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Hisayo Yamane, Megumi Ichiki, Ryutaro Tao, Tomoya Esumi, Keizo Yonemori, Takeshi Niikawa, and Hino Motosugi

factors that control fruit size and to elucidate the physiological changes caused by changes in the fruit size. The weight of persimmon ( Diospyros kaki Thunb.) fruit ranges from less than 60 g to more than 300 g depending on the cultivar. Somatic or

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Yuri Nakamura and Shozo Kobayashi

Restriction fragment length analyses of mitochondrial DNA (mtDNA) and chloroplast DNA (cpDNA) were carried out on 11 cultivars of Japanese persimmon (Diospyros kaki Thunb.) and five other Diospyros species. Total genomic DNA was digested with seven restriction endonucleases, Southern blotted, and hybridized with five mtDNA probes (PstI or SalI fragments of Brassica campestris L. mtDNA) and one cpDNA probe [pTBal, BamHI fragment of Nicotiana tabacum (L.) cpDNA]. All Japanese persimmon cultivars displayed identical mtDNA and cpDNA fragment patterns, while polymorphisms among species were observed using both mtDNA and cpDNA probes. A low degree of polymorphism was observed between D. kaki, D. oleifera Cheng., D. kuroiwai Nakai, D. virginiana L., and D. lotus L., suggesting that these species are closely related. A high degree of polymorphism was observed between D. rhombifolia Hemsl. and the other five species, indicating that this species is more distantly related to the other five.

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Wenhao Dai, Bingcheng Sheng, and Zhen Zhang

`Xiao Fang Shi' is a rare, dwarf cultivar of persimmon (Diospyros kaki Linn cv.) recently found north of Shanghai, China. The tree starts to bear fruit at 2 years of age, while standard trees start fruiting at 5 or 6 years of age. Dwarf and standard cultivars have about equal spring shoot growth, but the dwarf cultivar has little fall growth. To determine the mechanisms of dwarfness and early fruiting, enzyme-linked immunosorbant assay (ELISA) was used to analyze the endogenous indoleacetic acid (IAA), gibberellic acid (GA1+3) and abscicic acid (ABA) contents in leaves and shoot tips of dwarf (`Xiao Fang Shi') and standard (`Da Fang Shi' and `Zhu Sha Hong') persimmon. The measurement was done during the entire growing season. The results showed that IAA, GA1+3, and ABA contents were influenced by cultivars, ages of trees, and phenophases. The dwarf cultivar `Xiao Fang Shi' has lower IAA and GA1+3 but higher ABA contents than the two standard cultivars during the growing season. These correlations are especially evident when the fruit is ripening. The correlation coefficiency between contents of IAA and GA1+3 and tree height was 0.9704, while that between ABA content and tree height is –0.9697. The low IAA and GA, and high ABA contents may be responsible for little shoot growth of the dwarf cultivar in the fall.

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Akira Sugiura, Yoshiko Matsuda-Habu, Mei Gao, Tomoya Esumi, and Ryutaro Tao

Persimmon, or kaki ( Diospyros kaki ), has been widely grown in Japan since ancient times and is consumed as fresh or dried fruit. Because this plant seldom roots by cutting, practical propagation of selected cultivars has been exclusively

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Young-A Choi, R. Tao, K. Yonemori, and A. Sugiura

Multi-color genomic in situ hybridization (MCGISH) was performed for mitotic cells of the somatic hybrids of Diospyros kaki (2n = 6x = 90) and D. glandulosa (2n = 2x = 30). Total DNA of D. kaki and D. glandulosa were isolated and labeled with biotin-16-UTP and digoxigenin (DIG)-11-UTP, respectively. The labeled DNAs were used as probes to differentiate parental chromosomes. The biotin-labeled probe was detected with avidin-rhodamine, and the DIG-labeled probe was detected with anti-DIG-FITC (fluorescein isothiocyanate). Ninety chromosomes from D. kaki that showed reddish-orange and 30 chromosomes from D. glandulosa that showed greenish-yellow were observed under a fluorescence microscope. Some chromosomes showed cross-hybridization with both probes at their terminal or other chromosome regions. These results indicated that MCGISH could be used to analyze genomes of Diospyros species whose chromosomes are small and numerous.

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A. Yamada, R. Tao, and A. Sugiura

The efficacy of ploidy breeding using unreduced pollen in japanese persimmon (Diospyros kaki Thunb.) is not high because of the low frequency of unreduced pollen in most cultivars. This study was conducted in 2002 and 2003 to determine if the exposure to a low temperature before flowering could enhance the unreduced pollen formation in five cultivars of japanese persimmon including two cultivars that barely produce unreduced pollen under the field condition. The results showed that low-temperature treatment (4 °C for 48 hours) increased the occurrence of unreduced pollen at 15 to 17 and 17 to 18 days after the end of the low-temperature treatment in 2002 and 2003, respectively, in all five cultivars tested. Naturally occurring temperatures below 5 °C in the field also appeared to enhance the unreduced pollen formation in the cultivars that naturally produce unreduced pollen in the field.

