Prunus Mume) Germplasms Assessed by SSR Markers

Breeding Science 58: 401–410 (2008)

Genetic diversity in fruiting and flower-ornamental Japanese apricot (Prunus mume) germplasms assessed by SSR markers

Kyohei Hayashi1,2), Ko Shimazu2), Hideaki Yaegaki3), Masami Yamaguchi3), Hiroyuki Iketani1,3) and Toshiya Yamamoto*1,3)

1) Graduate School of Life and Environmental Sciences, University of Tsukuba, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8572, Japan 2) Wakayama Research Center of Agriculture, Forestry and Fisheries Fruit Tree Experiment Station, Laboratory of Japanese apricot, 1416-7 Higashi-honjyo, Minabe, Wakayama 645-0021, Japan 3) National Institute of Fruit Tree Science, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8605, Japan

The genetic diversity and relationships among 127 Japanese apricot (Prunus mume Siebold et Zucc.) germ- plasms, including 56 fruiting and 55 flower-ornamental cultivars derived from Japan, 8 germplasms from China, 7 germplasms from Taiwan and 1 germplasm from Thailand were assessed by SSR markers. Thirty- nine out of 58 SSR markers developed from peach and apricot could produce one or two amplified fragments in Japanese apricot, suggesting transferability across species. Fourteen SSR markers showing clear ampli- fication and high polymorphisms were chosen for further analysis. A total of 155 putative alleles were ob- served in Japanese apricot for 14 SSR loci with an average value of 11.1. The values of observed heterozy- gosity (HO) and expected heterozygosity (HE) ranged from 0.29 to 0.88 (mean value of 0.61) and 0.32 to 0.92 (mean value of 0.68), respectively. A phenogram for 127 Japanese apricot and 3 apricot germplasms showed 3 major clusters, 1) Bungo group of Japanese apricot and apricot, 2) germplasms from Taiwan and Thailand, 3) fruiting and flower-ornamental germplasms derived from Japan and China. In the present study, definite genetic differences were not found between fruiting and flower-ornamental groups, which supported the hy- pothesis that fruiting cultivars have been selected from flower-ornamentals.

Key Words: genetic diversity, Japanese apricot, Prunus mume, SSR.

Introduction Japanese apricot is generally classified into two types by purpose: ornamental and fruit production. The ornamental Japanese apricot (Chinese mei; Prunus mume Siebold et type exhibits extensive variations in flowers, i.e., flower Zucc.) belongs to the subfamily Prunoideae of the family size, petal (single, double) and color (white, pink, red). In Rosaceae and is cultivated in East Asian countries, e.g., the late Edo period (ca. 1800s), more than 300 ornamental Japan, China, Korea, Taiwan (Republic of China), Thailand, cultivars were documented in Japan (Mega et al. 1988, and Vietnam (Mehlenbacher et al. 1991, Horiuchi et al. Horiuchi et al. 1996). According to the traditional Japanese 1996). Japanese apricot originated from the mountainous classification (Mega et al. 1988, Horiuchi et al. 1996), the area of central China and may have been introduced into ornamental cultivars are divided into three groups (named as Japan ca. 2000 years ago (Yoshida and Yamanishi 1988, “kei”), i.e., Yabai, Koubai and Bungo. Cultivars belonging Mehlenbacher et al. 1991, Horiuchi et al. 1996). In East to the Yabai group are thought to be closely related with Asia, the trees bloom in late winter, typically late January or ancestral cultivars, including ‘Touji’, ‘Ukibotan’, and February, before the leaves appear. Therefore, Japanese apri- ‘Tairinryokugaku’. Cultivars of the Koubai group have red cot trees have been grown as early-blooming ornamentals, and flowers and branches, including ‘Benichidori’, ‘Ikuyonezame’ Japanese people have enjoyed their attractive and fragrant and ‘Kagoshimakou’. Cultivars of the Bungo group are char- flowers since the Nara period (8th century). In addition, their acterized by vigorous trees and large flowers, including fresh and processed fruits have been used for medicinal pur- ‘Musashino’, ‘Hakubotan’ and ‘Rinsibai’. Moreover, three poses for a long time. In recent years, the processed fruits groups are divided into seven sub-group (named as “shou”): have been recognized as important healthy foods in Japan, Yabai sub-group, Naniwa sub-group, Benifude sub-group for example, “Ume-boshi” pickle, “Ume-shu” liquors, juice, and Aojiku sub-group in the Yabai group; Koubai sub-group and cooked fruits (Faust et al. 1998). in the Koubai group; Bungo sub-group and Anzu sub-group in the Bungo group. In contrast, there are no or very few Communicated by M. Hirai reports on the genetic diversity of ornamental cultivars in Received May 26, 2008. Accepted October 1, 2008. Japanese apricot using molecular markers. The classifica- *Corresponding author (e-mail: [email protected]) tion system of group and sub-group (traditionally “kei” and 402 Hayashi, Shimazu, Yaegaki, Yamaguchi, Iketani and Yamamoto

