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Fruit shape is an important trait that attracts consumers, and the regulation of genes related to cell division is crucial for shaping multicellular organs. In Arabidopsis, MYB3R transcription factors, which harbor three imperfect repeats in the N-terminus, control organ growth by regulating cell division. However, the function of MYB3Rs in tomato remains unknown. Here, we characterized tomato SlMYB3R3, which was preferentially expressed in flowers and placed in a subclade with two Arabidopsis cell cycle suppressors (MYB3R3/5). slmyb3r3 knockout mutants were generated using the CRISPR/Cas9 system. Morphological observation of the slmyb3r3 mutants showed that fruits that were elongated and occasionally peanut-like in shape were formed, which was caused by significantly increased cell numbers in the longitudinal direction. Transcriptome and yeast one-hybrid assay results suggested that SlMYB3R3 acted as a suppressor of cell-cycle-related genes by binding to the mitosis-specific activator (MSA) motifs in their promoters. Taken together, knock out of the suppressor SlMYB3R3 leads to elongated fruit, which results from the altered cell division pattern at the ovary stage, by regulating cell-cycle-related genes in an MSA-dependent manner. Our results suggest that SlMYB3R3 and its orthologs have the potential to change fruit shape as part of the molecular breeding of fruit crops.

Bud dormancy helps woody perennials survive winter and activate robust plant development in the spring. For apple (Malus × domestica), short-term chilling induces bud dormancy in autumn, then prolonged chilling leads to dormancy release and a shift to a quiescent state in winter, with subsequent warm periods promoting bud break in spring. Epigenetic regulation contributes to seasonal responses such as vernalization. However, how histone modifications integrate seasonal cues and internal signals during bud dormancy in woody perennials remains largely unknown. Here, we show that H3K4me3 plays a key role in establishing permissive chromatin states during bud dormancy and bud break in apple. The global changes in gene expression strongly correlated with changes in H3K4me3, but not H3K27me3. High expression of DORMANCY-ASSOCIATED MADS-box (DAM) genes, key regulators of dormancy, in autumn was associated with high H3K4me3 levels. In addition, known DAM/SHORT VEGETATIVE PHASE (SVP) target genes significantly overlapped with H3K4me3-modified genes as bud dormancy progressed. These data suggest that H3K4me3 contributes to the central dormancy circuit, consisting of DAM/SVP and abscisic acid (ABA), in autumn. In winter, the lower expression and H3K4me3 levels at DAMs and gibberellin metabolism genes control chilling-induced release of dormancy. Warming conditions in spring facilitate the expression of genes related to phytohormones, the cell cycle, and cell wall modification by increasing H3K4me3 toward bud break. Our study also revealed that activation of auxin and repression of ABA sensitivity in spring are conditioned at least partly through temperature-mediated epigenetic regulation in winter., 果樹が季節に応答して休眠から目覚めるしくみを解明 --果樹の休眠と発芽におけるヒストン修飾の寄与を解析--. 京都大学プレスリリース. 2022-07-01.

MdPG1 encoding polygalacturonase in apple (Malus × domestica) is a key gene associated with fruit firmness and texture variations among apple cultivars. However, the causative variants of MdPG1 are still not known. In this study, we identified a SNPA/C variant within an ERF-binding element located in the promoter region of MdPG1. The promoter containing the ERF-binding element with SNPA, rather than the SNPC, could be strongly bound and activated by MdCBF2, a member of the AP2/ERF transcription factor family, as determined by yeast-one-hybrid and dual-luciferase reporter assays. We also demonstrated that the presence of a novel long non-coding RNA, lncRNAPG1, in the promoter of MdPG1 was a causative variant. lncRNAPG1 was specifically expressed in fruit tissues postharvest. lncRNAPG1 could reduce promoter activity when it was fused to the promoter of MdPG1 and a tobacco gene encoding Mg-chelatase H subunit (NtCHLH) in transgenic tobacco cells but could not reduce promoter activity when it was supplied in a separate gene construct, indicating a cis-regulatory effect. Our results provide new insights into genetic regulation of MdPG1 allele expression and are also useful for the development of elite apple cultivars.

