HORTSCIENCE 44(6):1562–1565. 2009. Table 1. Country of origin and the detected chloroplast DNA haplotype of 75 accessions of ·ananassa and seven accessions Maternal Lineages of the Cultivated of its wild relatives. Accessions Country Haplotype , Fragaria ·ananassa, F. ·ananassa cultivars in North America Allstar USA V Arking USA V Revealed by Chloroplast Blakemore USA V Cardinal USA V DNA Variation Chandler USA V Columbia USA C 1 Masanori Honjo , Sono Kataoka, Susumu Yui, and Masami Morishita Donner USA V National Agricultural Research Center for Tohoku Region, Nabeyashiki, 92 Douglas USA V Excelsior USA V Shimokuriyagawa, Morioka, Iwate 020-0123, Japan Fairfax USA V Fresno USA V Miyuki Kunihisa Geneva USA V National Institute of Vegetable and Tea Science, 3-1-1 Kannondai, Tsukuba, Hecker USA V Ibaraki 305-8666, Japan Holiday USA V Honeoye USA V Takayoshi Yano, Megumi Hamano, and Hiromichi Yamazaki Huxley USA V Lassen USA V National Agricultural Research Center for Tohoku Region, Nabeyashiki, 92 Linn USA V Shimokuriyagawa, Morioka, Iwate 020-0123, Japan Marshall USA V Missionary USA V Additional index words. , , haplotype, maternal Pajaro USA V inheritance, maternal origin Premier (Howard 17) USA V Raritan USA V Abstract. We analyzed sequence variation in chloroplast DNA (cpDNA) to investigate the Selva USA V origin of the cultivated strawberry, Fragaria ·ananassa. From analysis of two noncoding Sequoia USA V regions, trnL–trnF and trnR–rrn5, we found three haplotypes (V, C, and X) in F. Tioga USA V ·ananassa. Haplotype V corresponded to the haplotype of F. virginiana and was Tyee Canada V possessed by cultivars bred over a wide geographic range, including North America, Vibrant Canada V Europe, and Japan. Almost all the North American cultivars analyzed in this study Wiltguard USA V possessed haplotype V, suggesting a founder effect. Haplotype C corresponded to the F. ·ananassa cultivars in Europe Cambridge Favourite UK X haplotype of F. chiloensis and was detected mainly in Japanese cultivars. Haplotype X Cambridge Prizewinner UK V was found in only two English cultivars. This haplotype was positioned as intermediate Deutch Evern Germany V between haplotypes V and C in a median-joining network and was considered to be Merton Princess UK X representative of the process of differentiation between F. virginiana and F. chiloensis. Oranda Netherlands V Results of controlled crosses indicate that cpDNA haplotypes of F. ·ananassa are Redgauntlet UK V maternally inherited. These results verify that F. ·ananassa is an interspecific hybrid Senga Sengana Germany V between F. virginiana and F. chiloensis and indicate that traditional cultivars of F. F. ·ananassa cultivars in Japan ·ananassa have been derived from at least three maternal lineages. We demonstrate that Aiberry Japan V the cpDNA variation detected in this study can be used to verify parentage and for Akihime Japan V Asuka Wave Japan C extending hypotheses about June yellows, a leaf variegation disorder in strawberry. Bellerouge Japan V Benihoppe Japan V Decorouge Japan V Enrai Japan V Determining the historical pedigree of in that first hybridization was claimed to have Everberry Japan V cultivars is an important step in understand- been only five plants, which were introduced Fukuba Japan C ing the evolution of crop species (Matsuoka, from Chile to Europe in 1714 (Darrow, 1966). Harunoka Japan V 2005). It can also help to avoid inbreeding Systematic breeding began in England in the Haruyoi Japan V depression and to identify the origin of early 1800s and in North America in the mid- Hatsukuni Japan C agronomically important traits. 