“shou”) has not been assessed by molecular markers. abundance of alleles per locus (Weber and May 1989). In Cultivation for fruit production has a history of less than Prunus, a large number of SSR markers have been devel- one hundred years, which is rather shorter than that of oped from several stone fruit species, i.e., peach, cherry, and flower-ornamentals. About 100 cultivars for fruit production apricot (Aranzana et al. 2002, Cantini et al. 2001, Cipriani have been documented, which is a smaller number than that et al. 1999, Dirlewanger et al. 2002, Lopes et al. 2002, of ornamental cultivars (Mega et al. 1988, Horiuchi et al. Sosinski et al. 2000, Struss et al. 2002, Testolin et al. 2000, 1996). Many fruiting cultivars have been selected from in- Yamamoto et al. 2002, Yamamoto et al. 2003b). The Prunus digenous Japanese apricot and bred by controlled cross- reference genetic linkage map was established using F2 pollinations. Almost all fruiting cultivars have morphological population derived from a cross between almond ‘Texas’ characteristics of a single petal and white color of the flower, and peach ‘Earlygold’ (Dirlewanger et al. 2004, Howad et and the trees are generally more vigorous than those of orna- al. 2005). More than 400 SSR markers developed from mentals. Prunus are identified and distributed in 8 linkage groups in Japanese apricot is closely related with apricot (Prunus the Prunus reference genetic linkage map (Dirlewanger et al. armeniaca L.) and Japanese plum (Prunus salicina Lindl.) 2004, Howad et al. 2005). However, no SSR markers have and these three species belong to the same subgenus Prunus. been developed in Japanese apricot. Alternatively, it was re- It is considered that interspecific hybridizations among the ported that SSR markers could be transferable across species three species naturally occurred, especially for the combi- in the genus Prunus. SSR markers developed from peach nation between Japanese apricot and apricot because the could be used for studying genetic diversity in flowering flowering time of Japanese apricot and apricot sometimes cherries (Ohta et al. 2005) and parentage analysis in flower- overlaps (Yoshida and Yamanishi 1988, Mehlenbacher et al. ing cherries (Iketani et al. 2007). In Japanese apricot, SSR 1991, Tzonev and Yamaguchi 1999). It was suspected that markers developed from peach, sweet cherry and sour cherry cultivars in the Bungo group, such as ‘Bungo’, ‘Seiyoubai’ could be applicable for evaluating the genetic diversity of and ‘Taihei’ originated from interspecific hybridizations be- fruiting Japanese apricot derived from China (Gao et al. tween Japanese apricot and apricot, showing intermediate 2004). morphological characteristics in fruits, trees and leaves. In this study, 14 SSR markers chosen out of 58 SSR However, no or very few evidences have been shown for in- markers developed from peach and apricot were evaluated, terspecific hybridizations. It was reported that the chloro- in order to clarify the genetic variation of Japanese apricot. plast DNAs of the trnL-trnF region in Japanese apricot had Using the markers, we investigated the genetic variations of 200 bp of deletions compared with no deletion for the other 127 germplasms including Japanese fruiting cultivars, orna- Prunus including apricot (Ohta et al. 2006). ‘Seiyoubai’, mental cultivars and germplasms from Asian countries. We ‘Takadaume’, ‘Inabungo’, ‘Taihei’, ‘Fushida’ and ‘Kuroda’, also examined the genetic relationships for interspecific hy- which were classified as the Bungo group and had no dele- brids between Japanese apricot and apricot. tion in the trnL-trnF region, might be originated from inter- specific hybridization and their maternal parents might be Materials and Methods apricot (Ohta et al. 2006). Several classifications based on morphological studies Plant materials and DNA isolation have been proposed for Japanese apricot (Yoshida and A total of 127 Japanese apricot (Prunus mume Siebold et Yamanishi 1988, Mega et al. 1988, Horiuchi et al. 1996). Zucc.) germplasms were used in this study. Out of 111 germ- Recently, several molecular markers such as RAPD (Random plasms derived from Japan, 56 were cultivated for their Amplified Polymorphic DNA), AFLP (Amplified Fragment fruit production, and the other 55 were grown for ornamental Length Polymorphism) and SSR (simple sequence repeat, purpose. Eight, seven and one varieties originating from also known as microsatellites) have been used to identify China, Taiwan and Thailand, respectively, were used for and classify the genetic diversity of Japanese apricot analysis (Table 1). Three apricot (Prunus armeniaca L.) cul- (Shimada et al. 1994, Fang et al. 2005, Gao et al. 2004). tivars ‘Heiwa’, ‘Ogasawara’ and ‘Shinyo’ were also used as Yoshida and Yamanishi (1988) reported that Japanese apricot related species. Plant samples were maintained and collected and apricot cultivars were categorized into 10 types according from the Laboratory of Japanese Apricot in Wakayama to the characteristics of stones. Shimada et al. (1994) showed (Minabe, Wakayama), National Institute of Fruit Tree by RAPD analysis that 4 types were observed for Japanese Science (NIFTS) (Tsukuba, Ibaraki), Fukui Prefecture apricot. However, the genetic diversity of Japanese apricot is Horticultural Experiment Station (Mihama, Fukui) and still unclear and there is insufficient information on genetic Nagano Fruit Tree Experiment Station (Suzaka, Nagano). relationships. In addition, there are no reports that compare Leaves for DNA extraction were collected and stored at genetic relationships between fruiting cultivars and flower- –30°C until use. Genomic DNA was isolated from leaves by ornamentals by molecular markers. DNeasy Plant Mini Kit (Qiagen, Germany) according to the Recently, SSR markers have provided a more reliable protocols of the manufacturer. method for evaluating genetic diversity and constructing ge- netic maps because of the co-dominant inheritance and Genetic diversity in Japanese apricot by SSR markers 403

