Mitochondrial genome features and systematic evolution of diospyros kaki thunb 'Taishuu'.

BACKGROUND: 'Taishuu' has a crisp texture, abundant juice, and sweet flavor with hints of cantaloupe. The availability of mitochondrial genome data of Diospyros species is far from the known number of species. RESULTS: The sequencing data were assembled into a closed circular mitochondrial chromosome with a 421,308 bp length and a 45.79% GC content. The mitochondrial genome comprised 40 protein-coding, 24 tRNA, and three rRNA genes. The most common codons for arginine (Arg), proline (Pro), glycine (Gly), tryptophan (Trp), valine (Val), alanine (Ala), and leucine (Leu) were AGA, CCA, GGA, UGG, GUA, GCA, and CUA, respectively. The start codon for cox1 and nad4L protein-coding genes was ACG (ATG), whereas the remaining protein-coding genes started with ATG. There are four types of stop codons: CGA, TAA, TAG, and TGA, with TAA being the most frequently used stop codon (45.24%). In the D. kaki Thunb. 'Taishuu' mitochondrial genome, a total of 645 repeat sequences were identified, including 125 SSRs, 7 tandem repeats, and 513 dispersed repeats. Collinearity analysis revealed a close relationship between D. kaki Thunb. 'Taishuu' and Diospyros oleifera, with conserved homologous gene fragments shared among these species in large regions of the mitochondrial genome. The protein-coding genes ccmB and nad4L were observed to undergo positive selection. Analysis of homologous sequences between chloroplasts and mitochondria identified 28 homologous segments, with a total length of 24,075 bp, accounting for 5.71% of the mitochondrial genome. These homologous segments contain 8 annotated genes, including 6 tRNA genes and 2 protein-coding genes (rrn18 and ccmC). There are 23 homologous genes between chloroplasts and nuclei. Mitochondria, chloroplasts, and nuclei share two homologous genes, which are trnV-GAC and trnW-CCA. CONCLUSION: In conclusion, a high-quality chromosome-level draft genome for D. kaki was generated in this study, which will contribute to further studies of major economic traits in the genus Diospyros.

Genetic insights into the dissolution of dioecy in diploid persimmon Diospyros oleifera Cheng.

BACKGROUND: Dioecy, a sexual system of single-sexual (gynoecious/androecious) individuals, is rare in flowering plants. This rarity may be a result of the frequent transition from dioecy into systems with co-sexual individuals. RESULTS: In this study, co-sexual expression (monoecy and hermaphroditic development), previously thought to be polyploid-specific in Diospyros species, was identified in the diploid D. oleifeara historically. We characterized potential genetic mechanisms that underlie the dissolution of dioecy to monoecy and andro(gyno)monoecy, based on multiscale genome-wide investigations of 150 accessions of Diospyros oleifera. We found all co-sexual plants, including monoecious and andro(gyno)monoecious individuals, possessed the male determinant gene OGI, implying the presence of genetic factors controlling gynoecia development in genetically male D. oleifera. Importantly, discrepancies in the OGI/MeGI module were found in diploid monoecious D. oleifera compared with polyploid monoecious D. kaki, including no Kali insertion on the promoter of OGI, no different abundance of smRNAs targeting MeGI (a counterpart of OGI), and no different expression of MeGI between female and male floral buds. On the contrary, in both single- and co-sexual plants, female function was expressed in the presence of a genome-wide decrease in methylation levels, along with sexually distinct regulatory networks of smRNAs and their targets. Furthermore, a genome-wide association study (GWAS) identified a genomic region and a DUF247 gene cluster strongly associated with the monoecious phenotype and several regions that may contribute to andromonoecy. CONCLUSIONS: Collectively, our findings demonstrate stable breakdown of the dioecious system in D. oleifera, presumably also a result of genomic features of the Y-linked region.

Comparative Metabolomic and Transcriptomic Analyses Reveal Distinct Ascorbic Acid (AsA) Accumulation Patterns between PCA and PCNA Persimmon Developing Fruit.

