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Fruit ripening is a highly coordinated process involving molecular and biochemical changes that collectively determine fruit quality. The underlying metabolic programs and their transitions leading to fruit ripening remain largely under-characterized in blueberry (Vaccinium sp.), which exhibits atypical climacteric behavior. In this study, we focused on sugar, acid and anthocyanin metabolism in two rabbiteye blueberry cultivars, Premier and Powderblue, during fruit development and ripening. Concentrations of the three major sugars, sucrose (Suc), glucose (Glc), and fructose (Fru) increased steadily during fruit development leading up to ripening, and increased dramatically by around 2-fold in 'Premier' and 2- to 3-fold in 'Powderblue' during the final stage of fruit ripening. Starch concentration was very low throughout fruit development in both cultivars indicating that it does not serve the role of a major transitory carbon (C) storage form in blueberry fruit. Together, these patterns indicate continued import of C, likely in the form of Suc, throughout blueberry fruit development. Concentrations of the predominant acids, malate and quinate, decreased during ripening, and may contribute to increased shikimate biosynthesis which, in-turn, allows for downstream phenylpropanoid metabolism leading to anthocyanin synthesis. Consistently, anthocyanin concentrations were highest in fully ripened blue fruit. Weighted gene co-expression network analysis (WGCNA) was performed using a 'Powderblue' fruit ripening transcriptome and targeted fruit metabolite concentration data. A 'dark turquoise' module positively correlated with sugars and anthocyanins, and negatively correlated with acids (malate, quinate), was identified. Gene Ontology (GO) enrichment analysis of this module identified transcripts related to sugar, acid, and phenylpropanoid metabolism pathways. Among these, increased transcript abundance of a VACUOLAR INVERTASE during ripening was consistent with sugar storage in the vacuole. In general, transcript abundance of the glycolysis pathway genes was upregulated during ripening. The transcript abundance of PHOSPHOENOLPYRUVATE (PEP) CARBOXYKINASE increased during fruit ripening and was negatively correlated with malate concentration, suggesting increased malate conversion to PEP, which supports anthocyanin production during fruit ripening. This was further supported by the co-upregulation of several anthocyanin biosynthesis-related genes. Together, this study provides insights into important metabolic programs, and their underlying gene expression patterns during fruit development and ripening in blueberry., (© 2024. The Author(s).)

Blueberries ( Vaccinium spp.) are extremely sensitive to drought stress. Flavonoids are crucial secondary metabolites that possess the ability to withstand drought stress. Therefore, improving the drought resistance of blueberries by increasing the flavonoid content is crucial for the development of the blueberry industry. To explore the underlying molecular mechanism of blueberry in adaptation to drought stress, we performed an integrated analysis of the metabolome and transcriptome of blueberry leaves under drought stress. We found that the most enriched drought-responsive genes are mainly involved in flavonoid biosynthesis and plant hormone signal transduction pathways based on transcriptome data and the main drought-responsive metabolites come from the flavonoid class based on metabolome data. The UDP-glucose flavonoid 3-O-glucosyl transferase ( UFGT ), flavonol synthase ( FLS ), and anthocyanidin reductase ( ANR-2 ) genes may be the key genes for the accumulation of anthocyanins, flavonols, and flavans in response to drought stress in blueberry leaves, respectively. Delphinidin 3-glucoside and delphinidin-3-O-glucoside chloride may be the most important drought-responsive flavonoid metabolites. VcMYB1 , VcMYBPA1 , MYBPA1.2 , and MYBPA2.1 might be responsible for drought-induced flavonoid biosynthesis and VcMYB14, MYB14, MYB102 , and MYB108 may be responsible for blueberry leaf drought tolerance. ABA responsive elements binding factor ( ABF ) genes, MYB genes, bHLH genes, and flavonoid biosynthetic genes might form a regulatory network to regulate drought-induced accumulation of flavonoid metabolites in blueberry leaves. Our study provides a useful reference for breeding drought-resistant blueberry varieties.

Molecular hydrogen is beneficial for fruits quality improvement. However, the mechanism involved, especially cellular metabolic responses, has not been well established. Here, the integrated widely targeted metabolomics analysis (UPLC-MS/MS) and biochemical evidence revealed that hydrogen-based irrigation could orchestrate, either directly or indirectly, an array of physiological responses in blueberry (Vaccinium spp.) during harvesting stage, especially for the delayed senescence in harvested stage (4 °C for 12 d). The hubs to these changes are wide-ranging metabolic reprogramming and antioxidant machinery. A total of 1208 distinct annotated metabolites were identified, and the characterization of differential accumulated metabolites (DAMs) revealed that the reprogramming, particularly, involves phenolic acids and flavonoids accumulation. These changes were positively matched with the transcriptional profiles of representative genes for their synthesis during the growth stage. Together, our findings open a new window for development of hydrogen-based agriculture that increases the shelf-life of fruits in a smart and sustainable manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Yan Zeng, Xu Cheng, and Pathier Didier are employees and hold ownership interests (including patents) in Air Liquide (China) R&D Co., Ltd., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Postharvest blueberry fruit is prone to softening. Protein phosphorylation is an important post-translational modification that was involved in fruit softening. However, little is known about protein phosphorylation in postharvest blueberry fruit softening. The firmness, the apparent morphology, and cell structures of blueberry fruit were changed. As the decay rate of postharvest blueberry fruit increased, the soluble solid and titrable acid contents decreased significantly. Phosphoproteomic sequencing results showed that there were 4100 phosphorylated peptides, 5635 phosphorylated sites, and 1437 phosphorylated proteins and showed significant differences on 0 and 8 d. The GO database and KEGG pathway results indicated that these phosphorylated proteins were mainly involved in "biological process", "molecular function", "plant hormone signal transduction", and "metabolic pathways". Besides, a number of phosphorylated proteins were found in cell wall metabolism, plant hormone signaling, primary metabolism, energy metabolism, membrane and transport, ubiquitination-based proteins, and other metabolisms.

Blueberry (Vaccinium spp.) is among the most-consumed soft fruit and has been recognized as an important source of health-promoting compounds. Highly perishable and susceptible to rapid spoilage due to fruit softening and decay during postharvest storage, modern breeding programs are looking to maximize the quality and extend the market life of fresh blueberries. However, it is uncertain how genetically controlled postharvest quality traits are in blueberries. This study aimed to investigate the prediction ability and the genetic basis of the main fruit quality traits affected during blueberry postharvest to create breeding strategies for developing cultivars with an extended shelf life. To achieve this goal, we carried out target genotyping in a breeding population of 588 individuals and evaluated several fruit quality traits after 1 day, 1 week, 3 weeks, and 7 weeks of postharvest storage at 1°C. Using longitudinal genome-based methods, we estimated genetic parameters and predicted unobserved phenotypes. Our results showed large diversity, moderate heritability, and consistent predictive accuracies along the postharvest storage for most of the traits. Regarding the fruit quality, firmness showed the largest variation during postharvest storage, with a surprising number of genotypes maintaining or increasing their firmness, even after 7 weeks of cold storage. Our results suggest that we can effectively improve the blueberry postharvest quality through breeding and use genomic prediction to maximize the genetic gains in the long term. We also emphasize the potential of using longitudinal genomic prediction models to predict the fruit quality at extended postharvest periods by integrating known phenotypic data from harvest., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Genomic prediction is a modern approach that uses genome-wide markers to predict the genetic merit of unphenotyped individuals. With the potential to reduce the breeding cycles and increase the selection accuracy, this tool has been designed to rank genotypes and maximize genetic gains. Despite this importance, its practical implementation in breeding programs requires critical allocation of resources for its application in a predictive framework. In this study, we integrated genetic and data-driven methods to allocate resources for phenotyping and genotyping tailored to genomic prediction. To this end, we used a historical blueberry (Vaccinium corymbosun L.) breeding dataset containing more than 3000 individuals, genotyped using probe-based target sequencing and phenotyped for three fruit quality traits over several years. Our contribution in this study is threefold: (i) for the genotyping resource allocation, the use of genetic data-driven methods to select an optimal set of markers slightly improved prediction results for all the traits; (ii) for the long-term implication, we carried out a simulation study and emphasized that data-driven method results in a slight improvement in genetic gain over 30 cycles than random marker sampling; and (iii) for the phenotyping resource allocation, we compared different optimization algorithms to select training population, showing that it can be leveraged to increase predictive performances. Altogether, we provided a data-oriented decision-making approach for breeders by demonstrating that critical breeding decisions associated with resource allocation for genomic prediction can be tackled through a combination of statistics and genetic methods., (© 2024 The Author(s). The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Leaf economic spectrum (LES) relationships have been studied across many different plant lineages and at different organizational scales. However, the temporal stability of the LES relationships is largely unknown. We used the wild blueberry system with high genotypic diversity to test whether trait-trait relationships across genotypes demonstrate the same LES relationships found in the global database (GLOPNET) and whether they are stable across years. We studied leaf structure, photosynthesis, and leaf nutrients for 16 genotypes of two wild blueberry species semi-naturally grown in a common farm in Maine, USA, across 4 yr. We found substantial variation in leaf structure, physiology, and nutrient traits within and among genotypes, as well as across years in wild blueberries. The LES trait-trait relationships (covariance structure) across genotypes were not always found in all years. The trait syndrome of wild blueberries was shifted by changing environmental conditions over the years. Additionally, traits in 1 yr cannot be used to predict those of another year. Our findings show that LES generally holds among genotypes but is temporally unstable, stressing the significant influence of trait plasticity in response to fluctuating environmental conditions across years, and the importance of temporal dimensions in shaping functional traits and species coexistence., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Vaccinieae is a morphologically diverse and species-rich (∼1430 species) tribe in Ericaceae. Although the majority of diversity is tropical, Vaccinieae are best known for temperate crops (i.e., blueberries, cranberries, and lingonberries) in Vaccinium. Vaccinium itself (∼500 species) has been previously suggested as highly polyphyletic and taxonomic boundaries among many of the other genera in the tribe remain uncertain. We assessed the evolutionary history of Vaccinieae with phylogenomic analyses based on a target-enrichment dataset containing 256 low-copy nuclear loci and 210 species representing 30 of the 35 genera in the tribe and 25 of the 29 sections of Vaccinium. We conducted time-calibrated biogeographic analyses and diversification analyses to explore the area of origin and global dispersal history of the tribe. The analysis recovered a temperate North American origin for Vaccinieae approximately 30 million years ago. Tropical diversity of Vaccinieae was inferred to result from multiple, independent movements into the tropics from north-temperate ancestors. Diversification rate increases corresponded to radiation into the Andes and SE Asia. The pseudo-10-locular ovary evolved once in the tribe from the five-locular state, coinciding with the diversification of a major clade that includes most Asian Vaccinium and the group from which commercial blueberries are derived (V. sect. Cyanococcus). A reconstruction from available chromosome counts suggests that a major polyploid event predated the evolution of nearly half the diversity of Vaccinieae. The extent of polyphyly in Vaccinium documented here supports the need for a generic reclassification of the tribe., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Pollination is crucial to obtaining optimal blueberry yield and fruit quality. Despite substantial investments in seasonal beekeeping services, blueberry producers consistently report suboptimal pollinator visitation and fruit set in some cultivars. Flower morphology and floral rewards are among the key factors that have shown to contribute to pollinator attraction, however little is known about their relative importance for improving yield in the context of plant breeding. Clarifying the relationships between flower morphology, nectar reward content, pollinator recruitment, and pollination outcomes, as well as their genetic components, can inform breeding priorities for enhancing blueberry production. In the present study, we measured ten flower and nectar traits and indices of successful pollination, including fruit set, seed count, and fruit weight in 38 southern highbush blueberry genotypes. Additionally, we assessed pollinator visitation frequency and foraging behavior over two growing seasons. Several statistical models were tested to optimize the prediction of pollinator visitation and pollination success, including partial least squares, BayesB, ridge-regression, and random forest., Results: Random forest models obtained high predictive abilities for pollinator visitation frequency, with values of 0.54, 0.52, and 0.66 for honey bee, bumble bee, and total pollinator visits, respectively. The BayesB model provided the most consistent prediction of fruit set, fruit weight, and seed set, with predictive abilities of 0.07, -0.08, and 0.42, respectively. Variable importance analysis revealed that genotypic differences in nectar volume had the greatest impact on honey bee and bumble bee visitation, although preferences for flower morphological traits varied depending on the foraging task. Flower density was a major driving factor attracting nectar-foraging honey bees and bumble bees, while pollen-foraging bumble bees were most influenced by flower accessibility, specifically corolla length and the length-to-width ratio., Conclusions: Honey bees comprised the majority of pollinator visits, and were primarily influenced by nectar volume and flower density. Corolla length and the length-to-width ratio were also identified as the main predictors of fruit set, fruit weight, seed count, as well as pollen-foraging bumble bee visits, suggesting that these bees and their foraging preferences may play a pivotal role in fruit production. Moderate to high narrow-sense heritability values (ranging from 0.30 to 0.77) were obtained for all floral traits, indicating that selective breeding efforts may enhance cultivar attractiveness to pollinators., (© 2024. The Author(s).)

