Pollination Constant Non-astringent Cultivars Research Articles

Characterization of a highly polymorphic region closely linked to the AST locus and its potential use in breeding of hexaploid persimmon (Diospyros kaki Thunb.)

The pollination-constant, non-astringent (PCNA) type of persimmon is ideal for production because its fruits lose astringency at harvest regardless of seed formation. The PCNA trait in Japanese persimmons is controlled by a single locus, AST, and is recessive to the non-PCNA trait. Because cultivated persimmon is hexaploid, only the homozygous genotype with six recessive alleles is PCNA. A region tightly linked to AST has been used as a DNA marker for breeding. Three non-PCNA (A) alleles have been reported. Here, we show that the region linked to AST is highly polymorphic and includes microsatellites. By analyzing the size of PCR-amplified fragments, we distinguished 12 different A alleles from 14 non-PCNA cultivars and a Chinese PCNA ‘Luotian-tianshi.’ Then, using A fragment size, we assessed A allele inheritance in six non-PCNA × PCNA populations by analyzing segregation of each A allele in a population and segregation of progeny genotypes. By using A allele segregation analysis, we were able to estimate the copy number of each A allele in five non-PCNA parents but not in ‘Amahyakume.’ By analyzing progeny genotype segregation, we were able to estimate the ‘Amahyakume’ genotype. Our approach can be used not only for the selection of PCNA individuals in populations, but also for estimation of the copy number of A alleles in a possible non-PCNA parent. This would enable us to select non-PCNA parents with fewer A alleles, which would segregate more PCNA individuals in crosses with PCNA cultivars.

Breeding, genetics, and genomics of fruit trees.

Breeding, genetics, and genomics of fruit trees.

Persimmon breeding in Japan for pollination-constant non-astringent (PCNA) type with marker-assisted selection.

Oriental persimmon (Diospyros kaki) originated in Eastern Asia, and many indigenous cultivars have been developed in China, Japan, and Korea. These cultivars are classified into four groups based on their natural astringency loss on the tree and seed formation: pollination-constant non-astringent (PCNA), pollination-variant non-astringent (PVNA), pollination-constant astringent (PCA), and pollination-variant astringent (PVA). PCNA is the most desirable type because the fruit can be eaten without any postharvest treatment; therefore, one of the goals of our persimmon breeding programs is to release superior PCNA cultivars. The PCNA genotype is recessive to the other three non-PCNA genotypes, and PCNA-type F1 offspring are obtained exclusively from crosses among PCNA genotypes. Moreover, the number of superior PCNA cross-parents have been limited. In the late 1980s, inbreeding depression became obvious, especially in terms of fruit size, tree vigor, and productivity. To mitigate the inbreeding, a backcross program using PCNA [(non-PCNA × PCNA) × PCNA] was started in 1990. This process, however, was inefficient because only 15% of the offspring were PCNA, and all offspring had to be grown to the fruiting stage. Therefore, molecular markers linked to the PCNA locus were developed for discriminating PCNA offspring. A molecular marker linked to Chinese PCNA has also been developed.

Practical marker-assisted selection using two SCAR markers for fruit astringency type in crosses of ‘Taiten’ × PCNA cultivars in persimmon breeding