Table 1. Japanese apricot and apricot cultivars used in this study Ornamental characteristics Code a b c Collection site Cultivar or accession name Purpose Origin etc. Group d No. Flower color Flower type Others (JP accession No. ) Fa1 Orihime PF Saitama white single unknown Minabe (172776) Fa2 Koushuu Saisyou PF Nara white single unknown Minabe (113057) Fa3 Purple Queen PF Wakayama white single unknown Minabe Fa4 Hakuou PF Wakayama white single unknown Minabe Fa5 Maesawa PF Nagano white single unknown Minabe Fa6 Beniou PF Wakayama white single unknown Minabe Fa7 Kinugasa PF Wakayama white single unknown Minabe Fa8 Ryuukyou Koume PF Nagano white single unknown Minabe (172779) Fa9 Shinano Koume PF Nagano white single unknown Minabe Fa10 Kouyou Koume PF Nara white single unknown Minabe Fa11 Koushuu Shinkou PF Nara white single unknown NIFTS (113058) Fb1 Seiyoubai PF Hokkaido pink single B/ Minabe (172782) Fb2 Taihei PF old cultivar white single B/ Minabe (174241) Fb3 Bungo PF Oita pink single B/ Minabe Fb4 Fushida PF unknown white single B/ Minabe Fb5 Takadaume PF Fukushima white single B/ Minabe (174243) Fb6 Inabungo PF Nagano white double B/ Minabe Fd1 Nankou PF Wakayama white single unknown Minabe (172773) Fd2 Shirokaga PF old cultivar white single unknown Minabe (172785) Fd3 Kairyou Uchidaume PF Wakayama white single unknown Minabe (170661) Fd4 Benisashi PF Fukui white single unknown Minabe (113065) Fd5 Hanakami PF old cultivar white double unknown Minabe (170639) Fd6 Shinheidayu PF Fukui white single unknown Minabe Fd7 Kotsubu Nankou PF Wakayama white single unknown Minabe Fd8 Kaidarewase PF Wakayama white single unknown Minabe Fd9 Shirotama PF Wakayama white single unknown Minabe (113054) Fd10 Jizouume PF Wakayama white single unknown Minabe (172768) Fd11 Yousei PF Wakayama pink single unknown Minabe (174255) Fd12 Yakushiume PF Wakayama white single unknown Minabe (174252) Fd13 Gojirou PF Wakayama white single unknown Minabe (172766) Fd14 Kagajizou PF Ibaraki white single unknown Minabe Fd15 Gyokuei PF Tokyo white single unknown Minabe (170659) Fd16 Hachirou PF Ibaraki white single unknown Minabe Fd17 Oushuku PF Tokushima white single unknown Minabe (172777) Fd18 Kodama PF unknown white single unknown Minabe Fd19 Kinnyuuji PF Osaka white single unknown Minabe Fd20 Inazumi PF Toyama white single unknown Fukui (172767) Fd21 Rinshuu PF Nara white double unknown Minabe (170647) Fd22 Koshinoume PF Fukui white single unknown Fukui Fd23 Kensaki PF Fukui white single unknown Minabe (170644) Fd24 Tougorou PF Niigata pink single unknown Fukui (170651) Fd25 Juurou PF Kanagawa white single unknown Minabe (172769) Fd26 Tenjin PF unknown white single unknown Minabe Fd27 Toichi PF unknown white single unknown Minabe Fd28 Jorou PF Aichi white single unknown Minabe Fd29 Baigou PF Tokyo white single unknown Minabe (170653) Fd30 Yourou PF Wakayama pink single unknown Minabe (113055) Fd31 Gessekai PF Tokushima pink single unknown NIFTS (170658) Fd32 Gecchibai PF Miyazaki white double unknown NIFTS (170638) Fd33 Natsuka PF Aichi white single unknown NIFTS (172774) Fd34 Sugita PF Kanagawa white single unknown NIFTS (174238) Fd35 Muroya PF Ishikawa white single unknown NIFTS (172771) Fd36 Komukai PF Kanagawa white single unknown NIFTS (170646) Fd37 Shimosukeume PF unknown white single unknown NIFTS (172784) Fd38 Yatsubusa PF Akita white double unknown NIFTS Fd39 Aojiku PF, OF Nara white single green calyx Y/A Minabe 404 Hayashi, Shimazu, Yaegaki, Yamaguchi, Iketani and Yamamoto

Table 1. (continued) Ornamental characteristics Code a b c Collection site Cultivar or accession name Purpose Origin etc. Group d No. Flower color Flower type Others (JP accession No. ) C1 China Mume Wakayama 1 OF China white single unknown Minabe C2 China Mume Wakayama 2 OF China white single Y/N Minabe C3 China Mume Wakayama 3 OF China white single unknown Minabe C4 China Mume Wakayama 4 OF China white single unknown Minabe C5 China Mume Wakayama 5 PF China white single unknown Minabe C6 China Mume Wakayama 6 OF China white single unknown Minabe C7 China Mume Wakayama 7 PF China white single unknown Minabe C8 China Mume Wakayama 8 OF China white single unknown Minabe T1 Ellching PF Taiwan white single unknown Minabe T2 Taiwan Mume Wakayama 1 PF Taiwan white single unknown Minabe T3 Taiwan Mume Wakayama 2 PF Taiwan white single unknown Minabe T4 Taiwan Mume Wakayama 3 PF Taiwan white single unknown Minabe T5 Taiwan Mume 85-065 unknown Taiwan white single unknown NIFTS (229937) T6 Taiwan Yaseiume unknown Taiwan white single unknown NIFTS (174242) T7 Taiwan Mume 85491 OF Taiwan white single unknown NIFTS Thai Thailand Mume Wakayama 1 PF Thailand white single unknown Minabe La1 Mera OF unknown white single Y/Y Minabe La2 Yanagawashibori OF unknown pink double Y/Y Minabe La3 Tsukushikou OF unknown pink double Y/Y Minabe La4 Okina OF unknown white single Y/Y Minabe La5 Rinchigai OF unknown white double Y/Y Minabe La6 Gekkyuuden OF unknown white double Y/Y Minabe La7 Seiryuu Shidare OF unknown white single pendulous Y/Y Minabe La8 Jakoubai OF unknown white double Y/Y Minabe La9 Kankoubai OF unknown red single red branch Y/Y NIFTS (170662) La10 Mangetsu Shidare OF unknown white single pendulous Y/Y NIFTS (170671) La11 Michishirube OF unknown red single Y/Y NIFTS (170672) La12 Kasugano OF unknown white double Y/Y NIFTS (170663) La13 Tamabotan OF unknown white double Y/Y NIFTS (174244) La14 Touji OF unknown white single Y/Y NIFTS (174249) La15 Kenkyou OF unknown pink double Y/Y NIFTS (170664) La16 Tamagaki Shidare OF unknown pink single pendulous Y/Y NIFTS (174245) La17 Hitoe Ryokugaku OF unknown white single green calyx Y/A Minabe La18 Tairin Ryokugaku OF unknown white double green calyx Y/A NIFTS (113067) La19 Goshokou OF unknown pink double Y/N Minabe La20 Ukibotan OF unknown pink double Y/N Minabe La21 Fujibotan OF unknown pink double Y/N NIFTS (170657) La22 Chasenbai OF unknown white single Y/ Minabe Lb1 Kagoshimakou OF unknown red double red branch K/K Minabe Lb2 Kurohikari OF unknown red double red branch K/K Minabe Lb3 Morinoseki OF unknown pink single red branch K/K Minabe Lb4 Kurokumo OF unknown red double red branch K/K Minabe Lb5 Ikuyonezame OF unknown red double red branch K/K Minabe Lb6 Shinheike OF unknown pink double red branch K/K Minabe Lb7 Kinkou OF unknown red double red branch K/K Minabe Lb8 Benichidori OF unknown red single red branch K/K Minabe Lb9 Oosakazuki OF unknown red single red branch K/K Minabe Lb10 Koubai OF unknown red double red branch K/K NIFTS (170666) Lb11 Eikan OF unknown red double red branch K/ Minabe Lb12 Kanbai Shidare OF unknown red double red branch, K/ Minabe pendulous Lc1 Kurodaume OF unknown red double B/B Minabe Lc2 Musashino OF unknown red double B/B Minabe Lc3 Shirobotan OF unknown white double B/B Minabe (174237) Lc4 Makitateyama OF unknown red single B/B NIFTS (170670) Lc5 Kanshikou OF unknown red double B/A Minabe Genetic diversity in Japanese apricot by SSR markers 405