Background: The DELLA proteins, a class of GA signaling repressors, belong to the GRAS family of plant-specific nuclear proteins. Members of DELLA gene family encode transcriptional regulators with diverse functions in plant development and abiotic stress responses. To date, DELLAs have been identified in various plant species, such as Arabidopsis thaliana, Malus domestica, Populus trichocarpa, and other land plants. Most information of DELLA family genes was obtained from A. thaliana, whereas little is known about the DELLA gene family in blueberry., Results: In this study, we identified three DELLA genes in blueberry (Vaccinium darrowii, VdDELLA) and provided a complete overview of VdDELLA gene family, describing chromosome localization, protein properties, conserved domain, motif organization, and phylogenetic analysis. Three VdDELLA members, containing two highly conserved DELLA domain and GRAS domain, were distributed across three chromosomes. Additionally, cis-acting elements analysis indicated that VdDELLA genes might play a critical role in blueberry developmental processes, hormone, and stress responses. Expression analysis using quantitative real-time PCR (qRT-PCR) revealed that all of three VdDELLA genes were differentially expressed across various tissues. VdDELLA2 was the most highly expressed VdDELLA in all denoted tissues, with a highest expression in mature fruits. In addition, all of the three VdDELLA genes actively responded to diverse abiotic stresses. Based on qRT-PCR analysis, VdDELLA2 might act as a key regulator in V. darrowii in response to salt stress, whereas VdDELLA1 and VdDELLA2 might play an essential role in cold stress response. Under drought stress, all of three VdDELLA genes were involved in mediating drought response. Furthermore, their transiently co-localization with nuclear markers in A. thaliana protoplasts demonstrated their transcriptional regulator roles., Conclusions: In this study, three VdDELLA genes were identified in V. darrowii genome. Three VdDELLA genes were closely related to the C. moschata DELLA genes, S. lycopersicum DELLA genes, and M. domestica DELLA genes, respectively, indicating their similar biological functions. Expression analysis indicated that VdDELLA genes were highly efficient in blueberry fruit development. Expression patterns under different stress conditions revealed the differentially expressed VdDELLA genes responding to salt, drought, and cold stress. Overall, these results enrich our understanding of evolutionary relationship and potential functions of VdDELLA genes, which provide valuable information for further studies on genetic improvement of the plant yield and plant resistance., (© 2024. The Author(s).)

Background: Anthocyanins are water-soluble flavonoids in plants, which give plants bright colors and are widely used as food coloring agents, nutrients, and cosmetic additives. There are several limitations for traditional techniques of collecting anthocyanins from plant tissues, including species, origin, season, and technology. The benefits of using engineering microbial production of natural products include ease of use, controllability, and high efficiency., Results: In this study, ten genes encoding enzymes involved in the anthocyanin biosynthetic pathway were successfully cloned from anthocyanin-rich plant materials blueberry fruit and purple round eggplant rind. The Yeast Fab Assembly technology was utilized to construct the transcriptional units of these genes under different promoters. The transcriptional units of PAL and C4H, 4CL and CHS were fused and inserted into Chr. XVI and IV of yeast strain JDY52 respectively using homologous recombination to gain Strain A. The fragments containing the transcriptional units of CHI and F3H, F3'H and DFR were inserted into Chr. III and XVI to gain Strain B1. Strain B2 has the transcriptional units of ANS and 3GT in Chr. IV. Several anthocyanidins, including cyanidin, peonidin, pelargonidin, petunidin, and malvidin, were detected by LC-MS/MS following the predicted outcomes of the de novo biosynthesis of anthocyanins in S. cerevisiae using a multi-strain co-culture technique., Conclusions: We propose a novel concept for advancing the heterologous de novo anthocyanin biosynthetic pathway, as well as fundamental information and a theoretical framework for the ensuing optimization of the microbial synthesis of anthocyanins., (© 2024. The Author(s).)

Sulfur metabolism plays a major role in plant growth and development, environmental adaptation, and material synthesis, and the sulfate transporters are the beginning of sulfur metabolism. We identified 37 potential VcSULTR genes in the blueberry genome, encoding peptides with 534 to 766 amino acids. The genes were grouped into four subfamilies in an evolutionary analysis. The 37 putative VcSULTR proteins ranged in size from 60.03 to 83.87 kDa. These proteins were predicted to be hydrophobic and mostly localize to the plasma membrane. The VcSULTR genes were distributed on 30 chromosomes; VcSULTR3;5b and VcSULTR3;5c were the only tandemly repeated genes. The VcSULTR promoters contained cis -acting elements related to the fungal symbiosis and stress responses. The transcript levels of the VcSULTRs differed among blueberry organs and changed in response to ericoid mycorrhizal fungi and sulfate treatments. A subcellular localization analysis showed that VcSULTR2;1c localized to, and functioned in, the plasma membrane and chloroplast. The virus-induced gene knock-down of VcSULTR2;1c resulted in a significantly decreased endogenous sulfate content, and an up-regulation of genes encoding key enzymes in sulfur metabolism ( VcATPS2 and VcSiR1 ). These findings enhance our understanding of mycorrhizal-fungi-mediated sulfate transport in blueberry, and lay the foundation for further research on blueberry-mycorrhizal symbiosis.

Background: Blueberry fruit exhibit atypical climacteric ripening with a non-auto-catalytic increase in ethylene coincident with initiation of ripening. Further, application of ethephon, an ethylene-releasing plant growth regulator, accelerates ripening by increasing the proportion of ripe (blue) fruit as compared to the control treatment. To investigate the mechanistic role of ethylene in regulating blueberry ripening, we performed transcriptome analysis on fruit treated with ethephon, an ethylene-releasing plant growth regulator., Results: RNA-Sequencing was performed on two sets of rabbiteye blueberry ('Powderblue') fruit: (1) fruit from divergent developmental stages; and (2) fruit treated with ethephon, an ethylene-releasing compound. Differentially expressed genes (DEGs) from divergent developmental stages clustered into nine groups, among which cluster 1 displayed reduction in expression during ripening initiation and was enriched with photosynthesis related genes, while cluster 7 displayed increased expression during ripening and was enriched with aromatic-amino acid family catabolism genes, suggesting stimulation of anthocyanin biosynthesis. More DEGs were apparent at 1 day after ethephon treatment suggesting its early influence during ripening initiation. Overall, a higher number of genes were downregulated in response to ethylene. Many of these overlapped with cluster 1 genes, indicating that ethylene-mediated downregulation of photosynthesis is an important developmental event during the ripening transition. Analyses of DEGs in response to ethylene also indicated interplay among phytohormones. Ethylene positively regulated abscisic acid (ABA), negatively regulated jasmonates (JAs), and influenced auxin (IAA) metabolism and signaling genes. Phytohormone quantification supported these effects of ethylene, indicating coordination of blueberry fruit ripening by ethylene., Conclusion: This study provides insights into the role of ethylene in blueberry fruit ripening. Ethylene initiates blueberry ripening by downregulating photosynthesis-related genes. Also, ethylene regulates phytohormone-metabolism and signaling related genes, increases ABA, and decreases JA concentrations. Together, these results indicate that interplay among multiple phytohormones regulates the progression of ripening, and that ethylene is an important coordinator of such interactions during blueberry fruit ripening., (© 2024. The Author(s).)

