Kaki Thunb Research Articles

Influence of the extraction conditions on the carbohydrate and phenolic composition of functional pectin from persimmon waste streams

Persimmon fruit (Diospyros kaki Thunb.) production suffers great losses (15–20%) due to the inefficient overripening process. In order to valorise this waste, a compositional characterization of different fruit stages of the residues was done, and immature fruit was selected due to its high pectin and very high polyphenol content. A 3-level full factorial design was carried out to study the effect of temperature (70–95 °C) and low pH (0.5–1.5), on yield, degree of esterification, carbohydrate constituents, phenolic content and antioxidant capacity of the extracted pectin, and a complete polyphenol profile (UPLC-MS/MS) was performed on selected extracts. All responses could be accurately adjusted to the models (R2 > 80; lack of fit). Pectin yield, phenolic compounds and antioxidant activity ranged from 1.4 to 4.5%, 53.3 ± 2.27 to 111.7 ± 9.74 mg GAE/g pectin and 0.29 ± 0.01 to 2.77 ± 0.04 TEAC (Trolox μmol/mg pectin), respectively. A strong pectin-polyphenol interaction was found, which significantly enhanced acid resistance of both the pectin and polyphenol constituents, with optimum yield and polyphenol content at pH 1 and 95 °C. These new pectin-based ingredients might have a great potential as functional foods or natural food ingredients enhancing the quality and shelf-life.

The whole-genome sequence of the novel yeast species Metschnikowia persimmonesis isolated from medicinal plant Diospyros kaki Thunb.

The new yeast Metschnikowia persimmonesis KCTC 12991BP (KIOM G15050 strain) exhibits strong antimicrobial activity against some pathogens. This activity may be related to the medicinal profile of secondary metabolites that could be found in the genome of this species. Therefore, to explore its future possibility of producing some beneficial activities, including medicinal ability, we report high-quality whole-genome assembly of M. persimmonesis produced by PacBio RS II sequencer. The final draft assembly consisted of 16 scaffolds with GC content of 45.90% and comprised a fairly complete set (82.8%) of BUSCO result using Saccharomycetales lineage data set. The total length of the genome was 16.473 Mb, with a scaffold N50 of 1.982 Mb. Annotation of the M. persimmonesis genome revealed presence of 7029 genes and 6939 functionally annotated proteins. Based on the analysis of phylogenetic relationship and the average nucleotide identities, M. persimmonesis was proved to a novel species within the Metschnikowia genus. This finding is expected to significantly contribute to the discovery of high-value natural products from M. persimmonesis as well as for genome biology and evolution comparative analysis within Metschnikowia species.

In vitro genotoxicity and in vivo single-dose oral toxicity of polysaccharide fraction from the leaves of Diospyros kaki thunb

Background: Polysaccharides isolated from medicinal herbs are regarded as important bioactive materials due to their various physiological activities. In our previous study, the polysaccharide fraction (PLE0) was separated from pectinase-digested persimmon (Diospyros kaki Thunb.) leaves and showed immunostimulatory and anti-osteoporotic effects. However, there is currently no information regarding the toxicity of PLE0. Materials and Methods: As a toxicological evaluation, acute oral toxicity and in vitro genotoxicity assays (chromosomal aberration and bacterial reverse mutation tests) were performed. Results: In the single-dose acute oral toxicity test in female and male Sprague-Dawley (SD) rats, the median lethal dose of PLE0 was higher than 5000 mg/kg and adverse effects were not observed in terms of mortality and abnormal changes in clinical signs. Furthermore, chromosomal abnormality and bacterial reverse mutation tests showed that PLE0 displays no mutagenicity or clastogenicity. Conclusion: Based on these results, PLE0 was not mutagenic and did not induce chromosomal aberration in vitro under our experimental conditions and exhibited no acute oral toxicity at up to 5000 mg/kg in SD rats.