Table 1. (continued) Ornamental characteristics Code a b c Collection site Cultivar or accession name Purpose Origin etc. Group d No. Flower color Flower type Others (JP accession No. ) Lc6 Rinshibai OF unknown red double B/A Minabe Lc7 Mikaikou OF unknown red double red branch B/ Minabe Ld1 Gofuku Shidare OF unknown pink double pendulous unknown Minabe Ld2 Kansei Shidare OF unknown white single pendulous unknown Minabe Ld3 Asahitaki OF unknown white single pendulous unknown Minabe Ld4 Myoutobai OF unknown white double pendulous unknown Minabe Ld5 Suishinbai OF unknown pink single unknown Minabe Ld6 Hasegawashibori OF unknown white double unknown Minabe Ld7 Onikatsura OF unknown white double unknown Minabe Ld9 Unryuubai OF unknown white double unknown Minabe Ld8 Chouhanagata OF unknown pink double pendulous unknown NIFTS (174236) Ld10 Sarasa OF unknown white double unknown NIFTS (172781) Ld11 Yaezakikankou OF unknown red double unknown NIFTS Ld12 Tobiume OF Fukuoka pink double unknown NIFTS (174248) Ld13 Akananiwa OF unknown pink double unknown NIFTS (170652) Ld14 Issunbai OF unknown white double unknown NIFTS (170660) A1 Heiwa OF Nagano pink single NIFTS (174943) A2 Ogasawara OF Aomori pink single Nagano (174926) A3 Shinyo OF Nagano pink single NIFTS a Romanazation of Japanese names are followed the system of the Genebank of the National Institute of Agrobiological Sciences. b PF and OF denote processed fruits and ornamental flowers, respectively. c Group and subgroup of accesssions for ornamental flowers is denoted based on the Japanese traditional classification: Y/Y, Yabai group/Yabai subgroup; Y/A, Yabai/Aojiku; Y/N, Yabai/Naniwa; K/K, Koubai/Koubai; B/B, Bungo/Bungo; B/A, Bungo/Anzu. d Numbers indicated in parentheses are JP numbers from Genebank of the National Institute of Agrobiological Sciences.

SSR analysis scored at each locus. HE was calculated as unbiased formula Fifty-eight SSR markers, including 37 SSRs derived from allele frequencies; 1 − Σpi2 (i = 1 − m), where m is the from peach (Testolin et al. 2000, Sosinski et al. 2000, number of alleles at the target locus, and pi is the allele fre- Yamamoto et al. 2002) and 21 from apricot (Lopes et al. quency of the ith allele at the target locus. 2002), were used for genetic characterization (Table 2). PCR A phenogram of 130 germplasms including 3 apricot amplification was performed in a 20 µl solution of 10 mM cultivars was constructed using UPGMA (unweighted pair- Tris-HCl (pH 8.3), 50 mM KCl, 1.5–2.25 mM MgCl2, 0.01% group method using arithmetic average) based on the simi- gelatin, 0.2 mM each of dNTPs, 10 pmoles of each forward larities between genotypes estimated by Dice’s coefficient, primer labeled with fluorescent chemical (Fam/Tet/Hex or i.e., Dc = 2 nxy/(nx + ny), where nx and ny represent the puta- Fam/Vic/Ned) and unlabeled reverse primer, 10 ng of ge- tive SSR allele’s number of material X and Y, respectively, nomic DNA, and 0.5 unit of Taq polymerase (Invitrogen, and nxy represents the number of shared putative SSR al- USA). The PCR profile consisted of an initial denaturation lele’s number between X and Y. The program NTSYS-pc, for 4 min at 94°C followed by 35 cycles of 1 min at 94°C, 1 ver.2.01 (Rohlf 1998) was used to construct the phenogram. min at 50°C, 2 min at 72°C, and a final extension of 4 min at 72°C. Results The PCR products of each SSR locus were separated and detected using a PRISM 3100 DNA sequencer (Applied SSR amplification in Japanese apricot Biosystems, USA). The size of the amplified bands was deter- A total of 58 SSR markers developed from peach and mined on the basis of an internal standard DNA (GeneScan- apricot were evaluated and tested for amplification in 350TAMRA or 400HD-ROX, Applied Biosystems, USA) Japanese apricot, by using seven germplasms ‘Nankou’, with GeneScan software (Applied Biosystems, USA). ‘Shirokaga’, ‘Jizouume’, ‘Kagajizou’, ‘Benisashi’, ‘Orihime’ and NJ43 (F1 of ‘Nankou’ × ‘Jizouume’). Thirty-nine out of Data analysis 58 SSR markers produced 1 or 2 clear reproducible bands in The observed heterozygosity (HO) and the expected het- all Japanese apricot germplasms, showing successful cross- erozygosity (HE) were estimated for microsatellite loci in amplification in Japanese apricot. Twenty-five out of 37 115 germplasms except for 12 bud sports or synonyms using SSR markers (68%) developed in peach were shown to be CERVUS ver. 2.0 software (Marshall et al. 1998). HO was applicable in Japanese apricot. Fourteen out of 21 SSRs calculated as a ratio showing the heterozygous genotypes (67%) from apricot were also utilized in Japanese apricot, 406 Hayashi, Shimazu, Yaegaki, Yamaguchi, Iketani and Yamamoto