Pollination-constant, non-astringent (PCNA) type persimmons (Diospyros kaki Thunb.) steadily lose fruit astringency and are more desirable than other types of persimmons. The PCNA/non-PCNA trait is qualitatively controlled by AST/ast alleles at a single locus. Persimmons are hexaploid, and PCNA of Japanese origin and non-PCNA genotypes are denoted as aaaaaa and A-----, respectively, if AST gene denotes A and ast gene denotes a. ‘Taiten’ is an F1, non-PCNA persimmon offspring with genotype AAaaaa derived from PCNA×non-PCNA, and molecular markers linked AST alleles in ‘Taiten’ shows polymorphism as A2 and A3, so that the genotype of ‘Taiten’ can show A2A3aaaa using the alleles of molecular marker. Offspring from a backcross of ‘Taiten’בKanshu’ (PCNA) were evaluated. The astringency type was analyzed phenotypically, by fruit sensory and brown specks tests, and genotypically by SCAR markers with PCR primer pairs (E4/A2r for detecting the SCAR marker A2 and 7H9F/AST-R for both A2 and A3). The ratio of PCNA offspring was 43/251 (17.1%), and there was discrepancy between the phenotypic evaluation and SCAR marker estimation in only 3 offspring. The ratio was not significantly different from the theoretical ratio of nulliplex in autohexaploid. The PCNA offspring segregation ratio was 36/151 (23.8%) for ‘Fuyu’ (PCNA)בTaiten’ and 6/29 (20.7%) for ‘Tenjingosho’ (PCNA)בTaiten’ (nearly the same ratio reported for ‘Taiten’בKanshu’). Marker-assisted selection using SCAR markers is thus effective and practical in persimmon breeding.

TWO ASTRINGENT PERSIMMON CULTIVARS RELEASED BY NARO INSTITUTE OF FRUIT TREE SCIENCE, JAPAN

The national institute in Japan has conducted persimmon breeding since 1938, emphasizing the improvement of pollination constant non-astringent (PCNA) cultivars. However, PCNA cultivars of Japanese origin is a recessive trait, and its genetic resources are very limited, and repeated crossings within the narrow gene pool led to inbreeding, which hinders tree vigor, productivity, and fruit weight. Therefore, a strategy of obtaining PCNA offspring through the backcross (PCNA×non-PCNA)×PCNA has been going on since 1990. Two astringent selections from the first phase were released as new pollination variant astringent (PVA) cultivars ‘Taiten’ and ‘Taigetsu’ in 2007, both of which are derived from the cross of ‘Kurokuma’ (a local PVNA cultivar in Japan) × PCNA cultivar ‘Taishu’. Trees of both new cultivars are vigorous and productive. Crosses between each of those new cultivars and PCNA genotype yielded around one fifth PCNA offspring of the total. The ‘Taiten’ fruit ripens in late November, comparable to ‘Fuyu’ at the national institute in Akitsu. Its fruit weighs an average of 506 g. The flesh is soft, moderately coarse and very juicy. The soluble solids concentration in juice after removing astringency (SSC) averages 16.5%, which is comparable to ‘Fuyu’. Fruit cracking at the calyx or stylar end is rare. However, small areas of shallow concentric cracks on fruit skin (SCCS) occur in a considerable ratio of the fruit. The ‘Taigetsu’ fruit ripens in early November at Akitsu. Its fruit weighs an average of 459 g. The flesh is soft, moderately coarse and very juicy. The SSC averages 15.4%, which is comparable to ‘Hiratanenashi’. Fruit cracking at the calyx or stylar end is rare. SCCS are likely to occur in a considerable ratio of the fruit. Parthenocarpy in ‘Taigetsu’ is high.

Genetic differences among ‘Luotian-tianshi’ (Diospyros kaki Thunb.) genotypes native to China revealed by ISSR and IRAP markers

‘Luotian-tianshi’ (Diospyros kaki Thunb.), which has been geographically distributed only in Dabieshan Mountain around the junction of Hubei, Henan and Anhui Province in central China, is believed to be the first PCNA (pollination-constant non-astringent) persimmon native to China. An informative scenario about the genetic diversity and phylogenetic relationships of this native PCNA cultivar is still lacking. To understand the genetic diversity, we have effectively utilized ISSR and IRAP markers to predict the genetic differences among 43 ‘Luotian-tianshi’ genotypes collected from three major producing areas in China. A total number of 280 amplicons were observed ranging between 250 and 2000bp using ISSR and IRAP markers. ISSR markers (100%) revealed a higher polymorphism than the IRAP markers (97.33%). On the basis of cluster analysis in the UPGMA dendrogram, we observed that in case of ISSR the genotypes were clustered into two clusters, whereas three main clusters were obtained by IRAP, indicating that ‘Luotian-tianshi’ genotypes existed the genetic differences and some could be potential germplasms because of the significant genetic differences, like Acc11, Acc36, Acc40 and Acc41. ‘Luotian-tianshi’ was a cultivar group rather than a single cultivar, and mutation and crossover might have happened in the evolution.

