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Tomoya Esumi, Ryutaro Tao, and Keizo Yonemori

Japanese pear (Pyrus pyrifolia) and quince (Cydonia oblonga), both classified in the subfamily Maloideae, show differences in inflorescence architectures despite of the fact that they are genetically closely related. We previously isolated flowering related genes, LEAFY (LFY) and TERMINAL FLOWER 1 (TFL1) homologues, from these species and showed that they had two types of homologues for each gene. In this study, we examined the expression pattern of LFY and TFL1 homologues in these species by in situ hybridization and RT-PCR. The floral bud was dissected to small pieces under stereomicroscope; apical meristem, scales/bracts, pith, floral meristem, and inflorescence; and then used for RT-PCR. The LFY homologues were expressed in apical meristem and scales/bracts before the floral differentiation in both Japanese pear and quince. After floral differentiation, the expression was observed in floral meristem, scales/bracts and pith in both the species. The TFL1 homologues were strongly expressed in the apical meristem, but their expression was drastically decreased just before floral differentiation. It is considered that the decrease of expression of TFL1 homologues is a sign of floral initiation. The expression of TFL1 homologues was transiently increased at the beginning of floral differentiation in both species. Moreover, one of TFL1 homologues in Japanese pear was continuously expressed in the inflorescence part in the floral primordia, whereas expression of TFL1 homologues in quince almost completely disappeared after a solitary floral meristem was initiated. It was suggested that TFL1 homologues may also be involved in the inflorescence development of Japanese pear.

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Keizo Yonemori, Masayoshi Oshida, Fumio Fukuda, and Akira Sugiura

A method for collecting the vacuolar contents of intact tannin and parenchyma cells of persimmon (Diospyros kaki Thunb.) fruit using a micropipette was developed. Thin sections of the mesocarp tissue from mature persimmon fruit, `Miyazaki-mukaku' and `Hiratanenashi', were placed on a glass slide. Using a micromanipulator and an inverted microscope, a micropipette was inserted into a vacuole and its contents were withdrawn. A 5-nL sample of vacuole sap was collected per tannin cell from `Hiratanenashi' and 7 nL from `Miyazaki-mukaku', whereas only 2 nL was withdrawn from adjacent parenchyma cells. Analyses of the vacuolar sap revealed that the tannin cells of both cultivars contained 10% to 12% (m/v) of tannin as (+)-catechin equivalents and 10% to 13% (m/v) of soluble sugars, whereas the parenchyma cells contained trace amounts of tannins and ≈20% of soluble sugars. Tannin cells contain only a slight amount of sucrose, in contrast to a relatively large amount in parenchyma cells.

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Ayako Ikegami, Keizo Yonemori, Akira Sugiura, Akihiko Sato, and Masahiko Yamada

Japanese persimmon (Diospyros kaki Thunb.) cultivars are classified into four types depending on the nature of astringency loss of the fruit. The pollination-constant, non-astringent (PCNA) persimmons lose their astringency on the tree as the fruits develop. This PCNA trait is qualitatively inherited and recessive to the other three types, pollination-constant, astringent (PCA), pollination-variant, nonastringent (PVNA), and pollination-variant, astringent (PVA). In fact, crosses among Japanese PCNA cultivars yield only PCNA type in F1 generation as shown in recent breeding programs at the National Institute of Fruit Tree Science. Despite these previous results, we demonstrated here that non-PCNA (PVNA, PVA, and PCA) type offspring were derived at relatively high rates in the F1 generation from a cross between `Luo Tian Tian Shi', a PCNA accession from China, and the Japanese PCNA cultivar, `Taishu', despite the fact that `Luo Tian Tian Shi' was confirmed to be a true PCNA type by measuring tannin cell size, a principal morphological characteristic to distinguish PCNA cultivars from non-PCNA ones. When segregations of tannin cell size and tannin content in three progenies of the breeding populations derived from Chinese PCNA `Luo Tian Tian Shi' × Japanese PCNA `Taishu', Japanese PCNA `Shinshu' × Japanese PCNA `Taishu', and Japanese PVNA (non-PCNA) `Kurokuma' × Japanese PCNA `Taishu' were investigated, all offspring between Japanese PCNA cultivars contained only small tannin cells and were PCNA types, and those between Japanese PVNA × PCNA cultivars contained only large tannin cells and were non-PCNA types. However, hybrids between `Luo Tian Tian Shi' and `Taishu' segregated into populations of small and large tannin cells, indicating that `Luo Tian Tian Shi' is likely heterozygous for astringency. Therefore, Chinese PCNA `Luo Tian Tian Shi' should be different from Japanese PCNA cultivars in genetic makeup.

