Abstract
To select cultivars to produce doubled haploids (DHs) efficiently in an anther culture, a total of 28 apple cultivars (25 cultivars and 3 rootstock cultivars) were investigated to assess their callus formation rate, embryo formation rate, shoot formation rate from embryo, and efficiency of shoot multiplication and acclimatization. The callus formation rates of ‘Red Astrachan’, ‘Kinsei’, ‘Tsugaru’, ‘Golden Delicious’ (‘GD’), ‘American Summer Pearmain’ (‘ASP’), ‘Gala’, ‘Rome Beauty’, and ‘Jonathan’ were high, but ‘JM2’ and ‘King of Tompkins’ formed no callus. Regarding the embryo formation rates, ‘ASP’, ‘Rome Beauty’, ‘M.9’, and ‘Starking Delicious’ (‘SD’) were high, but ‘GD’, ‘JM2’, ‘King of Tompkins’, ‘Ralls Janet’, and ‘Smith’s Cider’ formed no embryo. Regarding the shoot formation rate from embryos, ‘Senshu’, ‘Tsugaru’, and ‘Sansa’ showed high values, but ‘Fuji’, ‘Jonathan’, and ‘Wijcik’ did not form shoots at all. The rate of shoot formation from anthers showed a high–low order of ‘Senshu’, ‘SD’, ‘ASP’, and ‘Tsugaru’. Multiplication and acclimatization of the shoots were not easy. Many individuals died. Eventually, only ‘Senshu’ and ‘SD’ acclimatized. Therefore, cultivars that produced DHs efficiently were ‘Senshu’ and ‘SD’. The results indicated that all processes of “embryo formation”, “shoot induction”, and “shoot multiplication and acclimatization” are important for obtaining DHs in apple anther cultures.
Introduction
No necessity exists for genetic fixation of fruit trees such as the apple (Malus × domestica Borkh.) because of the ability of vegetative propagation. However, there are great difficulties in fruit tree breeding and genetic analysis. Causes of the main difficulties for apple genetic analysis are self-incompatibility, a strong inbreeding depression, and a long juvenile period. For these reasons, obtaining homozygous pure lines solely through crossbreeding is almost impossible. Therefore, the production of a doubled haploid (DH) by anther culture improves apple genetic studies and breeding. Fertile DHs are invaluable for genome analysis (Yamamoto et al., 2015). The DHs are an appealing material for fruit tree breeding. Development of anther culture technology is an important research subject for efficient fruit tree breeding.
Anther cultures have been used for various plants species since haploid plants were obtained for the thorn-apple (Datura innoxia Mill.) (Guha and Maheshwari, 1964). In the apple, ‘Jonathan’ anthers including “uninucleate” stage pollen were cultivated in MS medium, and embryo formation was confirmed (Kubicki et al., 1975). Mii and Hasegawa (1981) conducted apple anther culture using seven cultivars. These anthers including “late uninucleate” to “binucleate” stage pollen were cultivated in MS medium under continuous light conditions. Then, the embryos from ‘Fuji’ and ‘Ralls Janet’ were formed. Hidano (1982) succeeded in adventitious embryo formation for ‘Fuji’, ‘Jonathan’, ‘Megumi’, and ‘Ralls Janet’ using “First pink” immediately before the stage of flower bud clustering including “uninucleate” stage pollen, under a 12 h light condition in MS medium. Xue and Niu (1984) obtained plantlets derived from anther cultures of several cultivars such as ‘Delicious’, ‘Ralls Janet’, and ‘Jonathan’ using “Pink” stage in flower cluster, which corresponds to the “uninucleate” stage in pollen. Wang and Ogata (1988) and Ogata and Wang (1989) achieved a high embryo formation rate (11.86%) of ‘M.9’ using anthers including “mid-uninucleate” to “late uninucleate” stage pollen in the N6 medium. They extended the cold pre-treatment (4°C) period of anthers for 16 days. They reported that continuous light suppressed embryo formation. Fujii (1989) obtained adventitious embryos derived from ‘Senshu’ using anthers including “uninucleate” stage pollen under continuous dark conditions. Fujii also succeeded in inducing plantlets by embryo cold treatment or cultivation of embryos in 1/2 MS medium supplemented with 5 μM Gibberellin A3 (GA3) and 5 μM 6-benzylaminopurine (BAP), and reported that 12 and one out of 13 strains were diploid and haploid, respectively. Hidano et al. (1994) conducted anther cultures using 32 cultivars, and reported that ‘Beauty of Bath’, ‘Monroe’, ‘Orei’, and some ‘Delicious’ strains showed a high embryo formation rate. Höfer et al. (1999) and Höfer (2004) obtained fertile plants from anther and microspore cultures of ‘Rene’ and ‘Alkmene’. Kadota et al. (2002) obtained regeneration shoots from ‘Fuji’, ‘Himekami’, ‘Kotoku’, ‘Starking Delicious’ (‘SD’), and ‘Tsugaru’, but only ‘SD’ grew into plantlets. Vanwynsberghe and Keulemans (2004) also regenerated plants from ‘Braeburn’. Okada et al. (2009) obtained fertile DHs derived from the anther culture of ‘Senshu’.
