Chinese Pollination-constant Non-astringent Research Articles

Recent Advances in Natural Deastringency and Genetic Improvement of Chinese PCNA Persimmon (Diospyros kaki)

Persimmon (Diospyros kaki) is a worldwide fruit cultivated mainly in the East Asia, Mediterranean, Caucasus, Latin America, and Oceania regions. This fruit contains abundant proanthocyanidins (PAs, also called condensed tannins), whose biosynthesis is the main cause of fruit astringency. As the original centre and top producing country, China has discovered a unique type with desirable natural deastringency, the Chinese pollination-constant non-astringent (C-PCNA) persimmon. Studies have revealed that the C-PCNA trait is controlled by a single and dominant locus, which differs from that of another type, the Japanese PCNA type, with recessive loci. In the C-PCNA type, accumulating evidence has shown that the astringency removal process involves two pathways (“dilution effect” and “coagulation effect”). Moreover, molecular marker-assisted selection (MAS) for the natural deastringency trait locus in C-PCNA has been used to test the non-astringency/astringency trait of hybrid offspring at the seedling stage. Importantly, persimmon can bear male flowers, female flowers, and perfect flowers, but sex-linked MAS has been developed for female-only persimmon. This sex-linked MAS, together with astringency-linked MAS and embryo rescue technology, may even shorten the conventional cross-breeding period of about 2–3 years. In addition, recently studies have established a stable genetic transformation system for persimmon transgenic improvement. Despite these efforts, how synthetic PAs and metabolism pathways lead to a deastringent trait remains unclear for persimmon. Thus, our review summarizes the latest research progress on the natural deastringency mechanism in C-PCNA, and we provide a new viewpoint for the genetic improvement of persimmon breeding in China.

Development of EST-SSR markers and their application in the genetic diversity of persimmon (Diospyros kaki Thunb.)

SSRs related to parent selection of D. kaki were validated by transcriptome sequencing, and genetic diversity of these markers among 76 persimmon germplasm resources was additionally investigated. The selection of hybrid parents is critical in cross-breeding plants for germplasm innovation and cultivating improved varieties. This study used a published RNA-Seq database of a persimmon cultivar ‘Zenjimaru’ to develop simple sequence repeat (SSR) markers to assist efficiently selection of appropriate persimmon cultivars for cross-breeding. A total of 154,741 unigenes with 41,999 SSR loci at a frequency of one per 2.30 kb were identified. Of the different SSR motif types, mononucleotide repeats (47.63%) were the most abundant, followed by dinucleotide repeats (38.23%). The main repeat motifs were A/T (42.26%) and AG/CT (24.72%). A total of 17,111 SSR primer pairs were designed, from which 100 selected loci were synthesized and preliminary screened with capillary electrophoresis by testing PCR products from 15 persimmon germplasm resources. The effectiveness of 13 primer pairs that produced products with distinct peaks at the predicted sizes was subsequently determined using 76 persimmon germplasm resources. A total of 111 alleles were identified by using 13 SSR markers, ranging from 2 to 17 with an average of 8.54 bands per locus. Additionally, different hybridized combinations could be selected according to a dendrogram based on these newly developed 13 SSR markers. And two androecious persimmons (“Yunjia-9” and “Luotian Yeshi-1”) were genetically closely related to Chinese pollination-constant non-astringent (C-PCNA) persimmons, which was significant for the cultivation of PCNA offspring. The identified SSR markers will not only promote persimmon cross-breeding programs by increasing the efficiency of parent selection but also assist in genetic resource identification and conservation.

Discovery of a millennial androecious germplasm and its potential in persimmon (Diospyros kaki Thunb.) breeding

Persimmon (Diospyros kaki Thunb.) can produce unisexual (male and female) and bisexual (hermaphrodite) flowers, showing relatively complex sexuality at the species level. In China, there are more than 1000 persimmon germplasms, but most are gynoecious cultivars that bear female flowers only. To date, few germplasms have been reported to generate male flowers, until seven androecious persimmons were recently found in the Dabieshan Mountains at the juncture of Hubei, Anhui and Henan Provinces in central China. Here, we report a new androecious germplasm located in Jiangxi Province that was cultivated by the first generation of the Luo family in the Song Dynasty nearly for a thousand years ago. Up to now, the millennial androecious germplasm has continuously borne only male flowers and the seeding plant still has a strong growth vigor. Investigation of the leaf and pollen morphology showed that this androecious germplasm is similar to ‘Male 2’ (androecious, discovered in Dabieshan Mountains) but different from two Japanese cultivars (‘Taishuu’ and ‘Zenjimaru’). Polymerase chain reaction (PCR) detection of the sex-linked marker DlSx-AF4S verified that the millennial androecious germplasm carries this sex-linked marker. Moreover, the RO2 marker, linked Chinese pollination constant non-astringent (C-PCNA) locus, was also present in the millennial androecious germplasm. Additionally, the genetic relationships between SSR markers showed that the millennial androecious germplasm is related to Dabieshan’s androecious genotypes and some persimmon cultivars of Chinese origin. Collectively, our results revealed that the ancient androecious persimmon found in Jiangxi Province is distinguished from previously reported androecious germplasms and has the potential to serve as a breeding resource for selecting sex-linked PCNA persimmons in the future.