Background: WOX genes are a class of plant-specific transcription factors. The WUSCHEL-related homeobox (WOX) family is a member of the homeobox transcription factor superfamily. Previous studies have shown that WOX members play important roles in plant growth and development. However, studies of the WOX gene family in blueberry plants have not been reported., Results: In order to understand the biological function of the WOX gene family in blueberries, bioinformatics were used methods to identify WOX gene family members in the blueberry genome, and analyzed the basic physical and chemical properties, gene structure, gene motifs, promoter cis-acting elements, chromosome location, evolutionary relationships, expression pattern of these family members and predicted their functions. Finally, 12 genes containing the WOX domain were identified and found to be distributed on eight chromosomes. Phylogenetic tree analysis showed that the blueberry WOX gene family had three major branches: ancient branch, middle branch, and WUS branch. Blueberry WOX gene family protein sequences differ in amino acid number, molecular weight, isoelectric point and hydrophobicity. Predictive analysis of promoter cis-acting elements showed that the promoters of the VdWOX genes contained abundant light response, hormone, and stress response elements. The VdWOX genes were induced to express in both stems and leaves in response to salt and drought stress., Conclusions: Our results provided comprehensive characteristics of the WOX gene family and important clues for further exploration of its role in the growth, development and resistance to various stress in blueberry plants., (© 2024. The Author(s).)

A high content of anthocyanin in blueberry (Vaccinium corymbosum) is an important indicator to evaluate fruit quality. Abscisic acid (ABA) can promote anthocyanin biosynthesis, but since the molecular mechanism is unclear, clarifying the mechanism will improve for blueberry breeding and cultivation regulation. VcbZIP55 regulating anthocyanin synthesis in blueberry were screened and mined using the published Isoform-sequencing, RNA-Seq and qRT-PCR at different fruit developmental stages. Blueberry genetic transformation and transgenic experiments confirmed that VcbZIP55 could promote anthocyanin biosynthesis in blueberry adventitious buds, tobacco leaves, blueberry leaves and blueberry fruit. VcbZIP55 responded to ABA signals and its expression was upregulated in blueberry fruit. In addition, using VcbZIP55 for Yeast one hybrid assay (Y1H) and transient expression in tobacco leaves demonstrated an interaction between VcbZIP55 and a G-Box motif on the VcMYB1 promoter to activate the expression of VcMYB1. This study will lay the theoretical foundation for the molecular mechanisms of phytohormone regulation responsible for anthocyanin synthesis and provide theoretical support for blueberry quality improvement., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

As a highly economic berry fruit crop, blueberry is enjoyed by most people and has various potential health benefits, many of which are attributed to the relatively high concentrations of flavonoids. To obtain more accurate and comprehensive transcripts, the full-length transcriptome of half-highbush blueberry ( Vaccinium corymbosum/angustifolium cultivar Northland) obtained using single molecule real-time and next-generation sequencing technologies was reported for the first time. Overall, 147,569 consensus transcripts (average length, 2738 bp; N50, 3176 bp) were obtained. After quality control steps, 63,425 high-quality isoforms were obtained and 5030 novel genes, 3002 long non-coding RNAs, 3946 transcription factor genes (TFs), 30,540 alternative splicing events, and 2285 fusion gene pairs were identified. To better explore the molecular mechanism of flavonoid biosynthesis in mature blueberry fruit, an integrative analysis of the metabolome and transcriptome was performed on the exocarp, sarcocarp, and seed. A relatively complete biosynthesis pathway map of phenylpropanoids, flavonoids, and proanthocyanins in blueberry was constructed. The results of the joint analysis showed that the 228 functional genes and 42 TFs regulated 78 differentially expressed metabolites within the biosynthesis pathway of phenylpropanoids/flavonoids. O2PLS analysis results showed that the key metabolites differentially accumulated in blueberry fruit tissues were albireodelphin, delphinidin 3,5-diglucoside, delphinidin 3-O-rutinoside, and delphinidin 3-O-sophoroside, and 10 structural genes (4 Vc4CLs , 3 VcBZ1s , 1 VcUGT75C1 , 1 VcAT , and 1 VcUGAT ), 4 transporter genes (1 VcGSTF and 3 VcMATEs ), and 10 TFs (1 VcMYB , 2 VcbHLHs , 4 VcWD40s, and 3 VcNACs ) exhibited strong correlations with 4 delphinidin glycosides. These findings provide insights into the molecular mechanisms of flavonoid biosynthesis and accumulation in blueberry fruit.

In blueberry ( Vaccinium corymbosum L.), a perennial shrub, flower bud initiation is mediated by a short-day (SD) photoperiod and buds bloom once the chilling requirement is satisfied. A plant factory with artificial lighting (PFAL) is a planting system that can provide a stable and highly efficient growing environment for blueberry production. However, the characteristics of bud differentiation of blueberry plants inside PFAL systems are poorly understood. To better understand flower bud initiation and the flowering mechanism of blueberry in PFAL systems, the anatomical structure of apical buds under SD conditions in a PFAL system was observed using the southern highbush cultivar 'Misty' and a transcriptomic analysis was performed to identify the candidate flowering genes. The results indicated that the apical bud of 'Misty' differentiated gradually along with SD time course and swelled obviously when chilling was introduced. A total of 39.28 Gb clean data were generated, and about 20.31-24.11 Mb high-quality clean reads were assembled, yielding a total of 17370 differentially expressed genes (DEGs), of which 9637 were up-regulated and 7733 were down-regulated. Based on the functional annotation, 26 DEGs were identified including 20 flowering-related and 6 low-temperature DEGs, out of which the expressive level of four flowering-related DEGs ( VcFT2 , VcFPA , VcFMADS1 , and VcCOP1 ) and two low-temperature-induced DEGs ( VcTIL-1 and VcLTI 65 -like) were confirmed by qRT-PCR with a good consistency with the pattern of transcriptome. Functional analysis indicated that VcFT2 was highly conserved with nuclear and cytoplasmic subcellular localization and was expressed mainly in blueberry leaf tissue. In Arabidopsis , ectopic overexpression of VcFT2 results in an early flowering phenotype, indicating that VcFT2 is a vital regulator of the SD-mediated flowering pathway in blueberry. These results contribute to the investigation of photoperiod-mediated flowering mechanisms of blueberry in PFAL systems.

