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

for the analysis of tannin contents. Three fruits were used for this analysis. A fruit skin-color index that indicates similar developmental stages of the fruits has been developed for HTN (National Agricultural Research Station, Tsukuba, Japan

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Richard J. McAvoy, Bernard B. Bible and Michael R. Evans

The early onset of bract necrosis in poinsettia (Euphorbia pulcherrima Willd. ex. Klotzch) is characterized by small dark-stained spots that precede the development of enlarged necrotic lesions. Electron micrographs of adaxial epidermal and subepidermal tissues with early symptoms of necrosis revealed large, electron-dense deposits in cell vacuoles. These spherical bodies resembled condensed tannins observed in the epidermal tissues of peach and apple fruit. Chemical analysis of bract tissues confirmed the presence of condensed tannins. Furthermore, there were higher concentrations of condensed tannin in bract samples with 2-mm-diameter lesions than in samples with lesions <0.5 mm (equivalent to catechin concentrations of 59 and 13 mg·g-1 fresh mass, respectively). No tannin bodies were observed in parallel samples of healthy-appearing bracts in which only trace concentrations of condensed tannins were measured (0.2 mg·g-1 fresh mass). The evidence suggests an association between condensed tannin accumulation in localized areas of the bract and the early appearance of bract necrosis symptoms.

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Travis Robert Alexander, Jacqueline King, Andrew Zimmerman and Carol A. Miles

(acid <0.45%, tannin >0.20%) and bittersharp (acid >0.45%, tannin >0.20%) cider apples ( Barker and Burroughs, 1953 ). Growers have historically selected cider apple cultivars for their fruit acid and tannin content levels ( Alwood, 1903 ; Barker, 1911

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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, 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.

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K. Yonemori, M. Oshida and A. Sugiura

In order to study the nature of tannins in vivo, we developed a method for collecting the vacuolar contents from intact tannin cells in persimmon fruit. We used a micropipette controlled with a MMS-77 micromanipulator system (Shimadzu Co., Kyoto, Japan) under an inverted microscope. Fruit flesh of mature persimmon fruit (cv. Miyazakimukaku) was cut into 300-μm-thick sections with a DSK-100 miaoslicer (Dosaka EM, Kyoto, Japan). The sections were then put on a glass slide, and a micropipette was inserted into a tannin cell to withdraw its contents. After determination of the sap volume collected, the sample was injected into a 25-μl drop of water on a glass slide. Then, the water-drop containing the tannin sample was transferred to a small microfuge tube and stored in a freezer until analysis. Based on calculations, we could collect approximately 7 to 12 nl of vacuolar contents per tannin cell. When tannin and sugar contents per tannin cell were determined, we found that tannin cells contain tannins at 10% to 15% as catechin equivalents (w/v) and 8% to 10% total sugars (w/v), while a whole fruit contains tannins at 1% to 1.5% as catechin equivalents and 10% to 13% total sugars on fresh weight basis. We are currently continuing more detailed analysis of tannin cell constituents.

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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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Frank M. Elia, George L. Hosfield, James D. Kelly and Mark A. Uebersax

A knowledge of the relative proportion of additive and nonadditive genetic variances for complex traits in a population forms a basis for studying trait inheritance and can be used as a tool in plant breeding. A North Carolina Design II mating scheme was used to determine the inheritance of cooking time, protein and tannin content, and water absorption among 16 genotypes of dry bean (Phaseolus vulgaris L.) representative of the Andean Center of Domestication. Heritability and the degree of dominance for the traits were also calculated to provide guidelines for adopting breeding strategies for cultivar development. Thirty-two progeny resulted from the matings and these were assigned to two sets of 16 progeny each. Variances due to general combining ability (GCA) and specific combining ability (SCA) were significant for the traits. The GCA was larger in all cases. Narrow-sense heritability for protein, tannin, water absorption, and cooking time averaged 0.88, 0.91, 0.77, and 0.90, respectively. Degree of dominance estimates indicted that the traits were governed by genes with partial dominance except, in one case, tannin had a degree of dominance value of zero, indicating no dominance. The phenotypic correlation (-0.82) between water absorption and cooking time justifies using the water absorption trait as an indirect selection method for cooking time. With regard to parent selection in crosses, significant differences between GCA females and GCA males suggested cytoplasmic influences on trait expression. Hence, the way a parent is used in a cross (i.e., as female or male) will offset trait segregation. Using fast-cooking bean cultivars in conjunction with fuel-efficient cooking methods may be the best strategy to conserve fuelwood and help reduce the rate of deforestation in East and Central Africa.

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Gregory Peck, Megan McGuire, Thomas Boudreau IV and Amanda Stewart

, most apples commercially grown in the United States have low YAN and polyphenol concentrations ( Thompson-Witrick et al., 2014 ). While both exogenous nitrogen and polyphenols (i.e., “enological tannins”) may be added to increase their concentration in

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Brianna L. Ewing, Gregory M. Peck, Sihui Ma, Andrew P. Neilson and Amanda C. Stewart

the sensory characteristics of ciders ( Thompson-Witrick et al., 2014 ; Valois et al., 2006 ). In the United Kingdom, apple cultivars are typically classified into four categories based on malic acid and tannin concentration ( Barker and Ettle, 1910