Persimmon fruit has a high nutritional value and significantly varies between pollination-constant astringent (PCA) and pollination-constant non-astringent (PCNA) persimmons. The astringency type affects sugar, flavonoids, and tannin accumulation and is well known in persimmon fruit. However, the impact of the fruit astringency type on ascorbic acid (AsA) accumulation is limited. In this study, typical PCA varieties ('Huojing' and 'Zhongshi5') and PCNA varieties ('Yohou' and 'Jiro') of persimmon fruit were sampled at four developing stages (S1-S4) to provide valuable information on AsA content variation in PCA and PCNA persimmon. Persimmon fruit is rich in ascorbic acid; the AsA content of the four varieties 'Zhongshi5', 'Huojing', 'Jiro', and 'Youhou' mature fruit reached 104.49, 48.69, 69.69, and 47.48 mg/100 g. Fruit of the same astringency type persimmon showed a similar AsA accumulation pattern. AsA content was significantly higher in PCA than PCNA fruit at S1-S3. The initial KEGG analysis of metabolites showed that galactose metabolism is the major biosynthetic pathway of AsA in persimmon fruit. There were significant differences in galactose pathway-related metabolite content in developing PCA and PCNA fruit, such as Lactose, D-Tagatose, and D-Sorbitol content in PCA being higher than that of PCNA. Combined gene expression and WGCNA analyses showed that the expression of the GME (evm.TU.contig4144.37) gene was higher in PCA-type than in PCNA-type fruit in S1-S3 and exhibited the highest correlation with AsA content (r = 690 **, p < 0.01). Four hub genes, including the DNA methylation gene, methyltransferase gene, F-box, and Actin-like Protein, were identified as potential regulators of the GME gene. These results provide basic information on how astringency types affect AsA accumulation and will provide valuable information for further investigation on AsA content variation in persimmon fruit.

Preferential transport activity of DkDTX5/MATE5 affects the formation of different astringency in persimmon.

Proanthocyanidins (PAs) are specialized metabolites that influence persimmon fruit quality. Normal astringent (A)-type and non-astringent (NA)-type mutants show significant variation in PA accumulation, but the influencing mechanism remains unclear. In this study, among the six identified DTXs/MATEs proteins associated with PA accumulation, we observed that allelic variation and preferential transport by DkDTX5/MATE5 induced variation in PA accumulation for A-type and NA-type fruit. The expression pattern of DkDTX5/MATE5 was correlated with PA accumulation in NA-type fruit. Upregulation and downregulation of DkDTX5/MATE5 promoted and inhibited PA accumulation, respectively, in the NA-type fruit. Interestingly, transporter assays of Xenopus laevis oocytes indicated that DkDTX5/MATE5 preferentially transported the PA precursors catechin, epicatechin, and epicatechin gallate, resulting in their increased ratios relative to the total PAs, which was the main source of variation in PA accumulation between the A-type and NA-type. The allele lacking Ser-84 in DkDTX5/MATE5 was identified as a dominantly expressed gene in the A-type and lost its transport function. Site-directed mutagenesis revealed that DkDTX5/MATE5 binds to PA precursors via Ser-84. These findings clarify the association between the transporter function of DkDTX5/MATE5 and PA variation, and can contribute to the breeding of new cultivars with improved fruit quality.

Ongoing Rapid Evolution of a Post-Y Region Revealed by Chromosome-Scale Genome Assembly of a Hexaploid Monoecious Persimmon (Diospyros kaki).

Plants have evolved sex chromosomes independently in many lineages, and loss of separate sexes can also occur. In this study, we assembled a monoecious recently hexaploidized persimmon (Diospyros kaki), in which the Y chromosome has lost the maleness-determining function. Comparative genomic analysis of D. kaki and its dioecious relatives uncovered the evolutionary process by which the nonfunctional Y chromosome (or Ymonoecy) was derived, which involved silencing of the sex-determining gene, OGI, approximately 2 million years ago. Analyses of the entire X and Ymonoecy chromosomes suggested that D. kaki's nonfunctional male-specific region of the Y chromosome (MSY), which we call a post-MSY, has conserved some characteristics of the original functional MSY. Specifically, comparing the functional MSY in Diospyros lotus and the nonfunctional "post-MSY" in D. kaki indicated that both have been rapidly rearranged, mainly via ongoing transposable element bursts, resembling structural changes often detected in Y-linked regions, some of which can enlarge the nonrecombining regions. The recent evolution of the post-MSY (and possibly also MSYs in dioecious Diospyros species) therefore probably reflects these regions' ancestral location in a pericentromeric region, rather than the presence of male-determining genes and/or genes controlling sexually dimorphic traits.