Dwarfing rootstocks are capable of high-density planting and are therefore urgently needed in the modern citrus cultivation system. However, little is known about the physiological relevance and molecular basis underlying citrus height. This study aimed to comprehensively analyze phytohormone, carbohydrate, and associated transcriptome changes in the stem of two weak growth rootstocks (‘TO’ and ‘FD’) relative to the vigorous ‘CC’ rootstock. The phenotypic observation revealed that the plant height, plant weight, and internode length were reduced in dwarfing rootstocks. Moreover, the contents of indole-3-acetic acid (IAA), trans-zeatin (tZ), and abscisic acid (ABA), were higher in TO and FD rootstocks, whereas the gibberellin 3 (GA3) content was higher in the CC rootstocks. The carbohydrate contents, including sucrose, fructose, glucose, starch, and lignin significantly decreased in both the TO and FD rootstocks. The full-length transcriptome analysis revealed a potential mechanism regulating dwarfing phenotype that was mainly related to the phytohormone signaling transduction, sugar and starch degradation, lignin synthesis, and cellulose and hemicellulose degradation processes. In addition, many transcription factors (TFs), long non-coding RNAs (lncRNAs), and alternative splicing (AS) events were identified, which might act as important contributors to control the stem elongation and development in the weak growth rootstocks. These findings might deepen the understanding of the complex mechanisms of the stem development responsible for citrus dwarfing and provide a series of candidate genes for the application in breeding new rootstocks with intensive dwarfing.

Persimmon (Diospyros kaki Thunb.) is becoming a fruit that is used worldwide because it contains high nutritional and medicinal value. However, the trait evaluation of persimmon is still needed and is critical for marketing and breeding, especially in China. Here, we evaluated thirteen quality indicators (fruit weight, horizontal length, vertical length, firmness, titratable acid content, vitamin C content, flavonoid content, anthocyanin content, soluble sugar content, pectinase activity, soluble protein content, tannin content, and tannin cell size) in six Chinese pollination-constant, astringent (PCA) persimmon cultivars (‘Jinxi 3′, ‘Ganfang 1′, ‘Poyang 5′, ‘Poyang 6′, ‘Yifeng 1′, and ‘Yifeng 3′) and a Japanese pollination-constant non-astringent (PCNA) persimmon cultivar (‘Youhou’). The quality indicator data were normalized and subjected to variant analysis and principal component analysis (PCA). The results showed that 10 of 13 indicators among the seven persimmon cultivars were significantly different, and the contribution rates of the first principal component reached 40.582%. The principal component comprehensive scores for ‘Poyang 5′ and ‘Ganfang 1′ ranked second and third, respectively, and were clustered with that of ‘Youhou.’ In addition, we also measured the expression levels of three stress resistance genes and three proanthocyanidin (PA, also called condensed tannins) pathway genes in these persimmon cultivars by quantitative reverse transcription PCR (qRT—PCR). The qRT—PCR analysis of DkCBF and DkWRKY3/8 showed low resistance to cold in ‘Ganfang 1′ but stronger resistance to anthracnose. Moreover, the expression of the PA pathway genes demonstrated that the PA content in ‘Ganfang 1′ was at a moderate level in the seven varieties. Together, our study revealed relatively comprehensive profiles of persimmon quality evaluation and demonstrated that ‘Ganfang 1′ may have the potential to be used as a breeding parent for future persimmon breeding programs.

Walnut (Juglans regia L.) plants typically flower after eight to ten years of juvenile growth. Precocious germplasm, also known as early-flowering or early-mature genotypes, have shortened juvenile phases of one to two years and are therefore crucial for enhancing breeding efficiency. However, such precocious germplasms are very limited. Here, we isolated and characterized the key flowering-time gene FLOWERING LOCUS C (FLC) in the precocious walnuts of the Xinjiang Uygur Autonomous Region. Sequence alignment showed that Juglans regia FLC (JrFLC)contained a conserved MINICHROMOSOME MAINTENANCE 1 (MCM1), AGAMOUS (AG), DEFICIENS (DEF), and SERUM RESPONSE FACTOR (SRF) (MADS)-box domain. Analysis of an FLC–green fluorescent protein (GFP) fusion protein revealed that JrFLC was localized to the nucleus. Gene expression analysis showed that JrFLC was specifically expressed during the bud dormancy stage of precocious walnut, and that expression levels gradually decreased as the ambient temperature warmed. Exogenous JrFLC overexpression in Arabidopsis thaliana delayed flowering and increased the total leaf number, suggesting a similar function of JrFLC as a floral repressor in walnut and in other plants. Together, these results showed that JrFLC played an important role in regulating the floral transition of Xinjiang precocious walnut. Further studies, including a detailed characterization of JrFLC, are expected to validate JrFLC as a strong target for genetic improvement in flowering time in walnut.