Research knowledge related to apple anther culture has accumulated gradually as described above. However these studies were conducted separately. Their results do not provide consistent or sufficient knowledge about callus and embryo formation or plant acclimatization in the anther culture. In this study, we investigated the callus formation rate, embryo formation rate, shoot formation rate, efficiency of shoot multiplication, and plantlet acclimatization of 28 apple cultivars, to elucidate the plant regenerative capacity which is expected to depend on the donor plant genotype in the procedures related to DH production (Tsukuni, 2006) by anther culture.
Materials and Methods
1. Plant materials
‘Akane’, ‘American Summer Pearmain’ (‘ASP’), ‘Fuji’, ‘Gala’, ‘Golden Delicious’ (‘GD’), ‘Granny Smith’, ‘Haruka’, ‘Indo’, ‘Jonathan’, ‘King of Tompkins’, ‘Kinsei’, ‘McIntosh’, ‘Orin’, ‘Ralls Janet’, ‘Red Astrachan’, ‘Rome Beauty’, ‘Sansa’, ‘Sekaiichi’, ‘Senshu’, ‘Smith’s Cider’, ‘SD’, ‘Tsugaru’, ‘Wijcik’, ‘Winesap’, and ‘Yoko’ belong to M. × domestica Borkh., ‘M.9’ belongs to M. pumila Mill. var. paradisiaca Schneid., and ‘JM2’ and ‘JM7’ were seedlings of M. prunifolia Borkh. ‘Seishi’ × ‘M.9’. Parentage and characteristics of these cultivars are shown in Table 1.
Flower bud clusters in apple cultivars ‘ASP’, ‘Fuji’, ‘Jonathan’, ‘Orin’, ‘Sansa’, ‘Senshu’, ‘SD’, ‘Tsugaru’, and ‘Wijcik’ were collected for three years from 2009–2011 (three-year test). The sampling days of flower bud clusters were April 28th to May 6th, May 3rd to 11th, and May 5th to 15th in 2009, 2010, and 2011, respectively. Those of ‘Akane’, ‘Fuji’, ‘Gala’, ‘GD’, ‘Granny Smith’, ‘Haruka’, ‘Indo’, ‘JM2’, ‘JM7’, ‘Jonathan’, ‘King of Tompkins’, ‘Kinsei’, ‘M.9’, ‘McIntosh’, ‘Ralls Janet’, ‘Red Astrachan’, ‘Rome Beauty’, ‘Sansa’, ‘Sekaiichi’, ‘Senshu’, ‘Smith’s Cider’, ‘SD’, ‘Tsugaru’, ‘Winesap’, and ‘Yoko’ were collected in 2012. The sampling days of flower bud clusters are shown in Table 1. All flowers of the 28 cultivars were collected from The Field Science Center, Faculty Agriculture, Iwate University and Apple Research Station, NARO Institute of Fruit Tree Science (Table 1).
2. Flower bud development stage and cold pre-treatment in collected flowers
Flower clusters for which the flower bud development stage was “First pink” (Zhang et al., 2013) were collected in each cultivar because these flowers mainly contain pollen of “early uninucleate” to “mid-uninucleate” stages. During cold pre-treatment, flower buds were placed in a cold (4°C) dark chamber for 25 days (Zhang et al., 2013). The anther culture was conducted using the anther of the lateral flowers.