Exogenous gibberellin delays maturation in persimmon fruit through transcriptional activators and repressors.

As the harvest season of most fruit is concentrated, fruit maturation manipulation is essential for the fresh fruit industry to prolong sales time. Gibberellin (GA), an important phytohormone necessary for plant growth and development, has also shown a substantial regulatory effect on fruit maturation; however, its regulatory mechanisms remain inconclusive. In this research, preharvest GA3 treatment effectively delayed fruit maturation in several persimmon (Diospyros kaki) cultivars. Among the proteins encoded by differentially expressed genes, 2 transcriptional activators (NAC TRANSCRIPTION FACTOR DkNAC24 and ETHYLENE RESPONSIVE FACTOR DkERF38) and a repressor (MYB-LIKE TRANSCRIPTION FACTOR DkMYB22) were direct regulators of GERANYLGERANYL DIPHOSPHATE SYNTHASE DkGGPS1, LYSINE HISTIDINE TRANSPORTER DkLHT1, and FRUCTOSE-BISPHOSPHATE ALDOLASE DkFBA1, respectively, resulting in the inhibition of carotenoid synthesis, outward transport of an ethylene precursor, and consumption of fructose and glucose. Thus, the present study not only provides a practical method to prolong the persimmon fruit maturation period in various cultivars but also provides insights into the regulatory mechanisms of GA on multiple aspects of fruit quality formation at the transcriptional regulation level.

Genetic basis of lineage-specific evolution of fruit traits in hexaploid persimmon.

Frequent polyploidization events in plants have led to the establishment of many lineage-specific traits representing each species. Little is known about the genetic bases for these specific traits in polyploids, presumably due to plant genomic complexity and their difficulties in applying genetic approaches. Hexaploid Oriental persimmon (Diospyros kaki) has evolved specific fruit characteristics, including wide variations in fruit shapes and astringency. In this study, using whole-genome diploidized/quantitative genotypes from ddRAD-Seq data of 173 persimmon cultivars, we examined their population structures and potential correlations between their structural transitions and variations in nine fruit traits. The population structures of persimmon cultivars were highly randomized and not substantially correlated with the representative fruit traits focused on in this study, except for fruit astringency. With genome-wide association analytic tools considering polyploid alleles, we identified the loci associated with the nine fruit traits; we mainly focused on fruit-shape variations, which have been numerically characterized by principal component analysis of elliptic Fourier descriptors. The genomic regions that putatively underwent selective sweep exhibited no overlap with the loci associated with these persimmon-specific fruit traits. These insights will contribute to understanding the genetic mechanisms by which fruit traits are independently established, possibly due to polyploidization events.

Allele-aware chromosome-level genome assembly of the autohexaploid Diospyros kaki Thunb.

Artificially improving persimmon (Diospyros kaki Thunb.), one of the most important fruit trees, remains challenging owing to the lack of reference genomes. In this study, we generated an allele-aware chromosome-level genome assembly for the autohexaploid persimmon 'Xiaoguotianshi' (Chinese-PCNA type) using PacBio CCS and Hi-C technology. The final assembly contained 4.52 Gb, with a contig N50 value of 5.28 Mb and scaffold N50 value of 44.01 Mb, of which 4.06 Gb (89.87%) of the assembly were anchored onto 90 chromosome-level pseudomolecules comprising 15 homologous groups with 6 allelic chromosomes in each. A total of 153,288 protein-coding genes were predicted, of which 98.60% were functionally annotated. Repetitive sequences accounted for 64.02% of the genome; and 110,480 rRNAs, 12,297 tRNAs, 1,483 miRNAs, and 3,510 snRNA genes were also identified. This genome assembly fills the knowledge gap in the autohexaploid persimmon genome, which is conducive in the study on the regulatory mechanisms underlying the major economically advantageous traits of persimmons and promoting breeding programs.