Table 2. Characteristics of Japanese apricot varieties using 14 SSR loci Linkage group and Fragment size No. of No. of a a SSR locus Origin HO HE position in Prunus Citation (bp) alleles genotypes reference map b UDP96-001 peach genomic DNA 109-137 6 14 0.69 0.71 G6 (29.5) Testolin et al. 2000 pchgms3 peach genomic DNA 172-202 14 32 0.70 0.78 G1 (37.5) Sosinski et al. 2000 MA007a peach genomic DNA 94-134 15 38 0.77 0.85 G2 (57.0) Yamamoto et al. 2002 MA010a peach genomic DNA 106-133 10 17 0.52 0.58 G7 (13.4) Yamamoto et al. 2002 MA017a peach genomic DNA 124-154 11 27 0.58 0.80 G8 (26.3) Yamamoto et al. 2002 MA040a peach genomic DNA 207-215 5 10 0.42 0.51 G6 (71.5) Yamamoto et al. 2002 MA053a peach genomic DNA 225-255 5 9 0.47 0.55 G4 (31.2) Yamamoto et al. 2002 M6a peach cDNA 183-237 16 20 0.34 0.47 G8 (34.7) Yamamoto et al. 2002 M7a peach cDNA 140-166 12 19 0.67 0.72 G8 Yamamoto et al. 2002 PaCITA4 apricot genomic DNA 142-182 17 44 0.87 0.89 G3 Lopes et al. 2002 PaCITA7 apricot genomic DNA 199-258 18 63 0.88 0.92 G1 c Lopes et al. 2002 PaCITA12 apricot genomic DNA 135-159 4 5 0.29 0.32 G6 (82.5) Lopes et al. 2002 PaCITA19 apricot genomic DNA 114-160 10 24 0.66 0.77 unknown Lopes et al. 2002 PaCITA21 apricot genomic DNA 217-246 12 27 0.66 0.73 G5 (33.5) Lopes et al. 2002 Average 11.1 24.9 0.61 0.68 a HO and HE were estimated for 115 individuals except 15 bud sports or synonyms. b Dirlewanger et al. 2004, Howad et al. 2005. c Dr. Hanada, personal communication. whose rate was almost the same as SSRs from peach. Four- 0.68. Three SSR markers showed high expected heterozy- teen SSR markers were chosen for further analysis because gosity of more than 0.80. of their reproducibility, clear shape for easy scoring, degree of polymorphism and position in the Prunus reference map. Evaluation of bud sports and synonym cultivars Seven SSRs (UDP96-001, pchgms3, MA007a, MA010a, One hundred and twenty seven germplasms could be dif- MA017a, MA040a, MA053a), two (M6a, M7a) and five ferentiated by 14 SSR markers except for 21 cultivars. These (PaCITA4, PaCITA7, PaCITA12, PaCITA19, PaCITA21) 21 cultivars were divided into 9 genotypes consisting 2 to 4 were derived from peach genomic DNAs, peach cDNAs and cultivars, which showed the same genotypes at all SSR loci apricot genomic DNAs, respectively (Table 2). (Table 3). Three cultivars ‘Koushuu Saishou’ (Fa2), ‘Purple Queen’ (Fa3) and ‘Hakuou’ (Fa4) had the same genotypes Genetic variation evaluated by SSR markers (Table 3). It was also found that 4 cultivars ‘Shirokaga’ A total of 155 putative alleles were obtained from 127 (Fd2), ‘Gojirou’ (Fd13), ‘Gyokuei’ (Fd15) and ‘Komukai’ Japanese apricot germplasms analyzed by 14 SSR markers, (Fd36) showed identical genotypes. The other 7 sets showed with an average value of 11.1. The number of alleles per lo- identical genotypes. cus ranged from 4 at PaCITA12 to 18 at PaCITA7 (Table 2). Further analysis by using the other 7 SSR markers and S- Four, five and five alleles were observed for the SSRs genotypes (self-incompatibility genotypes) could not differ- PaCITA12, MA040a and MA053a, respectively. In contrast, entiate ‘Shirokaga’, ‘Gojirou’, ‘Komukai’ and ‘Gyokuei’ 18, 17 and 16 alleles were obtained for the SSRs PaCITA7, (data not shown). PaCITA4 and M6a, respectively. A total of 349 SSR genotypes were obtained, ranging from 5 at PaCITA12 to 63 at PaCITA7 with the mean value Table 3. Japanese apricot cultivars showing identical SSR genotypes of 24.9 per marker (Table 2). Four SSR markers showed Genotype Cultivar name (Code No.) more than 30 genotypes, i.e., PaCITA7 (63 genotypes), 1 Shirokaga (Fd2), Gojirou (Fd13), Gyokuei (Fd15), Komukai PaCITA4 (44), MA007a (38) and pchgms3 (32). Two SSR (Fd36) markers PaCITA12 and MA053a generated 5 and 9 geno- 2 Koushuu Saishou (Fa2), Purple Queen (Fa3, bud spot of types, respectively, whose values were less than 10. Hakuou), Hakuou (Fa4, bud spot of Kousyuu-saisyou) The values of observed heterozygosity (HO) ranged from 3 Shinheidayu (Fd6), Jizouume (Fd10) 0.29 at PaCITA12 to 0.88 at PaCITA7, with an average val- 4 Kodama (Fd18), Kinnyuuji (Fd19) ue of 0.61 (Table 2). PaCITA7 and PaCITA4 showed values 5 Hanakami (Fd5), Rinshuu (Fd21) of 0.88 and 0.87 for HO, respectively, whereas PaCITA12 6 Ikuyonezame (Lb5), Kinkou (Lb7) and M6a generated rather small values of 0.29 and 0.34. The 7 Yanagawashibori (La2), Kasugano (La12) values of expected heterozygosity (HE) ranged from 0.32 at 8 Natsuka (Fd33), Shimosukeume (Fd37) PaCITA12 to 0.92 at PaCITA7, with an average value of 9 Hasegawashibori (Fd6), Sarasa (Fd10) Genetic diversity in Japanese apricot by SSR markers 407