3. Embryo induction medium and culture conditions
The embryo induction medium consisted of N6 medium (Chu et al., 1975) supplemented with 10 μM BAP, 0.5 μM naphthaleneacetic acid (NAA), and 5.0% sucrose. The culture were incubate in the dark (25°C). The details of composition of the embryo induction medium and other conditions of anther culture were identical to those reported previously (Zhang et al., 2013).
4. Investigation of embryo and callus formation rates
At 30 weeks from the beginning of anther culture, the embryos and calli were counted. Then the embryo and callus formation rates were calculated using the number of surviving anthers in the denominator (Tables 2 and 3).
5. Shoot induction from embryo
(1) Cold treatment medium
The embryos were transferred to hormone-free MS medium (Murashige and Skoog, 1962), for which the dosage of the nitrogen compound is half (1/2 MS) supplemented with 1.5% sucrose. The pH was adjusted to 5.7 with 0.1 M NaOH before adding 0.8% agar powder (for Plant Culture Medium; Wako Pure Chemical Industries Ltd., Osaka, Japan). Then, the medium was autoclaved for 20 min at 120°C under pressure of 1.2 kg·cm−2. After autoclaving, the medium was distributed into Petri dishes (90 × 15 mm), each containing 25 mL medium. These embryos underwent cold treatment in a cold (4°C) dark chamber for 3–5 months (Zhang et al., 2013). The subculture of the embryos was not conducted during the cold treatment period.
(2) Shoot induction medium
Medium for rooted embryos during cold treatment
Rooted embryos during cold treatment (Fig. 1A) were transferred to hormone-free 1/2 MS medium supplemented with 1.5% sucrose and 0.68% Bacto agar. Other procedures were the same as those described above.
Medium for non-rooted embryos during cold treatment
Non-rooted embryos during cold treatment were transferred to 1/2 MS medium supplemented with 1 mg·L−1 (4.4 μM) BAP, 1.5% sucrose, and 0.68% Bacto agar. Other procedures were the same as those described above.
6. Procedures for shoot multiplication and acclimatization
Regenerated shoots (Fig. 1B, C) were transferred to a shoot multiplication medium consisting of MS medium supplemented with 1 mg·L−1 (4.9 μM) indole-3-butyric acid (IBA), 10 mg·L−1 (44.4 μM) BAP, 3.0% sucrose, and 0.68% Bacto agar.
After shoot multiplication (Fig. 1D), the shoots were transferred to a root induction medium consisting of 1/2 MS medium supplemented with 0.5 mg·L−1 IBA, 1.5% sucrose, and 0.68% Bacto agar, or softwood grafting on rootstock (Fig. 1E). Rooted shoots (Fig. 1F) were transplanted in pots and were acclimatized in a greenhouse (Fig. 1G).
7. SSR and ploidy analysis
After acclimatization, SSR analysis was conducted to confirm whether the plants were derived from pollen grains. For the SSR analysis, total DNA was extracted from the leaves of ‘Senshu’, ‘SD’, and the acclimatized plants using a DNeasy Plants Mini Kit (QIAGEN Sciences, Gaithersburg, MD, USA). Thirty-five SSR markers showing a heterozygous genotype on the donor genotype ‘Senshu’ and ‘SD’ were used for identification. The details of SSR markers, other PCR conditions and the sequencing method of the PCR products of each SSR locus were identical to those described in a previous report (Okada et al., 2009).
The ploidy level of the plants was determined by flow cytometry. The methods for ploidy analysis were identical to those described in a previous report (Okada et al., 2009).
8. Statistical analysis
Analyses of cultivar differences were conducted by Tukey-HSD test (Table 2). The contribution of cultivars and years were calculated by two-way ANOVA (analysis of variance) with no replication (Table 2). The relation between the callus formation and embryo formation rates was analyzed by Peason’s correlation coefficient test (Fig. 3).
Results
For about 7 weeks from the beginning of anther culture, white to yellow calli (Fig. 2A) and white embryos (Fig. 2B, C) were observed. Callus and embryo formation were found to be of several types. In many cases, one embryo arose from one anther (Fig. 2B), in some other cases, some embryos arose from one anther (Fig. 2C). In another case, one embryo arose with the callus from one anther (Fig. 2D).