Main Conclusion: The expression peak of VcAP1.4, VcAP1.6, VcAP3.1, VcAP3.2, VcAG3, VcFLC2, and VcSVP9 coincided with the endo-dormancy release of flower buds. Additionally, GA4 +7 not only increased the expression of these genes but also promoted flower bud endo-dormancy release. The MIKC C -type MADS-box gene family is involved in the regulation of flower development. A total of 109 members of the MIKC C -type MADS-box gene family were identified in blueberry. According to the phylogenetic tree, these 109 MIKC C -type MADS-box proteins were divided into 13 subfamilies, which were distributed across 40 Scaffolds. The results of the conserved motif analysis showed that among 20 motifs, motifs 1, 3, and 9 formed the MADS-box structural domain, while motifs 2, 4, and 6 formed the K-box structural domain. The presence of 66 pairs of fragment duplication events in blueberry suggested that gene duplication events contributed to gene expansion and functional differentiation. Additionally, the presence of cis-acting elements revealed that VcFLC2, VcAG3, and VcSVP9 might have significant roles in the endo-dormancy release of flower buds. Meanwhile, under chilling conditions, VcAP3.1 and VcAG7 might facilitate flower bud dormancy release. VcSEP11 might promote flowering following the release of endo-dormancy, while the elevated expression of VcAP1.7 (DAM) could impede the endo-dormancy release of flower buds. The effect of gibberellin (GA 4+7 ) treatment on the expression pattern of MIKC C -type MADS-box genes revealed that VcAP1.4, VcAP1.6, VcAP3.1, VcAG3, and VcFLC2 might promote flower bud endo-dormancy release, while VcAP3.2, VcSEP11, and VcSVP9 might inhibit its endo-dormancy release. These results indicated that VcAP1.4, VcAP1.6, VcAP1.7 (DAM), VcAP3.1, VcAG3, VcAG7, VcFLC2, and VcSVP9 could be selected as key regulatory promoting genes for controlling the endo-dormancy of blueberry flower buds., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Colour change is an important event during fruit ripening in blueberry. It is well known that miR156/SPLs act as regulatory modules mediating anthocyanin biosynthesis and ethylene plays critical roles during colour change, but the intrinsic connections between the two pathways remain poorly understood. Previously, we demonstrated that blueberry VcMIR156a/VcSPL12 affects the accumulation of anthocyanins and chlorophylls in tomato and Arabidopsis. In this study, we first showed that VcMIR156a overexpression in blueberry led to enhanced anthocyanin biosynthesis, decreased chlorophyll accumulation, and, intriguingly, concomitant elevation in the expression of ethylene biosynthesis genes and the level of the ethylene precursor ACC. Conversely, VcSPL12 enhanced chlorophyll accumulation and suppressed anthocyanin biosynthesis and ACC synthesis in fruits. Moreover, the treatment with ethylene substitutes and inhibitors attenuated the effects of VcMIR156a and VcSPL12 on pigment accumulation. Protein-DNA interaction assays indicated that VcSPL12 could specifically bind to the promoters and inhibit the activities of the ethylene biosynthetic genes VcACS1 and VcACO6. Collectively, our results show that VcMIR156a/VcSPL12 alters ethylene production through targeting VcACS1 and VcACO6, therefore governing fruit colour change. Additionally, VcSPL12 may directly interact with the promoter region of the chlorophyll biosynthetic gene VcDVR, thereby activating its expression. These findings established an intrinsic connection between the miR156/SPL regulatory module and ethylene pathway., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Carotenoids and their derivates play critical physiologic roles in plants. However, these substrates and their metabolism have not been elucidated in fruit of blueberry (Vaccinium corymbosum). In this study, carotenoids and ABA were investigated by LC-MS and their biosynthesis were subject to proteomic analysis during fruit ripening. Activity of CCD1 and NCED1/3 were studied in vivo or in vitro. Also, effects of ethephon and 1-MCP on biosynthesis of carotenoid and ABA were investigated through the expression of corresponding genes using qPCR. As a result, carotenoid biosynthesis was prominently mitigated whereas its metabolism was enhanced during fruit ripening, which resulted in a decrease in the carotenoids. VcCCD1 could both cleave β-carotene, zeaxanthin and lutein at positions of 9, 10 (9', 10'), which was mainly responsible for the degradation of these carotenoids. Interestingly, in the situation of mitigation of carotenoid biosynthesis, ABA still rapidly accumulated, which was mainly attributed to the upregulated expression of VcNCED1/3. Notably, VcNCED1/3 also showed a cleavage activity of all-trans-zeaxanthin and a stereospecific cleavage activity of 9-cis-carotene to generate C15-carotenal. The C15-carotenal could be potentially converted to ABA through ZEP-independent ABA biosynthetic pathway during blueberry fruit ripening. Similar to a nature natural maturation, ethylene accelerated the carotenoid degradation and ABA biosynthesis trough downregulating the expression of genes in carotenoid biosynthesis and upregulating the expression of genes in ABA biosynthesis. These information help understand the regulation of carotenoids and ABA, and effects of ethylene on the regulation during blueberry fruit ripening., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Masson SAS.)

In vitro culture allows the production of numerous plants with both desirable and undesirable traits. To investigate the impact of the propagation method on highbush blueberry plants, an analysis was performed on four groups of differentially propagated plants: in vitro with axillary (TC-Ax) or adventitious shoots (TC-Ad), conventionally (SC) and using a mixed method (TC/SC). The analysis included plant features (shoot length and branching, chlorophyll and fluorescence and DNA methylation) and fruit properties (antioxidant compounds). The data obtained indicated significant differences between plants propagated conventionally and in vitro, as well as variations among plants derived from in vitro cultures with different types of explants. SC plants generally exhibited the lowest values of morphological and physiological parameters but produced fruits richest in antioxidant compounds. TC/SC plants were dominant in length, branching and fluorescence. Conversely, TC-Ax plants produced fruits with the lowest levels of antioxidant compounds. The methylation-sensitive amplified polymorphism (MSAP) technique was employed to detect molecular differences. TC-Ad plants showed the highest methylation level, whereas SC plants had the lowest. The overall methylation level varied among differentially propagated plants. It can be speculated that the differences among the analysed plants may be attributed to variations in DNA methylation.

Soil salinity is a major environmental factor constraining growth and productivity of highbush blueberry (Vaccinium corymbosum). Leaf Na+ content is associated with variation in salt tolerance among blueberry cultivars; however, the determinants and mechanisms conferring leaf Na+ exclusion are unknown. Here, we observed that the blueberry cultivar 'Duke' was more tolerant than 'Sweetheart' and accumulated less Na+ in leaves under salt stress conditions. Through transcript profiling, we identified a member of the high-affinity K+ transporter (HKT) family in blueberry, VcHKT1;1, as a candidate gene involved in leaf Na+ exclusion and salt tolerance. VcHKT1;1 encodes a Na+-preferential transporter localized to the plasma membrane and is preferentially expressed in the root stele. Heterologous expression of VcHKT1;1 in Arabidopsis (Arabidopsis thaliana) rescued the salt hypersensitivity phenotype of the athkt1 mutant. Decreased VcHKT1;1 transcript levels in blueberry plants expressing antisense-VcHKT1;1 led to increased Na+ concentrations in xylem sap and higher leaf Na+ contents compared with wild-type plants, indicating that VcHKT1;1 promotes leaf Na+ exclusion by retrieving Na+ from xylem sap. A naturally occurring 8-bp insertion in the promoter increased the transcription level of VcHKT1;1, thus promoting leaf Na+ exclusion and blueberry salt tolerance. Collectively, we provide evidence that VcHKT1;1 promotes leaf Na+ exclusion and propose natural variation in VcHKT1;1 will be valuable for breeding Na+-tolerant blueberry cultivars in the future., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Blueberry is a high-quality fruit tree with significant nutritional and economic value, but the intricate mechanism of sugar accumulation in its fruit remains unclear. In this study, the ripe fruits of blueberry cultivars 'Anna' and 'Misty' were utilized as experimental materials, and physiological and multi-omics methodologies were applied to analyze the regulatory mechanisms of the difference in sugar content between them. The results demonstrated that the 'Anna' fruit was smaller and had less hardness than the 'Misty' fruit, as well as higher sugar content, antioxidant capability, and lower active substance content. A total of 7067 differentially expressed genes (DEGs) (3674 up-regulated and 3393 down-regulated) and 140 differentially abundant metabolites (DAMs) (82 up-regulated and 58 down-regulated) were identified between the fruits of the two cultivars. According to KEGG analysis, DEGs were primarily abundant in phenylpropanoid synthesis and hormone signal transduction pathways, whereas DAMs were primarily enriched in ascorbate and aldarate metabolism, phenylpropanoid biosynthesis, and the pentose phosphate pathway. A combined multi-omics study showed that 116 DEGs and 3 DAMs in starch and sucrose metabolism (48 DEGs and 1 DAM), glycolysis and gluconeogenesis (54 DEGs and 1 DAM), and the pentose phosphate pathway (14 DEGs and 1 DAM) were significantly enriched. These findings suggest that blueberries predominantly increase sugar accumulation by activating carbon metabolism network pathways. Moreover, we identified critical transcription factors linked to the sugar response. This study presents new understandings regarding the molecular mechanisms underlying blueberry sugar accumulation and will be helpful in improving blueberry fruit quality through breeding.

Universal stress proteins (USPs) play essential roles in plant development, hormonal regulation, and abiotic stress responses. However, the characteristics and functional divergence of USP family members have not been studied in blueberry ( Vaccinium corymbosum ). In this study, we identified 72 VcUSP genes from the Genome Database for Vaccinium . These VcUSPs could be divided into five groups based on their phylogenetic relationships. VcUSPs from groups Ⅰ, Ⅳ, and Ⅴ each possess one UspA domain; group Ⅰ proteins also contain an ATP-binding site that is not present in group Ⅳ and Ⅴ proteins. Groups Ⅱ and Ⅲ include more complex proteins possessing one to three UspA domains and UspE or UspF domains. Prediction of cis-regulatory elements in the upstream sequences of VcUSP genes indicated that their protein products are likely involved in phytohormone signaling pathways and abiotic stress responses. Analysis of RNA deep sequencing data showed that 21 and 7 VcUSP genes were differentially expressed in response to UV-B radiation and exogenous abscisic acid (ABA) treatments, respectively. VcUSP41 and VcUSP68 expressions responded to both treatments, and their encoded proteins may integrate the UV-B and ABA signaling pathways. Weighted gene co-expression network analysis revealed that VcUSP22 , VcUSP26 , VcUSP67 , VcUSP68 , and VcUSP41 were co-expressed with many transcription factor genes, most of which encode members of the MYB, WRKY, zinc finger, bHLH, and AP2 families, and may be involved in plant hormone signal transduction, circadian rhythms, the MAPK signaling pathway, and UV-B-induced flavonoid biosynthesis under UV-B and exogenous ABA treatments. Our study provides a useful reference for the further functional analysis of VcUSP genes and blueberry molecular breeding.

