 19 sequence, the (O) haplotype of P. lannesiana was distinguished from the haplotypes of P. jamasakura by three unique single- nucleotide polymorphisms (SNPs) (positions 25, 101, and 171), which were also found in one of two ‘Somei-yoshino’ haplotypes (Fig. 5). By contrast, the other (K) haplotype was found to differ from the (E) haplotype of P. pendula by one base deletion at position 49 and one base substitu- tion of C to A at position 392 (Fig. 5). Consequently, we found that P. pendula f. ascendens ‘Komatsu-otome’ pos- sessed two haplotypes (K and E): one was the same haplo- Fig. 4 - Phylogenetic UPGMA tree of the intron 19 sequences in the PolA1 genes from 40 individuals in nine Cerasus species. Indi- viduals used are listed in Table 1. Fig. 5 - Haplotypes (H) of the intron 19 sequences in the PolA1 genes among ‘Somei-yoshino’ (KO), P. pendula (EE), ‘Komatsu-otome’ (KE), P. lannesiana (OO), and P. Jamasakura (YY), A: polymorphic bases and their positions in two haplotypes are shown, B: Sequence charts were produced using 21ex3P primer and converted to the complementary charts using the 4Peak software. 22 Adv. Hort. Sci., 2015 29(1): 17-23 type of ‘Somei-yoshino’ and the other was identical to that of a wild P. pendula individual. Except for cultivars derived from ‘Somei-yoshino,’ we found that ‘Komatsu-otome’ and two other trees (Nos. 142, 145) in Ueno Park had the same haplotype as ‘Somei-yoshino’ (Fig. 6). 4. Discussion Speciation of wild Cerasus species in Japan Comparing the sequences of intron 19 and exon 20 (Figs. 3 and 4), the nine wild species of Japanese Cerasus were clearly classified into two groups; the Jamasakura group of seven species (P. apetala, P. incisa, P. nipponica, P. jamasakura, P. sargentii, P. verecunda, and P. lannesi- ana) and the Pendula group of two species (P. pendula and P. maximowiczii). Although the former group had been grouped into sections, Apetalae, Incisae, and Sargentiel- la based on morphological differences (Kawasaki, 1966, 1991; Kobayashi, 1992; Ohba, 1992), all seven species shared the same 24-bp insertion within the long exon 20 (847 bp), suggesting that they originated from the same ancestor. The remaining two wild species, P. pendula and P. maximowiczii, contained the short exon 20 (823 bp), in common with three alien wild species (P. pseudo-serasus, P. campanulata, and P. cerasoides), and with P. armenia- ca (out-group). The results from the sequence analysis of exon 20 were thought to be more reliable than those for intron 19 for classifying the subgenus Cerasus, and poly- morphisms found in intron 19 will be useful for discrimi- nating among closely-related taxa and cultivars. Although the seven species in the Jamasakura group have clearly different phenotypes, such as the apetala flower of P. apetala and dwarf stature of P. incisa and P. nipponica, these species have formed a large hybridizing population because they share the same sequences for in- tron 19 and exon 20. The short intron 19 found in three in- dividuals (YM011, YM038, and YM277) of P. jamasakura might have been derived from an ancestral cryptic species. These results suggest that the classification of seven spe- cies in the Jamasakura group remains to be revised based on further molecular information. Origin of ‘Somei-yoshino’ ‘Somei-yoshino’ is the most popular flowering cherry cultivar in Japan and the rest of the world. Ever since Wil- son (1916) proposed a hypothesis for the hybrid origin of ‘Somei-yoshino,’ the biological and geographical origin of this cultivar has been disputed in Japan. In this study, we found that the O haplotype for intron 19 of P. lannesiana contained three unique SNPs, and these SNPs were also found in one of the two haplotypes (K and O) in ‘Somei- yoshino’ (Fig. 5). This indicates that the paternal parent of ‘Somei-yoshino’ was P. lannesiana or its cultivars. As P. lannesiana is endemic to the Izu Peninsula and the Izu Os- hima Islands, ‘Somei-yoshino’ may have originated on the the Izu Peninsula (Takenaka, 1962) or in Edo and Tokyo (Iwasaki, 1989), and not on Jeju Island, Korea (Park et al., 1984; Roh et al., 2007). The other (K) haplotype of intron 19 in ‘Somei-yoshino’ was identical to that (E) of P. pendula, except for two SNPs (Fig. 5). We also found that one of the two haplotypes (K and E) for intron 19 of ‘Komatsu-otome’ was the same as that of ‘Somei-yoshino.’ The original individual of ‘Komatsu- otome’ grows inside the Ueno Park, Tokyo (Fig. 6) and has a dwarf stature with pinkish flower petals. This implies that the maternal origin of ‘Somei-yoshino’ is a cultivar related to ‘Komatsu-otome.’ Out of five trees grown in the same posi- tion with ‘Somei-yoshino’ and ‘Komatsu-otome’ shown in Figure 6, two trees (Nos. 142, 145) contained K haplotype and three (Nos. 141, 142, 144) were hybrids between P. pen- dula f. ascendens and P. lannesiana var. speciosa. These results suggest that there were sufficient genetic resources to develop ‘Somei-yoshino.’ Because P. pendula and ‘Komatsu-otome’ bloom two weeks earlier than P. lannesiana, ‘Somei-yoshino’ and ‘Komatsu-misaki’ were probably produced in Tokyo through artificial hybridiza- tions between ‘Komatsu-otome’ or a related cultivar, and P. lannesiana or a related cultivar, before the end of the Edo Period (Iwasaki, 1989). Acknowledgements We would like to express our sincere thanks to Pro- fessor Dr. Toshio Ando of the Graduate School of Horti- culture, Chiba University and Dr. Akira Kobayashi of the Management Office of Tokyo Metropolitan Parks for their kind assistance during the course of this research. This work was supported by the Ocean Exposition Commemo- rative National Government Park Management Founda- tion, Okinawa, Japan. Fig. 6 - Haplotype of the PolA1 intron 19 of trees (Nos. 135-145) found around “Komatsu no miya” statue (S) in the Ueno Park. ‘Somei-yoshino’ (133, 134, 136, 138) and ‘Komatsu-otome’ (135) share a haplotype K, P. pendula (E), P. lannensiana (O). IY, KN and KZ are flowering cherry cultivars, ‘Ichi-yo,’ ‘Kan- zan,’ and ‘Kanzakura,’ respectively. 23 Nakamura et al., Origin of Prunus × yedoensis ‘Somei-yoshino’ based on sequence analysis of PolA1 gene References ALTSCHUL S.F., GISH W., MILLER W., MYERS E.W., LIP- MAN D.J., 1990 - Basic local alignment search tool. - J. Mol. Biol., 215: 403-410. DOYLE J.J., DOYLE J.L., 1987 - A rapid DNA isolation pro- cedure for small quantities of fresh leaf tissue. - Phytochem. Bull., 19: 11-15. HAYASHI Y., 1989 - Komatsu-otome. - Hana no tomo 36: 40-41 (in Japanese with Latin nomenclature. IKETANI H., KATSUKI T., KAWAHARA T., 2006 - Prunus × yedoensis ‘Somei-yoshino,’ a correct cultivar name for Yo- shino cherry. - J. Jpn. Bot., 81: 123-125. 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