Transcriptome and Metabolome Reveal Distinct Sugar Accumulation Pattern between PCNA and PCA Mature Persimmon Fruit.

Persimmon (Diospyros kaki) fruit have significant variation between pollination-constant non-astringent (PCNA) and pollination-constant astringent (PCA) persimmons. The astringency type affects not only the soluble tannin concentration but also the accumulation of individual sugars. Thus, we comprehensively investigate the gene expression and metabolite profiles of individual sugars to resolve the formation of flavor differences in PCNA and PCA persimmon fruit. The results showed that soluble sugar, starch content, sucrose synthase, and sucrose invertase were significantly different between PCNA and PCA persimmon fruit. The sucrose and starch metabolism pathway was considerably enriched, and six sugar metabolites involving this pathway were significantly differentially accumulated. In addition, the expression patterns of diferentially expressed genes (such as bglX, eglC, Cel, TPS, SUS, and TREH genes) were significantly correlated with the content of deferentially accumulated metabolites (such as starch, sucrose, and trehalose) in the sucrose and starch metabolism pathway. These results indicated that the sucrose and starch metabolism pathway maintained a central position of sugar metabolism between PCNA and PCA persimmon fruit. Our results provide a theoretical basis for exploring functional genes related to sugar metabolism and provide useful resources for future studies on the flavor differences between PCNA and PCA persimmon fruit.

Transcriptomic Interpretation on Explainable AI-Guided Intuition Uncovers Premonitory Reactions of Disordering Fate in Persimmon Fruit.

Deep neural network (DNN) techniques, as an advanced machine learning framework, have allowed various image diagnoses in plants, which often achieve better prediction performance than human experts in each specific field. Notwithstanding, in plant biology, the application of DNNs is still mostly limited to rapid and effective phenotyping. The recent development of explainable CNN frameworks has allowed visualization of the features in the prediction by a convolutional neural network (CNN), which potentially contributes to the understanding of physiological mechanisms in objective phenotypes. In this study, we propose an integration of explainable CNN and transcriptomic approach to make a physiological interpretation of a fruit internal disorder in persimmon, rapid over-softening. We constructed CNN models to accurately predict the fate to be rapid softening in persimmon cv. Soshu, only with photo images. The explainable CNNs, such as Gradient-weighted Class Activation Mapping (Grad-Class Activation Mapping (CAM)) and guided Grad-CAM, visualized specific featured regions relevant to the prediction of rapid softening, which would correspond to the premonitory symptoms in a fruit. Transcriptomic analyses to compare the featured regions of the predicted rapid-softening and control fruits suggested that rapid softening is triggered by precocious ethylene signal-dependent cell wall modification, despite exhibiting no direct phenotypic changes. Further transcriptomic comparison between the featured and non-featured regions in the predicted rapid-softening fruit suggested that premonitory symptoms reflected hypoxia and the related stress signals finally to induce ethylene signals. These results would provide a good example for the collaboration of image analysis and omics approaches in plant physiology, which uncovered a novel aspect of fruit premonitory reactions in the rapid-softening fate.

Full-length transcriptome profiling for fruit development in Diospyros oleifera using nanopore sequencing.

OBJECTIVES: Diospyros oleifera, one of the most economically important Diospyros species, is an ideal model for studying the fruit development of persimmon. While, the lack of whole-transcriptome has hindered the complex transcriptional regulation mechanisms of sugar and tannin during fruit development. DATA DESCRIPTION: We applied Oxford Nanopore Technologies to six developmental stage of fruit from D. oleifera for use in transcriptome sequencing. As a result of full-length transcriptome sequencing, 55.87 Gb of clean data were generated. After mapping onto the reference genome of D. oleifera, 51,588 full-length collapsing transcripts, including 2,727 new gene loci and 43,223 transcripts, were obtained. Comprehensively annotated, 38,086 of new transcripts were functional annotation, and 972 lncRNAs, 7,159 AS events were predicted. Here, we released the transcriptome database of D. oleifera at different stage of fruit development,which will provide a fundamention of to investigatethe transcript structure, variants and evolution of persimmon.