The regulatory role of gibberellin related genes DKGA2ox1 and MIR171f_3 in persimmon dwarfism

‘Nantongxiaofangshi’ (Diospyros kaki Thunb., D. kaki Thunb.) is a local cultivar of persimmon with dwarf-like traits in Jiangsu, China. Closely spaced planting afforded by dwarfism is usually one of the most important ways to promote fruit cultivation and production. However, the understanding of dwarfism in D. kaki Thunb. is very limited at the molecular level, which hinders the further increase of the fruit production. In this work, a persimmon transgenic system was successfully established, and the field experiments of grafting phenotype were carried out. The results showed that D. kaki Thunb. could be used as an interstock to induce dwarfing in grafted scions, and the dwarf character was better when interstock lengths were between 20 and 25 cm. Furthermore, the key genes related to dwarfism in D. kaki Thunb. were screened and verified, and subsequently, the regulatory role of related genes in persimmon dwarfism was figured out. It was found that the gene encoding gibberellin 2-oxidase-1 (DkGA2ox1) involved in GA biosynthesis was associated with the dwarfing in D. kaki Thunb. Overexpression of DkGA2ox1 in Diospyros lotus resulted in a typical dwarf phenotype. Meanwhile, the microRNA data showed that the miR171f_3 demonstrated the active involvement in GA pathway response in persimmon dwarfism. DkGA2ox1 and MIR171f_3, as two highly expressed genes in D. kaki Thunb. interstock, could be used as stimulus signals to affect the content of GA in scion, however, the specific transmission mechanism still needs to be further explored. Ultimately, the bioactive GA level was decreased, resulting in the scion dwarfism.

청도반시 추출물의 murine norovirus에 대한 항바이러스 효과

Norovirus infections are one of the most significant causes of foodborne outbreaks worldwide. This study aimed to investigate the antiviral effects of persimmon (Diospyros kaki Thunb. cv. Cheongdo-Bansi) extracts on murine norovirus (MNV). Persimmon extracts obtained by solvent extraction and squeezing, prior to freeze-drying were evaluated by determining the yield and tannin contents. The cytotoxicity and antiviral effect of persimmon extracts were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a time- of-addition assay, respectively. The yield and tannin content were in following order: methanol > water > ethanol > acetone > juice extracts and juice > acetone > water > ethanol > methanol extracts, respectively. The concentrations of methanol, ethanol, acetone, water, and juice extracts were determined to be 800, 800, 600, 200, and 400 μg/mL, respectively. The MNV titer was significantly reduced by 1.65 log plaque forming unit (PFU)/mL with 18% reduction in plaque formation in group pre-treated with water extract. Furthermore, the significant reductions in MNV titer by 2.14, 1.69, and 2.96 log PFU/mL were observed in groups co-treated with acetone, water, and juice extracts, respectively with plaque formation inhibition of 22-40%. However, there were no significant antiviral effects in the post-treated groups. This study suggests the potential use of persimmon extracts as anti-noroviral agents.

DkMYB14 is a bifunctional transcription factor that regulates the accumulation of proanthocyanidin in persimmon fruit

SUMMARYProanthocyanidins (PAs) are phenolic secondary metabolites that contribute to the protection of plant and human health. Persimmon (Diospyros kaki Thunb.) can accumulate abundant PAs in fruit, which cause a strong sensation of astringency. Proanthocyanidins can be classified into soluble and insoluble PAs; the former cause astringency but the latter do not. Soluble PAs can be converted into insoluble PAs upon interacting with acetaldehydes. We demonstrate here that DkMYB14, which regulates the accumulation of PA in persimmon fruit flesh, is a bifunctional transcription factor that acts as a repressor in PA biosynthesis but becomes an activator when involved in acetaldehyde biosynthesis. Interestingly, both functions contribute to the elimination of astringency by decreasing PA biosynthesis and promoting its insolubilization. We show that the amino acid Gly39 in the R2 domain and the ethylene response factor‐associated amphiphilic repression‐like motif in the C‐terminal of DkMYB14 are essential for the regulation of both PA and acetaldehyde synthesis. The repressive function of DkMYB14 was lost after the mutation of either motif, and all activities of DkMYB14 were eliminated following the mutation of both motifs. Our results demonstrate that DkMYB14 functions as both a transcriptional activator and a repressor, directly repressing biosynthesis of PA and promoting its insolubilization, resulting in non‐astringency in persimmon.

