Origins of Japanese flowering cherry (Prunus ...

Tree Genetics & Genomes DOI 10.1007/s11295-014-0697-1

ORIGINAL PAPER

Origins of Japanese flowering cherry (Prunus subgenus Cerasus) cultivars revealed using nuclear SSR markers Shuri Kato & Asako Matsumoto & Kensuke Yoshimura & Toshio Katsuki & Kojiro Iwamoto & Takayuki Kawahara & Yuzuru Mukai & Yoshiaki Tsuda & Shogo Ishio & Kentaro Nakamura & Kazuo Moriwaki & Toshihiko Shiroishi & Takashi Gojobori & Hiroshi Yoshimaru

Received: 13 February 2013 / Revised: 6 October 2013 / Accepted: 19 November 2013 # Springer-Verlag Berlin Heidelberg 2014

Abstract Japanese flowering cherry (Prunus subgenus Cerasus) cultivars, which are characterized by beautiful flowers, have been developed through hybridization among wild Prunus taxa. The long history of cultivation has caused significant confusion over the origins of these cultivars. We conducted molecular analysis using nuclear simple sequence repeat (SSR) polymorphisms to trace cultivar origins. Bayesian clustering based on the STRUCTURE analysis using SSR genotypes revealed that many cultivars originated from hybridization between two or more wild species. This suggests that morphological variations among flowering cherry cultivars probably arose through a complex sequence of hybridizations. Our findings generally supported estimates of

the origins of cultivars based on morphological study, although there were some exceptions. Keywords Prunus . Cerasus . Ornamental tree . Cultivars . SSR . Taxonomy

Introduction Flowering cherries (members of the Prunus subgenus Cerasus; Rosaceae) are the most popular ornamental trees in Japan and have been cultivated for more than 1,000 years (Flower Association of Japan 1982; Kuitert 1999). In Japan, more than

Communicated by A. Dandekar Electronic supplementary material The online version of this article (doi:10.1007/s11295-014-0697-1 ) contains supplementary material, which is available to authorized users. S. Kato Department of Tourism Science, Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachiouji, Tokyo 192-0397, Japan

Y. Mukai Department of Environmental Science, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan

S. Kato (*) : A. Matsumoto : K. Yoshimura Department of Forest Genetics, Forestry and Forest Products Research Institute, 1 Matsunosato, Tsukuba, Ibaraki 305-8687, Japan e-mail: [email protected]

Y. Tsuda Department of Evolutionary Functional Genomics, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D, 75236 Uppsala, Sweden

S. Kato e-mail: [email protected]

S. Ishio : K. Nakamura Tsukuba Research Institute, Sumitomo Forestry Co., Ltd., 3-2, Midorigahara, Tsukuba, Ibaraki 300-2646, Japan

T. Katsuki : K. Iwamoto : H. Yoshimaru Tama Forest Science Garden, Forestry and Forest Products Research Institute, 1833-81 Todorimachi, Hachioji, Tokyo 193-0843, Japan

K. Moriwaki RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan

T. Kawahara Shikoku Research Center, Forestry and Forest Products Research Institute, 2-915 Asakuranishimachi, Kochi 780-8077, Japan

T. Shiroishi : T. Gojobori National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan

Tree Genetics & Genomes

200 traditional cultivars are known (Kobayashi 1992), and they show diverse floral characteristics, including traits seldom found in the wild. Morphological studies on Japanese flowering cherry cultivars were initiated in the early twentieth century (Koidzumi 1913; Miyoshi 1916; Wilson 1916). Later works established a taxonomy for these cultivars (Flower Association of Japan 1982; Kawasaki 1993) that is now widely accepted (Ohba et al. 2007). The origins and pedigrees of the cultivars, which form the basis of the taxonomy, are of interest to many taxonomists, and the studies based on morphological observations have indicated that most cultivars originated from native Japanese taxa and hybrids between them (Kawasaki 1993; Koidzumi 1913; Miyoshi 1916; Kuitert 1999). Some of Japanese cultivars are also believed to be related to Prunus campanulata, which is distributed in Taiwan, southern China, and neighboring countries, and Prunus pseudocerasus, a native of China (Kawasaki 1993). These morphology-based predictions for origins of the cultivars need to be confirmed by analysis of their genetic background. Knowledge of the origins of cultivated plant species is important in understanding the history of the improvement, and developments in molecular markers have made it possible to analyze the genetic variation, which is one of the key criteria in finding the parental species (Burger et al. 2008). The genetic variations in the cultivated species of Prunus, including peach, Prunus persica; sweet cherry, Prunus avium; apricot, Prunus armeniaca; Japanese apricot, Prunus mume; and almond, Prunus dulcis, have been under intense investigation (Kole and Abbot 2012). For the Japanese flowering cherry, the parental species of some cultivars have been investigated by studies using molecular markers. Innan et al. (1995) used a DNA fingerprinting technique to determine the origin of the most popular cultivar, P. × yedoensis ‘Yedoensis’ (Somei-yoshino). Their findings indicated that this cultivar was produced through hybridization between Prunus lannesiana var. speciosa and Prunus pendula f. ascendens, but further studies may be required (Ohba et al. 2007). Recently, the origins of P. × kanzakura cultivars were estimated by analyses using amplified fragment length polymorphisms (AFLPs) (Ogawa et al. 2012) and nuclear simple sequence repeat (SSR) polymorphisms (Ohta et al. 2011). However, the origins of many other cultivars remain unclear, and an exhaustive survey of diverse cultivars is still required. And then, it will be much more effective to select a wide range of species as the parental candidates of cultivars. Such an approach may provide new insights into cultivar origins, which are not always predictable from morphological observations alone. In particular, it will increase the probability of identifying the correct parental species of cultivars, by avoiding the bias which can be introduced by fixing certain species as the parents on the basis of morphological traits. The objective of this study is to investigate the origins of Japanese flowering cherry cultivars. Our previous study