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Chinawat Yapwattanaphun, Suranant Subhadrabandhu, Chitose Honsho, and Keizo Yonemori

The phylogenetic relationships among 17 Garcinia species including G. mangostana (mangosteen) were analyzed by comparing sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA). Both parsimonious and neighbor joining (NJ) analyses revealed that G. mangostana is closely related to G. malaccensis believed to be a progenitor of mangosteen. Another suspected progenitor of mangosteen, G. hombroniana, was more distant from G. mangostana than G. malaccensis phylogenetically. Garcinia hombroniana formed a cluster with G. rostrata, G, speciosa and G. sizygiifolia, and this cluster was connected with a cluster of G. mangostana and G. malaccensis. The ITS sequence analysis showed that G. atroviridis, G. cowa, G. dulcis, G. malaccensis, G. mangostana, G. rostrata and G. vilersiana have nucleotide additivity (two different nucleotides at the same nucleotide position) at several sites in the ITS region. The occurrence of these species might be related to hybridization with ancestors, but the genomic compositions, even chromosome numbers, of these species are still unknown.

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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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Chitose Honsho, Keizo Yonemori, Akira Sugiura, Songpol Somsri, and Suranant Subhadrabandhu

Flower bud differentiation and the flowering habit of durian (Durio zibethinus Murray) `Mon Thong' from budbreak to anthesis were investigated at the Chantaburi Horticultural Research Center in Thailand. Clusters of flower buds appeared at the end of November on primary or secondary scaffold branches near where a flower cluster occurred the previous year. Anatomical observations revealed that the development of floral organs was acropetal; the five fused epicalyx forming a large, elongated envelope enclosing the sepals, petals, stamen and fused multi-carpellate pistil. Floral organ development was completed in early January. The mature flower bud more than doubled in size one day before anthesis, with anthesis starting around 1600 hr and ending ≈1900 hr. The anthers did not dehisce until the completion of flowering. This change induced heterostyly in this cultivar, which promoted out-crossing by reducing the possibility of self-pollination. Aromatic nectar that attracted insects to the flower was secreted during anthesis. This is the first report to have clarified the overall flowering process in durian and provides the basic information for elucidating reproductive biology of durian in future research.

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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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Ayako Ikegami, Keizo Yonemori, Akira Kitajima, Akihiko Sato, and Masahiko Yamada