Effects of 1-methylcyclopropene and aminoethoxyvinylglycine treatments on fruit quality and antioxidant metabolites in cold-stored ‘Sangjudungsi’ persimmons

After harvest, ‘Sangjudungsi’ persimmon (Diospyros kaki Thunb.) fruit quickly soften and develop physiological disorders during cold storage. The objective of this study was to evaluate the effectiveness of 1-methylcyclopropene (1-MCP) and aminoethoxyvinylglycine (AVG) treatments on fruit quality attributes and antioxidant metabolites in ‘Sangjudungsi’ persimmon fruit stored at 0.5 °C and 95% relative humidity over a 3-month period. The treatments caused a reduction in weight loss; in the middle of the cold storage, the soluble solids content was higher with the AVG treatment than with the 1-MCP treatment. Both treatments reduced the incidence and severity of peel blackening and fruit softening, whereas fruit decay was only affected by AVG. The content of total phenolic compounds was lower with AVG than with 1-MCP or in the untreated control during the same period. At the end of cold storage, the AVG treatment resulted in a greater total flavonoid and total antioxidant activity and a higher reduced glutathione level than with 1-MCP or in the untreated control. AVG likely evoked the least physiological and biochemical responses in persimmons, according to the results of a normalized heatmap matrix system. Correlation coefficient networking results showed that 1-MCP and AVG caused differential correlation responses between the fruit quality variables and targeted metabolites during cold storage. Overall, the results indicated that treatment of cold-stored persimmons with 1-MCP and AVG likely reduced the incidence of physiological disorders, although antioxidant metabolites were relatively less affected by both regulators.

Hydroxypropyl Methylcellulose-Based Edible Coatings Formulated with Antifungal Food Additives to Reduce Alternaria Black Spot and Maintain Postharvest Quality of Cold-Stored ‘Rojo Brillante’ Persimmons

Composite edible coatings based on hydroxypropyl methylcellulose (HPMC), as a polymeric phase, and oleic acid (OA) or beeswax (BW), as a hydrophobic phase, were formulated with different food additives as antifungal ingredients. HPMC–OA coatings containing 2% (w/v) sodium benzoate (SB), 1% ammonium carbonate (AC), 1% potassium carbonate (PC), 1% potassium bicarbonate (PBC), 1% sodium bicarbonate (SBC), 1% potassium silicate (PSi), 0.1% sodium methyl paraben (SMP) or 0.1% sodium ethyl paraben (SEP), and HPMC–BW coatings containing 2% sodium propionate (SP), 2% PBC, 2% SB or 0.1% SEP were evaluated for the control of Alternaria black spot (ABS) on Diospyros kaki Thunb. ’Rojo Brillante’ persimmons artificially inoculated with Alternaria alternata. After 14 days of incubation at 20 °C, HPMC–OA coatings formulated with PBC, PC or SEP were the most effective to reduce ABS incidence (61, 54, and 36% reduction, respectively, concerning uncoated control fruit) and severity (28, 12 and 22% reduction, respectively), while only HPMC–BW coatings formulated with SEP significantly reduced ABS incidence (50% reduction) and severity (36% reduction). HPMC–OA and HPMC–BW coatings containing 2% PBC or 0.1% SEP were selected to evaluate their effect on the weight loss, firmness and respiration rate of healthy ‘Rojo Brillante’ persimmons cold-stored at 1 °C and 90% relative humidity (RH) for 15 and 30 days, followed by 7 days of shelf life at 20 °C. HPMC–BW coatings were more effective in reducing fruit weight and firmness losses than HPMC–OA coatings, while all antifungal coatings significantly reduced fruit respiration. Overall, the HPMC–BW edible coating that contains SEP could be a promising postharvest treatment to control ABS and maintain the quality of cold-stored ‘Rojo Brillante’ persimmons.