clarified the clonal status of many cultivars using SSR analysis (Kato et al. 2012). Molecular marker methodology such as SSR analysis is a useful tool for tracing the origins of cultivars. We carried out a comparative analysis of genetic variations at SSR loci in cultivars, and in wild taxa considered to be potential parents of cultivars, using a Bayesian clustering approach in the STRUCTURE software.

Materials and methods Sample collection Ten Prunus taxa (eight Japanese taxa, P. pendula Maxim f. ascendens (Makino) Ohwi, Prunus apetala (Sieb. et Zucc.) Franch. et Savat. var. pilosa (Koidz.) Wilson, Prunus incisa Thunb. ex Murray var. incisa, P. incisa Thunb. ex Murray var. kinkiensis (Koidz.) Ohwi, Prunus sargentii Rehder, Prunus verecunda (Koidz.) Koehne, Prunus jamasakura Sieb. ex Koidz. and P. lannesiana (Carr.) Wilson var. speciosa (Koidz.) Makino, and two foreign taxa P. campanulata Maxim. and P. pseudocerasus Lindley) were used as parental candidates for tracing cultivar origins. We chose the taxa that are believed to be relevant to the breeding history of cultivars. Kawasaki (1993) pointed out based on his morphological observations that these taxa would bear a close relationship to the cultivars. The other Japanese native taxa, Prunus maximowiczii Ruprecht, P. apetala (Sieb. et Zucc.) Franch. et Savat. var. apetala, and Prunus nipponica Matsum., were also added to the list of parental candidates, although they were thought to be less contributed to the cultivar origins. These three taxa are expected to be genetically unrelated to the cultivars. We sampled 13 to 37 trees of each species (Online Resources 1 and 2), and their sampling locations are shown in Online Resource 3. A total of 311 trees were sampled, and they were selected as showing the archetypal characteristics of the respective species. We sampled 215 cultivars which we had previously surveyed (Kato et al. 2012). From one to three samples were tested for each cultivar. The identification numbers of samples used in this study are shown in Online Resource 4. DNA analysis DNA extraction and SSR genotyping followed the procedure described in Kato et al. (2012). We tested the 17 SSR markers used in Kato et al. (2012), AM287648, AM287842, AM288205, AM290339, DN553427, DN554499, DN556408, DW358868, DY640364, DY640849, DY646168, DY647422, DY652293, ASSR1, sk1-1, sk3-1, and sk4-1, as well as 14 additional SSR markers, BPPCT002, 005, 014, 026, 028, 034, 037, 040, and 041 (Dirlewanger et al. 2002), UDP96-001 and 97-402 (Cipriani et al. 1999), UDP98-