DkWRKY interacts with pyruvate kinase gene DkPK1 and promotes natural deastringency in C-PCNA persimmon

PAs, also known as condensed tannins, cause the astringency sensation in the persimmon fruit. The astringency of Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) can be naturally removed on the tree, but the regulatory mechanisms of deastringency remain to be elucidated. In our previous research, DkPK1 was shown to be involved in the natural loss of astringency of C-PCNA persimmon fruit. In the present study, yeast one-hybrid (Y1H) library screening using the DkPK1 promoter as baits identified two DkWRKY transcription factor genes (DkWRKY3 and -15). The transcript levels of both DkWRKY3 and -15 exhibited a positive correlation with the decrease in soluble proanthocyanidin (PA) content during the last developmental stage in C-PCNA persimmon. Multiple sequence analysis and subcellular localization confirmed that DkWRKY3 and -15 belonging to the group II and I families, respectively, were both located in the nucleus. Dual-luciferase and Y1H assays demonstrated that DkWRKY3 and -15 can transactivate the DkPK1 promoters. The combination of DkWRKY3 and -15 most likely produced an additive activation effect compared to a single activator on DkPK1, although the two transcriptional activators were not capable of interacting. Notably, DkWRKY3 and -15 showed ubiquitous expression in various organs and abundant upregulation in seeds. Furthermore, transient overexpression of both DkWRKY3 and -15 in persimmon leaves led to a significant decrease in the content of soluble PAs but a significant increase in the expression levels of the acetaldehyde metabolism-related DkPK, DkPDC and DkADH genes. Thus, we suggest that DkWRKY3 and -15 are the upstream regulators of DkPK1 and positively regulate the natural deastringency in C-PCNA persimmon.

Incomplete loss of astringency in pollination-constant non-astringent (PCNA) offspring derived from Japanese and Chinese PCNA persimmon

Incomplete loss of astringency in pollination-constant non-astringent (PCNA) offspring derived from Japanese and Chinese PCNA persimmon

DkPK Genes Promote Natural Deastringency in C-PCNA Persimmon by Up-regulating DkPDC and DkADH Expression.

The astringency of Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) can be naturally removed on the tree. This process is controlled by a single locus and is dominant against other types of persimmons; therefore, this variant is an important candidate for commercial cultivation and the breeding of PCNA cultivars. In our previous study, six full-length coding sequences (CDS) for pyruvate kinase genes (DkPK1-6) were isolated, and DkPK1 is thought to be involved in the natural deastringency of C-PCNA persimmon fruit. Here, we characterize the eight other DkPK genes (DkPK7-14) from C-PCNA persimmon fruit based on transcriptome data. The transcript changes in DkPK7-14 genes and correlations with the proanthocyanidin (PA) content were investigated during different fruit development stages in C-PCNA, J-PCNA, and non-PCNA persimmon; DkPK7 and DkPK8 exhibited up-regulation patterns during the last developmental stage in C-PCNA persimmon that was negatively correlated with the decrease in soluble PAs. Phylogenetic analysis and subcellular localization analysis revealed that DkPK7 and DkPK8 are cytosolic proteins. Notably, DkPK7 and DkPK8 were ubiquitously expressed in various persimmon organs and abundantly up-regulated in seeds. Furthermore, transient over-expression of DkPK7 and DkPK8 in persimmon leaves led to a significant decrease in the content of soluble PAs but a significant increase in the expression levels of the pyruvate decarboxylase (DkPDC) and alcohol dehydrogenase genes (DkADH), which are closely related to acetaldehyde metabolism. The accumulated acetaldehyde that results from the up-regulation of the DkPDC and DkADH genes can combine with soluble PAs to form insoluble PAs, resulting in the removal of astringency from persimmon fruit. Thus, we suggest that both DkPK7 and DkPK8 are likely to be involved in natural deastringency via the up-regulation of DkPDC and DkADH expression during the last developmental stage in C-PCNA persimmon.