Genetic Diversity and Association Analysis among Germplasms of Diospyros kaki in Zhejiang Province Based on SSR Markers

In subtropical to temperate regions, persimmon (Diospyros kaki Thunb.) is an economically important fruit crop cultivated for its edible fruits. Persimmons are distributed abundantly and widely in Zhejiang Province, representing a valuable resource for the breeding of new cultivars and studying the origin and evolution of persimmon. In this study, we elucidated the genetic structures and diversity patterns of 179 persimmon germplasms from 16 different ecologic populations in Zhejiang Province based on the analysis of 17 SSR markers. The results show that there was a medium degree of genetic diversity for persimmon found in Zhejiang Province. With the exception of the Tiantai Mountain and Xin’an River populations, we found extensive gene exchange had occurred among the other populations. The 179 D. kaki germplasms from the 16 populations could be separated into three distinct clusters (I, II, and III) with a higher mean pairwise genetic differentiation index (FST) (0.2714). Nearly all samples of Cluster-I were distributed inland. Cluster-II and Cluster-III contained samples that were widely distributed throughout Zhejiang Province including all samples from the coastal populations and the Northeast Plain populations. In addition, we performed association mapping with nine traits (fruit crude fiber content, fruit calcium content, fruit water content, fruit longitudinal diameter, fruit aspect ratio, seed width, seed length, leaf aspect ratio, and number of lateral veins) using these markers. This led to the identification of 13 significant marker–trait associations (MTAs; p < 0.00044, 0.1/228) using a general linear model, of which, six MTAs with a correlation coefficient (R2) >10% were consistently represented in the general linear model with p < 0.00044 in the two models. The genetic structures and diversity patterns of the persimmon germplasms revealed in this study will provide a reference for the efficient conservation and further utilization of persimmon germplasms. The MTAs identified in this study will be useful for future marker-assisted breeding of persimmon.

Persimmon-derived tannin ameliorates the pathogenesis of ulcerative colitis in a murine model through inhibition of the inflammatory response and alteration of microbiota

Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) induced by dysregulation of the immune response in the intestinal mucosa. Although the underlying mechanisms of UC development are not fully understood, disruption of gut microbiota, “dysbiosis”, is thought to lead to the development of IBD. Persimmon (Ebenaceae Diospyros kaki Thunb.)-derived tannin, which is a condensed polymeric tannin consisting of catechin groups, has antioxidant, anti-inflammatory, and antimicrobial activities. In this study, we assessed the effect of persimmon-derived tannin on a murine model of UC established by dextran sulfate sodium-induced colitis in female mice. Dietary supplementation of tannin significantly decreased disease activity and colon inflammation. A hydrolysate of tannin directly suppressed expression of inflammatory genes in macrophages in vitro. In faecal microbiota, the relative abundance of Bacteroides was increased significantly by tannin supplementation. Alpha-diversity indices in colitis-induced mice were significantly higher in the tannin diet group compared with the control diet group. Additionally, expansion of Enterobacteriaceae and Enterococcus, which is associated with disease progression of IBD, was remarkably suppressed in the tannin diet group. These results suggest that persimmon-derived tannin ameliorates colon inflammation in UC through alteration of the microbiota composition and immune response, which may be a promising candidate for IBD therapy.

Phytohormone and integrated mRNA and miRNA transcriptome analyses and differentiation of male between hermaphroditic floral buds of andromonoecious Diospyros kaki Thunb