Genetic relationships in Japanese apricot varieties served in Japanese apricot for 14 SSR loci. HO and HE A phenogram was constructed for 127 Japanese apricot ranged from 0.29 to 0.88 (mean 0.61) and 0.32 to 0.92 (mean germplasms and 3 apricot cultivars, in which the three major 0.68), respectively. Gao et al. (2004) reported 1 to 18 alleles clusters, A, B and C were found (Fig. 1). Cluster A consists per locus for 24 Japanese apricot cultivars in China by SSR of three apricot cultivars and 9 Japanese apricot cultivars tra- analysis, with an average value of 9.1. One to 9 alleles were ditionally classified as the Bungo group. Cluster B included obtained for 14 peach cultivars (Yamamoto et al. 2002) and almost all fruiting and flower-ornamental cultivars derived 3 to 12 alleles were identified for SSR analysis of 25 apricot from Japan and 8 germplasms from China. The cluster C in- cultivars (Lambert et al. 2004). In flowering cherry, the cluded 7 germplasms derived from Taiwan and one from number of alleles per locus ranged from 4 to 36 by SSR anal- Thailand (Fig. 1). ysis of 144 cultivars (Ohta et al. 2005). It was revealed that In the phenogram obtained, 51 fruiting cultivars and 51 Japanese apricot examined in this study showed sufficient flower-ornamental cultivars except for 9 Bungo group culti- genetic diversity and high heterozygosity. vars in Japan did not show distinct genetic differences, but Four cultivars ‘Shirokaga’, ‘Gojirou’, ‘Gyokuei’ and were mingled together in the same cluster B (Fig. 1). It ‘Komukai’, which showed identical SSR genotypes, had no seemed that fruiting and flower-ornamental cultivars were germination ability on pollen (Yaegaki et al. 2002, 2003) somewhat unevenly distributed in the cluster B. Some flow- and very similar morphological characteristics (data not ering cultivars formed 2 sub-clusters B-1 and B-2 in Fig. 1, shown). These results suggested that these 4 Japanese apri- which corresponded to the traditional classification of group cot cultivars might be synonyms of the oldest cultivar and subgroup (kei and shou). The B-1 sub-cluster consisted ‘Shirokaga’. ‘Purple Queen’, originating from a bud spot of of almost all Koubai group cultivars, which are character- ‘Hakuou’, showed different fruit characteristics with pur- ized by red flowers and red branches (Table 1). The B-2 sub- plish fruit skin. Although it was considered that ‘Hakuou’ cluster consisted of three cultivars belonging to Yabai originated from a bud spot of ‘Koushuu Saishou’, no differ- group/Aojiku subgroup (Table 1). Four flower-ornamental ences in morphological characteristics were observed. SSR cultivars of the Bungo group were separated into cluster A analysis could reconfirm ‘Purple Queen’ and ‘Hakuou’ as a (Fig. 1 and Table 1) bud sport and a synonym, respectively. No differences were detected for bud sport cultivars in peach with a rather small Discussion number of SSR markers (Yamamoto et al. 2003a, Mase et al. 2007); therefore, it will be necessary to use a large num- In this study, 39 out of 58 SSR markers (67%) developed ber of SSR markers or other DNA profiling methods such as from peach and apricot could produce one or two amplified RLGS (restriction landmark genomic scanning) analysis fragments in Japanese apricot and showed transferability (Mase et al. 2007) in order to identify bud sport mutants. across species. The transferability of SSR markers devel- Fruiting and flower-ornamental Japanese apricot culti- oped in Prunus was examined and reported within the genus vars in Japan were not genetically separated but were (Cipriani et al. 1999, Dirlewanger et al. 2002, Gao et al. grouped together by using SSR markers. Distribution of 2004, Ohta et al. 2005). Dirlewanger et al. (2004) showed fruiting and flower-ornamental cultivars in cluster B sug- that 93% of the tested SSR markers developed from peach gested presumably no relation to their origins, purposes and were transferable in apricot. Cipriani et al. (1999) described ornamental characteristics. The result did not contradict but that 76% of the SSR markers isolated from peach were suc- supported the hypothesis that fruiting cultivars have been cessfully utilized for apricot and Japanese plum, whose rate selected from flower-ornamentals. A large number of was similar to that obtained in this study. Gao et al. (2004) flower-ornamental cultivars belonging to the other groups reported that SSR markers developed from peach, sweet and sub-groups will help clarify the process by which the cherry and sour cherry were useful for Japanese apricot of present fruiting cultivars were established. fruit production in China and that 14 out of 24 SSR markers The subtropical germplasms from Taiwan and Thailand could be applicable to Japanese apricot germplasms. In this were distinguished into cluster C which was clearly separat- study, 39 SSR markers were found to be transferable in ed from the other two clusters (Fig. 1 and Table 1). It was re- Japanese apricot, only one of which (pchgms3) was investi- ported that cultivars from Taiwan were separated from other gated in the previous study (Gao et al. 2004). Therefore, a Japanese apricot cultivars by RAPD analysis (Shimada et al. total of 52 SSR markers were identified to be useful for 1994). Japanese apricot germplasms derived from Taiwan Japanese apricot. and Thailand showed unique characteristics such as early The 14 SSR markers chosen and used in this study are and long flowering time, late defoliation and low chilling distributed in 8 linkage groups in the reference map requirement for bud break (Yamane et al. 2006). It is con- (Dirlewanger et al. 2004, Howad et al. 2005). Since these sidered that these unique characteristics resulted from adapta- 14 SSR markers were independent or showed close linkage, tion to a subtropical climate. Our SSR analysis clearly it is considered that reliable results to evaluate genetic diver- displayed genetic differences between Japanese apricot sity could be generated. germplasms from southern subtropical regions and from A total of 155 putative alleles (average 11.1) were ob- northern regions of China and Japan. 408 Hayashi, Shimazu, Yaegaki, Yamaguchi, Iketani and Yamamoto

Fig. 1. A phenogram of 127 germplasms of Japanese apricot and 3 apricot cultivars constructed using the UPGMA method based on Dice’s coef- ficient. Accession names of Taiwan Mume Wakayama, Thailand Mume Wakayama and China Mume Wakayama are abbreviated as Taiwan MW, Thailand MW and China MW, respectively. Four Bungo group cultivars found in the cluster B, ‘Taihei’ (Fb2) ‘Shirobotan’ (Lc3), ‘Mikaikou’ (Lc7) and ‘Makitateyama’ (Lc4), are underlined. Genetic diversity in Japanese apricot by SSR markers 409

Eight germplasms from China were mingled with Japa- ture Horticultural Experiment Station) and the Nagano Fruit nese apricot cultivars derived from Japan and distributed in Tree Experiment Station for kindly providing samples. cluster B in the phenogram (Fig. 1). Gao et al. (2004) report- ed that five Japanese apricot cultivars from Japan could not Literature Cited be genetically separated from 19 germplasms from China.