1800s using a small number of native and Himiko Japan C The cultivated strawberry, Fragaria cultivated clones (Darrow, 1966). Most mod- Hokowase Japan V ·ananassa Duch., is one of the most eco- ern strawberry cultivars are the progeny of this Kitanokagayaki Japan V nomically important fruit crops in the world. relatively narrow range of germplasm, al- Kogyoku Japan V Kurume 34 Japan C It first arose from accidental hybridization though attempts have been made recently to Kurume 103 Japan V between two American octoploid species, F. increase genetic diversity by using wild ge- Miyazaki Japan V virginiana and F. chiloensis, in a European netic resources (Hancock et al., 2001; Luby Mo-ikko Japan V garden during the early to mid-1700s (Darrow, et al., 2008). On the basis of pedigree data, Morioka 16 Japan V 1966). The number of F. chiloensis involved Dale and Sjulin (1990) reported that the Morioka 26 Japan V majority of modern North American cultivars Morioka 30 Japan V came from only 17 cytoplasmic sources. Morioka 32 Japan V However, further tracing was impossible ow- Morioka 33 Japan V Received for publication 19 June 2009. Accepted ing to incomplete records. Morioka 34 Japan V for publication 12 Aug. 2009. Natsuakari Japan V Because chloroplast DNA (cpDNA) is Nyoho Japan V We thank Naoe Suzuki for technical assistance; unaffected by changes in ploidy, which can Rikiya Kimura, Setsuko Oki, Yukari Sakurai, and Ohishi Shikinari Japan V Keiko Iwabuchi for help in cultivation of plant complicate phylogenetic analysis, the ge- Reiko Japan V materials; and anonymous reviewers for their valu- nome is particularly useful for the phyloge- Sachinoka Japan C able comments. netic analysis of Fragaria. Potter et al. (2000) Sagahonoka Japan C 1To whom reprint requests should be addressed; examined the sequence variation in cpDNA e-mail [email protected]. among 14 species of Fragaria with various (Continued on next page)

1562 HORTSCIENCE VOL. 44(6) OCTOBER 2009 Table 1. (Continued) Country of origin and the Table 2. Chloroplast DNA primers used in the analysis of Fragaria ·ananassa, annealing temperature, and detected chloroplast DNA haplotype of 75 references. accessions of Fragaria ·ananassa and seven Annealing accessions of its wild relatives. Primer Sequence 5#-3# temp (C) References Accessions Country Haplotype trnL (UAA) GGTTCAAGTCCCTCTATCCC 50 Taberlet et al. (1991) Summer Candy Japan C trnF (GAA) ATTTGAACTGGTGACACGAG Taberlet et al. (1991) Summer Princess Japan V trnR (ACG) CGACACCGTGGTTCGTAGC 50 Yoshimura et al. (unpublished data) Summerberry Japan V rrn5 TGGTGTCCCAGGCGTAGAG Yoshimura et al. (unpublished data) Tochihitomi Japan C Tochiotome Japan C Toyonoka Japan C MgCl2, 0.9 units Taq polymerase, 0.2 mMof Table 3. Chloroplast DNA haplotype of seedlings Yachiyo Japan V each primer, and 10 ng template DNA in obtained from controlled cross-pollination of Wild relatives a total volume of 30 mL. Thermocycling cultivars of Fragaria ·ananassa. F. virginianaz V y conditions were as follows: 3 min at 94 C; Parentage (female · male) Haplotype F. virginiana V2 30 cycles of 30 s at 94 C, 45 s at 50 C, and F. chiloensis C Haplotype C · haplotype V ‘PI551445’ 45 s at 72 C; and a final extension step at Sagahonoka · Natsuakari, No.1 C F. vesca EMC Vesca 72 C for 5 min. The PCR was carried out in Sagahonoka · Natsuakari, No.2 C F. vesca UC1 Vesca a GeneAmp PCR System Model 9700 (Ap- Sagahonoka · Natsuakari, No.3 C F. vesca UC5 Vesca plied Biosystems, Foster City, CA) or a PCR Sagahonoka · Natsuakari, No.4 C F. nilgerrensisz Nil Thermal Cycler Dice (Takara, Tokyo, Japan). Sagahonoka · Natsuakari, No.5 C z Sagahonoka · Selva, No.1 C Germplasm conserved in the National The PCR products were purified with a QIA- Sagahonoka · Selva, No.2 C Agricultural Research Center for Tohoku region, quick PCR purification Kit (Qiagen GmbH, Haplotype V · haplotype C Morioka, Japan. Hilden, Germany). The DNA obtained was y Natsuakari · Sagahonoka, No.1 V Seeds were purchased from the B&T World sequenced with an ABI PRISM 310 Genetic Natsuakari · Sagahonoka, No.2 V Seeds, Paguignan, France, in 2002. Analyzer (Applied Biosystems) using a Big- Natsuakari · Sagahonoka, No.3 V Dye Terminator Cycle Sequencing Ready Selva · Sagahonoka V Reaction Kit (Applied Biosystems). Se- Haplotype V · haplotype X ploidy levels. Distinctive sequences for each