BackgroundPersimmon (Diospyros kaki Thunb.) has various labile sex types, and studying its sex differentiation can improve breeding efficiency. However, studies on sexual regulation patterns in persimmon have focused mainly on monoecy and dioecy, whereas little research has been published on andromonoecy. In order to reveal the sex differentiation regulation mechanism of andromonoecious persimmon, we performed histological and cytological observations, evaluated OGI and MeGI expression and conducted phytohormones assays and mRNA and small RNA transcriptome analyses of the male and hermaphroditic floral buds of the andromonoecious persimmon ‘Longyanyeshi 1’.ResultsStages 2 and 4 were identified as the critical morphological periods for sex differentiation of ‘Longyanyeshi 1’ by histological and cytological observation. At both stages, OGI was differentially expressed in male and hermaphroditic buds, but MeGI was not. This was different from their expressions in dioecious and monoecious persimmons. Meantime, the results of phytohormones assays showed that high IAA, ABA, GA3, and JA levels at stage 2 may have promoted male floral bud differentiation. However, high JA levels at stage 4 and high ZT levels at stages 2 and 4 may have promoted hermaphroditic floral bud differentiation. In these phytohormone biosynthesis and signaling pathways, 52 and 54 differential expression genes (including Aux/IAA, ARFs, DELLA, AHP, A-ARR, B-ARR, CYP735A, CRE1, PP2C, JAZ, MYC2, COI1, CTR1, SIMKK, ACO, and MPK6) were identified, respectively. During the development of male floral buds, five metacaspases genes may have been involved in pistil abortion. In addition, MYB, FAR1, bHLH, WRKY, and MADS transcription factors might play important roles in persimmon floral bud sex differentiation. Noteworthy, miR169v_1, miR169e_3, miR319_1, and miR319 were predicted to contribute to phytohormone biosynthesis and signaling pathways and floral organogenesis and may also regulate floral bud sex differentiation.ConclusionThe present study revealed the differences in morphology and phytohormones content between male and hermaphroditic floral buds of ‘Longyanyeshi 1’ during the process of sex differentiation, and identified a subset of candidate genes and miRNAs putatively associated with its sex differentiation. These findings can provide a foundation for molecular regulatory mechanism researching on andromonoecious persimmon.

First report of Passiflora latent virus(Passiflora latent virus;PLV) infecting persimmon in Korea.

Passiflora latent virus (PLV), a member of the genus Carlavirus in the family Betaflexiviridae has been reported in Passiflora species in Australia, Germany, Israel, the United States, and New Zealand (Tang et al., 2008). In September 2019, leaves showing a virus-like disease with mosaic, curling and necrosis were collected from ten persimmon (Diospyros kaki Thunb.) orchards in Gyeongsang province, Korea. Total RNA from a pooled sample of leaves from 21 trees was extracted using RNeasy Plant Mini Kit (Qiagen, Germany) and subjected to high throughput sequencing. After pre-processing and Ribo-Zero rRNA Removal, a cDNA library was prepared using an Illumina TruSeq Stranded Total RNA Kit and sequenced on an Illumina NovaSeq 6000 system (Macrogen Inc. Korea). De novo assembly of the 74,862,810 reads was performed using Trinity software (r20140717); the initially assembled 213,476 contigs were screened against the NCBI viral genome database using BLASTN. By these means, 12 contigs derived from PLV were identified. Contigs with lengths of 209 to 802 nt shared nt identities of 90.70 to 94.82% with PLV isolates, covering a total of 5,169 nt (~61.6% of the full PLV genome). Two additional viruses were also detected from the pooled sample: persimmon cryptic virus (PeCV) and persimmon virus A (PeVA). To confirm PLV infection, reverse transcription-polymerase chain reaction (RT-PCR) was performed using virus-specific primers, PLV-F (5'-ACACAAAACTGCGTGTTGGA-3') and PLV-R (5'-CAAGACCCACCTACCTCAGTGTG-3'), designed based on a 633 nt contig sequence in the polymerase gene. RT-PCR products of the expected 571 bp were obtained from two of 21 individual original samples; no asymptomatic plants were tested. Amplicons were cloned into the pGEM-T Easy Vector, and two clones per sample Sanger sequenced bidirectionally (BIONEER, Korea). The identical Sequence (GenBank LC556232) showed 99.65% nt identity to the contig, and 93.87% identity with the corresponding polymerase sequence of PLV-Rehovot isolate from passion fruit in Israel (MH379331). The two PLV positive samples showing leaf necrosis were also co-infected with PeVA, identified by RT-PCR using previously reported primers PeVAfor/ PeVArev (Morell et al., 2014), but not with PeCV (mixed with PeVA in only 1/21 plants; PeVA was found in 19/21 plants). None of the tested viruses were detected in two trees, displaying mosaic, and leaf curling, respectively. The foliar symptoms of PLV infection on passionfruit have been reported to vary throughout the year (Spiegel et al., 2007). No such observations in persimmon was possible, as the infected persimmon trees were removed and destroyed because they might pose a threat to the cultivation of passion fruits in Korea. To our knowledge, this is the first report of persimmon as a host of PLV anywhere in the world, and the first report of PLV in Korea in any host. A further survey is needed to determine possible presence of PLV on persimmon and Passiflora species.