412 and 98-416 (Testolin et al. 2000), and pchcms5 (Sosinski et al. 2000), which have been developed in P. persica. Statistical analyses The presence of null alleles was tested using Micro-Checker 2.2.3 (van Oosterhout et al. 2004). The linkage disequilibrium for all pairs of SSR markers was tested with GENEPOP version 4.1.3 (Raymond and Rousset 1995) using default parameters. In order to trace the origins of cultivars, we performed Bayesian clustering using the STRUCTURE 2.3.3 software package (hereafter, STRUCTURE analysis; Pritchard et al. 2010), which assigns individuals into K clusters by comparing their genotypes. Since STRUCTURE analysis can detect the ancestry and admixture of several demes within an individual’s genome, it has also been widely used in population genetic studies. The STRUCTURE analysis was performed in two steps. As a preliminary step, we carried out STRUCTURE analysis using only the individuals from the wild Prunus taxa, in order to select reference data suitable for tracing cultivar origin. The number of genetic clusters (K) was set to vary from 1 to 15. Each run consisted of a run length of 50,000 Markov chain Monte Carlo generations, after a burn-in period of 50,000 iterations. The runs were performed, without using prior information about the taxonomic name, under an admixture model with correlated allele frequencies. Fifty runs were performed for each value of K. The optimal value of K was selected by assessing the likelihood distribution (mean Ln P(K)) and ΔK values (Evanno et al. 2005) produced by Structure Harvester (Earl and vonHoldt 2012). Genetic relationships among clusters were evaluated based on net nucleotide distances between the clusters calculated in STRU CTURE (see documentation for the STRUCTURE program; Pritchard et al. 2010), and a neighbor-joining (NJ) tree based on the distances was generated with phylogeny inference package (PHYLIP) (Felsenstein 1989). Values of F, the amount of drift of each cluster from a common ancestral population (analogous to traditional FST values between each cluster and a common ancestral population), were also calculated in STRUCTURE (see documentation for the STRU CTURE program; Pritchard et al. 2010). Averaged membership coefficients (q) were obtained using CLUMPP 1.1.2 (Jakobsson and Rosenberg 2007), which implements the Greedy algorithm, to allow for label switching and to decide which of the clusters from each run corresponded to a specific label. The q values were obtained based on the outputs of the most likely 10 runs among the 50 replicates of the inferred K (i.e., those with the highest Ln P(K)), although the values of the distance between clusters and FST for each cluster were based on the run with a highest Ln P(K) value. If individuals were assigned to the clusters representing the taxonomic

classification predefined based on morphological traits, provided that q≥0.9, they were used as reference data in the next step. For the main step, we assessed the origins of cultivars using the USEPOPINFO option in the STRU CTURE program. Here, the STRUCTURE analysis was performed using pre-clustering information for all individuals of the selected reference data set (POPFLAG=1), and the 215 cultivars were treated as having unknown origin (POPFLAG=0). K was fixed at the optimal value decided in the previous step. In this analysis, allele frequencies were updated using only the reference data with POPFLAG=1 (option under the Advanced tab). The number of burn-in iterations and the total run length were the same as in the previous step. The program also estimated 90 % probability intervals for q values (a probability interval is the Bayesian analog of a confidence interval). In both steps of the STRUCTURE analysis, because in addition to tetraploid P. pseudocerasus, some cultivars were triploid (Iwatsubo et al. 2002, 2003, 2004), every individual was encoded as a formal tetraploid (PLOIDY parameter=4), assigning the two absent chromosome sets in diploids and the one absent chromosome set in triploids as “missing data” (RECESSIVEALLELE parameter=1). Because each sample was only treated as tetraploid in order to prepare the input file for the program, the ploidy level could be properly reflected in the analysis. This unfamiliar way has been applied in some studies (Cavallari et al 2010; Stöck et al 2010). To corroborate the results obtained by STRUCTURE analysis, phenetic clustering analysis, a general method for analyzing patterns of similarity, was also performed, although it must be noted that the method is not very effective (de Queiroz and Good 1997). The codominant SSR data were converted to a binary data matrix by treating absence of a defined allele as “0” and presence as “1”. The Jaccard coefficient, which does not consider the shared absence of a character as similarity (Jaccard 1908), was then computed from the binary data using FAMD 1.25 (Schlüter and Harris 2006). The distance matrix obtained was used to construct an Unweighted Pair Group Method (UPGMA) tree using the PHYLIP. The confidence limits on individual branches were estimated based on 1,000 bootstrap replications generated with FAMD.

Results A preliminary analysis of the data for 31 SSR loci obtained from the wild Prunus taxa using Micro-Checker detected deviations due to null alleles at five loci (AM290339, DY646168, BPPCT040, BPPCT041, and UDP97-402). No evidence of linkage disequilibrium was found. Therefore, the remaining 26 loci were included in subsequent analyses. Because the SSR markers which we tested for the first time