quencing data were aligned manually with (Morioka 32 · Sagahonoka) species, including F. virginiana and F. chi- CLUSTAL W (Thompson et al., 1994). In- · Cambridge Favourite V loensis, were found in the trnL–trnF region, Haplotype X · haplotype V sertions/deletions (indels) were generally and F. ·ananassa showed the same sequence Cambridge Favourite · Aiberry X placed so as to increase the number of as F. virginiana. Although this analysis Cambridge Favourite · X matching nucleotides in a sequence position. examined only one sample of F. ·ananassa Kitanokagayaki We determined cpDNA haplotypes from and therefore did not provide further infor- Cambridge Favourite x Morioka 32 X nucleotide substitutions and indels. To show Haplotype X · haplotype C mation about the involvement of other wild the relatedness of haplotypes, we constructed Cambridge Favourite · Sachinoka X species in the establishment of F. ·ananassa, a median-joining network (Bandelt et al., sequence variation in cpDNA may allow us 1999) with epsilon value set to zero using to infer the origin of each strawberry cultivar. the software Network 4.5.0.0 (http://www. Japanese cultivars and the American cultivar In this study, we investigated the mode fluxus-engineering.com). Columbia. We detected haplotype X in only of inheritance of cpDNA and then identified To confirm the mode of inheritance of two English cultivars, Cambridge Favourite the maternal origin of F. ·ananassa. We dis- cpDNA in F. ·ananassa, we performed and Merton Princess. cuss the accuracy of the reported pedigree of controlled cross-pollination between culti- All seedlings obtained from controlled cultivars and the relationship between vars possessing different haplotypes (Table cross-pollination possessed the same haplo- cpDNA haplotype and June yellows, a leaf 3). We examined the haplotype for one to five types as their maternal parents (Table 3). variegation disorder in strawberry. seedlings per cross. Discussion Materials and Methods Results The results of the controlled crosses We collected fresh leaves from 75 acces- We found two haplotypes (V and V2) in (Table 3) indicate that the cpDNA of F. sions of F. ·ananassa and from a total of F. virginiana that differed only in the number ·ananassa is maternally inherited, as it is in seven accessions from four related species, of tandem repeats of mononucleotides in the many angiosperms, including other Rosaceae F. virginiana, F. chiloensis, F. vesca, and F. intergenic spacer between trnR and rrn5 genera such as Prunus (Bouhadida et al., nilgerrensis, grown at the National Agricul- (Tables 1 and 4). Fragaria chiloensis, F. 2007), Malus (Matsumoto et al., 1997), and tural Research Center for Tohoku Region, vesca, and F. nilgerrensis possessed the Rubus (Moore, 1993). one of Japan’s official gene banks for straw- distinct haplotypes C, vesca, and nil, respec- Haplotypes V and C correspond to the (Table 1). Total DNA was extracted tively. In F. ·ananassa, we detected three haplotypes of F. virginiana and F. chiloensis, by using a modified PEG method (Rowland haplotypes (V, C, and X). Haplotype X respectively (Table 1). Furthermore, the se- and Nguyen, 1993) with Plant DNAzol Re- displayed the same sequence as haplotype C quences of the trnL–trnF region of each agent (Invitrogen, Carlsbad, CA), as de- in the spacer between trnL and trnF and as haplotype also corresponded to the sequences scribed by Sugimoto et al. (2005). Two haplotype V in the spacer between trnR and of most of F. virginiana and F. chiloensis noncoding regions of cpDNA were selected rrn5. The median-joining network analysis accessions analyzed by Potter et al. (2000), for polymerase chain reaction (PCR) ampli- placed haplotype X between haplotypes V respectively. Six of the seven accessions of fication, because previous studies revealed and C (Fig. 1). The nucleotide sequences of F. virginiana analyzed by Potter et al. (2000) that these regions contain intraspecific poly- these haplotypes will appear in