J. Japan. Soc. Hort. Sci. 63(3) : 543-551. 1994.

Classification of Mume (Prunus mume Sieb. et Zucc.) by RAPD Assay

Takehiko Shimada1, Takashi Haji2, Masami Yamaguchi2, Toshihide Takeda1, Keiichi Nomura1 and Masao Yoshida1 1Faculty of Agriculture, Kobe University, Nada-ku, Kobe 657 2Fruit Tree Research Station , Tsukuba, Ibaraki, 305

Summary The genetic relationship of mume cultivars was resolved by random amplified polymor- phic DNA (RAPD) assay using 95 decamer oligonucleotide primers. The heterozygosity within Prunus mume was confirmed by the numerous polymorphism of DNA fingerprints which exsist among cultivars. Representative cultivars were selected from four groups; 1) Ko-ume (small fruit), 2) Chuu-ume (medium fruit), 3) Ou-ume (large fruit), 4) hybrid between mume and apricot; which were generally classified by fruit size and morphological traits. Japanese mume cultivar were developed independently as indicated by the dissimilarity index between Taiwan and Japanese mume. The Ko-ume and Taiwan mume group with its narrow gene- tic variation are different from 'Bungo' which has characteristics of the apricot. Thus, they are genetically distant from 'Bungo' and apricot. Four flowering mume cultivars in this experiment are closely related to the fruiting mume but were classified into a segregate group, because a possibility exists that the fruiting mume may be a derivative of the flowering ones. The 3 groups of mume cultivars : 1) 'Muroya', 'Inazumi', and 'Tounoume'; 2) 'Komukai' and 'Gojirou'; and 3) 'Suzukishiro' and 'Taihei' consists of synonyms. Therefore, they could not be distinguished from each other with precise RAPD assay capable of detecting DNA polymorphisms. The possibility seems to be high that the cultivars within the 3 groups are identical. 'Takadaume' was shown as the nearest to apricot among mume culti- vars. 'AM2-1', 'AM2-2' and 'AM2-4' which are artificial hybrids between 'Jizouume' and 'Heiwa' were classified into the same cluster as 'Bungo'. It is sufficient evidence to prove that 'Bungo' is a hybrid between mume and apricot. It seems reasonable to suppose that RAPD assay has the potentiality to identify mume cultivars, considering the capabil- ity of discriminating these artificial hybrids. In conclusion, mume cultivars can be classified into seven groups; 1) Taiwan mume, 2) Ko-ume (small fruit), 3) Chuu-ume (medium fruit), 4) Ou-ume (large fruit) with white flow- er, 5) Ou-ume (large fruit) with pink flower, 6) Anzu-ume or Bungo-ume (apricot-mume hybrid), 7) Sumomo-ume (plum-mume hybrid). The DNA fingerprints of mume genome generated by RAPD assay reflect the own origin of mume cultivars. Hence, the method is expected to contribute to a mume breeding project.

acteristics (Tanaka, 1936; Yoshida and Kyotani Introduction 1971; Yoshida and Yamanishi, 1988). Recently The mume (Prunus mume Sieb. et Zucc.) origin- isozyme analysis has been used for classification ated in Southeast China and has been cultivated of cultivars (Aoki et al., 1972; Ujiie et al., 1991) since ancient time in Japan. The species is so gene- However, this method could not resolve relation- tically variant because there were many cultivars ship or identification among similar cultivars. with the apricot characteristics. Their classifica- Currently two new methods to detect DNA poly- tion has mainly been based on morphological char- morphisms were developed : 1) polymerase chain reaction (PCR) and 2) random amplified polymor- Received for publication 24 February. 1994. phic DNA (RAPD) assay (Williams et al., 1990). In

543 544 T. Shimada, T. Haji, M. Yamaguchi, T. Takeda, K. Nomura and M. Yoshida

Citrus (Matsuyama, 1993; Omura, 1993), and mic DNA of mume cultivars are reported. Malus (Harada, 1993), the PCR method with a nuc- Materials and Methods leotide primer were applied to cultivar classifica- tion, identification, and detection of mapping mar- 1. Plant materials kers. In mume, this method is suitable for classify- ing cultivars because it allows the high detection Fifty four cultivars and five selections planted of polymorphisms in their genome; besides the pro- in the Chiyoda experimental farm, Fruit Tree Re- cedure is simpler than that of isozyme analysis. search Station were used. In this paper, DNA polymorphisms and the phy- logenetic relationship by RAPD assay using geno-

Table 1. Genetic distance among Koume (small fruit) group.