Development of Molecular Markers Linked to the Allele Associated with the Non-astringent Trait of the Chinese Persimmon (Diospyros kaki Thunb.)

Persimmon cultivars are classified into four types depending on the nature of astringency loss of the fruit, namely pollination constant non-astringent (PCNA), pollination variant non-astringent (PVNA), pollination variant astringent (PVA), and pollination constant astringent (PCA). Among these four types, PCNA is the most important cultivar for persimmon breeding due to the stable loss of natural astringency on the tree. The trait of natural astringency loss is recessive in Japanese PCNA cultivars, while that in the Chinese PCNA cultivar, ‘Luo tian tian shi’, is dominant and the latter locus, termed CPCNA, is different from that in Japanese cultivars. In order to develop a molecular marker for the selection of the CPCNA-type cultivar, we performed amplified fragment length polymorphism (AFLP) in combination with bulked segregant analysis for F1 offspring derived from ‘Luo tian tian shi’, which was used as the maternal parent. A total of 384 primer combinations were tested, and three AFLP markers, namely EACT-MCCC-222 (RO1), EGGC-MCTC-309 (RO2), and EGCC-MCGA-105 (RO3), linked to the CPCNA dominant allele were obtained. Among these markers, EGGC/MCTC-309 (RO2) was converted into a sequence-characterized amplified region (SCAR) marker. PCR analysis using F1 offspring (n = 264) revealed that the relevance ratio of the SCAR marker was 94%. The polymorphism of the RO2 marker, which was strongly linked to the CPCNA dominant allele, was detected in only two Chinese PCNA cultivars, namely ‘Luo tian tian shi’ and ‘Tian bao gai’, among the Chinese, Korean, and Japanese cultivars tested. These results indicate that the RO2 marker contributes to marker-assisted selection for breeding programs of new PCNA cultivars having the CPCNA trait.

Segregations of Astringent Progenies in the F1 Populations Derived from Crosses between a Chinese Pollination-constant Nonastringent (PCNA) `Luo Tian Tian Shi', and Japanese PCNA and Pollination-constant Astringent (PCA) Cultivars of Japanese Origin

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.

Expression of Genes Involved in Proanthocyanidin Biosynthesis during Fruit Development in a Chinese Pollination-constant, Nonastringent (PCNA) Persimmon, `Luo Tian Tian Shi'

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.

Segregation of Astringency in F1 Progenies Derived from Crosses between Pollination-constant, Nonastringent Persimmon Cultivars

Japanese persimmon (Diospyros kaki Thunb.) cultivars are classified into four types depending on the nature of astringency loss of the fruit. The pollination-constant, non-astringent (PCNA) persimmons lose their astringency on the tree as the fruits develop. This PCNA trait is qualitatively inherited and recessive to the other three types, pollination-constant, astringent (PCA), pollination-variant, nonastringent (PVNA), and pollination-variant, astringent (PVA). In fact, crosses among Japanese PCNA cultivars yield only PCNA type in F1 generation as shown in recent breeding programs at the National Institute of Fruit Tree Science. Despite these previous results, we demonstrated here that non-PCNA (PVNA, PVA, and PCA) type offspring were derived at relatively high rates in the F1 generation from a cross between `Luo Tian Tian Shi', a PCNA accession from China, and the Japanese PCNA cultivar, `Taishu', despite the fact that `Luo Tian Tian Shi' was confirmed to be a true PCNA type by measuring tannin cell size, a principal morphological characteristic to distinguish PCNA cultivars from non-PCNA ones. When segregations of tannin cell size and tannin content in three progenies of the breeding populations derived from Chinese PCNA `Luo Tian Tian Shi' × Japanese PCNA `Taishu', Japanese PCNA `Shinshu' × Japanese PCNA `Taishu', and Japanese PVNA (non-PCNA) `Kurokuma' × Japanese PCNA `Taishu' were investigated, all offspring between Japanese PCNA cultivars contained only small tannin cells and were PCNA types, and those between Japanese PVNA × PCNA cultivars contained only large tannin cells and were non-PCNA types. However, hybrids between `Luo Tian Tian Shi' and `Taishu' segregated into populations of small and large tannin cells, indicating that `Luo Tian Tian Shi' is likely heterozygous for astringency. Therefore, Chinese PCNA `Luo Tian Tian Shi' should be different from Japanese PCNA cultivars in genetic makeup.