1. Callus formation rate
In the three-year test, all nine cultivars formed calli. ‘Tsugaru’ was the highest for callus formation from the average of three years of data (15.9%), ‘Tsugaru’ was followed by ‘ASP’ (10.2%), ‘Jonathan’ (9.0%), ‘Orin’ (5.3%), ‘SD’ (2.5%), ‘Senshu’ (2.1%), ‘Sansa’ (1.0%), ‘Wijcik’ (0.7%), and ‘Fuji’ (0.6%). The callus formation rate of ‘Tsugaru’ was significantly higher than ‘Fuji’, ‘Sansa’, ‘Senshu’, ‘SD’, and ‘Wijcik’; those of ‘ASP’ were also higher than those of ‘Fuji’ and ‘Wijcik’ (Table 2).
In 2012, ‘Red Astrachan’ (24.3%) was highest in 25 cultivars, followed by ‘Kinsei’ (22.2%), ‘GD’ (11.2%), ‘Gala’ (10.0%), ‘Rome Beauty’ (9.8%), ‘Senshu’ (5.5%), and ‘Tsugaru’ (4.7%) (Table 3). No callus formation was observed on ‘JM2’ or ‘King of Tompkins’.
2. Embryo formation rate
Table 2 presents the results of three-year tests. ‘ASP’ was the highest for embryo formation from the average of three years of data (14.4%), followed by ‘SD’ (6.2%), ‘Senshu’ (2.4%), ‘Jonathan’ (1.3%), ‘Tsugaru’ (0.5%), ‘Fuji’ (0.4%), and ‘Sansa’ and ‘Wijcik’ (0.2%). No embryo was formed on ‘Orin’ for two years. The embryo formation rate of ‘ASP’ was significantly higher than in the other eight cultivars.
In 2012, ‘Rome Beauty’ (6.4%) was the highest of 25 cultivars, followed by ‘M.9’ (5.7%), ‘SD’ (4.1%), ‘Red Astrachan’ (2.6%), ‘Kinsei’ (1.5%), and ‘Senshu’ (1.2%) (Table 3). No embryo formation was observed on ‘GD’, ‘JM2’, ‘King of Tompkins’, ‘Ralls Janet’, ‘Sansa’, or ‘Smith’s Cider’.
3. Shoot formation rate
The embryo formation rate and shoot formation rate are shown in Table 2. The shoot formation rate from embryos was in the order of ‘Senshu’ (21.55%), ‘Tsugaru’ (17.38%), ‘Sansa’ (16.67%), ‘SD’ (5.22%), and ‘ASP’ (1.55%). No shoot formation was observed on ‘Fuji’, ‘Jonathan’, ‘Orin’, or ‘Wijcik’.
The shoot formation rate from anthers was in the order of ‘Senshu’ (0.30%), ‘SD’ (0.27%), ‘ASP’ (0.25%), ‘Tsugaru’ (0.09%), and ‘Sansa’ (0.02%) (Table 2). In 2012, Only ‘Sekaiichi’, ‘Senshu’, and ‘SD’ regenerated shoots from embryos (data not shown).
4. Shoot multiplication and plantlet acclimatization
In the three-year test, 143 shoots derived from anther culture of ‘Senshu’ and 143 shoots derived from that of ‘SD’ were obtained. In ‘Senshu’, it succeeded in the multiplication of 56 out of 143 shoots, and 25 plantlets out of 56 shoots acclimatized successfully. In ‘SD’, it succeeded in the multiplication of 18 out of 143 shoots; 1 plantlet out of 18 shoots was acclimatized. The shoots from ‘ASP’, ‘Sansa’, and ‘Tsugaru’ did not multiply. All shoots were dead.
In the acclimatized plants, only one allele of donor cultivars was amplified in each sample by SSR marker analysis, and this result confirmed that all individuals had a haploid origin. The ploidy analysis data showed that the plants were diploid (data not shown).
Discussion
In anther cultures, the induction of plantlets depends strongly on the donor plant genotype such as wheat (Last and Brettell, 1990), barley (Hayes et al., 2003; Huang, 1985), and Cruciferae (Ferrie, 2003; Sato, 2000). Therefore, selecting cultivars showing high efficiency for shoot regeneration by anther culture is important for plant breeding and the development of breeding technology.