Table 2. Genetic distance among Chuu-ume (medium fruit) group. J. Japan. Soc. Hort. Sci. 63 (3) : 543-551. 1994. 545

the standard. The solution was adjusted to 3 ng •E 2. DNA extraction 1 with sterile water ƒÊ‚Œ . Total DNA was isolated from fresh leaves of 3. PCR condition and electrophoresis mume and apricot cultivars by the method of Doyle and Doyle (1 9 87). DNA concentration was PCR were performed in 10 ƒÊl solution contain- estimated by a mini-gel method using A -DNA as ing 10 ng genomic DNA, 10 mM Tris-HCl (pH

Table 3. Genetic distance among Ou-ume (large fruit) group.

Table 4. Genetic distance among Anzu-ume or Bungo-ume (apricot-mume hybrid) group. 546 T. Shimada, T. Haji, M. Yamaguchi, T. Takeda, K. Nomura and M. Yoshida

8.0), 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, Results and Discussion 0.1 mM each of dATP, dTTP, dGTP, dCTP

(Takara) and 0.2 unit of Taq DNA polymerase 1. Detection of polymorphic amplified bands using 95 (Amlitaq(R), , Perkin-Elmer-Cetus). Amplification was decamer primers carried out in a BioOven (BioTherm Co.) prog- ramed for 45 cycles of 10 sec. at 94 •Ž 1 minute Ninety five decamer primers with different GC at 33 •Ž, 1 minute and 50 seconds at 73 •Ž, fol- content (10 primers with 40%, 10 primers with lowed by one cycle of 2 minutes at 73 •Ž. The en- 50%, 55 primers with 60%, 20 primers with 70% tire reaction mixtures were loaded on a 2% Sea- GC content) were surveyed in this experiment.

Kem(R) agarose (FMC) gel in TAE buffer (0.04 M Each primer constructed specific fingerprints. It Tris-acetate and 0.001 M EDTA) containing was independent between sequence of primer and ethidium bromide, and the amplified DNA frag- number of polymorphisms. But some primers, of ments were electrophoresed and the resulting gel which GC content of fast half part from the fifth photographed under UV light. ends were high, effected the number of amplified bands. So if many cultivars were subjected to 4. Data analysis RAPD analysis, screening of primers is required

Dissimilarity index between each pair of acces- to avoid waste of labor and experimental cost. sions is the number of different bands divided by The frequency of DNA polymorphisms by RAPD the total number of polymorphic bands. The data assay was sufficiently high (1.7 RAPDs per prim- were subjected to cluster analysis using group er) to survey the phylogeny of P. mume . Primer average method. The genetic distant (d) was calcu- screening makes the number of RAPDs per primer lated according to Swarfford and Olson (1990), higher. Comparing the number of amplified DNA and Packer et al. (1991). fragments of mume genome with those of peach (P. persica) and apricot (P. armeniaca), mume genome was the easiest to classify. From many annealing sites of primer, it was reconfirmed that mume had

Fig. 1. Dendrogram of Ko-ume (small-fruit) group based on the data from polymorphic fragments generated by 10-mer oligonucleotide primers. * : Control cultivar. J. Japan. Soc. Hort. Sci. 63(3) : 543-551. 1994. 547

Fig. 2. Dendrogram of Chuo-uwe (midium fruit) group based on the data from polymorphic fragments generated by 10-mer oligonucleotide primers. * : Control cultivar .