異なる地域での甘ガキ‘次郎’の自然脱渋過程の解析

When grown in colder northern districts of Japan, pollination constant/non-astringent (PCNA) persimmon fruits often remain astringent at harvest season.Fruit growth and changes in soluble tannin content in flesh of PCNA persimmon cv. Jiro were compared between fruits from Yamagata (colder, northern district) and from Kyoto (warmer, middle district) with reference to changes of mean temperature in both districts in 1983 and from 1985 to 1987.Loss of astringency occurred before early or middle of August in Kyoto. In Yamagata, however, it varied with years; in some years the fruit had an astringent taste even in late September. This is suggested to be mainly due to the difference in mean temperature between the two areas during the period after bloom, especially in late stage of fruit growth. In Yamagata, the high mean temperature accelerated fruit growth in early stage, resulting in faster decrease of soluble tannin content. Larger fruit contained less soluble tannin than smaller fruit harvested on the same day at the same place. In the colder area, a little astringent taste is sometimes retained in the fruit at the later stage, after August. No clear relationship was observed between complete disappearance of the astringency and the mean temperature in the later stage.In Kyoto, fruit growth and soluble tannin content were investigated on several PCNA cultivars. There was no clear relationship between fruit growth and soluble tannin content. Cultivars retaining astringent taste at the late stage tend to contain more tannin substances at the immature stage. The rate of removal of soluble tannin, however, was not different among the cultivars.

カキ果実のタンニン細胞の発育過程と自然脱渋との関連について

This study was carried out to elucidate the mechanism of natural removal of astringency in pollination constant non-astringent (PCNA) type fruits of Japanese persimmon, especially focusing on development of the tannin cells.1. Development of the tannin cells in plastic-embedded flesh sections was observed using an image analysis system. The tannin cells in PCNA type fruits stopped their enlargement at the end of June, whereas those in pollination variant nonastringent (PVNA) and astringent (PVA), and pollination constant astringent (PCA) type fruits continued to increase in size until the end of July. Thus, final size of tannin cells was much smaller in the former fruits than the latter. Number of tannin cells per unit area was little variable in all 4 type fruits at any stage, so that in PCNA cultivars the area occupied by tannin cells reduced with time after tannin cells stopped their development. Such a decreasing process was coincident with the decreasing process of soluble tannins in PCNA type fruits.2. In PCNA and PVNA cultivars, the tannin cells of fresh pericarp sections stained with ferric chloride were observed in order to clarify whether or not tannins coagulated in early August. At this time soluble tannins in non-astringent type fruits were negligibly low in contents. The observation revealed that tannins in PCNA type fruits hardly coagulated by this time, although those in PVNA type fruits completely changed into insoluble complexes.3. These results suggested that the decrease of astringency of PCNA type fruits on trees might be mainly attributable to the reduction in relative area occupied by tannin cells, but not their coagulation. Such a reduction in area occupied by tannin cells started when development of tannin cells stopped, as mentioned above.