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Infection of tomato leaves and fruits by necrotrophic fungal pathogen Botrytis cinerea causes considerable economic loss. Melatonin has multiple physiological functions and is considered a potential biocontrol agent for its positive effects on plant innate immunity. In tomato, melatonin reduces Botrytis-caused post-harvest fruit decay by activating jasmonic acid (JA) signaling. In this study, we analyzed the effects of melatonin on resistance to Botrytis and associated defense mechanisms in tomato leaves. Treatment of detached tomato leaves with melatonin at a concentration as low as 100 µM induced resistance to Botrytis based on reduced lesion size and fungal growth. Melatonin-induced resistance to gray mold was associated with activation of a complex set of defense mechanisms in tomato leaves. First, melatonin suppressed Botrytis-induced cell death and accumulation of reactive oxygen species (ROS), which was correlated with increased activities of ROS-scavenging enzymes and enhanced expression of their corresponding genes. Second, melatonin treatment induced callose synthase genes and promoted rapid and strong callose deposition in tomato leaves. Melatonin also induced other anti-microbial genes encoding chitinases, β-1,3-glucanases, phenylalanine ammonialyases and polyphenol oxidases. Third, melatonin increased Botrytis-induced expression of genes encoding SlWRKY33, SlMYC2 and SlERF transcription factors, which activate defense-related genes expression through JA and ethylene (ET) signaling pathways. By contrast, melatonin suppressed salicylic acid (SA) signaling based on reduced expression of the SlPR1 marker gene, which is known to antagonize defense against Botrytis. These results collectively indicate that exogenous melatonin orchestrates multiple signaling pathways to activate a complex set of defense mechanisms against Botrytis in tomato leaves.

The main units of persimmon proanthocyanidins (PAs) are composed of flavan-3-ols including epigallocatechin gallate (EGCG) and gallocatechin gallate (GCG). Precise quantification of GCG is challenging due to its trace amounts in persimmon. In this study, to establish the optimal UHPLC-Q-Exactive Orbitrap/MS technique for the determination of PAs monomer composition in persimmon fruit flesh of different astringency types, mass spectrometry and chromatographic conditions were optimized. The results showed that when operating in negative ion mode, using a T3 chromatographic column (a type of C18 column with high-strength silica), acetonitrile as the organic phase, a 0.1% mobile phase acid content, and a mobile phase flow rate of 0.2 mL/min, the chromatographic peak shape and resolution of the PAs monomer composition improved. Additionally, there was no tailing phenomenon observed in the chromatographic peaks. At the same time, the intra-day and inter-day precision, stability, and recovery of the procedure were good. The relative standard deviation (RSD) of stability was less than 5%. The intra-day precision was in the range of 1.14% to 2.36%, and the inter-day precision ranged from 1.03% to 2.92%, both of which were less than 5%. The recovery rate ranged from 94.43% to 98.59% with an RSD less than 5%. The results showed that the UHPLC-Q-Exactive Orbitrap/MS technique established in this study can not only be used for the quantification of EGCG and GCG in persimmon fruit flesh but also be suitable for analyzing other PAs monomer compositions, providing robust support for the related research on persimmon PAs.

The tomato is a fruit vegetable rich in nutritional and medicinal value grown in greenhouses and fields worldwide. It is severely sensitive to heat stress, which frequently occurs with rising global warming. Predictions indicate a 0.2 °C increase in average surface temperatures per decade for the next three decades, which underlines the threat of austere heat stress in the future. Previous studies have reported that heat stress adversely affects tomato growth, limits nutrient availability, hammers photosynthesis, disrupts reproduction, denatures proteins, upsets signaling pathways, and damages cell membranes. The overproduction of reactive oxygen species in response to heat stress is toxic to tomato plants. The negative consequences of heat stress on the tomato have been the focus of much investigation, resulting in the emergence of several therapeutic interventions. However, a considerable distance remains to be covered to develop tomato varieties that are tolerant to current heat stress and durable in the perspective of increasing global warming. This current review provides a critical analysis of the heat stress consequences on the tomato in the context of global warming, its innate response to heat stress, and the elucidation of domains characterized by a scarcity of knowledge, along with potential avenues for enhancing sustainable tolerance against heat stress through the involvement of diverse advanced technologies. The particular mechanism underlying thermotolerance remains indeterminate and requires further elucidatory investigation. The precise roles and interplay of signaling pathways in response to heat stress remain unresolved. The etiology of tomato plants’ physiological and molecular responses against heat stress remains unexplained. Utilizing modern functional genomics techniques, including transcriptomics, proteomics, and metabolomics, can assist in identifying potential candidate proteins, metabolites, genes, gene networks, and signaling pathways contributing to tomato stress tolerance. Improving tomato tolerance against heat stress urges a comprehensive and combined strategy including modern techniques, the latest apparatuses, speedy breeding, physiology, and molecular markers to regulate their physiological, molecular, and biochemical reactions.

