Metabolomic selection for enhanced fruit flavor.

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.

Genome-wide prediction and functional analysis of WOX genes in blueberry.

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.

Ethylene promotes fruit ripening initiation by downregulating photosynthesis, enhancing abscisic acid and suppressing jasmonic acid in blueberry (Vaccinium ashei).

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.

Characterization of the MIKCC-type MADS-box gene family in blueberry and its possible mechanism for regulating flowering in response to the chilling requirement.

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 MIKCC-type MADS-box gene family is involved in the regulation of flower development. A total of 109 members of the MIKCC-type MADS-box gene family were identified in blueberry. According to the phylogenetic tree, these 109 MIKCC-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 (GA4+7) treatment on the expression pattern of MIKCC-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.

The miR156/SPL12 module orchestrates fruit colour change through directly regulating ethylene production pathway in blueberry.

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.

Na+-preferential ion transporter HKT1;1 mediates salt tolerance in blueberry.

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.

De novo biosynthesis of anthocyanins in Saccharomyces cerevisiae using metabolic pathway synthases from blueberry.

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.

Transcriptome-Based Identification of Candidate Flowering-Associated Genes of Blueberry in a Plant Factory with Artificial Lighting (PFAL) under Short-Day-Length Conditions.

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.

Integrated Untargeted Metabolome, Full-Length Sequencing and Transcriptome Analyses Reveal the Mechanism of Flavonoid Biosynthesis in Blueberry (Vaccinium spp.) Fruit.

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.

Genome-Wide Identification of the Sulfate Transporters Gene Family in Blueberry (Vaccinium spp.) and Its Response to Ericoid Mycorrhizal Fungi.

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.

Identification of R2R3-MYB family in blueberry and its potential involvement of anthocyanin biosynthesis in fruits.

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.

Genome-wide analysis of blueberry B-box family genes and identification of members activated by abiotic stress.

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.

Modulation of defense genes and phenolic compounds in wild blueberry in response to Botrytis cinerea under field conditions.

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.

Autopolyploid inheritance and a heterozygous reciprocal translocation shape chromosome genetic behavior in tetraploid blueberry (Vaccinium corymbosum).

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.

Genome-wide identification of GH9 gene family and the assessment of its role during fruit abscission zone formation in Vaccinium ashei.

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.

Comprehensive Analysis of Highbush Blueberry Plants Propagated In Vitro and Conventionally.

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.

Transcriptomic and Metabolomic Profiling Reveals the Variations in Carbohydrate Metabolism between Two Blueberry Cultivars.

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.

Characterization and Functional Analysis of Chalcone Synthase Genes in Highbush Blueberry (Vaccinium corymbosum).

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.

Genome-Wide Analysis of the Universal Stress Protein Gene Family in Blueberry and Their Transcriptional Responses to UV-B Irradiation and Abscisic Acid.

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.

Full-length fruit transcriptomes of southern highbush (Vaccinium sp.) and rabbiteye (V. virgatum Ait.) blueberry.

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.