For tree crops, shortening the juvenile phase is a vital strategy to advance fruit bearing and enhance the breeding efficiency. Walnut (Juglans regia L.) seedlings usually take at least eight to 10 years to flower, but early-flowering (EF) types can flower one or two years after planting. In this study, RNA sequencing (RNA-Seq) and microRNA sequencing (miRNA-Seq) were used for a transcriptome-wide analysis of gene and miRNA expression in hybrids of the Xinjiang EF walnut variety ‘Xinwen 81’ and later-flowering (LF) walnut. Based on a high-quality chromosome-scale reference genome, a total of 3009 differentially expressed genes (DEGs) were identified, of which 933 were upregulated (accounting for 31%) and 2076 were downregulated (accounting for 69%). DEGs were functionally annotated, and the flowering-related genes FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), and LEAFY (LFY) showed remarkable upregulation in EF compared with in the LF walnut. In addition, miRNAs associated with floral transition were screened as candidates for flowering time regulation in the walnut. This work provides new insights into walnut floral transition, which may ultimately contribute to genetic improvement of the walnut.

MicroRNAs (miRNAs) are endogenous noncoding small RNAs containing 21–24 nucleotides (nt) that regulate gene expression precisely and efficiently at the posttranscriptional level through the negative regulation of target messenger RNA (mRNA) expression, such as translational inhibition or degradation. Likewise, as a controlling element, miRNA itself is regulated by a variety of factors when performing its basic purposes, such as SNP detection, miRNA purging, methylation, and the circadian clock in model crops. In current years, miRNA-mediated controls have been intensely investigated in horticultural plants, leading to the discovery of numerous novel mechanisms that exhibit significantly greater mechanistic complexity and distinctive regulatory properties than those explored in model species. In fruit crops, miRNAs play a crucial role corresponding to various biological, metabolic functions and environmental challenges, including growth, expansion, response to biotic and abiotic stress, signaling of growth hormones, and the regulation of secondary product metabolism. In this study, we appraisal the current improvement of small regulatory RNA research in fruit crops, emphasizing miRNA mechanisms and their correlation with key trait rule. Considering that miRNAs engaged in the regulation of all aspects of fruit tree life activities, we focus here on their biosynthesis, target genes, function and regulatory network, as well as the mechanistic connection among them, to provide a theoretic base and breakthrough for upcoming exploration on miRNAs in fruit plants.

Background Proanthocyanidins (PAs) are important plant secondary metabolites that confer flavor, nutritional value, and resistance to pathogens. Persimmon is one of the PA richest crops. Mature fruits can be inedible because of the astringency caused by high PA levels and need to go through a de-astringency treatment before consumption. The molecular basis for PA accumulation is poorly known, particularly transcriptional regulators. We characterised three genotypes (‘Luotiantianshi’ (LT), ‘Mopanshi’ (MP), and ‘Youhou’ (YH)) with different PA accumulation patterns using an approach that combined PacBio full-length sequencing and Illumina-based RNA sequencing to build high-quality full-length transcriptomes. Additionally, we analysed transcriptome dynamics of the three genotypes (LT, MP, and YH) at four key fruit developmental stages. Results A total of 96,463 transcripts were obtained. We identified 80,075 protein-coding sequences (CDSs), 71,137 simple sequence repeats (SSRs), and 27,845 long noncoding RNAs (lncRNAs). Pearson correlation coefficient (PCC), principal component analysis (PCA), and differentially expressed transcripts (DETs) analyses indicated that the four different developmental stages within a genotype exhibited similar transcriptome activities. A total of 2,164 transcripts specific to each fruit developmental stage were detected. The transcripts specific to early stages were attributed to phenylpropanoid and flavonoid biosynthesis. Co-expression network analyses revealed MEbrown and MEblue modules were strongly associated to PA accumulation. From these two modules, 20 hub TFs are potential regulators for PA accumulation. Among them, Cluster_78388 (SBP protein), Cluster_63454 (bZIP protein), and Cluster_66595 (MYB protein) appear to involve in the PA biosynthesis in Chinese genotypes. Conclusions This is the first high-quality reference transcriptome for commercial persimmon. Our work provides insights into the molecular pathways underlying PA accumulation and enhances our global understanding of transcriptome dynamics throughout fruit development.