Japanese apricot cultivated in Japan was introduced from Aranzana,M.J., J.Garcia-Mas, J.Carbo and P.Arús (2002) Develop- ment and variability analysis of microsatellite markers in peach. China several times (Yoshida and Yamanishi 1988). In this Plant Breed. 121: 87–92. study, SSR analysis also supported the hypothesis that Cantini, C., A.F. Iezzoni, W.F. Lamboy, M. Boritzki and D. Struss Japanese apricot cultivated in Japan was introduced from (2001) DNA fingerprinting of tetraploid cherry germplasm using northern China. In order to understand comprehensive ge- simple sequence repeats. J. Am. Soc. Hortic. Sci. 126: 205–209. netic diversity in Japanese apricot, it will be necessary to Cipriani, G., G. Lot, W.G. Huang, M.T. Marrazzo, E. Peterlunger and analyze a large number of indigenous as well as cultivated R. Testolin (1999) AC/GT and AG/CT microsatellite repeats in Japanese apricot accessions distributed in China. peach [Prunus persica (L) Batsch]: Isolation, characterization and Out of 13 cultivars, 9 Bungo group cultivars were clearly cross-species amplification in Prunus. Theor. Appl. Genet. 99: 65– separated from other Japanese apricot cultivars and formed 72. the same cluster A as three apricot cultivars (Fig. 1). It was Dirlewanger, E., P. Cosson, M. Tavaud, M.J. Aranzana, C. Poizat, suspected that Bungo group cultivars, which have morpho- A.Zanetto, P. Arús and F. Laigret (2002) Development of microsatellite markers in peach [Prunus persica (L.) Batsch] and logical traits characterized by apricot, for example, late their use in genetic diversity analysis in peach and sweet cherry flowering and harvesting time, and large flower and fruit (Prunus avium L.). Theor. Appl. Genet. 105: 127–138. size, might originate from interspecific hybridizations be- Dirlewanger, E., E. Graziano, T. Joobeur, F. Garriga-Caldere, P. Cosson, tween Japanese apricot and apricot (Yoshida and Yamanishi W. Howad and P. Arús (2004) Comparative mapping and marker- 1988, Mehlenbacher et al. 1991, Tzonev and Yamaguchi assisted selection in Rosaceae fruit crops. Proc. Natl. Acad. Sci. 1999). SSR analysis suggested that many cultivars of Bungo USA 101: 9891–9896. group would be genetically influenced by apricot. Although Fang, J., Y. Qiao and Z. Zhang (2005) Genotyping fruiting mei (Prunus four Bungo group cultivars, i.e., ‘Taihei’ (Fb2) ‘Shirobotan’ mume Sieb. et Zucc.) cultivars using amplified fragment-length (Lc3), ‘Mikaikou’ (Lc7) and ‘Makitateyama’ (Lc4), showed polymorphism markers. HortScience 40: 325–328. morphological characteristics influenced by apricot, they Faust,M., D.Surányi and F.Nyujtó (1998) Origin and dissemination of In were classed as cluster B. This result suggested a possibility apricot. : Janick,J. (ed.) Horticultural Reviews, Volume 22, John Wiley & Sons, Inc. pp. 225–266. that complicated hybridizations (not simple F1 hybrids) Gao, Z.H., Z.J. Shen, Z.H. Han, J.G. Fang, Y.M. Zhang and Z. Zhang might have occurred. Further analysis will be necessary to (2004) Microsatellite markers and genetic diversity in Japanese evaluate to what extent apricot influenced these cultivars apricot (Prunus mume). HortScience 39: 1571–1574. genetically. Since ‘Seiyoubai’, ‘Takadaume’, ‘Inabungo’, Horiuchi, S., M. Yoshida, H. Kariya, T. Nakamura, H. Hasebe, T. Suzaki ‘Fushida’ and ‘Kurodaume’ included in group A had no and T. Sakitani (1996) Nihonnoume Sekainoume. Yokendo. deletion in the trnL-trnF region, the same as apricot, they Howad, W., T. Yamamoto, E. Dirlewanger, R. Testolin, P. Cosson, were presumably originated from interspecific hybridization G.Cipriani, A.J. Monforte, L. Georgi, A.G. Abbot and P. Arús with apricot as maternal parents. (2005) Mapping with a few plants: using selective mapping for In conclusion, SSR analysis could identify Japanese apri- microsatellite saturation of the Prunus reference map. Genetics cot into three major clusters, i.e., Bungo group of Japanese 171: 1305–1309. apricot and apricot, subtropical germplasms derived from Iketani,H., S.Ohta, T.Kawahara, T.Katsuki, N.Mase, Y.Sato and T.Yamamoto (2007) Analyses of clonal status in ‘Somei-yoshino’ Taiwan and Thailand, and germplasms from Japan and and confirmation of genealogical record in other cultivars of China. Fruiting and flower-ornamental cultivars in Japan Prunus × yedoensis by microsatellite markers. Breed. Sci. 57: 1–6. were not genetically separated but were grouped together, sup- Lambert, P., L.S. Hagen, P. Arús and J.M. Audergon (2004) Genetic porting the two hypotheses that Japanese apricot cultivated linkage maps of two apricot cultivars (Prunus armeniaca L.) com- in Japan had been introduced from China and that fruiting pared with the almond Texas × peach Earlygold reference map of cultivars had been selected from flower-ornamentals. Infor- Prunus. Theor. Appl. Genet. 108: 1120–1130. mation obtained in the present study will be efficiently uti- Lopes, M.S., K.M. Sefc, M. Laimer and A. Da Camara Machado (2002) lized for cultivar identification, DNA profiling, genetic Identification of microsatellite loci in apricot. Mol. Ecol. Notes 2: study and Japanese apricot breeding programs. 24–26. Mase, N., H. Iketani and Y. Sato (2007) Analysis of bud sport cultivars Prunus persica Acknowledgements of peaches ( (L.) Batsch) by simple sequence repeats (SSR) and restriction landmark genomic scanning (RLGS). J. Jpn. Soc. Hortic. Sci. 76: 20–27. We are grateful to Mr. T. Imai, Drs. S. Terakami, Y. Ban, Marshall, T.C., J. Slate, L. Kruuk and J.M. Pemberton (1998) Statistical T. Kimura, T. Moriguchi and T. Hayashi for their valuable confidence for likelihood-based paternity inference in natural pop- suggestions and useful discussion. We are also grateful to ulations. Mol. Ecol. 7: 639–655. Ms. E. Ueda and T. Iida for technical assistance with SSR Mehlenbacher, S.A., V. Cocin and L.F. Hough (1991) Apricots analysis. Finally, we thank Dr. T. Watanabe (Fukui Prefec- (Prunus). In: James, N. Moore and James, R. Ballington Jr. (eds.) 410 Hayashi, Shimazu, Yaegaki, Yamaguchi, Iketani and Yamamoto