the DNA Data corresponded to haplotype V in our study and morphisms (Potter et al., 2000; Honjo et al., Bank of Japan under accession numbers one to haplotype C. Also, six of the seven unpublished data), specifically the spacer AB514801 to AB514816. accessions of F. chiloensis corresponded to between trnL and trnF and the spacer be- We found haplotype V in 61 cultivars of haplotype C and one to haplotype V. Three of tween trnR and rrn5. The primers used are F. ·ananassa originating from a diverse geo- the seven accessions of the diploid species listed in Table 2. The PCR reaction mix graphic range, namely, North America, F. vesca also corresponded to haplotype V. contained 1 · PCR buffer [10 mM TrisÁHCl Europe, and Japan. All North American Thus, F. virginiana predominantly possesses (pH 8.3), 50 mM KCl, 100 mM each dNTP, cultivars except ‘Columbia’ possessed hap- haplotype V and F. chiloensis haplotype 0.02% Triton X-100, 0.01% gelatin], 1.5 mM lotype V. We found haplotype C in 11 C. Haplotype V can be regarded as more

HORTSCIENCE VOL. 44(6) OCTOBER 2009 1563 Table 4. Substitutions, indels and repeat variation in trnL–trnF and trnR–rrn5 regions of chloroplast DNA in some Fragaria species. Gene region/sequence position trnL–trnF trnR–rrn5 Haplotype 100 117 126 186–188 266–272 433 162–171 173 180 V C T A — — T AAAAAAAAAA G G V2 C T A — — T AAAAAAAAA G G C C T A — — C AAAAAAAAAA T G X C T A — — C AAAAAAAAAA G G Vesca C T A — CATATCA T AAAAAAAAAA G G Nil T C C ATA — T AAAAAAAAAA G T

they or their progeny possessed haplotype V (Table 1). The nine are ‘Missionary’, ‘Mar- shall’, ‘Hudson Bay’ (ancestor of ‘Sequoia’ and ‘Wiltguard’), ‘Middlefield’ (ancestor of ‘Chandler’, ‘Douglas’, ‘Hecker’, and ‘Selva’), ‘Chesapeake’ (ancestor of ‘Allstar’ and ‘Linn’), ‘Aberdeen’ (ancestor of ‘Holiday’ and ‘Raritan’), ‘Neunan’ (ancestor of ‘Pajaro’ Fig. 1. Median-joining network of the chloroplast and ‘Tyee’), ‘Ettersburg 450’ (ancestor of DNA haplotypes of Fragaria ·ananassa (C, V, ‘Honeoye’ and ‘Vibrant’), and ‘Streamliner’ X), F. virginiana (V, V2), F. chiloensis (C), F. (ancestor of ‘Geneva’). It is apparent that there vesca (vesca), and F. nilgerrensis (nil) based on has been a strong founder effect in the spread the sequences of two noncoding regions. Each of haplotype V in North America. letter corresponds to a haplotype, and the size Of the Japanese cultivars analyzed in this of the circle is proportional to the haplotype’s frequency. study, 11 (28%) possessed haplotype C (Table 1), indicating that cultivars with F. chiloensis maternal origin are more common Fig. 2. Reported maternal pedigree of strawberry cultivars leading to Japanese flagship cultivars ancestral than haplotype C judging from the in Japan than in North America. Strawberry Tochiotome and Sachinoka. The numbers in- relationship with F. vesca. The accession that breeding in Japan began with the establish- dicate the year released and the bold letters contained haplotype C despite being classi- ment of the cultivar Fukuba in 1899. We indicate the chloroplast DNA haplotype of the fied as F. virginiana was considered to be found that ‘Fukuba’ possesses haplotype C, cultivars. Bold and thin lines connecting acces- the result of introgression, and the accession which probably explains the relatively high sions indicate a relationship between mother that contained haplotype V despite being clas- frequency of haplotype C in Japanese culti- and progeny and that between grandmother and sified as F. chiloensis was inferred to be vars, because its lineage has frequently been grandchild, respectively. originated from a population that may have used for breeding in Japan. been established earlier in the evolution of We found haplotype X in only two culti- strawberry leaves (Darrow, 1966; Jamieson the two species (Potter et al., 2000). Although vars, Cambridge Favourite and Merton Prin- and Sanford, 1996). Jeong et al. (1988) and haplotype X was not found in wild species, cess (Table 1). Both cultivars