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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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Takashi Akagi, Yumi Takeda, Keizo Yonemori, Ayako Ikegami, Atsushi Kono, Masahiko Yamada and Shinya Kanzaki

Persimmon (Diospyros kaki Thunb.) is generally hexaploid, and a single AST locus controls the pollination-constant non-astringency trait on each of six corresponding chromosomes. The pollination-constant non-astringent (PCNA) genotype is nulliplex and requires homozygous recessive alleles (ast) at the AST locus. There are several non-PCNA cultivars/selections that could be cross parents; however, the probability of yielding nulliplex offspring depends on the number of recessive alleles (ast). In genotyping for the AST locus in hexaploid persimmon, in contrast to the situation in diploid plants, we need to detect the AST/ast allele dosage; this cannot be detected by common codominant markers. In this study, we detected the allele dosage of Mast, which is a marker allele strongly linked to the ast allele among cultivars, by quantitative real-time polymerase chain reaction (qPCR) using three reference sites, actin (DkAct), anthocyanin reductase (DkANR), and L5R, whose sequences are conserved in the genome of persimmon cultivars. Based on the allele dosage of the Mast, AST/ast genotypes were estimated for 63 non-astringent cultivars/selections, of which only five cultivars/selections were estimated to be simplex or duplex. The quantitative genotyping method using qPCR may be generally effective for polyploid plants.

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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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Wichan Eiadthong, Keizo Yonemori, Shinya Kanzaki, Akira Sugiura, Naoki Utsunomiya and Suranant Subhadrabandhu

The phylogenetic relationships among 14 Mangifera L. species including three economically important species, i.e., common mango (M. indica L.), horse mango (M. foetida Lour.) and kwini (M. odorata Griff.), were analyzed by comparing 217 amplified fragment length polymorphism (AFLP) markers. The unweighted pair grouping method using arithmetic averages (UPGMA) and neighbor-joining (NJ) method were used and two outgroup taxa, cashew nut (Anacardium occidentale L.) and gandaria (Bouea macrophylla Griff.), were added to both analyses. The common mango was closely related to banana mango (M. sylvatica Roxb.), M. laurina Bl., and M. oblongifolia Hook.f. Intraspecific variation among seven cultivars of common mango was much smaller than interspecific variation and these cultivars were classified into one M. indica group using both methods. Mangifera macrocarpa Bl., M. foetida, and M. odorata were also related to M. indica in both UPGMA and NJ trees, although these three species are classified into a different subgenus (subgenus Limus) from the subgenus Mangifera to which M. indica belongs. Also, in both UPGMA and NJ trees, M. gedebe Miq. and M. griffithii Hk.f. were placed in distant positions among the Mangifera species tested, indicating these two species are related distantly to M. indica. The AFLP technique was confirmed to be useful for phylogenetic analysis.

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Nobuhito Mitani, Atsushi Kono, Masahiko Yamada, Akihiko Sato, Shozo Kobayashi, Yusuke Ban, Toshihito Ueno, Mikio Shiraishi, Shinya Kanzaki, Tomoyuki Tsujimoto and Keizo Yonemori

Persimmon (Diospyros kaki Thunb) is hexaploid, and the pollination-constant, non-astringent (PCNA)/non-PCNA trait of Japanese origin is qualitatively controlled by the AST/ast alleles at a single locus and the PCNA trait is recessive to the non-PCNA trait. To avoid inbreeding depression led by repeated crosses among PCNA genotypes, non-PCNA genotypes should be used as cross parents. The marker-assisted selection system has been developed for the selection of PCNA offspring in the progeny derived from the cross of non-PCNA ‘Taigetsu’ (non-PCNA ‘Kurokuma’ × PCNA ‘Taishu’) to PCNA ‘Kanshu’. The primer pairs E8.5/E9r and 7H9F/AST-R were used for detecting the molecular markers A1 and A3, respectively, which link AST alleles. Complete agreement was found between the sequence-characterized amplified region (SCAR) marker genotype and fruit astringency phenotype of the 48 offspring. The result confirmed that the marker-assisted selection using those markers was highly practical. In a larger offspring population (522 offspring) from the same cross, offspring segregated into 100 with both markers, 162 with only A1, 179 with A3, and 81 with neither, and this segregation ratio was significantly different from 2:3:3:2, which is the segregation ratio of random chromosome assortment in autohexaploid. The percentage of offspring expected to be PCNA was 15.5% (81 of 522), which was slightly lower than 20%.