Background: B-box (BBX) proteins play important roles in regulating plant growth, development, and abiotic stress responses. BBX family genes have been identified and functionally characterized in many plant species, but little is known about the BBX family in blueberry (Vaccinium corymbosum)., Result: In this study, we identified 23 VcBBX genes from the Genome Database for Vaccinium (GDV). These VcBBXs can be divided into five clades based on gene structures and conserved domains in their encoded proteins. The prediction of cis-acting elements in the upstream sequences of VcBBX genes and protein-protein interactions indicated that VcBBX proteins are likely involved in phytohormone signaling pathways and abiotic stress responses. Analysis of transcriptome deep sequencing (RNA-seq) data showed that VcBBX genes exhibited organ-specific expression pattern and 11 VcBBX genes respond to ultraviolet B (UV-B) radiation. The co-expression analysis revealed that the encoded 11 VcBBX proteins act as bridges integrating UV-B and phytohormone signaling pathways in blueberry under UV-B radiation. Reverse-transcription quantitative PCR (RT-qPCR) analysis showed that most VcBBX genes respond to drought, salt, and cold stress. Among VcBBX proteins, VcBBX24 is highly expressed in all the organs, not only responds to abiotic stress, but it also interacts with proteins in UV-B and phytohormone signaling pathways, as revealed by computational analysis and co-expression analysis, and might be an important regulator integrating abiotic stress and phytohormone signaling networks., Conclusions: Twenty-three VcBBX genes were identified in blueberry, in which, 11 VcBBX genes respond to UV-B radiation, and act as bridges integrating UV-B and phytohormone signaling pathways according to RNA-seq data. The expression patterns under abiotic stress suggested that the functional roles of most VcBBX genes respose to drought, salt, and cold stress. Our study provides a useful reference for functional analysis of VcBBX genes and for improving abiotic stress tolerance in blueberry., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Key Message: The genomic location and stage-specific expression pattern of GH9 genes reveal their critical roles during fruit abscission zone formation in Vaccinium ashei. Glycosyl hydrolase family 9 (GH9) cellulases play a crucial role in both cellulose synthesis and hydrolysis during plant growth and development. Despite this importance, there is currently no study on the involvement of GH9-encoding genes, specifically VaGH9s, in abscission zone formation of rabbiteye blueberries (Vaccinium ashei). In this study, we identified a total of 61 VaGH9s in the genome, which can be classified into 3 subclasses based on conserved motifs and domains, gene structures, and phylogenetic analyses. Our synteny analysis revealed that VaGH9s are more closely related to the GH9s of Populus L. than to those of Arabidopsis, Vitis vinifera, and Citrus sinensis. In silico structural analysis predicted that most of VaGH9s are hydrophilic, and localized in cell membrane and/or cell wall, and the variable sets of cis-acting regulatory elements and functional diversity with four categories of stress response, hormone regulation, growth and development, and transcription factor-related elements are present in the promoter sequence of VaGH9s genes. Transcriptomic analysis showed that there were 22 differentially expressed VaGH9s in fruit abscission zone tissue at the veraison stage, and the expression of VaGH9B2 and VaGH9C10 was continuously increased during fruit maturation, which were in parallel with the increasing levels of cellulase activity and oxidative stress indicators, suggesting that they are involved in the separation stage of fruit abscission in Vaccinium ashei. Our work identified 22 VaGH9s potentially involved in different stages of fruit abscission and would aid further investigation into the molecular regulation of abscission in rabbiteye blueberries fruit., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Chalcone synthase (CHS) is the first key enzyme-catalyzing plant flavonoid biosynthesis. Until now, however, the blueberry CHS gene family has not been systematically characterized and studied. In this study, we identified 22 CHS genes that could be further classified into four subfamilies from the highbush blueberry ( Vaccinium corymbosum ) genome. This classification was well supported by the high nucleotide and protein sequence similarities and similar gene structure and conserved motifs among VcCHS members from the same subfamily. Gene duplication analysis revealed that the expansion of the blueberry CHS gene family was mainly caused by segmental duplications. Promoter analysis revealed that the promoter regions of VcCHSs contained numerous cis -acting elements responsive to light, phytohormone and stress, along with binding sites for 36 different types of transcription factors. Gene expression analysis revealed that Subfamily I VcCHSs highly expressed in fruits at late ripening stages. Through transient overexpression, we found that three VcCHSs ( VcCHS13 from subfamily II; VcCHS8 and VcCHS21 from subfamily I) could significantly enhance the anthocyanin accumulation and up-regulate the expression of flavonoid biosynthetic structural genes in blueberry leaves and apple fruits. Notably, the promoting effect of the Subfamily I member VcCHS21 was the best. The promoter of VcCHS21 contains a G-box (CACGTG) and an E-box sequence, as well as a bHLH binding site. A yeast one hybridization (Y1H) assay revealed that three anthocyanin biosynthesis regulatory bHLHs (VcAN1, VcbHLH1-1 and VcbHLH1-2) could specifically bind to the G-box sequence (CACGTG) in the VcCHS21 promoter, indicating that the expression of VcCHS21 was regulated by bHLHs. Our study will be helpful for understanding the characteristics and functions of blueberry CHSs.

Background: Blueberries (Vaccinium corymbosum) are regarded as "superfoods" attributed to large amounts of anthocyanins, a group of flavonoid metabolites, which provide pigmentation in plant and beneficial effects for human health. MYB transcription factor is one of vital components in the regulation of plant secondary metabolism, which occupies a dominant position in the regulatory network of anthocyanin biosynthesis. However, the role of MYB family in blueberry responding to anthocyanin biosynthesis remains elusive., Results: In this study, we conducted a comprehensive analysis of VcMYBs in blueberry based on the genome data, including phylogenetic relationship, conserved motifs, identification of differentially expressed MYB genes during fruit development and their expression profiling, etc. A total of 437 unique MYB sequences with two SANT domains were identified in blueberry, which were divided into 3 phylogenetic trees. Noticeably, there are many trigenic and tetragenic VcMYBs pairs with more than 95% identity to each other. Meanwhile, the transcript accumulations of VcMYBs were surveyed underlying blueberry fruit development, and they showed diverse expression patterns, suggesting various functional roles in fruit ripening. More importantly, distinct transcript profiles between skin and pulp of ripe fruit were observed for several VcMYBs, such as VcMYB437, implying the potential roles in anthocyanin biosynthesis., Conclusions: Totally, 437 VcMYBs were identified and characterized. Subsequently, their transcriptional patterns were explored during fruit development and fruit tissues (skin and pulp) closely related to anthocyanin biosynthesis. These genome-wide data and findings will contribute to demonstrating the functional roles of VcMYBs and their regulatory mechanisms for anthocyanins production and accumulation in blueberry in the future study., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Vaccinium corymbosum (highbush blueberry) in British Columbia relies on honeybee pollination for a reliable fruit set. As floral volatiles may help explain pollinator preference for blueberry, we surveyed components of variation for volatiles using gas chromatography-mass spectrometry (GC/MS). Principal component analysis of GC chromatogram peaks revealed a grouping of cultivars by a biosynthetic pathway that also corresponds with their known pedigree. To identify genetic variance, we identified 34 chemicals with adequate sample sizes. We estimated "natural heritability" (using uncontrolled crosses in natural environments) in two ways: (1) as "clonal repeatability," which is equivalent to broad-sense heritability and is an upper bound for narrow-sense heritability, and (2) "marker-based heritability," which serves as a lower bound for narrow-sense heritability. Both methods indicate that heritability is relatively low, ca. 15%, and variable among traits. This is expected as the floral volatile release is changeable and dependent on environmental conditions. It might be possible to use highly heritable volatiles for breeding.

Different light wavelengths display diverse effects on fruit quality formation and anthocyanin biosynthesis. Blueberry is a kind of fruit rich in anthocyanin with important economic and nutritional values. This study explored the effects of different light wavelengths (white (W), red (R), blue (B) and yellow (Y)) on fruit quality and gene expression of anthocyanin biosynthesis in blueberry. We found that the B and W treatments attained the maximum values of fruit width, fruit height and fruit weight in blueberry fruits. The R treatment attained the maximum activities of superoxide dismutase (SOD) and peroxidase (POD), and the Y treatment displayed the maximum contents of ascorbic acid (AsA), glutathione (GSH) and total phenol in fruits, thus improving blueberry-fruit antioxidant capacity. Interestingly, there were differences in the solidity-acid ratio of fruit under different light-wavelength treatments. Moreover, blue light could significantly improve the expression levels of anthocyanin biosynthesis genes and anthocyanin content in fruits. Correlation and principal component analysis showed that total acid content and antioxidant enzymes were significantly negatively correlated with anthocyanin content in blueberry fruits. These results provide new insights for the application of light wavelength to improve blueberry fruit quality and anthocyanin content.

Botrytis blight is an important disease of wild blueberry [(Vaccinium angustifolium (Va) and V. myrtilloides (Vm))] with variable symptoms in the field due to differences in susceptibility among blueberry phenotypes. Representative blueberry plants of varying phenotypes were inoculated with spores of B. cinerea. The relative expression of pathogenesis-related genes (PR3, PR4), flavonoid biosynthesis genes, and estimation of the concentration of ten phenolic compounds between uninoculated and inoculated samples at different time points were analyzed. Representative plants of six phenotypes (brown stem Va, green stem Va, Va f. nigrum, tall, medium, and short stems of Vm) were collected and studied using qRT-PCR. The expression of targeted genes indicated a response of inoculated plants to B. cinerea at either 12, 24, 48 or 96 h post inoculation (hpi). The maximum expression of PR3 occurred at 24 hpi in all the phenotypes except Va f. nigrum and tall stem Vm. Maximum expression of both PR genes occurred at 12 hpi in Va f. nigrum. Chalcone synthase, flavonol synthase and anthocyanin synthase were suppressed at 12 hpi followed by an upregulation at 24 hpi. The expression of flavonoid pathway genes was phenotype-specific with their regulation patterns showing temporal differences among the phenotypes. Phenolic compound accumulation was temporally regulated at different post-inoculation time points. M-coumaric acid and kaempferol-3-glucoside are the compounds that were increased with B. cinerea inoculation. Results from this study suggest that the expression of PR and flavonoid genes, and the accumulation of phenolic compounds associated with B. cinerea infection could be phenotype specific. This study may provide a starting point for understanding and determining the mechanisms governing the wild blueberry-B. cinerea pathosystem., (© 2023. The Author(s).)