in this study provided additional genotypic data for the cultivars, the clonal patterns of cultivars described by Kato et al. (2012) were slightly modified. We therefore altered some of the clone identifiers (Online Resource 4). Genotypes with small differences at only one or two loci were distinguished by assigning branch numbers and formally treated as a single clone. In the STRUCTURE analysis using only the data obtained from wild Prunus taxa (preliminary step), the optimal value of K was 7 or 11 (Fig. 1a). Bar charts for the proportions of q values are therefore presented for K=7 and 11 (Fig. 1b). For K = 7, species in three groupings, consisting of two taxa (P. campanulata and P. pseudocerasus), five taxa (P. apetala var. apetala, P. apetala var. pilosa, P. incisa var. incisa, P. incisa var. kinkiensis, and P. nipponica), and two taxa (P. sargentii and P. verecunda), respectively, could not be separated, and only four taxa, P. maximowiczii, P. pendula f. ascendens, P. jamasakura, and P. lannesiana var. speciosa, were individually distinguishable. For K= 11, six additional taxa, P. campanulata, P. pseudocerasus, P. apetala var. apetala, P. nipponica, P. sargentii, and P. verecunda, were distinguishable, but the remaining three taxa, P. apetala var. pilosa, P. incisa var. incisa, and P. incisa var. kinkiensis, still could not be distinguished. The clusters representing distinguishable taxa showed higher F values and were clearly different from the other clusters (Fig. 1c). The proportions of q values were also summarized for each taxon (Table 1). The clusters most probably representing individual taxa were named on the basis of the corresponding taxonomic names. The taxa which could not be distinguished were formally treated as a single species. Of 311 individuals sampled, 270 and 243 were adopted as reference individuals in the cases where K= 7 and 11, respectively. In the STRUCTURE analysis in which the 215 cultivars were treated as being of unknown origin (main step), the individuals stringently selected from wild Prunus taxa were used as the source of reference data. In this step, the K value was fixed at 7 or 11, and the q values for each cultivar and the 90 % probability intervals were inferred (Online Resource 5). When the probability intervals for a q value were above zero, a significant association with the species represented by the cluster was supported. The morphology-based estimates of cultivar origins are also included as a guide (Online Resource 5). The information sources were obtained from descriptions in ten scientific papers listed in the “Sample collection” section of Kato et al. (2012). Bar charts for the proportions of q values are presented and juxtaposed with the UPGMA dendrogram based on the Jaccard coefficient (Fig. 2). Many cultivars were found to be admixtures of two or more clusters and were generally grouped according to individual cultivated taxa. A few cultivated taxa, including P. pendula and P. lannesiana var. speciosa, contained genetic components of only a single cluster.

Fig. 1 STRUCTURE analysis of 311 individuals collected from 13 Prunus taxa. a Values of mean Ln P(K) and ΔK. The highest ΔK value was obtained at K=7, indicating the existence of seven genetic clusters. The ΔK value was also high at K=11, and the values of mean Ln P(K) plateaued at K=11 or above, implying the existence of 11 clusters. b Bar charts for the proportions of q values when K=7 and 11. The individuals marked with an asterisk were excluded from the reference data set used for tracing the origins of cultivars. c Genetic relationships among clusters when K=7 and 11. The cluster names are defined in Table 1. NJ trees were generated based on net nucleotide distances between the clusters calculated using the STRUCTURE program. The net nucleotide distance is the average probability that the two alleles in a pair from two different clusters are different (see documentation for the STRUCTURE program; Pritchard et al. 2010). The value of F, the amount of drift for each cluster from a common ancestral population (analogous to traditional FST values between each cluster and a common ancestral population, see documentation for STRUCTURE program; Pritchard et al. 2010), is shown in parentheses

Discussion Identification of wild Prunus taxa using SSR markers Although in many cases each taxon of wild Prunus was assigned to a specific cluster with a high value of q (q≥0.9), some were not (Table 1). In P. pseudocerasus, some individuals had admixed origins from more than two clusters (Fig. 1b). T his may be because the samples of P. pseudocerasus were derived from cultivated trees collected from botanical gardens and nursery companies (Online Resource 2), implying that their origins are uncertain. Nevertheless, P. pseudocerasus could be clearly separated from the other taxa when K=11, as supported by the high F value (0.56) of the corresponding cluster, although it was indistinguishable from P. campanulata when K= 7. Therefore, there is little doubt that our approach is capable of detecting the genetic component that is derived from P. pseudocerasus. In three taxa, P. apetala var. pilosa, P. incisa var. incisa, and P. incisa var. kinkiensis, some individuals had origins admixed with the cluster corresponding to P. sargentii and P. verecunda (sar/ver) when K=7 (Fig. 1b). Ohta et al. (2005) revealed that two taxa in the section Sargentiella, P. sargentii and P. verecunda, and two sections, Apetalae and Incisae, were closely related to each other (the taxonomic system of the sections was defined according to Kawasaki 1991). Our study also showed that these taxa may be difficult to distinguish clearly, given the relatively low F values of the clusters corresponding to them (Fig. 1c). The other taxa appeared more distantly related to each other (Ohta et al. 2005). The clusters corresponding to these taxa showed higher F values (Fig. 1c), suggesting that our ability to detect their genetic components is sufficient. Although P. maximowiczii was included among the parental candidates for tracing cultivar origins as a precaution, almost no genetic association with the cultivars was detected


a

K=7

b -20000

0

0.2

K = 11 0.4

0.6

0.8

10

0.2

0.4

0.6

0.8

1 max1

-22000

max2 max3 max4 max5 max6

Mean Ln P ( K)

*

P. maximowiczii

max7

-24000

*

max8

cam1 cam2

-26000

cam3 cam4

-28000 -30000

* *

* *

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* *

* *

* * * *

* * * * *

cam5 cam6 cam7 cam8 cam9 cam10 pse1 pse2 pse3 pse4 pse5 pse6 pse7 pse8 pse9 pse10 pse11 pse12 pse13 pse14 pse15 pen1