Expression patterns of the genes involved in condensed tannin (CT) biosynthesis during fruit development was investigated in a Chinese pollination-constant, nonastringent (PCNA) persimmon (Diospyros kaki Thunb.) `Luo Tian Tian Shi'. The transcript levels of phenylalanine ammonia-lyase (PAL) and dihydroflavonol reductase (DFR) in `Luo Tian Tian Shi' were detected at high levels throughout the fruit growth. Chalcone synthase (CHS) and flavonol 3-hydroxylase (F3H) also continued to be transcribed during fruit growth, although their levels decreased earlier than PAL and DFR. In contrast, expression levels of these genes declined into undetectable levels at an early stage of fruit development in Japanese PCNA persimmon. In addition, anthocyanidin reductase (ANR), which encodes a key enzyme of the proanthocyanidin biosynthesis, was transcribed at high levels in `Luo Tian Tian Shi' during fruit growth, but not in Japanese PCNA persimmon. By contrast, the expression of D. kaki serine carboxypeptidase-like protein 1 (DkSCPL1) that was obtained from suppression subtractive hybridization (SSH) analysis between artificially astringency-removed fruit and astringent fruit in a different experiment, declined earlier than the other flavonoid biosynthesis genes in `Luo Tian Tian Shi', coincident with the termination of the tannin cell development. In the F1 progeny of the cross between `Luo Tian Tian Shi' and Japanese PCNA `Taishu', similar expression patterns were obtained among segregated PCNA and astringent offspring. These results indicate that Chinese PCNA is different from Japanese PCNA in expression of the genes involved in CT biosynthesis. In conclusion, we clarified that expression of the genes (PAL to ANR, but not SCPL) involved in flavonoid biosynthesis was continuous in the Chinese PCNA cultivar, despite the termination of tannin cell development.

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Hisayo Yamane, Yukinobu Kashiwa, Tomomi Ooka, Ryutaro Tao, and Keizo Yonemori

To understand the molecular basis of the endodormancy of buds of perennial plants, we searched for the genes that are expressed preferentially in endodormant lateral buds of the deciduous fruit tree japanese apricot (Prunus mume Sieb. et Zucc.) using suppression subtractive hybridization with mirror orientation selection (SSH/MOS). We generated two SSH/MOS libraries containing gene pools that are expressed preferentially in endodormant buds in comparison with paradormant or ecodormant buds to search for the genes that are upregulated by endodormancy induction or down-regulated by endodormancy release, respectively. Differential screening and sequencing indicated that genes involved in gibberellin metabolism, stress resistance, cell wall modification, and signal transduction, such as transcription factors, are upregulated in endodormant buds. After a further expression survey and full-length cDNA cloning, we found that a gene similar to the SVP/AGL24-type MADS-box transcription factor showed endodormancy-associated expression. Seasonal expression analysis suggested that the SVP/AGL24 homolog in japanese apricot might be involved in endodormancy regulation of its lateral buds.

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Keizo Yonemori, Ayako Ikegami, Sai Eguchi, Akira Kitajima, Shinya Kanzaki, Akihiko Sato, and Masahiko Yamada

There is a non-astringent type of persimmon that loses its astringency naturally on trees, despite the absence of seeds. This type is called pollination-constant and non-astringent (PCNA)-type. PCNA-type was thought to have originated in Japan as a mutant that terminates tannin accumulation at an early stage of fruit development. This trait is confirmed to be recessive and the PCNA-type must be homozygous in all alleles. In fact, crossing among PCNA-type individuals yields only the PCNA-type in F1, while crossing between PCNA-type and non-PCNA-type yields only the non-PCNA-type. However, a new PCNA cultivar, `Luo Tian Tian Shi', was reported in 1982 to exist in Luo Tian County, China, and this PCNA-type seemed to have different mechanisms to be PCNA-type. Our previous report showed that a crossing between `Luo Tian Tian Shi' and Japanese PCNA `Taishu' yielded both PCNA-type and astringent-type in F1, indicating that the trait of PCNA in `Luo Tian Tian Shi' may be dominant. To confirm this hypothesis, we made crossings between `Luo Tian Tian Shi' and Japanese astringent-type `Yotsumizo' or `Iwasedo', and top-grafted these seedlings for earlier fruiting. As we had some fruits in a total of 25 F1 individuals last year, we investigated segregation of astringency among these individuals. We also confirmed the parental relationships of these progenies by analyzing several SSR markers. We confirmed the segregation of PCNA-type and astringent-type in F1. No mistake of artificial pollination was shown in all individuals by SSR markers. Thus, we concluded that the trait of astringency-loss in `Luo Tian Tian Shi' is dominant and the use of this cultivar as a parent will open a new window for breeding PCNA-type persimmon.