Proanthocyanidins (PAs) are secondary metabolites that contribute to the protection of the plant and also to the taste of the fruit, mainly through astringency. Persimmon (Diospyros kaki) is unique in being able to accumulate abundant PAs in the fruit flesh. Fruits of the nonastringent (NA)-type mutants lose their ability to produce PA at an early stage of fruit development, while those of the normal astringent (A) type remain rich in PA until fully ripened. The expression of many PA pathway genes was coincidentally terminated in the NA type at an early stage of fruit development. The five genes encoding the Myb transcription factor were isolated from an A-type cultivar (Kuramitsu). One of them, DkMyb4, showed an expression pattern synchronous to that of the PA pathway genes in A-and NA-type fruit flesh. The ectopic expression of DkMyb4 in kiwifruit (Actinidia deliciosa) induced PA biosynthesis but not anthocyanin biosynthesis. The suppression of DkMyb4 in persimmon calluses caused a substantial down-regulation of the PA pathway genes and PA biosynthesis. Furthermore, analysis of the DNA-binding ability of DkMyb4 showed that it directly binds to the MYBCORE cis-motif in the promoters of the some PA pathway genes. All our results indicate that DkMyb4 acts as a regulator of PA biosynthesis in persimmon and, therefore, suggest that the reduction in the DkMyb4 expression causes the NA-type-specific down-regulation of PA biosynthesis and resultant NA trait.

Sexuality of crops affects both cultivation and breeding systems. Cultivated persimmon (Diospyros kaki Thunb) has a morphologically well-characterized polygamous or gyonodioecious sexual system. However, the genetic basis of sexuality in D. kaki has yet to be characterized. Here, we used dioecious D. lotus L., a diploid wild relative species to hexaploid or nonaploid D. kaki, as a model to clarify the genetic basis of sexuality in Diospyros and to develop molecular markers associated with the sexuality of individuals. Using 62 F 1 offspring segregated into distinct male/female phenotypes, we found two amplified fragment-length polymorphism markers, DlSx-AF4 and DlSx-AF7, which cosegregated with maleness. This could suggest that the sexuality of D. lotus is controlled by a single gene/haploblock, and the male is dominant over the female. Thus, D. lotus's sexuality can be described as the heterogametic male type, the XY-type, as reported for most other dioecious plant species. For unknown reasons, segregation of the phenotype of a sequence-characterized amplified region marker developed from DlSx-AF4 (DlSx-AF4S) and/or the male/female phenotype in two different crosses in D. lotus showed an apparent bias towards femaleness and better fitted 1:2 than 1:1, which is the theoretical segregation for a single genetic locus or haploblock in diploid D. lotus. DlSx-AF4S could distinguish D. kaki cultivars with female and male flowers from cultivars with only female flowers, strongly indicating that the same genetic system controls D. kaki's sexuality and that DlSx-AF4S could be used as a genetic marker for sexuality in D. kaki breeding programs.

Persimmon (Diospyros kaki Thunb.) is one of the major tree crops in East Asia and is generally hexaploid. A single ASTRINGENCY (AST) locus controls the astringency/ non-astringency (A/NA) trait of persimmon fruit, one of the most important traits for consumption, on each of the six corresponding chromosomes. Although several molecular approaches are in progress to elucidate the molecular mechanisms of astringency trait in persimmon, the distinct polysomic behavior of the AST locus remains to be solved. The aim of this study was to perform fine genotyping of a highly polymorphic marker locus linked to the AST locus, detect the allele pairing in ten segregated F 1 lines derived from hybridization of A-type × NA-type cultivars, and identify the basis of hexaploid inheritance at the AST locus in persimmon. The results showed that persimmon cultivars frequently produce aneuploid offspring bearing an extra chromosome with the AST locus, with the incidence of aneuploidy varying among the cultivars. On the examination of hexasomic behavior in persimmon cultivars, the ratios of individuals bearing each allele pair segregated from A-type parents showed a good fit to the expected ratios in an autohexaploid inheritance model, except for cvs. Luo-tiantian-shi and Sa-gok-shi which fitted to an autoallohexaploid inheritance model. These results suggest variable hexasomic behavior among persimmon cultivars.

Persimmon fruits accumulate a large amount of proanthocyanidin (PA). Fruits of the mutant non-astringent (NA) type lose their ability to accumulate PA at an early stage of fruit development, whereas fruits of the normal astringent (A) type sustain PA accumulation until ripening. This allelotype is determined by the genotype of a single ASTRINGENCY (AST) locus. It is possible that the reduction in PA accumulation in NA-type fruits is due to phenological down-regulation of DkMyb4 (a PA regulator) and the resultant down-regulation of structural genes in the PA pathway. In this study, attempts were made to identify the regulatory mechanisms of phenological PA accumulation in A- and NA-type fruits, focusing particularly on the effects of ambient temperature. Continuous cool temperature conditions caused sustained expression of DkMyb4 in NA-type fruits, as well as in A-type fruits, resulting in increased expression of PA pathway genes and PA accumulation. However, the expression of some A/NA phenotypic marker genes was not significantly affected by the cool temperature conditions. In addition, PA composition in NA-type fruits exposed to cool temperatures differed from that in A-type fruits. These results indicate that a cool ambient temperature may have induced DkMyb4 expression and resultant PA accumulation, but did not directly affect the expression of the AST gene.

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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%.

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