P. campanulata P. pseudocerasus

pen2 pen3

pen4

-32000

pen5

0

2

4

6

pen6

8 10 12 14

pen7 pen8

K

P. pendula f.ascendens

pen9 pen10 pen11 pen12 ape1 ape2

60

ape3 ape4 ape5 ape6

*

50

ape7

P. apetala var. apetala

ape8 ape9 ape10

40

*

K

* *

* * * * *

ape11 ape12 ape13 ape14 ape15 ape16 ape17 ape18 ape19

* *

30

*

ape20

P. apetala var. pilosa

ape21 ape22 inc1

20

* * * ** * * * *

10

* * * **

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0

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*

0

2

4

6

8 10 12 14

c

inc5 inc6 inc7 inc8

inc10

inc12 inc13

inc15 nip1 nip2 nip3 nip4 nip5 nip6

nip8

P. nipponica

*

jam

* *

(0.15)

K=7

P. incisa var. kinkiensis

inc14

nip7

jam

P. incisa var. incisa

inc9

inc11

* * *

K

inc2 inc3 inc4

* *

nip9 nip10

sar1

*

*

sar2 sar3

sar/ver

sar4

ape/inc/nip

sar/ver

sar5

ape/inc/nip

(0.08)

sar6

max

* * * * *

(0.23)

*

*

(0.05)

max

lan.spe

an.spe

(0.42)

sar7 sar8 sar9 sar10 sar11 sar12 sar13 sar14

P. sargentii

sar15 sar16 sar17 sar18 sar19 sar20 sar21

cam/pse

ver1

cam/pse

ver2 ver3

(0.37)

*

ver4 ver5 ver6 ver7 ver8 ver9

pen

ver10

pen

*

(0.37)

* * * *

cam (0.56)

ver11 ver12 ver13 ver14 ver15

P. verecunda

ver16 ver17 ver18 ver19

cam

* *

K = 11

* *

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pse

*

*

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ver20

jam1 jam2 jam3

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jam4 jam5 jam6 jam7 jam8

max

max

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(0.24)

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P. jamasakura

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*

jam12 jam13

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jam14 jam15

pen

jam16 jam17

pen

ver

jam18

ver

ape.pil/inc (0.05)

ape.pil/inc

(0.10)

*

(0.38)

jam19 lan1

*

*

ape (0.18)

lan2 lan3

ape

lan.spe an.spe

(0.44)

sar

lan4

nip

sar nip (0.13) (0.11)

*

jam

(0.15)

lan5

*

jam

lan6

*

0.1

*

P. lannesiana var. speciosa

lan7

lan8

0.1

(Fig. 2). This finding supports initial assumptions based on morphological classification. For the other taxa, P. apetala var. apetala and P. nipponica, which are unlikely to contribute to

cultivar origins, it is still not possible to draw definite conclusions, given that our ability to detect their genetic components may still be insufficient. Our preliminary conclusions about

20

15

15

30

24

15

13

16

23

35

37

36

32

P. maximowiczii

P. campanulata

P. pseudocerasus

P. pendula f. ascendens

P. apetala var. apetala

P. apetala var. pilosa

P. incisa var. incisa

P. incisa var. kinkiensis

P. nipponica

P. sargentii

P. verecunda

P. jamasakura

P. lannesiana var. speciosa

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0.01

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0.01

0.01

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0.01

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0.99

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–

0.03

0.86

0.02

–

pen

cam/pse

max

K=7

0.01

0.01

0.01

0.01

0.98

0.80

0.74

0.94

0.98

–

0.02

0.01

–

ape/inc/nip

0.01

0.01

0.97

0.97

0.01

0.13

0.16

0.04

–

–

0.05

0.01

0.01

sar/ver

0.01

0.96

0.01

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0.01

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32

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33

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7

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Reference

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0.96

–

cam

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max

K=11

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0.01

0.01

–

–

0.01

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0.83

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–

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0.94

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–

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0.01

0.01

0.07

0.81

0.88

0.80

0.01

–

0.08

–

0.01

ape.pil/inc

–

0.01

0.01

0.01

0.87

0.07

0.02

0.11

0.02

–

–

0.01

–

nip

0.01

0.01

0.03

0.94

0.01

0.03

0.02

0.03

0.01

–

–

–

–

sar

–

0.01

0.93

0.02

0.01

0.01

0.01

0.03

0.01

–

–

–

0.01

ver

0.01

0.94

0.01

0.01

0.01

0.03

0.03

0.01

–

–

–

0.01

–

jam

0.96

0.01

–

0.01

0.01

0.01

0.02

–

–

–

0.03

–

–

lan.spe

28

28

29

28

15

9

9

9

21

30

7

12

18

Reference

P. campanulata; P. pseudocerasus; P. pendula f. ascendens; P. apetala var. apetala; P. apetala var. pilosa; P. incisa var. incisa; P. incisa var. kinkiensis; P. nipponica; P. sargentii; ver P. verecunda; jam P. jamasakura; P. lannesiana var. speciosa