a heterozygous genome. Taiwan mume was genetically different from Japanese mume cultivars was established by 2. Classification of "Ko-ume (small fruit)" and "T isozyme analysis (Ujiie et al., 1991). The same aiwan mume" group conclusion was obtaiend by the RAPD assay. Twelve cultivars belonging to this group and 3. Classification of "Chuu-ume (medium fruit)" group four cultivars ('Tamaume' X 'Koushiusaishou', 'Tamau me', 'Bungo' X 'Koushiuoujiku' and 'Bungo Eleven cultivars belonging to this group and (single)') for control were analyzed using 95 prim- five cultivars ('Issunbai', 'Taiwan yasei ume', 'Tou- ers. Discernible bands among all cultivars were ji', 'Kankoubai', 'Koubai') for control were analy- detected. Out of 969 different bands observed, 60 zed using 95 primers. Out of 784 different bands, reproducible polymorphic bands generated by 36 48 reliable polymorphic bands generated by 31 primers were selected for cluster analysis. The primers were chosen and subjected to the cluster discrimination of cultivar from the others was analysis. Different DNA fragment appeared among possible using at least 8 bands. the gels of 'Muroya', 'Inazumi' and 'Tounoume' in The dissimilarity index was calculated for each spite of the many assays conducted. The discri- pair of all cultivars, and the data subjected to mination among the other cultivars was possible cluster analysis. These cultivars were divided into with a minimum of 5 bands. The small variation two groups. The average genetic distance (d) be- reflects a local specification for the Wakayama tween Taiwan mume group and Japanese Ko-ume and Hokuriku districts. The average genetic dis- (small fruit) group was 0.38, which is farther 0.24 tance among each subcluster was 0.26. 'Taiwan of the Taiwan mume and 0.23 of the Japanese Ko- yasei ume' was genetically distant (d = 0.53) to ume (small fruit) group. Moreover 'Bungo' was these cultivars, indicating that it evolved apart genetically more distant to these groups (d= 0.49). from the present Japanese cultivars. The flowering These findings suggest that Taiwan mume and Ko- mume, 'Touji', 'Kankoubai', 'Koubai' and 'Issunbai', ume have few characteristics of apricot. That are classified separately, even though their re- 548 T. Shimada, T. Haji, M. Yamaguchi, T. Takeda, K. Nomura and M. Yoshida

Fig. 3. Dendrogram of Ou-ume (large fruit) group based on the data from polymorphic fragments generated by 10-mer oligonuc- leotide primers. * : Control cultivar.

Fig. 4. Dendrogram of Anzu-ume or Bungo-ume (apricot-mume hybrid) group based on the data from polymorphic fragments generated by 10-mer oligonucleotide primers. * : Control cultivar. J. Japan. Soc. Hort. Sci. 63(3) : 543-551. 1994. 549 lationship to the fruiting mume is close, consider- 'Jizouume' and 'Heiwa' were classified into the ing their morphological characteristics and genetic 'Bungo' subcluster . Our assay gives credence that 'Bungo' might be natural hybrid between mume distances obtained here. We, therefore, believe fruiting cultivars in Japan are derivatives of the and apricot. However, Fumoto (1972) reported flowering mumes. that the demonstrating the hybrid parentage of 'Bungo' could be difficult if the patterns of seed 4. Classification of "Ou-ume (large fruit)" group protein among 'Bungo', 'Koushiusaishou' and apri- Twelve cultivars belonging to this group and cot are compared. Among mume cultivars 'Taka- four cultivars ('Takadaume', 'Suzukishiro', 'Taihei', daume' is genetically the nearest to apricots; it is 'Jizouume') for control were analyzed with 95 classified into the apricot group. Haji (1993) clas- primers. Out of 1197 different bands, 55 reliable sified mume, plum, and apricot cultivars by length bands generated by 28 primers were selected and of peduncles. He reported that this was an impor- subjected to cluster analysis. The numerous ampli- tant index with which 'Takadaume' was rated a fied bands within this group is that the highly close relative to the apricot. These results were heterozygous genomic DNA made their annealing consistent with our data. sites with random primers increase. There was no Different polymorphic bands among three F1 difference among the polymorphic bands between hybrids were detected by RAPD assay. Consider- Gojirou' and 'Komukai', 'Suzukishiro' and 'Taihei'.' ing this result, the degree of polymorphism detec- These cultivars appear extremely similar and are tion by this method should have sufficient preci- generally considered synonyms, and the DNA fing- sion to identify mume cultivars. erprints confirm this possibility. Against it, the 6. Phylogenetic studies in mume difference in polymorphic banding between 'G yokuei' and 'Baigou' were detected even though Mume classification has been researched by discrimination between them was impossible by many methods, but they were influeced by en- isozyme band patterns. The dendrogram generally vironmental factors so that it was difficult to dis- divided the white flower and the pink flower criminate between closely related species and cul- group. 'Takadaume' was genetically distant to tivars. RAPD assay directly reflects the structural these cultivars (d = 0.62). "Ou-ume (large fruit)" differences of genome and it was not affected by seemed to have inherited apricot characters, but it environmental factors. It has the potentiality to was demonstrated to be different from 'Taka- study the phylogeny of species and should be the daume', the so-called "apricot-mume". Among powerful tool to identify the mume cultivars. Japanese cultivars, many have some characters of In conclusion, we believe that mume cultivars "arpicot" , so discrimination between mume-apricot should be divided into seven groups; 1) Taiwan and apricot-mume should be quite difficult. The mume, 2) Ko-ume (small fruit), 3) Chuu-ume detection of bands specific to apricot enabled us to (medium fruit), 4) Ou-ume (large fruit) with white discriminate among them. flower, 5) Ou-ume (large fruit) with pink flower, 6) Anzu-ume or Bungo ume (apricot-mume hybrid), 5. Classification of "Anzu-ume or Bungo-ume (mume- and 7) Sumomo-ume (plum-mume hybrid). The arpicot hybrid)"group DNA fingerprints of mume genome generated by Twelve cultivars belonging to this group and RAPD assay reflects the origin of mume cultivars. four varieties ('Shirokaga', 'Jizouume', 'Mai-huang- Hence, this method is expected to assist in the zhun-xing', 'Heiwa') were chosen and analyzed identification and resolve the relationship among with 95 primers. Out of 792 different bands, 31 mume and apricot cultivars. reliable polymorphic bands generated by 19 prim- Acknowledgement ers were chosen and subjected to the cluster analysis. Morphological characteristics of apricot We greatly thank JSPS Fellowships for Japanese often could be observed in these cultivars. The de- Junior Scientists. This work was also supported in ndrogram was roughly classified into two groups part by a Grant in Aid (No. 04454053) for Scien- (apricot group and mume group). 'AM2-1', tific Research from the Ministry of Education, Sci- 'AM2-2' and 'AM2-4' which are hybrids between ence, and Culture, Japan. 550 T. Shimada, T. Haji, M. Yamaguchi, T. Takeda, K. Nomura and M. Yoshida