Diospyros L. is the largest, most widely distributed, and economically important genus of the family Ebenaceae. However, various researchers disagree on the number of species and geographical distribution of Diospyros in China. For the purpose of providing a scientific basis for the effective protection and rational use of Diospyros, the present study involved a comprehensive review of the literature, electronic databases, and herbaria as well as field investigations. The results showed as the following. (1) While 174 scientific names have been published for various taxa of Diospyros in China, by screening and excluding synonyms, 65 species names were considered acceptable; these account for about 12% of the total species of Diospyros globally. (2) A review of herbarium specimens documented that Diospyros occurs in 28 administrative regions of China, more than the literature records. Species richness decreases from south to north in China while Guangxi, Yunnan, Guangdong, and Hainan have the greatest number of species. (3) Among the 65 relatively reliable Diospyros species, 45 species are endemic to China and 18 species are stenochoric. Some species may have become extinct as a result of environmental degradation and the status of some germplasm remain unresolved. In brief, Chinese Diospyros includes abundant, China-specific, and widely distributed features. Nevertheless, comprehensive species delimitation and the analysis of phylogenetic relationships will still require further study. Keywords: Diospyros L., Ebenaceae, species number, geographic distribution

Walnut (Juglans regia L.) is an important nut fruit crop mainly grown for its high nutritional and medicinal value. In walnut fruit, the pellicle is the main source of polyphenols (such as proanthocyanidins), which are natural bioactive compounds but also cause astringency and bitterness for walnut fruit consumption. However, the gene regulatory networks of phenolic biosynthetic pathways remain largely unknown in walnut pellicles. Here, we performed RNA sequencing (RNA-seq) to identify differentially expressed genes (DEGs) associated with pellicle development in walnut. In this study, seven developmental stages (8-, 9-, 11-, 13-, 15-, 17-, and 19-week after pollination) of ‘Xinwen179’ pellicle tissues were harvested to conduct further transcriptome-wide profiles. Via RNA-seq, we explored several key DEGs involved in the phenolic biosynthetic pathway, such as dihydroflavonol-4-reductase (DFR), leucoanthocyanidin reductase (LAR), anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR), which are dynamically expressed at developmental stages of the walnut pellicle. Among them, ANR may directly contribute to proanthocyanidins accumulation during walnut development. Taken together, our preliminary investigation on DEGs associated with pellicle development will not only elucidate the gene regulatory networks of the phenolic biosynthetic pathway for pellicle development, but also contribute to the broad spectrum of RNA-seq data resources for further genetic improvement of walnut.

Persimmon (Diospyros kaki Thunb.) is an important fruit crop in East Asia and has great commercial potential due to the high demand for fresh edible fruits or processed dried persimmon, as well as its being a source of raw materials for the chemical industry. Genetic engineering has shown great potential in persimmon fruit improvement and compensated for the limitations of conventional breeding. In this study, we developed an efficient transformation system and in vivo regeneration protocol by using the leaf disc culture of ‘Gongcheng Shuishi’ persimmon through the optimization of different basic media and hormone combinations. Based on the secondary induction system on 1/2DKW (DKW medium) + ZT (2.0 mg/L) + TDZ (0.5 mg/L) and MS (1/2 N) + ZT (2.0 mg/L) + IAA (0.1 mg/L), the callus induction rate and adventitious bud induction rate were 96.0% and 94.0%, respectively, and RNA interference of DkANR (ANR, anthocyanidin reductase) was achieved after transformation, thus resulting in a reduction in proanthocyanidin accumulation. The GFP (green fluorescent protein) fluorescence signal was visualized in the transgenic seedlings with GFP gene cotransformation. A total of 17.9% of the positive regenerated transgenic seedlings with empty vector were confirmed by both hyg gene and 35S promoter PCR analysis. Southern blotting assays showed that 20 positive transgenic persimmon seedlings were obtained from 135 regenerated transformants derived from DkANRi. Altogether, a reliable and highly efficient transformation system was achieved in persimmon, which may contribute to gene function identification and genetic improvement in the future.