We defined each taxon as being represented by the cluster in which the q value is largest. The cluster names were determined on the basis of the corresponding taxonomic names. The q value of the cluster most probably representing each taxon is shown in bold face; the individuals assigned into a cluster with q≥0.9 were used as references for tracing the origins of cultivars. En dashes indicate values less than 0.01

N

Wild taxon

Table 1 Inferences from STRUCTURE analysis on 13 wild Prunus taxa when K=7 or 11



Fig. 2 UPGMA dendrogram based on Jaccard’s coefficient and inferences from STRUCTURE analysis on 215 cultivars. The scale of the tree represents the dissimilarity index, and bootstrap values higher than 50 % are given at the branch nodes. For the STRUCTURE analysis, the bar charts displayed vertically were constructed using the proportions of the q values when K=7 and 11, respectively (the q values are also shown in Online Resource 5). The clusters defined in Table 1 are identified by

different colors. The scientific name for each cultivar and the Japanese name written in kanji characters are shown on the right-hand side of the corresponding bar chart. The names of cultivated taxa consisting of more than eight clones are identified by individual colors. The remaining taxa are represented in white, and cultivars in these taxa are identified by their taxonomic names in parentheses. Clone identifiers, details of which are provided in Online Resource 4, are also shown in parentheses


each cultivar are as follows, based mainly on the results shown in Fig. 2 and Online Resource 5. Origins of the P. lannesiana cultivars, which constitute a large proportion of Japanese flowering cherry cultivars Morphological studies have suggested that P. lannesiana cultivars originated from P. lannesiana var. speciosa (Koidzumi 1913; Kawasaki 1993). This prediction was verified by our STRUCTURE analysis with both K=7 and K=11, and about half the genomes of cultivars in this cultivated taxon were placed in the P. lannesiana var. speciosa cluster (lan.spe). A significant association with P. jamasakura was also detected for almost every cultivar, and about 30 % of their genomes were placed in the P. jamasakura cluster (jam). However, it appeared that P. lannesiana cultivars were not simply hybrids between P. lannesiana var. speciosa and P. jamasakura, since other wild taxa were also definitely related. Although it was previously considered that few cultivars of P. lannesiana had been related to P. apetala var. pilosa, P. incisa var. incisa, or P. incisa var. kinkiensis (Kawasaki 1993), our study confirmed that some cultivars of P. lannesiana contained genetic components derived from these three taxa. STRU CTURE results with both K=7 and K=11 supported significant association with P. apetala var. pilosa, P. incisa var. incisa, and/or P. incisa var. kinkiensis for the following P. lannesiana cultivars: ‘Kariginu’, ‘Okinazakura’, ‘Sirayuki’, ‘Nigrescens’, and ‘Shibayama’ (Cer031a and b, Cer032c, Cer033, and 034), which are probably synonymous or closely related; ‘Multiplex’, ‘Ojochin’, and ‘Senriko’ (Cer039-i and iii), which are probably derived from bud sport mutants or synonymous; ‘Matsumae-hayazaki’ (Cer058-i and ii); ‘Matsumaesarasa’ (Cer059); and ‘Multipetala’ (Cer068). It may be that the compact tree forms in some P. lannesiana cultivars are derived from two sections, Apetalae and Incisae, known as dwarf wild cherries. In P. lannesiana ‘Nigrescens’ and ‘Shibayama’ (Cer032a-i and ii, b and c, and Cer034), a significant association with P. pendula f. ascendens was also detected. It is possible that P. × subhirtella cultivars, which are believed to be hybrids between P. pendula f. ascendens and P. incisa var. incisa (Ohwi 1973; Kawasaki 1993, see below), played a part in their development. More surprisingly, a significant association with P. nipponica was detected for three cultivars of P. lannesiana: the two synonymous cultivars ‘Chosiuhizakura’ and ‘Kenrokuen-kumagai’ (Cer007) and ‘Mirabilis’ (Cer066). We did not expect this result because Kawasaki (1993) pointed out that P. nipponica, commonly known as Japanese alpine cherry, has been linked with only a few artificially generated cultivars. However, it seems reasonable to assume that our finding is not entirely erroneous because individuals of P. nipponica produce flowers with rose pink petals, which could influence the flower color of P. lannesiana cultivars. In the past, P. sargentii