Blueberry (Vaccinium corymbosum L.), a woody perennial bush in the genus Vaccinium, is an economically important and popular fruit crop worldwide. Development the superior cultivars, which including excellent fruit traits, not only means higher yielding and economic efficiency, but also produce fruit that to meet the preferences of different consumers. Excavating fruit quality-related genes, studying their functions, and using transgenic or molecular-assisted breeding are beneficial to the development of excellent blueberry varieties. Genetic transformation is an excellent way to study the function of genes in plants, however, it is a labor-intensive and time-consuming process to genetically transform many woody plants, including blueberry. Virus-induced gene silencing (VIGS) provides an efficient approach to knock-down the expression of target genes for functional analysis. In this study, tobacco rattle virus induced genes silencing (TRV-VIGS) was established in blueberry fruits using the VcANS gene as a reporter. The silenced sector of the skin of blueberry fruits injected with pTRV2 (plasmid Tobacco Rattle Virus, TRV-RNA2)::VcANS remained green or white at 25 days after agroinfiltration. In agroinfiltrated materials, the VcANS transcript levels were much lower in fruits with phenotypic changes (delayed color change) than in those infiltrated with the pTRV2 empty vector. Silencing of VcANS also affected the expression of other genes involved in the anthocyanin synthesis pathway. The experimental results support that VcANS can be used as an effective marker gene for VIGS system. In addition, the TRV-VIGS system has been successfully established in blueberry fruits, which provided an effective verification method for functional identification of unknown genes in blueberry fruits., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Understanding chromosome recombination behavior in polyploidy species is key to advancing genetic discoveries. In blueberry, a tetraploid species, the line of evidences about its genetic behavior still remain poorly understood, owing to the inter-specific, and inter-ploidy admixture of its genome and lack of in depth genome-wide inheritance and comparative structural studies. Here we describe a new high-quality, phased, chromosome-scale genome of a diploid blueberry, clone W85. The genome was integrated with cytogenetics and high-density, genetic maps representing six tetraploid blueberry cultivars, harboring different levels of wild genome admixture, to uncover recombination behavior and structural genome divergence across tetraploid and wild diploid species. Analysis of chromosome inheritance and pairing demonstrated that tetraploid blueberry behaves as an autotetraploid with tetrasomic inheritance. Comparative analysis demonstrated the presence of a reciprocal, heterozygous, translocation spanning one homolog of chr-6 and one of chr-10 in the cultivar Draper. The translocation affects pairing and recombination of chromosomes 6 and 10. Besides the translocation detected in Draper, no other structural genomic divergences were detected across tetraploid cultivars and highly inter-crossable wild diploid species. These findings and resources will facilitate new genetic and comparative genomic studies in Vaccinium and the development of genomic assisted selection strategy for this crop., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Background/aim: This study evaluated the effect of blueberry leaf hot water extract (BLEx) on Sjögren's syndrome (SS)-like lacrimal hyposecretion in male non-obese diabetic (NOD) mice., Materials and Methods: NOD or BALB/c mice were fed 1% BLEx or control (AIN-93G) for 2 weeks from the age of 4 to 6 weeks. Pilocarpine-induced tear volume was measured using a phenol red-impregnated thread. The lacrimal glands were evaluated histologically by H&E staining. The IL-1β and TNF-α levels in the lacrimal gland tissue were measured by ELISA. The mRNA expression levels of secretion-related proteins were measured by real-time PCR. LC3 I/II and arginase 1 expression levels were measured by western blot., Results: After feeding with BLEx, pilocarpine-induced tear secretion in NOD mice was increased. In contrast, the mRNA expression levels of the cholinergic muscarinic M3 receptor, aquaporin 5, and ion channels related to lacrimal secretion were not changed by BLEx administration. In addition, the protein expression of arginase 1, which was recently reported to be involved in tear hyposecretion in NOD mice, was also not improved by BLEx administration. Although infiltration in the lacrimal gland of NOD mice was not decreased, the levels of TNF-α and the autophagy-related protein LC3 were significantly suppressed by BLEx treatment., Conclusion: BLEx treatment may ameliorate lacrimal hyposecretion in NOD mice by delaying the progression of autoimmune disease by suppressing autophagy in lacrimal glands., (Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Vaccinium is a large genus of shrubs that includes a handful of economically important berry crops. Given the numerous hybridizations and polyploidization events, the taxonomy of this genus has remained the subject of long debate. In addition, berries and berry-based products are liable to adulteration, either fraudulent or unintentional due to misidentification of species. The availability of more genomic information could help achieve higher phylogenetic resolution for the genus, provide molecular markers for berry crops identification, and a framework for efficient genetic engineering of chloroplasts. Therefore, in this study we assembled five Vaccinium chloroplast sequences representing the economically relevant berry types: northern highbush blueberry (V. corymbosum), southern highbush blueberry (V. corymbosum hybrids), rabbiteye blueberry (V. virgatum), lowbush blueberry (V. angustifolium), and bilberry (V. myrtillus). Comparative analyses showed that the Vaccinium chloroplast genomes exhibited an overall highly conserved synteny and sequence identity among them. Polymorphic regions included the expansion/contraction of inverted repeats, gene copy number variation, simple sequence repeats, indels, and single nucleotide polymorphisms. Based on their in silico discrimination power, we suggested variants that could be developed into molecular markers for berry crops identification. Phylogenetic analysis revealed multiple origins of highbush blueberry plastomes, likely due to the hybridization events that occurred during northern and southern highbush blueberry domestication., (© 2022. The Author(s).)

Background: Blueberries (Vaccinium sp.) are native to North America and breeding efforts to improve blueberry fruit quality are focused on improving traits such as increased firmness, enhanced flavor and greater shelf-life. Such efforts require additional genomic resources, especially in southern highbush and rabbiteye blueberries., Results: We generated the first full-length fruit transcriptome for the southern highbush and rabbiteye blueberry using the cultivars, Suziblue and Powderblue, respectively. The transcriptome was generated using the Pacific Biosciences single-molecule long-read isoform sequencing platform with cDNA pooled from seven stages during fruit development and postharvest storage. Raw reads were processed through the Isoseq pipeline and full-length transcripts were mapped to the 'Draper' genome with unmapped reads collapsed using Cogent. Finally, we identified 16,299 and 15,882 non-redundant transcripts in 'Suziblue' and 'Powderblue' respectively by combining the reads mapped to Northern Highbush blueberry 'Draper' genome and Cogent analysis. In both cultivars, > 80% of sequences were longer than 1,000 nt, with the median transcript length around 1,700 nt. Functionally annotated transcripts using Blast2GO were > 92% in both 'Suziblue' and 'Powderblue' with overall equal distribution of gene ontology (GO) terms in the two cultivars. Analyses of alternative splicing events indicated that around 40% non-redundant sequences exhibited more than one isoform. Additionally, long non-coding RNAs were predicted to represent 5.6% and 7% of the transcriptomes in 'Suziblue' and 'Powderblue', respectively. Fruit ripening is regulated by several hormone-related genes and transcription factors. Among transcripts associated with phytohormone metabolism/signaling, the highest number of transcripts were related to abscisic acid (ABA) and auxin metabolism followed by those for brassinosteroid, jasmonic acid and ethylene metabolism. Among transcription factor-associated transcripts, those belonging to ripening-related APETALA2/ethylene-responsive element-binding factor (AP2/ERF), NAC (NAM, ATAF1/2 and CUC2), leucine zipper (HB-zip), basic helix-loop-helix (bHLH), MYB (v-MYB, discovered in avian myeloblastosis virus genome) and MADS-Box gene families, were abundant. Further we measured three fruit ripening quality traits and indicators [ABA, and anthocyanin concentration, and texture] during fruit development and ripening. ABA concentration increased during the initial stages of fruit ripening and then declined at the Ripe stage, whereas anthocyanin content increased during the final stages of fruit ripening in both cultivars. Fruit firmness declined during ripening in 'Powderblue'. Genes associated with the above parameters were identified using the full-length transcriptome. Transcript abundance patterns of these genes were consistent with changes in the fruit ripening and quality-related characteristics., Conclusions: A full-length, well-annotated fruit transcriptome was generated for two blueberry species commonly cultivated in the southeastern United States. The robustness of the transcriptome was verified by the identification and expression analyses of multiple fruit ripening and quality-regulating genes. The full-length transcriptome is a valuable addition to the blueberry genomic resources and will aid in further improving the annotation. It will also provide a useful resource for the investigation of molecular aspects of ripening and postharvest processes., (© 2022. The Author(s).)

Background: DNA binding with one finger (Dof) proteins are plant-specific transcription factor (TF) that plays a significant role in various biological processes such as plant growth and development, hormone regulation, and resistance to abiotic stress. The Dof genes have been identified and reported in multiple plants, but so far, the whole genome identification and analysis of Dof transcription factors in blueberry ( Vaccinium corymbosum L.) have not been reported yet., Methods: Using the Vaccinium genome, we have identified 51 VcDof genes in blueberry. We have further analyzed their physicochemical properties, phylogenetic relationships, gene structure, collinear analysis, selective evolutionary pressure, cis-acting promoter elements, and tissue and abiotic stress expression patterns., Results: Fifty-one VcDof genes were divided into eight subfamilies, and the genes in each subfamily contained similar gene structure and motif ordering. A total of 24 pairs of colinear genes were screened; VcDof genes expanded mainly due to whole-genome duplication, which was subjected to strong purifying selection pressure during the evolution. The promoter of VcDof genes contains three types of cis-acting elements for plant growth and development, phytohormone and stress defense responsiveness. Expression profiles of VcDof genes in different tissues and fruit developmental stages of blueberry indicated that VcDof2 and VcDof45 might play a specific role in anthesis and fruit growth and development. Expression profiles of VcDof genes in different stress indicated that VcDof1 , VcDof11 , and VcDof15 were highly sensitive to abiotic stress. This study provides a theoretical basis for further clarifying the biological function of Dof genes in blueberry., Competing Interests: The authors declare there are no competing interests., (©2022 Li et al.)