Persimmon proanthocyanidin (PA) biosynthesis is controlled by structural genes and regulated by transcription factors (TFs). MicroRNAs are a key factor involved in regulating gene expression at the posttranscriptional level whose functions in persimmon PA biosynthesis are poorly understood. Here, we identified a microRNA, miR858b, that putatively targets two R2R3-MYB TFs, DkMYB19 and DkMYB20. DkMYB19, DkMYB20, and miR858b showed divergent expression patterns during fruit development, and the interaction between miR858b and DkMYB19 or DkMYB20 was experimentally validated by 5′ RNA ligase-mediated RACE, LUC enzyme activity analysis, and GFP signal detection. The DkMYB19 localized to the nucleus as well as the cytoplasm and DkMYB20 localized to the nucleus. The overexpression of miR858b led to the downregulation of DkMYB19 and DkMYB20, which reduced the content of PA, whereas a reduction in miR858b activity upregulated DkMYB19 and DkMYB20, resulting in a high content of PA in leaves transiently expressing a small tandem target mimic construct for blocking miR858 (STTM858b) in vivo. The transient transformation of miR858b in fruit discs in vitro also reduced the content of PA, while the content of PA increased under the transient transformation of fruit discs with STTM858b, DkMYB19, or DkMYB20. A similar phenomenon was observed upon the overexpression of miR858b in wild-type (WT) Arabidopsis and DkMYB19 or DkMYB20 in persimmon leaf calli. These findings suggested that miR858b repressed the expression of DkMYB19 and DkMYB20, which contributed to the PA accumulation in 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.

It is crucial to develop a rapid technique for identifying sexuality in the seedling stage of persimmon (Diospyros kaki Thunb.), and the elimination of male progeny has been regarded as an important strategy for enhancing breeding efficiency. In this study, phenotype characterization and genotyping of the male-linked OGI marker were carried out using 205 accessions, including persimmon cultivars, F1 progeny and nine related Diospyros species. All persimmon cultivars displayed consistent results regarding OGI amplification and sex phenotype. A total of 143 F1 progeny were derived from 11 crosses, among which 95 individuals had flowered. In the flowering full-sib families, the amplification of the OGI marker in agreement with the sex phenotype was obtained in 85 plants (89.5%). The segregation of OGI in ‘Huashi 1’ × ‘Luotian Tianshi’ and ‘Huashi 1’ × Male 3 F1 populations fit a 1 : 1 ratio. Furthermore, high OGI transferability was observed in nine related species. Overall, the results indicated that the OGI locus could be used to distinguish male from female persimmon plants at an early stage.

The androecious genotype of Diospyros spp . ‘Male 8’, which is distributed in Dabie Mountain area of the central China, was probably derived from the hybrids or natural variation of Chinese pollination constant and non-astringent (CPCNA) persimmon. Its application potential as the breeding parent of pollination constant and non-astringent (PCNA) is undefined. In this study, we identified the astringency type of a hybrid individual (H8-2) derived from the cross between ‘Huashi 1’ (pollination variant and astringent, PVA) and ‘Male 8’. Through comparison with the known genotypes of CPCNA, Japanese PCNA (JPCNA) and non-PCNA, the results of soluble/insoluble tannin content and tannin cell size measure showed that H8-2 was a PCNA genotype with the characteristic of natural deastringency of CPCNA. According to the currently known genetic features of PCNA persimmon, a new individual of PCNA may be generated in F 1 population when two JPCNA genotypes are crossed or when the CPCNA genotype is used as a parent. Thus, the study verifies that androecious genotype ‘Male 8’ carries the dominant gene locus that controls the non-astringent trait of CPCNA, indicating its potential to be used as pollen donor for the genetic improvement of PCNA persimmon.

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.

Proanthocyanidins (PAs) are secondary metabolites that contribute to important plant traits like fruit astringency. Persimmon (Diospyros kaki Thunb.) is unique for accumulating abundant PAs in its fruit flesh. Two non-astringent-type mutants, namely, Japanese pollination-constant and non-astringent (J-PCNA) and Chinese PCNA (C-PCNA), were unable to accumulate PA naturally on the tree before harvest. It was clear that the decreased expression profile of a Myb transcription factor (TF) DkMyb4 in persimmon fruits results in the PCNA trait in J-PCNA mutant. However, the mechanism leading to the PCNA trait in C-PCNA mutant remains unclear. In this study, with the purpose of figuring out whether or not there was a certain Myb TF resulting in PCNA trait in C-PCNA mutant, a gene homologous to DkMyb4 was isolated from C-PCNA cultivar ‘Luotian-tianshi’, named DkPA1. Bioinformatics analysis showed that DkPA1 shared high similarity to DkMyb4 in amino acid level, and that DkPA1 was phylogenetically closer to PA-related Myb TFs. Expression pattern of DkPA1 coincided with expression of PA pathway genes, also consistent with PA accumulation model in both C-PCNA and non-PCNA fruit during fruit development. Though being absent in J-PCNA, we found there were MYBCORE cis-motifs in promoter of PA pathway Leucoanthocyanidin reductase (DkLAR) gene isolated from C-PCNA and non-PCNA, which was reported as a key cis-element for PA biosynthesis and target of Myb TFs. These results suggested that DkLAR was regulated by DkPA1 in C-PCNA and non-PCNA, it played much more important role in C-PCNA and non-PCNA PA biosynthesis than that in J-PCNA.