individuals, which also produce beautiful flowers with rose pink petals, were believed to make a major contribution to many cultivars of P. lannesiana (Miyoshi 1916; Kawasaki 1993). However, a significant association with P. sargentii was detected only for the two synonymous cultivars ‘Kongosan’ and ‘Purpurea‘(Cer017) and for ‘Oshusatozakura’ (Cer070) among the P. lannesiana cultivars, an observation differing from the anticipated result. A significant association with P. verecunda was also detected, though only in ‘Kodaiji’ (Cer046), ‘Gosho-odora’ (Cer063), and ‘Ohta-zakura’ (Cer069), among the P. lannesiana cultivars, again confounding expectations. However, it is not at present possible to draw definite conclusions because it appears difficult to clearly distinguish two taxa of section Sargentiella, P. sargentii and P. verecunda, and two sections, Apetalae and Incisae, as pointed out above. Several other cultivars also showed genetic components which differed from the expected result. The Mazakura cultivars, which were informally defined by Kawasaki (1993), have been assumed to be related to P. pseudocerasus because they share the common trait of aerial roots. However, our results revealed no association with the species for the Mazakura cultivars (Cer035 to 039). The morphological trait of aerial roots, initially derived from P. pseudocerasus, appears to be retained, no matter how many times other species are crossed with a line (Flower Association of Japan 1982; Kawasaki 1993). Thus, a large part of the genetic components that are derived from P. pseudocerasus might have disappeared from the genomes of these cultivars as a result of repeated crossings. Alternatively, the samples we used to provide reference data for P. pseudocerasus may have represented a narrow and biased range of genetic variation, potentially yielding misleading results. This possibility is consistent with the fact that the P. pseudocerasus samples were collected from botanical gardens and nursery companies and some of them were clonally derived (Online Resource 2). Origins of the cultivars believed to have resulted from hybridization between two or more species based on morphological studies Prunus × subhirtella cultivars are believed to be hybrids between P. pendula f. ascendens and P. incisa var. incisa (Ohwi 1973; Kawasaki 1993). Although a significant association with the former was verified, association with the latter was not (Cer135 to 144) because three taxa, P. apetala var. pilosa, P. incisa var. incisa, and P. incisa var. kinkiensis, were indistinguishable (Fig. 1). Thus, we can currently speculate no further. Almost all the P. × subhirtella cultivars were closely related, according to the UPGMA dendrogram (Fig. 2). Prunus × yedoensis is a hybrid between P. pendula f. ascendens and P. lannesiana var. speciosa, according to the taxonomic definition. The P. × yedoensis cultivars


mostly fitted this definition. However, a significant association with P. jamasakura was also detected in the cases of P. × yedoensis ‘America’ (Cer169), ‘Kurama-zakura’ (Cer175), ‘Morioka-pendula’ (Cer178), ‘Pendula’ (Cer180), ‘Perpendens’ (Cer181), ‘Pilosa’ (Cer183), and ‘Sasabe-zakura’ (Cer186), though not for the other cultivars. This was further emphasized by the UPGMA dendrogram, which shows two major groups, excluding P. × yedoensis ‘Sasabe-zakura’ (Fig. 2). Iketani et al (2007) investigated P. × yedoensis cultivars but were unable to determine the origin of P. × yedoensis ‘Perpendens’. The present study strongly suggests that some cultivars of P. × yedoensis, including ‘Perpendens’, are also related to P. jamasakura. The most famous of the P. × yedoensis cultivars, ‘Yedoensis’, has been studied intensively (Takenaka 1963; Innan et al. 1995), and its origin is currently believed to be as a hybrid between P. pendula f. ascendens and P. lannesiana var. speciosa. Our findings support this belief, although there was also a small and nonsignificant association with P. jamasakura (Cr194). Recently, Ogawa et al. (2012) showed that P. × kanzakura ‘Atami-zakura’, which is treated as a part of P. × kanzakura ‘Kanzakura’ in Kato et al (2012), is a hybrid between P. jamasakura and P. campanulata, and P. × kanzakura ‘Kawazu-zakura’ is a hybrid between P. lannesiana var. speciosa and P. campanulata. Ohta et al. (2011) reported similar results for P. × kanzakura ‘Kawazu-zakura’. Our findings (Cer204-i and ii and Cer207) strongly support their findings, and it appears certain that the P. × kanzakura cultivars are hybrids between P. campanulata and P. lannesiana var. speciosa and/or P. jamasakura. It has been suggested that there are several cultivars, except for P. lannesiana, that are related to the early flowering species P. pseudocerasus. In our study, a significant association with P. pseudocerasus was observed for all the cultivars of P. × introrsa, P. × miyoshii, and P. × takenakae (Cer213 to 221), but not for Prunus ‘Kobuku-zakura’ (Cer195), which was close to P. × yedoensis. In the P. × introrsa cultivars, apart from ‘Myoshoji’ (Cer216), a significant association with P. campanulata was also shown. The ‘Hina-zakura’ (Cer213) and ‘Yayoi-zakura’ (Cer219) cultivars of P. × introrsa were artificially generated (Tamura and Iyama 1989), suggesting that it may be easy to create hybrids between P. pseudocerasus and P. campanulata, both of which are early flowering. Association with two or more species was also verified for other cultivars, suggesting that they have been developed through complex crossing. Origins of the cultivars believed to be derived from a single species on the basis of morphological studies All clones of P. pendula ‘Pendula’ (Cer145 to 163), with the exception of Cer160, contained genetic components derived solely from P. pendula f. ascendens. This is reasonable