Transcriptomic profiling analysis to identify genes associated with PA biosynthesis and insolubilization in the late stage of fruit development in C-PCNA persimmon.

PA-enhanced content causes astringency in persimmon fruit. PCNA persimmons can lose their astringency naturally and they become edible when still on the tree, which allows for conserves of physical and financial resources. C-PCNA persimmon originates in China. Its deastringency trait primarily depends on decreased PA biosynthesis and PA insolubilization at the late stage of fruit development. Although some genes and transcription factors that may be involved in the deastringency of C-PCNA persimmon have been reported, the expression patterns of these genes during the key deastringency stage are reported less. To investigate the variation in PA contents and the expression patterns of deastringency-related genes during typical C-PCNA persimmon 'Xiaoguo-tianshi' fruit development and ripening, PA content and transcriptional profiling were carried out at five late stages from 70 to 160 DAF. The combinational analysis phenotype, PA content, and DEG enrichment revealed that 120-140 DAF and 140-160 DAF were the critical phases for PA biosynthesis reduction and PA insolubilization, respectively. The expression of PA biosynthesis-associated genes indicated that the downregulation of the ANR gene at 140-160 DAF may be associated with PA biosynthesis and is decreased by inhibiting its precursor cis-flavan-3-ols. We also found that a decrease in acetaldehyde metabolism-associated ALDH genes and an increase in ADH and PDC genes might result in C-PCNA persimmon PA insolubilization. In addition, a few MYB-bHLH-WD40 (MBW) homologous transcription factors in persimmon might play important roles in persimmon PA accumulation. Furthermore, combined coexpression network analysis and phylogenetic analysis of MBW suggested that three putative transcription factors WD40 (evm.TU.contig1.155), MYB (evm.TU.contig8910.486) and bHLH (evm.TU.contig1398.203), might connect and co-regulate both PA biosynthesis and its insolubilization in C-PCNA persimmon. The present study elucidated transcriptional insights into PA biosynthesis and insolubilization during the late development stages based on the C-PCNA D. kaki genome (unpublished). Thus, we focused on PA content variation and the expression patterns of genes involved in PA biosynthesis and insolubilization. Our work has provided additional evidence on previous knowledge and a basis for further exploration of the natural deastringency of C-PCNA persimmon.

DkmiR397 Regulates Proanthocyanidin Biosynthesis via Negative Modulating DkLAC2 in Chinese PCNA Persimmon.

Persimmon fruits accumulate a large amount of proanthocyanidins (PAs), which makes an astringent sensation. Proanthocyanidins (PAs) are the polymers of flavan-3-ols stored in plant vacuoles under laccase activation. A laccase gene, DkLAC2, is putatively involved in PAs biosynthesis and regulated by microRNA (DkmiR397) in persimmon. However, the polymerization of PAs in association with miRNA397 still needs to be explored in persimmon. Here, we identified pre-DkmiR397 and its target gene DkLAC2 in 'Eshi 1' persimmon. Histochemical staining with GUS and dual luciferase assay both confirmed DkmiR397-DkLAC2 binding after co-transformation in tobacco leaves. Diverse expression patterns of DkLAC2 and DkmiR397 were exhibited during persimmon fruit development stages. Moreover, a contrasting expression pattern was also observed after the combined DkLAC2-miR397 transformation in persimmon leaves, suggesting that DkmiR397 might be a negative regulator of DkLAC2. Similarly, the transient transformation of DkmiR397 in persimmon fruit discs in vitro also reduced PA accumulation by repressing DkLAC2, whereas the up-regulation of DkLAC2 increased the accumulation of PAs by short tandem target mimic STTM-miR397. A similar expression pattern was observed when overexpressing of DkLAC2 in Arabidopsis wild type (WT) and overexpression of DkLAC2, DkmiR397 in persimmon leaf callus. Our results revealed that the role of DkmiR397 repressed the expression of DkLAC2 concerning PA biosynthesis, providing a potential target for the manipulation of PAs metabolism in persimmon.

Reinvention of hermaphroditism via activation of a RADIALIS-like gene in hexaploid persimmon.