were bred in Rose (1992) suggested that inheritance of the median-joining network suggests that England, ‘Cambridge Favourite’ in 1947 from shade-adapted chloroplasts from F. virginiana plants belonging to haplotype X could be a cross between (‘Etter seedling’ · ‘Avant is one of the possible causes of June yellows. classified as either F. virginiana or F. chi- Tout’) · ‘Blakemore’ and ‘Merton Princess’ Our study included a number of cultivars in loensis (Fig. 1). The sequence of haplotype in 1956 to 1957 (Darrow, 1966). This sug- which June yellows has occurred (Hughes, X appears to reflect an intermediate point in gests that presumably few individuals pos- 1989): ‘Howard 17 (Premier)’, ‘Blakemore’, the process of differentiation between F. vir- sessing haplotype X were collected and ‘Pajaro’, ‘Senga Sengana’, ‘Tyee’, and ‘Cam- giniana and F. chiloensis. These results con- transferred to England and used for breeding. bridge Favourite’. All but one possessed firm that F. ·ananassa is an interspecific Chloroplast DNA variation can be used haplotype V, which originates from F. virgin- hybrid of F. virginiana and F. chiloensis to verify maternal parentage. ‘Kurume 34’ is iana. The exception, ‘Cambridge Favourite’, (Darrow, 1966) and indicate that traditional a valuable accession because it is the source possessed haplotype X. In contrast, June cultivars of F. ·ananassa derive from at least of several commercially important Japanese yellows has so far not been reported from three maternal lineages. cultivars, including ‘Toyonoka’, ‘Sachinoka’, cultivars possessing haplotype C in Japan, Haplotype V is widely distributed, and and ‘Tochiotome’ (presently the most com- although seedlings related to ‘Howard 17 appears to be predominant in North America monly grown cultivar in Japan). ‘Kurume 34’ (Premier)’ developed the condition, just as (Table 1). Dale and Sjulin (1990) reported is long believed to have been bred from they do in North America and Europe that 134 cultivars released in North America ‘Yachiyo’ · ‘Donner’ (Honda et al., 1976). (Hanaoka et al., 1964). These factors appear between 1960 and 1987 were derived from, However, we found that ‘Kurume 34’ and its to support the arguments of Jeong et al. (1988) at most, 17 maternal founding clones. Of those descendants possess haplotype C, whereas and Rose (1992). Also, Rose (1992) raised the inferred 17 lineages, three were identified both ‘Yachiyo’ and ‘Donner’ possess haplo- possibility that June yellows is an example of their native clones from pedigree data. Our type V (Table 1; Fig. 2). This suggests that hybrid variegation that arises after interspe- results support that pedigree: ‘Columbia’, some of the flagship cultivars in Japan are cific crosses in plants with biparental plastid which was recorded to be derived from F. descended from different maternal origin from inheritance, resulting in a nonharmonious in- chiloensis ‘Reedsport’, possessed haplotype what has hitherto been reported. teraction between the plastids of one parent C, and ‘Arking’ and ‘Cardinal’, which were Although the contribution of cytoplasmic and the hybrid genome after the ‘‘sorting out’’ derived from F. virginiana ‘The Native genes to agronomically important traits of the of plastids that are compatible. However, we Iowa’, possessed haplotype V. Although the strawberry is not well understood, several obtained no evidence of cpDNA inheritance other 14 founding clones were impossible to studies have implicated the cytoplasmic or from the male parent to progeny. Further trace further on the basis of the pedigree data, chloroplast genome in June yellows (Hughes, analysis of the chloroplast and nuclear genes our results suggest that nine of these 14 1989; Jeong et al., 1988; Rose, 1992), which that play a major role in plastome–genome originated from F. virginiana, because either is a progressive and deleterious chlorosis of incompatibility (Yao and Cohen 2000) may

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