because P. pendula ‘Pendula’ is considered to be the weeping type of P. pendula f. ascendens (Hara 1950; Kawasaki 1993). The AFLP analysis carried out by Ogawa et al (2012) did not similarly detect any difference between these two taxa. Prunus pendula ‘Plena-rosea’ (Cer164), which produces double flowers with rose pink petals, also contained genetic components derived purely from P. pendula f. ascendens and was found to be closely related to ‘Pendula’, as shown by the high bootstrap value in the UPGMA dendrogram (Fig. 2). However, this was not the case for P. pendula ‘Ujou-shidare’ (Cer165), and significant associations with three taxa, P. apetala var. pilosa, P. incisa var. incisa, and/or P. incisa var. kinkiensis were shown for this cultivar, suggesting that it might be closely related to P. × subhirtella. All cultivars of P. lannesiana var. speciosa (Cer077 to 087) except ‘Formosa’ (Cer078) and ‘Semiplena’ (Cer084) contained genetic components derived solely from wild individuals. In addition, P. sargentii ‘Kushiroyae’ (Cer108), P. verecunda cultivars (Cer109 to 113) with the exception of ‘Kanzashi-zakura’ (Cer111), P. apetala var. pilosa ‘Multipetala’ (Cer115), P. incisa var. kinkiensis ‘Viridicalyx’ (Cer125 and 126), and P. campanulata ‘Ryukyu-hizakura’ (Cer203) contained genetic components derived mainly from wild individuals. However, many, though not all, cultivars of P. jamasakura (Cer088 to 107) were strongly influenced by P. lannesiana var. speciosa, to the extent that they could be regarded as P. lannesiana cultivars. This was also the case for P. incisa var. incisa cultivars (Cer116 to 124). Some of the cultivars derived from a single species are characterized by double or chrysanthemum flowers, which probably result from minor mutations in a small number of functional genes. These morphological traits, which are originally derived from wild taxa, are believed to be the primary sources of floral variation among Japanese flowering cherry cultivars. Estimation of cultivar origin in Japanese flowering cherry using SSR analysis Most cultivars were assigned to two or more clusters in the STRUCTURE analysis using SSR data, suggesting that their diverse morphologies have been acquired through a complex breeding process. The fact that many wild species have been involved in the formation of cultivars is very unique because the other cultivated species of Prunus have originated from single species in many cases (Kole and Abbot 2012). Exceptions are cultivars produced through hybridization events in apricot (Maghuly et al. 2005), Japanese apricot (Hayashi et al. 2008), and almond (Zeinalabedinia et al. 2010), although the genetic origins appear to be less complicated than those of Japanese flowering cherry cultivars. However, the current results should not be accepted unquestioningly because some of the wild taxa used as reference were indistinguishable or difficult to distinguish from one


another. In order to gain a better of understanding the breeding process, more SSR markers are required. In particular, if three taxa, P. apetala var. pilosa, P. incisa var. incisa, and P. incisa var. kinkiensis, could be distinguished, further insights and more definite answers to the question of cultivar origins could be provided. Nevertheless, the three taxa, P. pendula f. ascendens, P. jamasakura, and P. lannesiana var. speciosa, from which many cultivars have been generated, were clearly distinguishable, as supported by high F values for the corresponding clusters (Fig. 1c). Findings related to the cultivars associated with these taxa therefore represent reasonably accurate conclusions, and we believe that the SSR analysis we conducted has provided many insights into cultivar origins, which could not be predicted from morphological observations. The findings of this study will need to be confirmed through more detailed examination of the morphology of each cultivar. Additionally, other marker systems that have a better resolution than SSR markers should be explored in future studies. For example, single nucleotide polymorphisms (SNPs) are abundant throughout the genome and would provide high density genetic markers that could potentially provide a better resolution (Gupta et al. 2001). Fernández i Marti et al. (2012) reported that SNPs had a greater power than SSR markers in assessing the genetic relationship of sweet cherry cultivars. The use of SNPs in Japanese flowering cherry cultivars will also allow for more intensive analysis of cultivar origins. Acknowledgments This research was supported by research grant #200904 of the Forestry and Forest Products Research Institute and the Grant-in-Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology, Japan, grant number 24380087. We thank members of staff of the Shinjuku Gyoen National Garden for granting us permission to investigate and collect materials. We also thank the editor and anonymous reviewers for their constructive suggestions.

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