Codification and description of growth stages in persimmon (Diospyros kaki Thunb.) using the extended BBCH scale

Persimmon (Diospyros kaki Thunb.) is one of the most important fruit that is grown commercially worldwide including the East Asia and Mediterranean regions, and it has been cultivated for its fruit with high nutritional values. Phenology of fruits has attracted the interest of many breeders, growers, and researchers for accurately schedule timing of horticultural managements, but detailed and specific scale for persimmon, especially for the pollination-constant non-astringent (PCNA) and androecious persimmons, remain non-available. In this study, we characterized persimmon phenological growth stages in four persimmon cultivars containing two important morphological traits of sexuality and astringency using the BBCH (Biologische Bundesanstalt, Bundessortenamt Chemische Industrie) scale. The principal growth stages of persimmon were divided into the vegetative and reproductive phenology. We first investigated the vegetative phenology, including: bud development stage (0), leaf development stage (1), shoot development stage (3), and senescence and rest stage (9). The consequent reproductive phenology contained the four following stages, inflorescence emergence stage (5), flowering stage (6), fruit development stage (7), and fruit maturity stage (8). To better understand these phenological growth stages, we next provided illustrative photos with codification and description in persimmon. In addition, the phenological records over two growing seasons in the National Field Genebank for Persimmon (NFGP) were used to draw a schematic representation of principal growth stages. Correct identification of phenological stages is of great importance for the characterization and management in persimmon. Thus, this study will provide the dissemination of knowledge of persimmon cultivars among growers and researchers.

Comparative transcriptome analysis reveals the mechanism involving ethylene and cell wall modification related genes in Diospyros kaki fruit firmness during ripening

Rapid loss of firmness is a major handicap for persimmon (Diospyros kaki Thunb.) transportation and retail. The present study employed a comparative transcriptomic approach to elucidate the mechanism involving ethylene and cell wall modification related genes in fruit firmness control of two cultivars during post harvest ripening. In contrast to the short shelf life cultivar (Mopan), the long shelf life cultivar (Yoho) kept high firmness during ripening. Extensive loss of firmness in Mopan drove an intense transcriptional activity. Globally, Mopan and Yoho shared very few common differentially expressed structural genes and regulators. Yoho strongly repressed the expression of ACC synthase and several classes of cell wall degradation genes at the onset of ripening and only induced them during late ripening period. Various ERF, WRKY, MYB, bHLH transcription factors were found highly active during fruit ripening. Overall, this study generates novel gene resources as important tools for extending persimmon shelf life.

Transcriptome Analysis Revealed the Roles of Carbohydrate Metabolism on Differential Acetaldehyde Production Capacity in Persimmon Fruit in Response to High-CO2 Treatment.

Persimmon (Diospyros kaki Thunb.) fruit is unique due to the continuous accumulation of soluble tannins during fruit development in most cultivars, which causes undesired astringency. High-CO2 treatment was the most effective widely used method for astringency removal. However, differential effects of high-CO2 treatment between cultivars were observed and the molecular basis remained inclusive. Previously, one cultivar ("Luoyangfangtianshengshi," LYFTSS) showed rapid deastringency, while two cultivars ("Shijiazhuanglianhuashi," SJZLHS; "Laopige," LPG) showed slow deastringency in response to high-CO2 (95% CO2) treatment. In this study, the metabolites (acetaldehyde and ethanol) related to deastringency were further analyzed and both acetaldehyde and ethanol were higher in SJZLHS and LYFTSS than that in LPG, where acetaldehyde was undetectable. Based on the RNA-seq data, the weighted gene coexpression network analysis (WGCNA) revealed that one module, comprised of 1773 unigenes, significantly correlated with the contents of acetaldehyde and ethanol (P < 0.001). Further analysis based on the acetaldehyde metabolism pathway indicated that the differentially expressed structural genes, including previously characterized DkADH and DkPDC and also their upstream members (e.g., PFK, phosphofructokinase), showed positive correlations with acetaldehyde production. Quantitative analysis of the precursor substances indicated that sucrose, glucose, and fructose exhibited limited differences between cultivar except for malic acid. However, the content of malic acid is much less than the total soluble sugar content. To verify the correlations between these genes and acetaldehyde production, the fruit from 14 more cultivars were collected and treated with high CO2. After the treatment, acetaldehyde contents in different cultivars ranked in 30.4-255.5 μg/g FW. Real-time polymerase chain reaction (PCR) and correlation analysis indicated that the EVM0002315 (PFK) gene, belonging to carbohydrate metabolism, was significantly correlated with acetaldehyde content in fruit. Thus, it could be proposed that the differentially expressed carbohydrate metabolism related genes (especially PFK) are the basis for the variance of acetaldehyde production among different persimmon cultivars.