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Shinya Kanzaki, Keizo Yonemori, Akira Sugiura, Akihiko Sato, and Masahiko Yamada

Japanese persimmon (Diospyros kaki Thunb.) cultivars are classified into four types depending upon the nature of astringency loss of the fruit. Among them, the pollination-constant and nonastringent (PCNA) type is the most desirable for fresh fruit consumption due to the trait of stable loss of astringency on the tree with fruit development. Lack of tannin accumulation is the main cause of natural astringency loss in PCNA-type fruit, and is qualitatively inherited. The PCNA trait is recessive to the non-PCNA trait. In this study, we investigated amplified fragment length polymorphism (AFLP) markers for the trait of natural astringency loss of PCNA-type fruit using bulked segregant analysis (BSA) for efficient selection of PCNA type plants in a breeding population. A total of 128 primer combinations were tested and one AFLP marker was found to be linked to the dominant allele controlling the trait for astringency. This marker, EACC/MCTA-400, was absent in all of the PCNA-type plants tested, whereas it was present in about half of the non-PCNA-type plants tested. However, RFLP analysis using this marker enabled the detection of the other dominant allele, and all PCNA-type plants could be distinguished from the non-PCNA-type plants. Application of this marker system will be useful for the selection of PCNA-type plants in persimmon breeding.

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Akira Sugiura, Takeshi Ohkuma, Young A Choi, Ryutaro Tao, and Mihoko Tamura

To produce nonaploid Japanese persimmon (Diospyros kaki L.f.) by artificial hybridization, we surveyed the natural occurrence of unreduced (2n) pollen among hexaploid cultivars and sorted them from normal reduced (n) pollen. The sorted 2n pollen was crossed with a hexaploid female cultivar and the resultant embryos were rescued by in vitro culture techniques to obtain plantlets. Three out of six male-flower-bearing cultivars (2n = 6x = 90) produced 2n pollen at rates of 4.8% to 15.5% varying with the cultivar, which was estimated by both pollen size and flow cytometry. After sorting giant (2n) from normal pollen grains by using nylon mesh, they were crossed with a hexaploid female cultivar. The seeds obtained from pollination with normal pollen were perfect, but those obtained from pollination with giant pollen were mostly imperfect, with embryo growth being suspended at the globular stage. Although the rate of survival was very low, some embryos at the globular stage were rescued successfully and grown in vitro. Both flow cytometric analysis and chromosome counting proved that the plantlets obtained were nonaploid.

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Young A Choi, Ryutaro Tao, Keizo Yonemori, and Akira Sugiura

5S ribosomal DNA (rDNA) was visualized on the somatic metaphase chromosome of persimmon (Diospyros kaki) and ten wild Diospyros species by fluorescent in situ hybridization (FISH). The digoxigenin (DIG)-labeled 5S rDNA probe was hybridized onto the chromosomes and visualized by incubation with anti-DIG-fluorescein isothiocyanate (FITC). Strong signals of 5S rDNA probe were observed on several chromosomes of Diospyros species tested. Furthermore, multicolor FISH using 5S and 45S rDNA probes differently labeled with DIG and biotin, revealed separate localization of the two rDNA genes on different chromosomes of Diospyros species tested, suggesting that 5S and 45S rDNA sites can be used as chromosome markers in Diospyros. The number of 5S rDNA sites varied with the Diospyros species. More 5S rDNA sites were observed in four diploid species native to Southern Africa than in three Asian diploid species. The former had four or six 5S rDNA sites while the latter had two. Three Asian polyploidy species had four to eight 5S rDNA sites. Among the Asian species, the number of 5S rDNA sites seemed to increase according to ploidy level of species. These features of 5S rDNA sites were very similar to those of 45S rDNA sites in Diospyros. Phylogenetic relationship between D. kaki and wild species tested are discussed based on the number and chromosomal distribution of 5S and 45S rDNA.

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S.K. Kang, H. Motosugi, K. Yonemori, and A. Sugiura

Microcomputer-based thermal analysis (TA) was conducted on dormant mixed buds of Japanese persimmon (`Hiratanenashi'). The exotherms of buds were detected by thermoelectric modules. Flower buds of peach (Prunus persica Batsch cv. Shimizuhakuto) were also analyzed. When TA was used on a whole excised bud, including bud scales, the persimmon buds had only one exotherm at –14.3 °C, while the peach buds had high and low exotherms at –8.4 °C and –14.1 °C, respectively. However, when the exotherm was measured for the primordium, with the bud scales and transitional leaves removed, each primordium showed only one exotherm at –20.7 °C in persimmon and –11 °C in peach. Determination of killing temperature by visual observation, electrolyte leakage method, and triphenyltetrazolium chloride test revealed that the primordium of the persimmon bud was killed at about –14 °C as the excised whole bud or as the whole bud attached to the branch segment. Using the same method, the naked primordium was killed between –22 °C and –25 °C as the primordium was cooled. The peach primordium was killed at –14 °C when examined as a whole bud and at –11 °C as a naked primordium. Furthermore, the exotherm temperatures of persimmon buds and stem segments were measured at appropriate intervals during the two winter seasons 1993–95. Exotherm temperatures of persimmon buds were always higher than the low-temperature exotherm (LTE) temperatures of the stem segments and lower than the high-temperature exotherm (HTE) temperatures of the stem segments. LT50 of persimmon buds almost coincided with the exotherm temperatures of buds. A postulated role of bud scales in supercooling is discussed.