1. Relation between callus and embryo
Nakayama et al. (1971, 1972) reported differences among cultivars in terms of the callus formation rate in apple anther cultures. Mii and Hasegawa (1981), Hidano et al. (1994), and Tsukuni (2006) also reported callus formation differences among cultivars. Reportedly, calli and embryos derive from the same cell (Hidaka, 1990; Hidano et al., 1994; Matsumoto, 1990). The callus formation rate of ‘Orin’, which formed no embryos, was higher than that of ‘Senshu’ and ‘SD’, which exhibited a higher embryo formation rate (Table 2). ‘Orin’ did not form embryos in this experiment, but Tsukuni et al. (2005) reported that the embryo formation rate of ‘Orin’ was 4.6%. These results suggest that the cultivars that showed a high callus formation rate and low embryo formation rate such as ‘Kinsei’, ‘Orin’, and ‘Red Astrachan’ were able to control the differentiation direction of the microspore with the change of culture conditions. Our study revealed two cases of the timing of callus and embryo formation from the same anther. One case showed the same time formation of callus and embryos. In another case, the callus formed earlier and the embryos formed later (Fig. 2D) (Zhang et al., 2013). Many cultivars formed calli within the early period after culture start and then decreased as the cold pre-treatment period lengthened. On the other hand, the embryo formation rate increased after callus formation was suppressed (Zhang et al., 2013). Therefore, it is possible that the embryo formation rate of these cultivars can be improved by extension of the cold pre-treatment period.
Results from experiments conducted in 2012 showed a significant correlation between the callus formation rate and the embryo formation rate at the 1% level (P = 0.0069) (Fig. 3). The total value of the callus formation rate and embryo formation rate is shown in Table 3. The cultivars showing a high total value are considered to possess high potential ability for embryo formation. The total value rate is an important indicator of potential ability for regeneration in pollen cells. However, the existence of cultivars in which the embryo formation rate was not high although the sum of the callus and embryo formation rate was high such as ‘Gala’ and ‘GD’ (Table 3), suggested that other factors may be involved in embryo formation because the R2 value was only 28.7% (Fig. 3).
2. Factors affecting the callus and the embryo formation rate
(1) Environmental factors
The embryo formation rate changed greatly over three years (Table 2). The embryo formation rate in 2010 was extremely low, except for ‘ASP’. The rate in 2009 was higher than in 2011. The embryo formation rates of ‘ASP’ and ‘SD’ in 2009 were, respectively, 26.7% and 10.7%. The value of 2009 was almost double, about 1.5 times greater than that of 2011. From the results of two-way ANOVA, significant differences were observed on the main effect of year for the callus formation rate and the shoot formation rate from anthers (Table 2). This indicated that the reactivity of the pollen received environmental effects in the anther culture. Probably the weather conditions during the flower bud development period and the differences in nutritional conditions on the flower bud in each year influence the embryo formation rate. The length from bud break to sampling time probably influences the development of flower buds and the reactivity of the pollen (Tsukuni et al., 2005). It is necessary to investigate the relation between weather conditions before anthesis and callus and embryo formation rates.
(2) Genetic factors
The highest embryo formation rate was in ‘ASP’ followed by ‘SD’, ‘Senshu’, and ‘Jonathan’; this order did not change for three years (Table 1). Significant differences were observed on the main effect of cultivars on the callus formation rate, embryo formation rate, and the shoot formation rate from embryos (Table 2). These results indicate that genetic factors influence the embryo formation rate. The maximum embryo formation rate in ‘ASP’ was reported by Hidano et al. (1994) and Tsukuni et al. (2005). That of ‘SD’ was reported by Kadota et al. (2002) and by Tsukuni et al. (2005).
Many cultivars which showed a callus formation rate over about 5% were ‘GD’ and its progenies, such as ‘Gala’, ‘Kinsei’, ‘Orin’, ‘Senshu’, and ‘Tsugaru’ (Table 2). The parentages of ‘ASP’, ‘Jonathan’, ‘Red Astrachan’, and ‘Rome Beauty’ were unknown (Table 1). ‘ASP’, ‘Jonathan’, ‘Kinsei’, ‘M.9’, ‘Red Astrachan’, ‘Rome Beauty’, ‘Senshu’, and ‘SD’ showed high embryo formation rates of over 1% (Table 2). ‘SD’ is a mutant of ‘Delicious’. ‘Kinsei’ and ‘Senshu’ are progenies of ‘GD’ and ‘Delicious’. ‘GD’ and ‘Delicious’ may have the ability to produce progenies with high pollen reactivity in anther cultures. It is necessary to investigate the progenies of the cultivars which showed high embryo formation rates.