sis and Breeding in Fruit Trees : 66-73. Literature Cited Packer, C., D. A. Gilbert, A. E. Pusey and S. J. O'Brien. Aoki, N., T. Tonosaki and M. Fuzita. 1972. Phylogene- 1991. A molecular genetic analysis of kinship and tic relationships among cultivars of mume (Prunus cooperation in Africa lions. Nature 351 : mume Sieb. et Zucc.) and apricot (Prunus armeniaca 562-565. L.) as determined by zymography. The bulletin of Shimada, T., K. Hosaka, T. Nakanishi, T. Ozaki, M. the Faculty of Agriculture, Hirosaki University. Yoshida, J. Yamamoto and K. Fujimoto. 1992. No. 18 : 107-113. (In Japanese with English Classification and parent determination by RAPD summary). in mume. J. Japan. Soc. Hort. Sci. 61 (Suppl. 1) : Doyle, J. J. and J. L. Doyle. 1987. A rapid DNA isola- 140-141. (In Japanese). tion procedure for small quantities of fresh leaf Swarfford, D. L. and G. J. Olson. 1990. Phylogeny re- tissue. Phytochemical Bulletin 19 : 11-15. construction. In : D. M. Hillis and C. Moritz (eds.). Fumoto, J. 1972. Studies on relationships of mume and Molecular systematics Sinauer Associates, Sunder- apricot characteristics of Bungo. Doctoral Thesis. land, USA. Kyoto University, Kyoto. (In Japanese). Tanaka, Y. 1936. A monograph of Prunus mume with Haji, T., M. Yamaguchi, M. Yoshida and T. Shimada. reference to its fruit varieties. Contrib. from Hort. 1993. Studies on relationships between Japanese Inst., Taihoku Imp. Univ. No. 15 : 1-44. (In plum (Prunus salicina Lindley), mume (P. mume Japanese with English summary). Sieb. et Zucc.) and apricot (P. armeniaca L.). Inters- Ujiie, T., K. Shimura and H. Kyotani. 1991. Cultivar pecific and intraspecific differences in the length identification of Prunus mume by means of isozy- of flower stalk. J. Japan. Soc. Hort. Sic. 62 (Suppl. me banding patterns. J. Japan. Soc. Hort. Sci. 60 1) : 90-91. (In Japanese). (Suppl. 2) : 166-167. (In Japanese). Harada, T., T. Sato, R. Ishikawa, M. Niizeki and K. Williams, J. G. K., A. R. Kubelik, K. J. Livak, J. A. Saito. 1993. DNA amplification fingerprinting de- Rafalski and S. V. Tingey. 1990. DNA polymorph- tect genetic variation and paternity in Malus isms amplified by arbitrary primers are useful Techniques on Gene Diagnosis and Breeding in genetic markers. Nucleic Acids Res. 18 : Fruit Trees : 81-87. 6531-6535. Matsuyama, T., M. Omura and T. Akihama. 1993. Yoshida, M. and H. Kyotani. 1971. Classification of DNA fingerprinting in Citrus cultivars. Techni- mume and apricot cultivars. I. Morphological clas- ques on Gene Diagnosis and Breeding in Fruit sification. Abstr. Japan. Soc. Hort. Sci. Spring Trees : 26-30. Meet. 12-13. (In Japanese). Omura, M., T. Hidaka, H. Nesumi, T. Yoshida and I. Yoshida, M. and H. Yamanishi. 1988. Apricot cultivars Nakamura. 1993. PCR markers in Citrus identi- in Japan. Acta Hortic. 209 : 69-81. fication and mapping. Techniques on Gene Diagno- J. Japan. Soc. Hort. Sci. 63(3) : 543-551. 1994. 551