In flowering plants, different lineages have independently transitioned from the ancestral hermaphroditic state into and out of various sexual systems1. Polyploidizations are often associated with this plasticity in sexual systems2,3. Persimmons (the genus Diospyros) have evolved dioecy via lineage-specific palaeoploidizations. More recently, hexaploid D. kaki has established monoecy and also exhibits reversions from male to hermaphrodite flowers in response to natural environmental signals (natural hermaphroditism, NH), or to artificial cytokinin treatment (artificial hermaphroditism, AH). We sought to identify the molecular pathways underlying these polyploid-specific reversions to hermaphroditism. Co-expression network analyses identified regulatory pathways specific to NH or AH transitions. Surprisingly, the two pathways appeared to be antagonistic, with abscisic acid and cytokinin signalling for NH and AH, respectively. Among the genes common to both pathways leading to hermaphroditic flowers, we identified a small-Myb RADIALIS-like gene, named DkRAD, which is specifically activated in hexaploid D. kaki. Consistently, ectopic overexpression of DkRAD in two model plants resulted in hypergrowth of the gynoecium. These results suggest that production of hermaphrodite flowers via polyploidization depends on DkRAD activation, which is not associated with a loss-of-function within the existing sex determination pathway, but rather represents a new path to (or reinvention of) hermaphroditism.

Population Analysis of Diospyros lotus in the Northwestern Caucasus Based on Leaf Morphology and Multilocus DNA Markers.

Diospyros lotus is the one of the most frost-tolerant species in the Diospyros genera, used as a rootstock for colder regions. Natural populations of D. lotus have a fragmented character of distribution in the Northwestern Caucasus, one of the coldest regions of Diospyros cultivation. To predict the behavior of D. lotus populations in an extreme environment, it is necessary to investigate the intraspecific genetic diversity and phenotypic variability of populations in the colder regions. In this study, we analyzed five geographically distant populations of D. lotus according to 33 morphological leaf traits, and the most informative traits were established, namely, leaf length, leaf width, leaf index (leaf to length ratio) and the length of the fourth veins. Additionally, we evaluated the intraspecific genetic diversity of D. lotus using ISSR and SCoT markers and proposed a new parameter for the evaluation of genetic polymorphism among populations, in order to eliminate the effect of sample number. This new parameter is the relative genetic polymorphism, which is the ratio of polymorphism to the number of samples. Based on morphological and genetic data, the northernmost population from Shkhafit was phenotypically and genetically distant from the other populations. The correspondence between several morphological traits (leaf width, leaf length and first to fifth right vein angles) and several marker bands (SCoT5, SCoT7, SCoT30: 800-1500 bp; ISSR13, ISSR14, ISSR880: 500-1000 bp) were observed for the Shkhafit population. Unique SCoT and ISSR fragments can be used as markers for breeding purposes. The results provide a better understanding of adaptive mechanisms in D. lotus in extreme environments and will be important for the further expansion of the cultivation area for persimmon in colder regions.

Interspecific Hybridization and Island Colonization History, not Rarity, Most Strongly Affect the Genetic Diversity in Diospyros a Clade of Mascarene-Endemic Trees.

Many factors shape the genetic diversity of island-endemic trees, with important implications for conservation. Oceanic island-endemic lineages undergo an initial founding bottleneck during the colonization process and subsequently accumulate diversity following colonization. Moreover, many island endemics occur in small populations and are further threatened by anthropogenic factors that cause population declines, making them susceptible to losses in genetic diversity through genetic drift, inbreeding, and bottlenecks. However, life-history traits commonly found in trees, such as outcrossing mechanisms, long lifespans, and a propensity for interspecific hybridization, may help buffer against losses of genetic variation. To assess the relative importance of colonization history, rarity, and distribution in shaping genetic diversity of island-endemic trees, we conducted a comparative population genomic analysis of 13 species of Diospyros (Ebenaceae) endemic to the Mascarene Islands that differ in island colonization history, distribution, population size, and IUCN threat status. We genotyped 328 individuals across the islands using 2b-RADseq, compared genetic diversity both among and within species, and assessed patterns of genetic structure. Genetic diversity did not vary significantly by IUCN status, but we found that species that co-occur with others on the same intermediate-aged island (Mauritius) had much greater genetic diversity than those that occur solitarily on an island (Réunion and Rodrigues), likely because of greater interspecific hybridization among species with overlapping distributions and processes related to time since island colonization. Results presented here were used to determine priority localities for in situ and ex situ conservation efforts to maximize the genetic diversity of each Mascarene Diospyros species.