Molecular cloning and functional characterization of DkMATE1 involved in proanthocyanidin precursor transport in persimmon (Diospyros kaki Thunb.) fruit

Persimmon fruits accumulate a large amount of proanthocyanidins (PAs) in “tannin cells” during development that cause the sensation of astringency due to coagulation of oral proteins. Pollination-constant non-astringent (PCNA) is a spontaneous mutant persimmon phenotype that loses its astringency naturally on the tree at maturity; while the more common non-PCNA fruits remain rich in PAs until they are fully ripened. Here, we isolated a DkMATE1 gene encoding a Multidrug And Toxic Compound Extrusion (MATE) family protein from the Chinese PCNA (C-PCNA) ‘Eshi 1’. Expression patterns of DkMATE1 were positively correlated with the accumulation of PAs in different types of persimmons fruits during fruit development. An analysis of the inferred amino acid sequences and phylogenetic relationships indicated that DkMATE1 is a putative PA precursor transporter, and subcellular localization assays revealed that DkMATE1 is localized in the vacuolar membrane. Ectopic expression of the DkMATE1 in Arabidopsis tt12 mutant supported that DkMATE1 could complement its biological function in transporting epicatechin 3′-O-glucoside as a PAs precursor from the cytoplasm to vacuole. Furthermore, the transient over-expression and silencing of DkMATE1 in ‘Mopanshi’ persimmon leaves resulted in a significant increase and a decrease in PA content, respectively. The analysis of cis-elements in DkMATE1 promoter regions indicated that DkMATE1 might be regulated by DkMYB4, another well-known structural gene in persimmon. Overall, our results show that DkMATE1 may be an essential PA precursor membrane transporter that plays an important role in PA biosynthesis in persimmon.

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.

Isolation and Characterization of DkPK Genes Associated with Natural Deastringency in C-PCNA Persimmon.

Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) is considered to be an important germplasm resource for the breeding of PCNA cultivars, though its molecular mechanisms of astringency removal remain to be elucidated. Previously, we showed that the abundance of pyruvate kinase gene transcripts increased rapidly during astringency removal in C-PCNA persimmon fruit. Here, we report the full-length coding sequences of six novel DkPK genes from C-PCNA persimmon fruit isolated based on a complementary DNA (cDNA) library and transcriptome data. The expression patterns of these six DkPK genes and correlations with the soluble proanthocyanidin (PA) content were analyzed during various fruit development stages in different types of persimmon, with DkPK1 showing an expression pattern during the last stage in C-PCNA persimmon that was positively correlated with a decrease in soluble PAs. Phylogenetic analysis revealed that DkPK1 belongs to cytosolic-1 subgroup, and subcellular localization analysis confirmed that DkPK1 is located in the cytosol. Notably, tissue expression profiling revealed ubiquitous DkPK1 expression in different persimmon organs, with the highest expression in seeds. Furthermore, transient over-expression of DkPK1 in persimmon leaves resulted in a significant decrease in the content of soluble PAs but a significant increase in the transcript levels of pyruvate decarboxylase genes (DkPDC1, -3, -4, -5), which catalyze the conversion of pyruvate to acetaldehyde. Thus, we propose that an acetaldehyde-based coagulation effect reduces the content of soluble PAs. Taken together, our results suggest that DkPK1 might be involved in the natural removal of astringency at the last developmental stage in C-PCNA persimmon. This is the first report to identify several novel full-length DkPK genes as well as their potential roles in the natural loss of astringency in C-PCNA persimmon.

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.

Effectiveness of the RO2 marker for the identification of non-astringency trait in Chinese PCNA persimmon and its possible segregation ratio in hybrid F1 population

An important breeding target for persimmon is to achieve elite cultivars of the pollination constant non-astringent (PCNA) type. The RO2 SCAR marker has been established to identify the trait of Chinese PCNA type by Ikegami et al. [J. Jpn. Soc. Hort. Sci. 80 (2011) 150–155]. Here, the informativeness of RO2 was tested in all Chinese PCNA types and two F1 populations bred from crosses between a non-PCNA maternal parent and either a Chinese PCNA type or an androecious male parent. The RO2 marker was detected in ten Chinese PCNA types and six androecious genotypes, but not in any of the non-PNCA and Japanese PCNA types. The segregation behavior of RO2 in both F1 populations was consistent with a 1:1 ratio as previously reported by Ikegami et al. The presence of the RO2 marker among androecious material indicates that they also have potential as donor of non-astringency trait of Chinese PCNA type.

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.