‘Dar Sofiyivky’ as a new Ukrainian cultivar of persimmon (Diospyros L.)

Aim. Creation of new domestic persimmon cultivars (Diospyros spp.) adapted to the soil and climatic conditions of Ukraine will contribute to the introduction of new fruit plants into the industrial horticulture that combines high productivity and fruit quality, enriches the assortment of food products and improves the components of rational alimentation. Methods. Field studies of the persimmon species, cultivar and form collection, hybridization and station testing of hybrid seedlings have been conducted in the orchards of State Enterprise Experimental Facility “Novokakhovska” of Institute of Rice of the National Academy of Agrarian Sciences of Ukraine (Kherson region). In vitro seed germination of and two-year hybrid persimmon seedlings growth has been carried out in the Department of Genetics, Breeding and Reproductive Biology of Plants of the National Dendrological Park "Sofiyivka" of the National Academy of Sciences of Ukraine. The rest of the experiments and data statistical analysis have been done using generally accepted methods in breeding. Results. Advantages of D virginiana L. in hybridization with D. kaki Thunb. to increase the persimmon adaptability to unfavorable wintering factors have been confirmed in the extreme temperature conditions of the winters of 2005–2006 and 2011–2012. In the hybrid progeny from free pollination of the ‘Kolhospnytsia’ cultivar with the American persimmon D. virginiana in the genealogy, a new cultivar ‘Dar Sofiyivky’ has been selected. It combines increased winter hardiness with good fruit quality, early fruit production and early ripeness. In 2019, the cultivar ‘Dar Sofiyivky’ has been included in the State Register of Plant Varieties suitable for distribution in Ukraine. In vitro technologies have been used in the tissue culture laboratory of the “Sofiyivka” Dendrological Park for germination of interspecific hybrids seeds, which have insufficient germination in the field. A initial hybrid seedling has been obtained, from which the ‘Dar Sofiyivky’ genealogy is derived. Conclusions. Horticultural plant breeding, in particular persimmons (Diospyros spp.), in areas with ecologically different contrasting conditions within the framework of a single program to increase the adaptability of plants and the quality of fruits is promoted to accelerate breeding and expand the northern border cultivation of thermophilic fruit crops.

Development of Kompetitive Allele Specific PCR markers for identification of persimmon varieties using genotyping-by-sequencing

Persimmon ( Diospyros kaki Thunb.) is the most widely cultivated species of the genus Diospyros . In this study, genetic diversity and variations in persimmon genotypes were investigated using single nucleotide polymorphism (SNP) markers identified by genotyping-by-sequencing (GBS) analysis. Ninety-five persimmon accessions grown in the Pear Research Institute, National Institute Horticultural and Herbal Science, were sequenced using the Illumina Hiseq2500 platform and polymorphic SNPs were detected to develop molecular markers. These reliable SNPs were analyzed using the Kompetitive Allele Specific PCR (KASP) assay to discriminate among persimmon genotypes. GBS generated a total of 447,495,724 trimmed reads, of which 89.7% were raw reads. After demultiplexing and sequence quality trimming, 108,876,644 clean reads were mapped to the reference transcriptome. An average of 1,146,070 genotype reads were mapped. Filtering of raw SNPs in each sample led to selection of a total of 1,725,401 high-quality SNPs. The number of homozygous and heterozygous SNPs ranged from 1,933 to 6,834 and from 846 to 5,927, respectively. Of the 49 SNPs selected for development of an identification system for persimmons, 15 SNPs were used in the KASP assay to analyze 32 persimmon accessions. These KASP markers discriminated among all accessions. How to cite: Ma KB, Yang SJ, Jo YS, et al. Development of Kompetitive Allele Specific PCR markers for identification of persimmon varieties using genotyping-by-sequencing. Electron J Biotechnol 2021;49. https://doi.org/10.1016/j.ejbt.2020.11.003