RAPD分 析 法 に よ る ウ メ(Prunes mume Sieb. et Zucc,)の 品 種 分 類

島 田 武 彦1・ 土 師 岳2・ 山 口 正 己2・ 武 田 敏 秀1・ 野 村 啓 一1・ 吉 田 雅 夫1

1神戸 大 学 農 学 部 657 神 戸 市 灘 区

2果 樹 試 験 場 305 茨 城 県 つ くば 市

摘 要

95種 類 の10m erの オ ペ ロ ン プ ラ イ マ ー を用 い て, RAPD分 析 法 は 近 縁 な 品 種 問 で もDNA多 型 を 十 分 に RAPD分 析 法 に よ り59品 種 ・系 統 の ウ メ の 品 種 分 類 検 出 で き る の で,従 来 の 方 法 で は 識 別 で き な か っ た 異 を 行 った.ウ メ を 小 梅 ・台 湾 梅 品 種 群,中 梅 品 種 群, 名 同 品種 や 同 名 異 品 種 を識 別 す る こ と も 可 能 で あ る と 大 梅 品 種 群,杏 梅 品 種 群 に分 け,各 々 の 品 種 群 につ い 推 察 さ れ る. て 分 類 を行 っ た.小 梅 と 台 湾 梅 の グ ル ー プ は 遺 伝 的 変 RAPD分 析 の 結 果,実 ウ メ は 次 の7つ の グ ル ー プ に 異 が 小 さ く,ア ン ズ の 形 質 を ほ と ん ど含 ん で い な か っ 分 類 で き た.1)台 湾 梅 品 種 群,2)小 梅 品 種 群,3) た.台 湾 野 生 梅 は 中梅 品 種 群 と は 遠 縁 で,明 ら か に 異 中 梅 品 種 群,4)大 梅(白 花)品 種 群,5)大 梅(桃 な っ て い た.‘室 谷’と‘藤 之梅’,‘小 向’ と‘古 城’,‘鈴 花)品 種 群,6)杏 梅 品 種 群,7)李 梅 品 種 群.花 ウ メ 木 白’と‘太 平’で は 相 互 の 識 別 が で き ず,こ れ ら は 異 は 実 ウ メ と遺 伝 的 特 性 は 近 い もの と考 え られ,中 梅 品 名 同 品 種 で あ る 可 能 性 が 高 い と考 え ら れ る‘ 豊 後’は 種 群,大 梅 品 種 群,杏 梅 品 種 群 の い ず れ か に属 す る も ウ メ と ア ン ズ の 雑 種 で あ る こ とが 証 明 さ れ た.‘ 高 田 の と推 察 さ れ る. 梅’は ア ン ズ に か な り近 縁 で あ る こ とが 確 か め られ た.