Objectives: Several clinical trials have been conducted worldwide to evaluate the efficacy of honey against stomatitis. However, it is unclear which types of honey are effective at preventing and/or treating stomatitis. This study aimed to evaluate the potencies of several types of honey in preventing and/or curing aphthous stomatitis in in vitro studies., Methods: The following experiments were performed: H 2 O 2 -induced cytotoxicity and mucosal cell migration in a scratch assay using buccal mucosa squamous carcinoma (HO-1-N-1) cells and the cellular expression of heme oxygenase-1 (HO-1) mRNA encoding an enzyme involved in protection against oxidative stress by real-time RT-PCR analysis, and liquid-liquid extraction and UHPLC analysis in order to examine the active components of honey., Results: Of the 13 types of honey used, Canadian blueberry honey exhibited the protective effect on H 2 O 2 -induced cytotoxicity and enhanced cell migration. In addition, blueberry honey increased the expression of HO-1 mRNA with and without cotreatment with H 2 O 2 . With regard to active components of blueberry honey, the water-soluble components with a mass of >10 kDa showed a cytoprotective effect, but they have not been identified., Conclusion: Canadian blueberry honey, but not the other types of honey, prevents H 2 O 2 -induced oxidation of cells, probably through activation of the antioxidant and cytoprotective enzyme HO-1. Blueberry honey also enhanced cell migration, which may be relevant to wound healing. The results of this study suggest the possibility of prophylactic and therapeutic effects of Canadian blueberry honey on human stomatitis that could complement existing treatments., Competing Interests: Conflicts of interest The authors are employees of API Co., Ltd., (Copyright © 2022 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

Vaccinium darrowii is a subtropical wild blueberry species that has been used to breed economically important southern highbush cultivars. The adaptive traits of V. darrowii to subtropical climates can provide valuable information for breeding blueberry and perhaps other plants, especially against the background of global warming. Here, we assembled the V. darrowii genome into 12 pseudochromosomes using Oxford Nanopore long reads complemented with Hi-C scaffolding technologies, and we predicted 41 815 genes using RNA-sequencing evidence. Syntenic analysis across three Vaccinium species revealed a highly conserved genome structure, with the highest collinearity between V. darrowii and Vaccinium corymbosum. This conserved genome structure may explain the high fertility observed during crossbreeding of V. darrowii with other blueberry cultivars. Analysis of gene expansion and tandem duplication indicated possible roles for defense- and flowering-associated genes in the adaptation of V. darrowii to the subtropics. Putative SOC1 genes in V. darrowii were identified based on phylogeny and expression analysis. Blueberries are covered in a thick cuticle layer and contain anthocyanins, which confer their powdery blue color. Using RNA sequencing, we delineated the cuticle biosynthesis pathways of Vaccinium species in V. darrowii. This result can serve as a reference for breeding berries whose colors are appealing to customers. The V. darrowii reference genome, together with the unique traits of this species, including its diploid genome, short vegetative phase, and high compatibility in hybridization with other blueberries, make V. darrowii a potential research model for blueberry species., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: To study the protective effect of anthocyanins extracted from Vaccinium Uliginosum (VU) on retinal 661W cells against microwave radiation induced retinal injury., Methods: 661W cells were divided into 6 groups, including control, model [661W cells radiated by microwave (30 mW/cm 2 , 1 h)] and VU groups [661W cells pretreated with anthocyanins extracted from VU (25, 50, 100 and 200 µg/mL, respectively) for 48 h, and radiated by microwave 30 mW/cm 2 , 1 h]. After treatment with different interventions, the cell apoptosis index (AI) was determined using Heochst staining; contents of malonaldehyde (MDA), glutataione (GSH), and activity of superoxide dismutase (SOD) were measured. mRNA expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1(HO-1) were detected by real time quantitative polymerase chain reaction, and the expression of HO-1 protein was examined by Western blot analysis. Nucleus and cytoplasm were separated and Nrf2 protein expression was further verified by Western blot analysis., Results: There was significant difference in AI among the groups (F=322.83, P<;0.05). Compared with the control group, AI was significantly higher in the model group and was lower in 4 VU-pretreated groups (P<;0.05). Linear regression analysis showed the decline of AI was in a dose-dependent manner with VU treatment (r=0.8419, P<;0.05). The MDA and GSH contents of 661W cells in VU-treated groups were significantly lower than the model group (P<;0.05). Compared with the model group, the SOD activity in the VU-treated groups (50, 100 and 200 µg/mL) was significantly higher (all P<;0.05). The Nrf2 and HO-1 mRNA expressions were slightly increased after irradiation, and obviously increased in 100 µg/mL VU-treated group. After irradiation, the relative expressions of HO-1 and Nrf2 proteins in nucleus were slightly increased (P<;0.05), and the changes in cytoplasm were not obvious, whereas it was significantly increased in both nucleus and cytoplasm in the VU treatment groups., Conclusions: Anthocyanins extracted from VU could reduce apoptosis, stabilize cell membrane, and alleviate oxidant injury of mouse retinal photoreceptor 661W cells. The mechanism might be through activating Nrf2/HO-1 signal pathway and inducing HO-1 transcription and translation., (© 2021. The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg.)

Four non-pathogenic strains isolated from the galls on blueberry plants (Vaccinium corymbosum) were characterized by using polyphasic taxonomic methods. Based on 16S rRNA gene phylogeny, strains were clustered within the genus Agrobacterium. Furthermore, multilocus sequence analysis (MLSA) based on the partial sequences of atpD, recA and rpoB housekeeping genes and whole-genome-based phylogeny indicated that the strains studied form a novel Agrobacterium species. Analyses showed that the strains belong to "rubi" sub-clade of Agrobacterium genus and their closest relatives are Agrobacterium rubi and "Agrobacterium bohemicum". Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) comparisons between genome sequences of representative strains B7.6 T and B19.1.4, and their closest relatives, confirmed the distinct phylogenetic position of studied strains, because obtained values were considerably below the proposed thresholds for the species delineation. The four strains studied were phenotypically distinguishable from other species of the genus Agrobacterium. Overall, polyphasic characterization showed that the strains studied represent a novel species of the genus Agrobacterium, for which the name Agrobacterium vaccinii sp. nov. is proposed. The type strain of A. vaccinii is B7.6 T (=CFBP 8740 T  = LMG 31849 T )., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Background: Blueberry is one of the most important fruit crops worldwide. Anthocyanin is an important secondary metabolites that affects the appearance and nutritive quality of blueberries. However, few studies have focused on the molecular mechanism underlying anthocyanin accumulation induced by light intensity in blueberries., Results: The metabolic analysis revealed that there were 134 significantly changed metabolites in the natural light compared to the control, and flavone, flavonol, and anthocyanins were the most significantly increased. Transcriptome analysis found 6 candidate genes for the anthocyanin synthesis pathway. Quantitative reverse transcription PCR (qRT-PCR) results confirmed changes in the expression levels of genes encoding metabolites involved in the flavonoid synthesis pathways. The flavonoid metabolic flux in the light intensity-treatment increased the accumulation of delphinidin-3-O-arabinoside compared to under the shading-treatment. Furthermore, we performed qRT-PCR analysis of anthocyanin biosynthesis genes and predicted that the gene of VcF3'5'H4 may be a candidate gene for anthocyanin accumulation and is highly expressed in light intensity-treated fruit. Through the co-expression analysis of transcription factors and anthocyanin synthesis pathway genes, we found that the VcbHLH004 gene may regulate VcF3'5'H4, and then we transformed VcbHLH004 heterologously into tomato to verify its function., Conclusion: These results provide novel insights into light intensity regulation of blueberry anthocyanin accumulation and represent a valuable data set to guide future functional studies and blueberry breeding., (© 2022. The Author(s).)

Background: Auxin responsive factor (ARF) family is one of core components in auxin signalling pathway, which governs diverse developmental processes and stress responses. Blueberry is an economically important berry-bearing crop and prefers to acidic soil. However, the understandings of ARF family has not yet been reported in blueberry., Results: In the present study, 60 ARF genes (VcARF) were identified in blueberry, and they showed diverse gene structures and motif compositions among the groups and similar within each group in the phylogenetic tree. Noticeably, 9 digenic, 5 trigenic and 6 tetragenic VcARF pairs exhibited more than 95% identity to each other. Computational analysis indicated that 23 VcARFs harbored the miRNA responsive element (MRE) of miR160 or miR167 like other plant ARF genes. Interestingly, the MRE of miR156d/h-3p was observed in the 5'UTR of 3 VcARFs, suggesting a potentially novel post-transcriptional control. Furthermore, the transcript accumulations of VcARFs were investigated during fruit development, and three categories of transcript profiles were observed, implying different functional roles. Meanwhile, the expressions of VcARFs to different pH conditions (pH4.5 and pH6.5) were surveyed in pH-sensitive and tolerant blueberry species, and a number of VcARFs showed different transcript accumulations. More importantly, distinct transcriptional response to pH stress (pH6.5) were observed for several VcARFs (such as VcARF6s and VcARF19-3/19-4) between pH-sensitive and tolerant species, suggesting their potential roles in adaption to pH stress., Conclusions: Sixty VcARF genes were identified and characterized, and their transcript profiles were surveyed during fruit development and in response to pH stress. These findings will contribute to future research for eliciting the functional roles of VcARFs and regulatory mechanisms, especially fruit development and adaption to pH stress., (© 2022. The Author(s).)