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

Apricot is a widely cultivated fruit tree of the drupe family, and its sweet/bitter kernel traits are important indicators of the quality and merchantability of apricots. The sweetness/bitterness traits were mainly determined by amygdalin content. However, the lack of high-quality genomes has limited insight into the traits. In this study, a high-quality genome of ‘Xiaobaixing’ was obtained by using single-molecule sequencing and chromosome-conformation capture techniques, with eight chromosomes of 0.21 Gb in length and 52.80% repetitive sequences. A total of 29,157 protein-coding genes were predicted with contigs N50 = 3.56 Mb and scaffold N50 = 26.73 Mb. Construction of phylogenetic trees of 15 species of Rosaceae fruit trees, with ‘Xiaobaixing’ differentiated by 5.3 Ma as the closest relative to ‘Yinxiangbai’. GO functional annotation and KEGG enrichment analysis identified 227 specific gene families to ‘Xiaobaixing’, with 569 expansion-gene families and 1316 contraction-gene families, including the significant expansion of phenylalanine N-monooxygenase and β-glucosidase genes associated with amygdalin synthesis, significant contraction of wild black cherry glucoside β-glucosidase genes, amygdalin β-glucosidase genes, and β-glucosidase genes, and significant enrichment of positively selected genes in the cyanogenic amino acid metabolic pathway. The 88 bHLH genes were identified in the genome of ‘Xiaobaixing’, and ParbHLH66 (rna-Par24659.1) was found to be a key gene for the identification of sweet/bitter kernels of apricots. The amino acid sequence encoded by its gene is highly conserved in the species of Prunus mume, Prunus dulcis, Prunus persica, and Prunus avium and may be participating in the regulation of amygdalin biosynthesis, which provides a theoretical foundation for the molecular identification of sweet/bitter kernels of apricots.

Abstract 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.

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.

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.

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.

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.

Semantic segmentation of mitochondria from electron microscopy (EM) images is an essential step to obtain reliable morphological statistics about mitochondria. However, automatically delineating plenty of mitochondria of varied shapes from complex backgrounds with sufficient accuracy is challenging. To address these challenges, we develop a hierarchical encoder-decoder network (HED-Net), which has a three-level nested U-shape architecture to capture rich contextual information. Given the irregular shape of mitochondria, we introduce a novel soft label-decomposition strategy to exploit shape knowledge in manual labels. Rather than simply using the ground truth label maps as the unique supervision in the model training, we introduce additional subcategory-aware supervision by softly decomposing each manual label map into two complementary label maps according to mitochondria's ovality. The three label maps are integrated with our HED-Net to supervise the model training. While the original label map guides the network to segment all the mitochondria of varied shapes, the auxiliary label maps guide the network to segment subcategories of mitochondria of circular shape and elliptic shape, respectively, which are much more manageable tasks. Extensive experiments on two public benchmarks show that our HED-Net performs favorably against state-of-the-art methods.

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 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.

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 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.

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.

Husk and pellicle as the agri-food waste in the walnut-product industry are in soaring demand because of their rich polyphenol content. This study investigated the differential compounds related to walnut polyphenol between husk and pellicle during fruit development stage. By using ultra-high performance liquid chromatography-quadrupole-orbitrap (UHPLC-Q-Orbitrap), a total of 110 bioactive components, including hydrolysable tannins, flavonoids, phenolic acids and quinones, were tentatively identified, 33 of which were different between husk and pellicle. The trend of dynamic content of 16 polyphenols was clarified during walnut development stage by high-performance liquid chromatography (HPLC). This is the first time to comprehensive identification of phenolic compounds in walnut husk and pellicle, and our results indicated that the pellicle is a rich resource of polyphenols. The dynamic trend of some polyphenols was consistent with total phenols. The comprehensive characterization of walnut polyphenol and quantification of main phenolic compounds will be beneficial for understanding the potential application value of walnut and for exploiting its metabolism pathway.

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 F1 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.