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Akira Kitajima, Atsu Yamasaki, Tsuyoshi Habu, Bannarat Preedasuttijit and Kojiro Hasegawa

Satsuma mandarin (Citrus unshiu Marcow.) chromosomes were stained with Giemsa and fluorochromes chromomycin A3 (CMA)/4′,6-diamidino-2-phenyindole (DAPI). Eighteen chromosomes were categorized into eight groups by the position and relative size of the CMA (+) region and relative length of chromosome. Ponkan (C. reticulata Blanco) DNA labeled with Dig-rhodamine (red) and pummelo [C. maxima (Burm.) Merr.] DNA labeled with biotin-fluorescein isothiocyanate (green) were used as genomic in situ hybridization (GISH) probes. GISH signals were detected on CMA (+) regions and other heterochromatin blocks. The chromosomes were categorized into 12 groups by the coloration and size of GISH signals with relative length of chromosomes. GISH allowed six pairs of speculated homozygous and six individual heterozygous chromosomes of satsuma mandarin to be identified unambiguously. In 10 chromosomes with distinct GISH signals on the CMA (+) regions, red GISH signals were detected on nine chromosomes, indicating that satsuma mandarin is closely related to ponkan. Two colors (red and green) of GISH signals were detected on type C chromosome and three different colors (red, green, and yellow) were detected on type A, indicating that pummelo is involved in the origin of satsuma mandarin. The origins of types A and C chromosomes in satsuma mandarin were also discussed. This article demonstrates that GISH is a powerful tool for chromosome identification and karyotyping in citrus.

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Atsu Yamasaki, Akira Kitajima, Norihiro Ohara, Mitsutoshi Tanaka and Kojiro Hasegawa

The factors of seedless expression in Citrus kinokuni hort. ex Tanaka ‘Mukaku Kishu’, ‘Southern Yellow’ {‘Tanikawa Buntan’ pummelo [an uncertain hybrid with C. maxima (Burm.) Merr.] × ‘Mukaku Kishu’}, and the hybrid seedling BSY lines of ‘Southern Yellow’ × Bu1-7 pummelo [chance seedling of ‘Suisho Buntan’ pummelo (C. maxima)] were investigated histologically. ‘Mukaku Kishu’, ‘Southern Yellow’, BSY18, and BSY21 are completely seedless, whereas others are seedy. The percentage of abnormal embryo sacs and fertilization in seedless cultivars and lines showed no differences from seedy ones, indicating that the seedlessness of ‘Mukaku Kishu’ and its progenies was not involved in abnormal embryo sacs and unfertilized ovules. In seedy cultivars and lines, embryos developed to the cotyledon stage by 14 weeks after pollination. In the seedless cultivars and lines, however, embryos only developed to the early stages of zygote or embryo development. These results indicate that the seedless expression of the seedless progenies of ‘Mukaku Kishu’ coincides with ‘Mukaku Kishu’ and is caused by an arrested seed development at the early stage.

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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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Tsuyoshi Habu, Fumio Kishida, Miki Morikita, Akira Kitajima, Toshiaki Yamada and Ryutaro Tao

Japanese apricot (Prunus mume Sieb. et Zucc.) exhibits S-RNase-based gametophytic self-incompatibility as do other Prunus species. Both self-incompatible and self-compatible Japanese apricot cultivars are grown commercially in Japan. These self-compatible cultivars are shown to have a common S-haplotype called S f that contains S f-RNase and SFB f (S-haplotype-specific F-box protein). This study describes a simple and rapid detection of SFB f, in Japanese apricot, based on loop-mediated isothermal amplification (LAMP) method. A set of 4 primers, F3, B3, FIP, and BIP primer, were designed from the exon and the putative inserted sequence of SFB f. Optimal reaction time at 63 C was determined to be 90 minutes. It appeared that the LAMP method combined with the ultrasimple DNA extraction efficiently detected SFB f. The advantage of the marker-assisted selection of self-compatibility based on the LAMP method was discussed.

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

Pollination-constant and non-astringent (PCNA)-type persimmon has probably originated from astringent (non-PCNA)-type as a mutant that terminates condensed tannin accumulation at an early stage of fruit development. This trait is confirmed to be recessive and is controlled by a single locus. Since PCNA-type fruit stops tannin accumulation at an early stage, comparison of the gene expressions between PCNA- and non-PCNA-type will reveal the genes conferring condensed tannin accumulation in persimmon fruit. We performed suppression subtractive hybridization (SSH) analysis for detecting differentially expressed genes in non-PCNA-type fruit using BC1 offspring from a cross between PCNA `Fuyu' and non-PCNA “275-13” (F1 progeny derived from non-PCNA `Aizumishirazu' × PCNA `Taishu'). Fruits from seven individuals of PCNA or non-PCNA offspring in BC1 were sampled at early two stages of fruit development and total RNA was extracted by hot borate method from each fruit of different stage. Then, RNA was pooled as PCNA or non-PCNA bulk at two stages and cDNA was synthesized from each bulk for SSH analysis. A total of 5000 clones expressed differentially in non-PCNA-type fruit were picked from SSH library of two stages and 198 positive clones confirmed by differential screening were sequenced. The homologous sequences for the genes involved in flavonoid biosynthesis (CHS, CHI, F3H, F3'5'H, DFR, UFGT, and ANS) were obtained from the clones. The genes that are not considered to be involved in flavonoid biosynthesis so far (SCPL and DHQ) were also detected with high frequencies. We will discuss the role of these genes for condensed tannin accumulation in persimmon fruit.

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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.

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Ayako Ikegami, Sai Eguchi, Keizo Yonemori, Masahiko Yamada, Akihiko Sato, Nobuhito Mitani and Akira Kitajima

Pollination-constant and nonastringent (PCNA) is one of the most desirable traits in persimmons as this type of cultivar loses its astringency while still on the tree before harvest. Among Japanese PCNA cultivars, the trait is qualitatively inherited and recessive to pollination-constant, astringent (PCA), pollination-variant, nonastringent (PVNA), and pollination-variant, astringent (PVA) types. However, in a previous trial, both astringent and nonastringent types segregated in the F1 population that resulted from a cross between a Chinese PCNA `Luo Tian Tian Shi' and a Japanese PCNA cultivar. Because of the unusual segregation, in this study, we crossed another Japanese PCNA `Okugosho' with `Luo Tian Tian Shi' to confirm the segregation of astringent types by measuring the tannin cell size and tannin concentration at harvest. Previously, we found that astringent types have larger tannin cells than PCNA-type. The F1 hybrid progenies from the cross segregated into both PCNA and astringent-type individuals in approximately 1:1 ratio. Likewise, the F1 population from the astringent-type `Yotsumizo' and `Iwasedo' × `Luo Tian Tian Shi' were ascertained to contain both PCNA and astringent types, which indicates that the PCNA trait of `Luo Tian Tian Shi' was dominant. Thus, this Chinese cultivar has the potential to become an important parental material for future breeding of PCNA persimmons.