Although they are staple foods in cuisines globally, many commercial fruit varieties have become progressively less flavorful over time. Due to the cost and difficulty associated with flavor phenotyping, breeding programs have long been challenged in selecting for this complex trait. To address this issue, we leveraged targeted metabolomics of diverse tomato and blueberry accessions and their corresponding consumer panel ratings to create statistical and machine learning models that can predict sensory perceptions of fruit flavor. Using these models, a breeding program can assess flavor ratings for a large number of genotypes, previously limited by the low throughput of consumer sensory panels. The ability to predict consumer ratings of liking, sweet, sour, umami, and flavor intensity was evaluated by a 10-fold cross-validation, and the accuracies of 18 different models were assessed. The prediction accuracies were high for most attributes and ranged from 0.87 for sourness intensity in blueberry using XGBoost to 0.46 for overall liking in tomato using linear regression. Further, the best-performing models were used to infer the flavor compounds (sugars, acids, and volatiles) that contribute most to each flavor attribute. We found that the variance decomposition of overall liking score estimates that 42% and 56% of the variance was explained by volatile organic compounds in tomato and blueberry, respectively. We expect that these models will enable an earlier incorporation of flavor as breeding targets and encourage selection and release of more flavorful fruit varieties., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Dark septate endophytes (DSEs) have attracted much attention due to their positive roles in plant growth as well as resistance to various abiotic stresses. However, there are no reports on the molecular mechanisms of DSE fungi to improve salt tolerance in plants. In this study, the blueberry seedlings inoculated with T010, a beneficial DSE fungus reported previously, grew more vigorously than the non-inoculated control under salt stress. Physiological indicators showed that T010 inoculation increased antioxidant activities of blueberry roots. To explore its molecular mechanism, we focused on the bZIP TFs VabZIP12, who was highly up-regulated with T010 inoculation under salt stress. Further studies showed that VabZIP12, as a transcription activator, could combine both G-Box 1 and G-Box 2 motifs. Moreover, overexpression of VabZIP12 enhanced salt stress tolerance through increasing the activities of the enzymatic antioxidants in the transgenic Arabidopsis with up-regulation the related genes. These results indicated that the induction of VabZIP12 contribute to improving the tolerance of blueberry to salt stress by T010 inoculation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: The present study aimed to determine whether the ozonation process affects the flavonoid biosynthesis in highbush blueberry (Vaccinum corymbosum L.) fruit. Flavanone 3β-hydroxylase (F3H) was used as a marker of the flavonoid biosynthesis pathway. The activity of F3H, the expression of gene encoding F3H and the antioxidant status in blueberries treated with ozone at a concentration of 15 ppm for 30 min, every 12 h of storage, and maintained at 4 °C for 4 weeks were investigated., Results: The results showed that ozonation process increases the expression of the F3H gene after 1 week of storage, which translates into a higher catalytic capacity of protein, as well as a higher content of flavonoids and total antioxidant potential of ozonated blueberries compared to non-ozonated fruits., Conclusion: The present study provides experimental evidence indicating that ozone treatment in proposed process conditions positively affects flavonoid metabolism in highbush blueberry fruit leading to the maintainance of the high quality of the fruit during storage. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Basic helix-loop-helix proteins (bHLHs) play very important roles in the anthocyanin biosynthesis of many plant species. However, the reports on blueberry anthocyanin biosynthesis-related bHLHs were very limited. In this study, six anthocyanin biosynthesis-related bHLHs were identified from blueberry genome data through homologous protein sequence alignment. Among these blueberry bHLHs, VcAN1, VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3 were clustered into one group, while VcbHLH1-1 and VcbHLH1-2 were clustered into the other group. All these bHLHs were of the bHLH-MYC&#95;N domain, had DNA binding sites and reported conserved amino acids in the bHLH domain, indicating that they were all G-box binding proteins. Protein subcellular location prediction result revealed that all these bHLHs were nucleus-located. Gene structure analysis showed that VcAN1 gDNA contained eight introns, while all the others contained seven introns. Many light-, phytohormone-, stress- and plant growth and development-related cis -acting elements and transcription factor binding sites (TFBSs) were identified in their promoters, but the types and numbers of cis -elements and TFBSs varied greatly between the two bHLH groups. Quantitative real-time PCR results showed that VcAN1 expressed highly in old leaf, stem and blue fruit, and its expression increased as the blueberry fruit ripened. Its expression in purple podetium and old leaf was respectively significantly higher than in green podetium and young leaf, indicating that VcAN1 plays roles in anthocyanin biosynthesis regulation not only in fruit but also in podetium and leaf. VcbHLH1-1 expressed the highest in young leaf and stem, and the lowest in green fruit. The expression of VcbHLH1-1 also increased as the fruit ripened, and its expression in blue fruit was significantly higher than in green fruit. VcbHLH1-2 showed high expression in stem but low expression in fruit, especially in red fruit. Our study indicated that the anthocyanin biosynthesis regulatory functions of these bHLHs showed certain spatiotemporal specificity. Additionally, VcAN1 might be a key gene controlling the anthocyanin biosynthesis in blueberry, whose function is worth exploring further for its potential applications in plant high anthocyanin breeding.

Flowering and fruit set are important traits affecting fruit quality and yield in rabbiteye blueberry (Vaccinium ashei). Intense efforts have been made to elucidate the influence of vernalization and phytohormones on flowering, but the molecular mechanisms of flowering and fruit set remain unclear. To unravel these mechanisms, we performed transcriptome analysis to explore blueberry transcripts from flowering to early fruit stage. We divided flowering and fruit set into flower bud (S2), initial flower (S3), bloom flower (S4), pad fruit (S5), and cup fruit (S6) based on phenotype and identified 1,344, 69, 658, and 189 unique differentially expressed genes (DEGs) in comparisons of S3/S2, S4/S3, S5/S4, and S6/S5, respectively. There were obviously more DEGs in S3/S2 and S5/S4 than in S4/S3, and S6/S5, suggesting that S3/S2 and S5/S4 represent major transitions from buds to fruit in blueberry. GO and KEGG enrichment analysis indicated these DEGs were mostly enriched in phytohormone biosynthesis and signaling, transporter proteins, photosynthesis, anthocyanins biosynthesis, disease resistance protein and transcription factor categories, in addition, transcript levels of phytohormones and transporters changed greatly throughout the flowering and fruit set process. Gibberellic acid and jasmonic acid mainly acted on the early stage of flowering development like expression of the florigen gene FT, while the expression of auxin response factor genes increased almost throughout the process from bud to fruit development. Transporter proteins were mainly associated with minerals during the early flowering development stage and sugars during the early fruit stage. At the early fruit stage, anthocyanins started to accumulate, and the fruit was susceptible to diseases such as fungal infection. Expression of the transcription factor MYB86 was up-regulated during initial fruit development, which may promote anthocyanin accumulation. These results will aid future studies exploring the molecular mechanism underlying flowering and fruit set of rabbiteye blueberry., Competing Interests: The authors have declared that no competing interests exist.

Background: Monilinia blight caused by Monilinia vaccinii-corymbosi (Reade) Honey (M.vc) is a major disease of wild blueberry that can result in severe crop losses in the absence of an integrated disease management programme. The fungus causes blight in the emerging floral and vegetative buds, but the degree of susceptibility varies among the different wild blueberry phenotypes, ranging from the highly susceptible V. a. f. nigrum to the moderately susceptible V. angustifolium and the least susceptible V. myrtilloides., Results: The present study evaluated the defense responses of these major phenotypes during their primary infection (floral buds) with M.vc. The temporal expression profiles of PR genes (PR3 and PR4) and the flavonoid pathway structural genes (CHS, ANS, ANR, DFR and FLS) were analysed. The PR3 and PR4 gene expression profiles revealed that V. myrtilloides responded to M.vc infection by activating the expression of both PR genes. V. a. f. nigrum, on the other hand, failed to activate these genes, while V. angustifolium, exhibited an intermediate response. Our study with the flavonoid pathway genes indicated variability in activation of the genes during post-infection time points with ANS and ANR in V. myrtilloides, FLS in V. angustifolium and no response observed in V. a. f. nigrum., Conclusions: Altogether, this study highlights that the degree of phenotype susceptibility is associated with the timely activation of host defense responsive genes. Data obtained in this study provided a starting point for a better understanding of the wild blueberry- M. vaccinii-corymbosi pathosystem., (© 2021. The Author(s).)

Blueberry (Vaccinium ssp.) is a perennial shrub belonging to the family Ericaceae, which is highly tolerant of acid soils and heavy metal pollution. In the present study, blueberry was subjected to cadmium (Cd) stress in simulated pot culture. The transcriptomics and rhizosphere fungal diversity of blueberry were analyzed, and the iron (Fe), manganese (Mn), copper (Cu), zinc (Zn) and cadmium (Cd) content of blueberry tissues, soil and DGT was determined. A correlation analysis was also performed. A total of 84 374 annotated genes were identified in the root, stem, leaf and fruit tissue of blueberry, of which 3370 were DEGs, and in stem tissue, of which 2521 were DEGs. The annotation data showed that these DEGs were mainly concentrated in a series of metabolic pathways related to signal transduction, defense and the plant-pathogen response. Blueberry transferred excess Cd from the root to the stem for storage, and the highest levels of Cd were found in stem tissue, consistent with the results of transcriptome analysis, while the lowest Cd concentration occurred in the fruit, Cd also inhibited the absorption of other metal elements by blueberry. A series of genes related to Cd regulation were screened by analyzing the correlation between heavy metal content and transcriptome results. The roots of blueberry rely on mycorrhiza to absorb nutrients from the soil. The presence of Cd has a significant effect on the microbial community composition of the blueberry rhizosphere. The fungal family Coniochaetaceae, which is extremely extremelytolerant, has gradually become the dominant population. The results of this study increase our understanding of the plant regulation mechanism for heavy metals, and suggest potential methods of soil remediation using blueberry., (© 2021. The Author(s).)