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  • Author or Editor: Masahiko Yamada x
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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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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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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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‘Porotan’ is a Japanese chestnut cultivar (Castanea crenata Sieb. et Zucc.) that was selected from offspring of the cross 550-40 × ‘Tanzawa’ and released in 2006. Its nut is distinguished by a pellicle that is easy to peel after roasting; previously, all Japanese chestnut cultivars were thought to have a pellicle that was difficult to peel. Both 550-40 and ‘Tanzawa’ are Japanese chestnuts, and 550-40 is a selection descended from ‘Tanzawa’. Both 550-40 and ‘Tanzawa’ have a pellicle that is difficult to peel. Among 59 offspring of a cross of 550-40 × ‘Tanzawa’, 12 had an easy-peeling pellicle and 47 had a difficult-peeling pellicle; this ratio is not significantly different from the 1:3 expected ratio for monogenic inheritance based on a chi-square test at P = 0.05. A half-diallel cross without selfings was made among ‘Porotan’, ‘Tanzawa’, and ‘Tsukuba’. All the offspring from ‘Tanzawa’ × ‘Tsukuba’ and from ‘Tsukuba’ × ‘Porotan’ had a difficult-peeling pellicle; in contrast, 39 offspring from ‘Tanzawa’ × ‘Porotan’ segregated in a ratio of 19 difficult-peeling pellicle to 20 easy-peeling pellicle, which is not significantly different from the expected 1:1 ratio for monogenic segregation based on a chi-square test at P = 0.05. These results suggest that the easy-peeling pellicle trait of ‘Porotan’ is controlled by a major recessive gene at a single locus. We designated the pellicle peelability locus as P/p. According to this model, the ‘Tsukuba’ genotype is homozygous-dominant (PP), the ‘Tanzawa’ genotype is heterozygous (Pp), and the ‘Porotan’ genotype is homozygous-recessive (pp).

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Loquat canker, caused by Pseudomonas syringae pv. eriobotryae, is a serious disease of loquat [Eriobotrya japonica (Thunb.) Lindl.] in some countries such as Japan. Therefore, improved canker resistance is an important objective for loquat breeding. The resistance to loquat canker Group C in descendants of ‘Shiromogi’ was expressed only in homozygotes with a recessive gene at a single locus, which was designated pse-c/pse-c. ‘Champagne’, which is distantly related to ‘Shiromogi’, is another cultivar with resistance to Group C. The inheritance of this resistance in progenies of crosses between ‘Champagne’ and susceptible cultivars was examined. The offspring seedlings from 14 crosses between ‘Champagne’ (pse-c/pse-c) and 12 susceptible cultivars (Pse-c/Pse-c or Pse-c/pse-c) were classified into two types of resistant and susceptible. All of the hybrid progenies between ‘Champagne’ (pse-c/pse-c) and Pse-c/Pse-c parents showed two types of resistant and susceptible. The proportion of resistant offspring showed great differences significantly, depending on the hybrid combinations. It ranged from 0.203 to 0.596 with an average of 0.407. It indicated that the resistance was controlled by one or more additional genes or loci other than the Pse-c (pse-c) locus. In addition, the proportion of resistant offspring from crosses between ‘Champagne’ (pse-c/pse-c) and Pse-c/pse-c parents ranged from 0.463 to 0.701 (and averaged 0.601), which seriously deviated from the segregation of 1:1, indicating that the segregation was both Mendelian and polygenic in a threshold character. The proportion of resistant seedlings cannot be predicted by the phenotype and the genotype in the Pse-c (pse-c) locus. Therefore, the general combining ability of ‘Champagne’ resulting from the additional gene effect was estimated, which was 0.407 and 0.101 for ‘Champagne’ × Pse-c/Pse-c and ‘Champagne’ × Pse-c/pse-c cultivars, respectively. The gene effect of susceptible cultivars ranged from –0.204 (‘Yougyoku’) to +0.189 (‘Togoshi’) for ‘Champagne’ × Pse-c/Pse-c cultivars and from –0.138 (‘Taisho’) to +0.089 (‘Nagasakiwase’) for ‘Champagne’ × Pse-c/pse-c cultivars.

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We evaluated the nut harvesting date (NHD), nut weight (NW), pericarp splitting (PS), and infestation by insects (II) in eight cultivars/selections of Japanese chestnut, including a Japanese–Chinese hybrid, over 6 years. Data were analyzed by analysis of variance (without transformation for NHD, after log-transformation for NW and PS, and after square root transformation for II). The among-tree variance accounted for only 1.1% to 8.5% of the total variance. The variance component resulting from residual factors for the tree × year interaction and sampling errors was the largest component for NW, PS, and II, accounting for 46% to 54% of the total environmental variance. Because tree replication is costly and time-consuming in chestnut breeding, increasing the number of yearly repetitions is more efficient than increasing the number of tree replicates. Broad-sense heritability was 0.84 for NHD, 0.27 for NW, 0.48 for PS, and 0.17 for II in evaluations with one tree without yearly repetition. It increased to 0.91 for NHD, 0.40 for NW, 0.62 for PS, and 0.29 for II in evaluations with one tree in 2 years. For NHD, the heritabilities are sufficiently high to distinguish genetic differences among cultivars/selection. In contrast, the low heritability of II suggests that this trait should not be evaluated with an emphasis on the initial selection stage but rather with an emphasis on the secondary selection stage based on testing at several locations with a large number of yearly and tree replications.

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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 M ast , 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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To aid the breeding of citrus (Citrus sp.) for high carotenoid content, we assayed the fruit flesh of 48 cultivars and selections within a parental population consisting of both old and new cultivars and selections at two locations in Japan. The mean total carotenoid (CAR) content across all 48 cultivars and selections over the two locations was 26.59 μg·g−1 fresh weight (FW). The most prominent carotenoid was β-cryptoxanthin [BCR (12.09 μg·g−1 FW)] followed by violaxanthin [VIO (8.04 μg·g−1 FW)], ζ-carotene (2.27 μg·g−1 FW), phytoene (1.86 μg·g−1 FW), and β-carotene (0.96 μg·g−1 FW). Broad-sense heritabilities of CAR, BCR, and VIO were 0.80 or greater based on a sample of five fruit on one tree per location in one time sampling for 1 year in a location, which were revealed to be large enough for gauging the genetic variation. The mean CAR and BCR contents in a cultivar and selection group in advanced generations were nearly the same as in the initial population, suggesting no or little selection pressure on carotenoid content in the citrus breeding so far. High carotenoid contents in cultivars and selections released or selected recently, which have high fruit qualities, suggest their high potential for combining high fruit quality and high carotenoid content in breeding. We showed that the critical phenotypic value used in selecting hybrid seedlings can be determined from the estimate of environmental variance.

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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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