Genome Sequence Resource of the Causal Agent of Persimmon Anthracnose, Colletotrichum horii Strain SD010 from China.

Colletotrichum horii is a main causal agent of persimmon (Diospyros kaki) anthracnose and is distributed widely in persimmon-producing areas of the world. Here, we report the first high-quality draft genome sequence of C. horii strain SD010. This will provide a reference for understanding adaptive evolution of genome structure, genes, and population diversity among members of the C. gloeosporioides species complex, and also help in understanding the mechanisms of host-pathogen interactions and improve management strategies of anthracnose.

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.

Genomics-based discrimination of 2n gamete formation mechanisms in polyploids: a case study in nonaploid Diospyros kaki 'Akiou'.

Unreduced gametes (2n gametes), possessing double the haploid genome, whatever ploidy that happens to be, are a common source of ploidy variation in plant populations. First and second division restitution (FDR and SDR) are the dominant mechanisms of 2n gamete production; all else being equal, FDR gametes have a higher degree of heterozygosity, thus they are advantageous in breeding. The discrimination of these mechanisms from the consequence of hybridization is challenging, especially in higher polyploids, and usually requires information on centromere location. In this study, we propose a genotyping-based strategy to uncover the mechanisms of 2n gamete formation in progeny that has a higher ploidy than its parents. Simulation of 2n gamete production revealed that FDR and SDR pathways can be discriminated based on allele transmission patterns alone without information on centromere location. We applied this strategy to study the formation mechanism of a nonaploid Diospyros kaki 'Akiou', which was bred via hybridization between D. kaki hexaploid cultivars. The result demonstrated that 'Akiou' was derived from the fertilization of a normal female gamete by a 2n male gamete and that this 2n gamete was produced through FDR. Consequently, the distinct duplex transmission pattern in the FDR gamete enabled us to infer the genomic characteristics of polyploid persimmon. The method could be tested only for the plant being polypoid, which allows for the ability to discriminate causes of 2n gamete formation using allele dosage in progeny, and will be useful in future studies of polyploid genomics.

The regulation of DKGA2ox1 and miR171f_3 in scion dwarfing with Diospyros kaki Thunb. cv. 'Nan-tong-xiao-fang-shi' as interstocks.

MAIN CONCLUSION: High-throughput sequencing and yeast one and two-hybrid library screening reveal that DKGA2ox1 and miR171f_3 are involved in the regulation of scion dwarfing with 'Nan-tong-xiao-fang-shi' as interstocks. Diospyros kaki Thunb. cv. Nan-tong-xiao-fang-shi ('Nan-tong-xiao-fang-shi') interstocks play a critical role in the scion dwarfing. However, the understanding of the molecular signaling pathways that regulate the scion dwarfing with 'Nan-tong-xiao-fang-shi' as interstocks remain unclear. In this work, the regulatory network in the scion dwarfing with 'Nan-tong-xiao-fang-shi' as interstocks was identified. Using a yeast one-hybrid library screening, luciferase activity analysis, luciferase complementation imaging assays and GFP signal detection, a SPL transcription factor named Diospyros kaki SPL (DKSPL), potentially functioning as a transcriptional activator of the Diospyros kaki GA2ox1 (DKGA2ox1) gene, was identified as a key stimulating factor in the persimmon growth and development. The DKSPL was found in the nucleus, and might play a role in the transcriptional regulation system. A microRNA named miR171f_3 was identified, which might act as a negative regulator of Diospyros kaki SCR (DKSCR) in persimmon. The interactions between DKSCR and seven proteins were experimentally validated with a yeast two-hybrid library screening. Compared to the non-grafted wildtype persimmon, the tissue section of graft union healed well due to the increased expression of cinnamyl-alcohol dehydrogenase. These results indicate that DKGA2ox1 and miR171f_3 may co-promote the scion dwarfing by plant hormone signal transduction pathways.