Genetic resources of temperate fruit and nut crops I, The Inter- Weber, J.K. and P.E. May (1989) Abundant class of human DNA poly- national Society for Horticultural Science, Wageningen, pp. 65–107. morphisms which can be typed using the polymerase chain reac- Mega, K., E. Tomita, S. Kitamura, S. Saito and S. Mizukami (1988) tion. Am. J. Hum. Genet. 44: 388–397. Ume. In: Aoba, T. et al. (ed.) The Grand Dictionary of Horticulture, Yaegaki, H., T. Haji and M. Yamaguchi (2002) Cultivar differences of Shogakukan, Tokyo, pp. 289–300. quantity, rate of strained pollen and rate of germinated pollen in Ohta, S., T. Katsuki, T. Tanaka, T. Hayashi, Y. Sato and T. Yamamoto Japanese apricot (Prunus mume Sieb. et Zucc.) cultivars. Bull. (2005) Genetic variation in flowering cherries (Prunus subgenus Natl. Inst. Fruit Tree Sci. 1: 47–53. cerasus) characterized by SSR markers. Breed. Sci. 55: 415–424. Yaegaki, H., T. Haji, Y. Nakamura, M. Miyake, K. Nishimura, H. Ohta, S., K. Hayashi, H. Yaegaki, N. Mitsui, M. Omura, C. Nishitani and Kyotani and M. Yamaguchi (2003) Variental and yearly variation T. Yamamoto (2006) Genetic relationship among fruiting and in fruit and endocarp weights and their ratio in Japanese apricot flower-Japanese apricot characterized by chloroplast DNA markers. (Prunus mume. Sieb. et Zucc.) cultivars. J. Jpn. Soc. Hortic. Sci. DNA Polymorphism 14: 138–140. 72: 546–550. Rohlf, F.J. (1998) NTSYS, numerical taxonomy and multivariate anal- Yamamoto, T., K. Mochida, T. Imai, Y.Z. Shi, I. Ogiwara and T. Hayashi ysis system, Ver. 2.01. Exeter Publishing, Ltd., Setauket, New (2002) Microsatellite markers in peach [Prunus persica (L) York. Batsch] derived from an enriched genomic and cDNA libraries. Shimada, T., T. Haji, M. Yamaguchi, T. Takeda, K. Nomura and Mol. Ecol. Notes 2: 298–301. M.Yoshida (1994) Classification of Mume (Prunus mume Sieb. et Yamamoto, T., K. Mochida and T. Imai, T. Haji, H. Yaegaki, M. Zucc.) by RAPD assay. J. Jpn. Soc. Hortic. Sci. 63: 543–551. Yamaguchi, N. Matsuda, I. Ogiwara and T. Hayashi (2003a) Parent- Sosinski, B., M. Gannavaraqu, L.D. Hanger, L.E. Beck, G.J. King, age analysis in Japanese peaches using SSR markers. Breed. Sci. C.D.Ryder, S. Rajapakse, W.V. Baird, R.E. Ballard and A.G.Abbott 53: 35–40. (2000) Characterization of microsatellite markers in peach [Prunus Yamamoto, T., K. Mochida and T. Hayashi (2003b) Shanhai Suimitsuto, persica (L.) Batsch]. Theor. Appl. Genet. 101: 421–428. one of the origins of Japanese peach cultivars. J. Jpn. Soc. Hortic. Struss, D., M. Boritzki, R. Karle and A.F. Iezzoni (2002) Microsatellite Sci. 72: 116–121. markers differentiate eight Giessen cherry rootstocks. HortScience Yamane, H., Y. Kashiwa, E. Kakehi, K. Yonemori, H. Mori, K. Hayashi, 37: 191–193. K. Iwamoto, R. Tao and I. Kataoka (2006) Differential expression of Testolin, R., T. Marrazzo, G. Cipriani, R. Quarta, I. Verde, M.T. Dettori, dehydrin in flower buds of two Japanese apricot cultivars requiring M. Pancaldi and S. Sansavini (2000) Microsatellite DNA in peach different chilling requirements for bud break. Tree Physiol. 26: (Prunus persica L. Batsch) and its use in fingerprinting and testing 1559–1563. the genomic origin of cultivars. Genome 43: 512–520. Yoshida, M. and H. Yamanishi (1988) Apricot cultivars in Japan. Acta Tzonev, R. and M. Yamaguchi (1999) Investigation on some far-east Hortic. 209: 69–81. Prunus species: Phenology. Acta Hortic. 488: 239–241.