Multidrug and toxic compound extrusion (MATE) proteins are the most recently identified family of multidrug transporters. In plants, this family is remarkably large compared to the human and bacteria counterpart, highlighting the importance of MATE proteins in this kingdom. Here 33 Unigenes annotated as MATE transporters were found in the blueberry fruit transcriptome, of which eight full-length cDNA sequences were identified and cloned. These proteins are composed of 477–517 residues, with molecular masses ~54 kDa, and theoretical isoelectric points from 5.35 to 8.41. Bioinformatics analysis predicted 10–12 putative transmembrane segments for VcMATEs, and localization to the plasma membrane without an N-terminal signal peptide. All blueberry MATE proteins shared 32.1–84.4% identity, among which VcMATE2, VcMATE3, VcMATE5, VcMATE7, VcMATE8, and VcMATE9 were more similar to the MATE-type flavonoid transporters. Phylogenetic analysis showed VcMATE2, VcMATE3, VcMATE5, VcMATE7, VcMATE8 and VcMATE9 clustered with MATE-type flavonoid transporters, indicating that they might be involved in flavonoid transport. VcMATE1 and VcMATE4 may be involved in the transport of secondary metabolites, the detoxification of xenobiotics, or the export of toxic cations. Real-time quantitative PCR demonstrated that the expression profile of the eight VcMATE genes varied spatially and temporally. Analysis of expression and anthocyanin accumulation indicated that there were some correlation between the expression profile and the accumulation of anthocyanins. These results showed VcMATEs might be involved in diverse physiological functions, and anthocyanins across the membranes might be mutually maintained by MATE-type flavonoid transporters and other mechanisms. This study will enrich the MATE-based transport mechanisms of secondary metabolite, and provide a new biotechonology strategy to develop better nutritional blueberry cultivars.

Cranberries (Vaccinium macrocarpon Ait.), renowned for their excellent health benefits, are an important berry crop. Here, we performed transcriptome sequencing of one cranberry cultivar, from fruits at two different developmental stages, on the Illumina HiSeq 2000 platform. Our main goals were to identify putative genes for major metabolic pathways of bioactive compounds and compare the expression patterns between white fruit (W) and red fruit (R) in cranberry. In this study, two cDNA libraries of W and R were constructed. Approximately 119 million raw sequencing reads were generated and assembled de novo, yielding 57,331 high quality unigenes with an average length of 739 bp. Using BLASTx, 38,460 unigenes were identified as putative homologs of annotated sequences in public protein databases, including NCBI NR, NT, Swiss-Prot, KEGG, COG and GO. Of these, 21,898 unigenes mapped to 128 KEGG pathways, with the metabolic pathways, secondary metabolites, glycerophospholipid metabolism, ether lipid metabolism, starch and sucrose metabolism, purine metabolism, and pyrimidine metabolism being well represented. Among them, many candidate genes were involved in flavonoid biosynthesis, transport and regulation. Furthermore, digital gene expression (DEG) analysis identified 3,257 unigenes that were differentially expressed between the two fruit developmental stages. In addition, 14,473 simple sequence repeats (SSRs) were detected. Our results present comprehensive gene expression information about the cranberry fruit transcriptome that could facilitate our understanding of the molecular mechanisms of fruit development in cranberries. Although it will be necessary to validate the functions carried out by these genes, these results could be used to improve the quality of breeding programs for the cranberry and related species.

Background: Caffeic acid O-methyltransferases (COMTs) play an important role in the diversification of natural products, especially in the phenylalanine metabolic pathway of plant. The content of COMT genes in blueberry and relationship between their expression patterns and the lignin content during fruit development have not clearly investigated by now. Results: Ninety-two VcCOMTs were identified in Vaccinium corymbosum. According to phylogenetic analyses, the 92 VcCOMTs were divided into 2 groups. The gene structure and conserved motifs within groups were similar which supported the reliability of the phylogenetic structure groupings. Dispersed duplication (DSD) and whole-genome duplication (WGD) were determined to be the major forces in VcCOMTs evolution. The results showed that the results of qRT-PCR and lignin content for 22 VcCOMTs, VcCOMT40 and VcCOMT92 were related to lignin content at different stages of fruit development of blueberry. Conclusion: We identified COMT gene family in blueberry, and performed comparative analyses of the phylogenetic relationships in the 15 species of land plant, and gene duplication patterns of COMT genes in 5 of the 15 species. We found 2 VcCOMTs were highly expressed and their relative contents were similar to the variation trend of lignin content during the development of blueberry fruit. These results provide a clue for further study on the roles of VcCOMTs in the development of blueberry fruit and could promisingly be foundations for breeding blueberry clutivals with higher fruit firmness and longer shelf life.

As a small fruit rich in anthocyanins, blueberry (Vaccinium corymbosum × angustifolium) has become a focus of research in recent years for identifying genes related to anthocyanin transport and stress resistance mechanisms based on transcriptome sequencing. However, the lack of validated, stably expressed reference genes greatly limits the functional study of blueberry genes. Therefore, in this study, we selected 14 candidate reference genes from a blueberry transcriptome database and used three algorithms (geNorm, NormFinder and BestKeeper) to evaluate the expression stability of these genes in various organs at different fruit developmental stages under five abiotic stress conditions. EF1α, EIF and TBP were observed to be the most stable and were thus chosen as reference genes for quantitative real‐time PCR. Measurement of the relative expression of VcMATE1 (European Nucleotide Archive accession number KF875433) in blueberry further verified the reliability of these reference genes, which may have great utility for determining the accuracy of gene expression analyses in future research on blueberry.

Real-time fluorescent quantitative PCR (qRT-PCR) is often chosen as an effective experimental method for analyzing gene expression. However, an appropriate reference gene as a standard is needed to obtain accurate gene expression data. To date, no internal reference genes have been reported for research on cranberries. Expanding the selection of internal reference genes for cranberry will enable reliable gene expression analysis, and, at the same time, can also lay a solid foundation for revealing the biological characteristics of cranberry. Here, we selected ten candidate reference gene families and used three statistical software tools—geNorm, NormFinder and BestKeeper—to evaluate their expression stability under the influence of different experimental factors. The results showed that protein phosphatase 2A regulatory subunit (PP2A) or RNA helicase-like 8 (RH 8) was the best choice for an internal reference gene when analyzing different cranberry cultivars. In two sample sets comprising different cranberry organs and three abiotic stress treatments, sand family protein (SAND) was the best choice as a reference gene. In this study, we screened genes that are stably expressed under the influence of various experimental factors by qRT-PCR. Our results will guide future studies involving gene expression analysis of cranberry.