(3) Culture conditions
Wang et al. (2004) reported that embryos were induced by cold treatment for three weeks at 4°C in five out of six wheat cultivars. However, all six cultivars formed embryos during treatment for four days at 33°C. Cultivars which show difficulty inducing embryos by cold treatment may induce embryos by high-temperature treatment. In our anther culture using ‘ASP’, ‘GD’, ‘Senshu’, and ‘Tsugaru’, which have high embryo formation ability, we succeeded in the formation of calli and embryos by high-temperature pre-treatment; however, both callus and embryo formation rates of high-temperature pre-treatment were inferior to those of cold pre-temperature (data not shown). These facts show that apple pollen can react to the different pre-treatment stimulation. It is necessary to investigate high-temperature pre-treatment using cultivars for which the reactivity of pollen is low for cold pre-treatment. For the medium composition, the embryo formation rate of ‘Fuji’ rose from 1.5% to 20% by changing the NAA concentration from 0.5 μM to 0.2 μM in the case where the BAP concentration was 10 μM in the embryo induction medium (Tsukuni, 2006). These results indicate that the embryo formation rate could be further improved by changing the conditions of the pre-treatment of flower buds and the culture conditions of anthers.
3. Relation between the embryo formation rate and shoot formation rate
Cultivars show differences in the shoot formation rate in apple anther cultures (Table 2). The embryo formation rate of ‘Senshu’ (2.4%) was lower than that of either ‘ASP’ (14.4%) or ‘SD’ (6.2%). However, the shoot formation rate of ‘Senshu’ was the highest. Therefore, the shoot formation rate from anthers in ‘Senshu’, which is affected by the embryo formation rate and shoot formation rate from embryos, was higher (0.30%) than that of ‘ASP’ (0.25%) or ‘SD’ (0.27%). Actually, ‘ASP’ showed the highest embryo formation rate, but the shoot formation rate from embryos was low. Therefore, the final shoot formation rate was lower than that of ‘Senshu’ or ‘SD’. These results demonstrate that shoot regeneration capability is extremely important for obtaining DHs by anther culture. For the shoot formation rates from embryos, significant differences were observed on the main effect of cultivar, but not on the main effect of year (Table 2). These results indicate that the genotype influences shoot formation from the embryo, while the environmental conditions at flower bud cluster sampling do not have any influence after the process of embryogenesis.
4. Difficulty of shoot multiplication and plantlet acclimatization
Not all shoots were able to multiply or acclimatize. Many individuals died during these procedures. In fact, many individuals died after a few days without growth. Some exhibited decreased shoot multiplication capacity after 2–3 subcultures, stopped growing and died.
Primula is highly heterozygous because of its heteromorphic self-incompatibility; therefore, there are many non-lethal, but harmful, recessive genes in primula genomes in the heterozygous form. When the seedlings are raised by selfing, the plants become weak because the genes appear as a phenotype (Ishihama et al., 2006). In the apple, the seedlings raised by selfing also showed morphological abnormalities and devigor. It was assumed that the growth difficulties in apple DHs is due to remarked inbreeding depression.
5. Obtaining fertile plants
The individuals maintained in vitro, which achieved multiplication of 56 and 18 strains derived from ‘Senshu’ and ‘SD’, respectively, must be tested to assess their acclimatization, induction of precocious flowering, and selection of fertile strains. Okada et al. (2009) raised DHs derived from ‘Senshu’ and reported that the fertile strains accounted for 3 out of 12 acclimatized DHs. These DHs must be used efficiently as materials for breeding and genetic analysis.
Most previous studies of apple anther cultures observed only embryo induction from the anther. Of course, the selection of cultivars with high embryo formation ability is important for DH production. When aiming to obtain DHs from numerous and diverse cultivars, however, selecting the cultivars showing a high shoot formation rate is important, and just one must be selected for the embryo formation rate. Furthermore, it must be considered that cultivar differences exist for the efficiency of shoot multiplication and acclimatization. These facts indicate that all processes in embryo formation, shoot formation from embryo, and multiplication and acclimatization are important to obtain good DHs.
Acknowledgements
The authors are grateful to M.S. Hidemi Oshino of the NARO Institute of Fruit Tree Science for her technical assistance.
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