Leucoanthocyanidin reductase (LAR) converts leucoanthocyanidin to (+)-catechin, a precursor of proanthocyanidins abundant in Japanese persimmon (Diospyros kaki Thunb.) fruits. A putative LAR gene (DkLAR) was isolated by rapid amplification of cDNA ends from young fruits. The full-length cDNA of DkLAR gene was 1 356 bp long and encoded an open reading frame of 349 residues. The deduced DkLAR protein was closely related to the homolog in other plant species. The expression of the DkLAR gene in Chinese pollination-constant non-astringent (PCNA) genotype was coincident with the tannin cell development, but was not in Japanese PCNA and Chinese pollination-variant astringent (PCA) genotypes.

Four molecular markers, including inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP), sequence-specific amplified polymorphism (SSAP), and amplified fragment length polymorphism (AFLP), were compared in terms of their informativeness and efficiency for analysis of genetic relationships among 28 genotypes in the genus Diospyros. The results were as follows: (1) the highest level of detected polymorphism were observed for IRAP; (2) AFLP was the most efficient marker system due to the simultaneous detection of abundant polymorphism markers per single reaction; (3) the marker index (MI) value was lower for SSAP than for AFLP, but SSAP had a higher level of detected polymorphism than AFLP; (4) the correlation coefficients of similarity were statistically significant for all four marker systems; (5) the four molecular markers yielded similar phylogenetic trees. To our knowledge, this was the first detailed report of a comparison of performance among three retrotransposon-based molecular markers (IRAP, REMAP, SSAP) and the AFLP technique (DNA-based molecular marker) on a set of samples of Diospyros. The results provide guidance for future efficient use of these molecular methods in the genetic analysis of Diospyros.

A modified method of inter-retrotransposon amplified polymorphism (IRAP) with primers annealing to the conserved reverse transcriptase sites instead of the conserved long-terminal repeat (LTR) regions and retrotransposon–microsatellite amplified polymorphism (REMAP) markers were used to investigate genetic relationships of some Japanese persimmon ( Diospyros kaki Thunb.) genotypes from Japan and China and the related species. In total, 117 scorable amplification bands ranging from 250 to 2000 bp were generated by IRAP and REMAP analysis, among which 101 (86%) were polymorphic, with an average of six polymorphic bands per primer combination. The analysis of genetic relationships of 27 genotypes revealed that the Japanese, Chinese genotypes and related species were distinctly separated. The phylogenetic trees constructed by unweighted pair-group method with arithmetic averages (UPGMA) and Wagner parsimony cluster methods agreed well with the geographic origin information. The Japanese persimmon genotypes from Japan and China may have different genetic background, suggesting a long independent domestication, intercrossing and breeding history in both regions. ‘Baogaitianshi’ and ‘Xiaobaogaitianshi’ had a high similarity coefficient, implying that they possibly have the bud sport relationships. The related species were clearly clustered in a separated group, suggesting that they are very closely related.

Sequence-related amplified polymorphism (SRAP) marker was employed to analyze genetic relationships of 10 Chinese, 11 Japanese varieties of persimmon and its 6 relatives. Twenty out of 72 primer combinations could amplify clearly and consistently. Just only one primer set could discriminate all the genotypes used in this study. The genetic relationships were analyzed using unweighted pair-group method of arithmetic average cluster analysis (UPGMA) and principal coordinated analysis (PCOA). The results showed: Chinese varieties were grouped into two clusters, while the Japanese varieties were grouped into one cluster with two subgroups. The persimmon relatives were clustered together. Probably, Chinese and Japanese Pollination-constant and Non-astringent (PCNA) persimmons had different genetic background. ‘Eshi No.1’ from Hubei province was close to Male type 6. ‘Xiangxitianshi’ from Xiangxi municipality, Hunan province, had a high similarity coefficient with ‘Maekawa-Jirou’ and was clustered in the Japanese varieties group, indicating that ‘Xiangxitianshi’ maybe close to the Japanese variety in terms of genetic background. The Chinese Pollination-constant and Astringent (PCA) and PCNA persimmons were not distinctly distinguished implying that the non-astringent trait of Chinese PCNA type might result from the PCA type.