Tree growth, productivity, and fruit quality of ‘Fuyu’ persimmon trees onto different dwarfing rootstocks

To investigate the dwarfing effect of candidate rootstocks on persimmon (Diospyros kaki Thunb.), we compared the growth of ‘Fuyu’ scions grafted onto ‘Hourakudai’ and S22 clone dwarfing rootstocks with that on a standard D. kaki rootstock, ‘Aogaki’ seedlings, in unpruned and non-bearing trees. In addition, we compared the growth, fruit quality, and yield efficiency of ‘Fuyu’ scions grafted onto three candidate dwarfing rootstocks (‘Hourakudai’, No. 3, and S22 clones) with those of ‘Aogaki’ seedlings grown under conventional cultivation (pruned and fruited). In unpruned and non-bearing trees, grafts on S22 and ‘Hourakudai’ rootstocks reached about 75 % of the height of trees on the standard rootstock. Tree canopy volumes on the S22 and ‘Hourakudai’ rootstocks were about 67 % and 42 %, respectively, of those on the standard rootstock. The high positive correlation between TCSA and tree canopy volume was observed for not only between trees grown under unpruned and non-bearing but also trees grown under pruned and bearing. Tree canopy volume was a better indicator of dwarfing capacity of rootstocks than tree height or tree canopy area. In conventional cultivation, trees grafted on all three candidate dwarfing rootstocks had smaller canopy area and canopy volume than those grafted onto the standard rootstock, from 2 to 11 years after planting. Although differences in tree growth between the three dwarf rootstocks were observed, the differences tended to decrease as tree age advanced. The quality of fruit produced (fruit weight, soluble solids content, and skin color) by trees on dwarf rootstocks did not differ from that produced on standard rootstocks, although annual variations occurred. Trees on S22 rootstock had the lowest yield owing to decreased vigor. Trees on ‘Hourakudai’ rootstock had the highest yield efficiency per trunk cross-sectional area of scions and per tree canopy volume. ‘Hourakudai’ softwood cuttings showed the highest rooting rate (80 %). These results identify ‘Hourakudai’ as a promising new rootstock for growing dwarfed ‘Fuyu’ trees, owing to the high yield efficiency without a decrease in fruit quality.

Pollination effectiveness of European honeybee, Apis mellifera (Hymenoptera: Apidae), in an Oriental persimmon, Diospyros kaki (Ericales: Ebenaceae), orchard

Most common cultivars of Oriental (or Japanese) persimmon, Diospyros kaki Thunb. (Ericales: Ebenaceae), set mostly female flowers and require pollen from male flowers of other cultivars for pollination. Growers often introduce the European honeybee, Apis mellifera L. (Hymenoptera: Apidae), into their orchards to promote pollination. Here, we investigated the pollination effectiveness of A. mellifera for D. kaki ‘Saijo,’ by monitoring flower visitors, analyzing pollen grains on bees’ body surfaces, and comparing the number of mature seeds in fruits among years with different pollinator availabilities. Apis mellifera and the bumblebee Bombus ardens ardens Smith (Hymenoptera: Apidae) were the major visitors for 3 years, although their dominance varied among years. The number of mature seeds was positively correlated with the number of B. ardens ​ardens visiting D. kaki flowers, but not with that of A. mellifera. Apis mellifera might be less efficient because visitors to female flowers carried significantly fewer pollen grains on their body surfaces than those of B. ardens ​ardens. Analysis of pollen loads of honeybees captured at their nest entrance suggested their preference for red clover, Trifolium pratense L. (Fabales: Fabaceae), and Toxicodendron spp. (Sapindales: Anacardiaceae), over D. kaki as a pollen source in our study site. The effectiveness of A. mellifera on D. kaki pollination should be carefully evaluated considering the effects of coexisting floral and pollinator species.

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.