Introduction: The WRKY transcription factor (TF) family is one of the largest TF families in plants and is widely involved in responses to both biotic and abiotic stresses.Methods: To clarify the function of the WRKY family in blueberries, this study identified the WRKY genes within the blueberry genome and systematically analyzed gene characteristics, phylogenetic evolution, promoter cis-elements, expression patterns, and subcellular localization of the encoded products.Results: In this study, 57 VcWRKY genes were identified, and all encoding products had a complete WRKY heptapeptide structure and zinc-finger motif. The VcWRKY genes were divided into three subgroups (I-III) by phylogenetic analysis. Group II was divided into five subgroups: IIa, IIb, IIc, IId, and IIe. 57 VcWRKY genes were distributed unevenly across 32 chromosomes. The amino acids ranged from 172 to 841, and molecular weights varied from 19.75 to 92.28 kD. Intra-group syntenic analysis identified 12 pairs of duplicate segments. Furthermore, 34 cis-element recognition sites were identified in the promoter regions of VcWRKY genes, primarily comprising phytohormone-responsive and light-responsive elements. Comparative syntenic maps were generated to investigate the evolutionary relationships of VcWRKY genes, revealing the closest homology to dicotyledonous WRKY gene families. VcWRKY genes were predominantly expressed in the fruit flesh and roots of blueberries. Gene expression analysis showed that the responses of VcWRKY genes to stress treatments were more strongly in leaves than in roots. Notably, VcWRKY13 and VcWRKY25 exhibited significant upregulation under salt stress, alkali stress, and saline-alkali stress, and VcWRKY1 and VcWRKY13 showed notable induction under drought stress. Subcellular localization analysis confirmed that VcWRKY13 and VcWRKY25 function within the nucleus.Conclusion: These findings establish a foundation for further investigation into the functions and regulatory mechanisms of VcWRKY genes and provide guidance for selecting stress-tolerant genes in the development of blueberry cultivars.

Blueberries are recognized worldwide as one of the most important healthy foods due to their anthocyanins, which have special antioxidant properties. They have become a highly produced and valuable fruit crop. Most blueberry varieties are rich in anthocyanins, which impart a beautiful blue color; however, there are currently several blueberry varieties with different colors worldwide, and these special-colored varieties are the key to analyzing the coloring mechanism of blueberry fruit. Fruit color could be seen as an important nutritional quality trait in terms of marketing. In this study, a combination of transcriptomic and metabolomic analyses was performed on three representative blueberry varieties (‘Pink Popcorn’, ‘Chandler’, and ‘Black Pearl’) with pink, blue, and black fruits, respectively. The metabolomic results showed that the delphinium pigment is the dominant anthocyanin, which is the prerequisite for the formation of fruit color in blueberries. We identified 18 candidate structural genes in the anthocyanin biosynthesis pathway that were significantly up-regulated during three stages of fruit ripening in ‘Black Pearl’ and ‘Chandler’, but these were not found to be significantly expressed in ‘Pink Popcorn’ after combining the transcriptomic analysis results. The non-expression of the VcANS gene may lead to the pink color of the mature fruit of ‘Pink Popcorn’. The phylogenetic tree, heatmap analysis, and WGCNA analysis identified a candidate transcription factor, VcMYBA, which may regulate the differences between black and blue fruits in blueberries by regulating the expression level of multiple structural genes in the anthocyanin biosynthesis pathway. These results provide new insights into the mechanisms of anthocyanin accumulation and coloration in blueberries during fruit ripening and can help support production practices to improve fruit quality characteristics. The key candidate genes that regulate the fruit color differences among different blueberry varieties have the potential to enhance the antioxidant properties and quality characteristics of blueberries through future genomic editing.

Blueberry belongs to the Vaccinium genus and is a highly popular fruit crop with significant economic importance. It was not until the early twentieth century that they began to be domesticated through extensive interspecific hybridization. Here, we collected 220 Vaccinium accessions from various geographical locations, including 154 from the United States, 14 from China, eight from Australia, and 29 from Europe and other countries, comprising 164 Vaccinium corymbosum, 15 Vaccinium ashei, 10 lowbush blueberries, seven half-high blueberries, and others. We present the whole-genome variation map of 220 accessions and reconstructed the hundred-year molecular history of interspecific hybridization of blueberry. We focused on the two major blueberry subgroups, the northern highbush blueberry (NHB) and southern highbush blueberry (SHB) and identified candidate genes that contribute to their distinct traits in climate adaptability and fruit quality. Our analysis unveiled the role of gene introgression from Vaccinium darrowii and V. ashei into SHB in driving the differentiation between SHB and NHB, potentially facilitating SHB’s adaptation to subtropical environments. Assisted by genome-wide association studies, our analysis suggested VcTBL44 as a pivotal gene regulator governing fruit firmness in SHB. Additionally, we conducted whole-genome bisulfite sequencing on nine NHB and 12 SHB cultivars, and characterized regions that are differentially methylated between the two subgroups. In particular, we discovered that the β-alanine metabolic pathway genes were enriched for DNA methylation changes. Our study provides high-quality genetic and epigenetic variation maps for blueberry, which offer valuable insights and resources for future blueberry breeding.

The Agrobacterium tumefaciens-mediated transformation for blueberries remains less efficient than is desirable. A new leaf callus regeneration and genetic transformation system was investigated in blueberries in this study. The leaf explants of cv. ‘Legacy’ and ‘Northland’ were used to establish the stable callus induction system when placed on the woody plant medium (WPM) supplemented with 1.0 mg·L⁻¹ 2, 4-D, 0.4 mg·L⁻¹ 6-BA for 30 d; then, the callus was sub-cultured in the proliferation medium supplemented with 1.5 mg·L⁻¹ 2, 4-D, 0.4 mg·L⁻¹ 6-BA in the darkness at 25 °C every 30 days. The co-cultivation of callus with A. tumefaciens was operated on WPM plus 100 μM acetosyringone for 4 days; then, the transferred callus was grown in WPM supplemented with 1.5 mg·L⁻¹ 2,4-D, 0.4 mg·L⁻¹ 6-BA, 50 mg·L⁻¹ hygromycin, and 200 mg·L⁻¹ cefotaxime. The VcCHS transgenic blueberry callus with both GFP signal and Hyg resistance was obtained from the transformed callus of cv. ‘Northland’. The rate of GFP signal detected in the transformed callus was as high as 49.02%, which was consistent with the PCR assay. Collectively, this study provides a highly efficient genetic transformation system in blueberry callus and a powerful approach for the molecular breeding of blueberries.

New Varieties of Blueberry Released by US in 2018 and Analysis of Breeding Trends

Vaccinium L. is an important fruit tree with nutritional, medicinal, and ornamental values. However, the mitochondrial (mt) genome of Vaccinium L. remains largely unexplored. Vaccinium carlesii Dunn is an endemic wild resource in China, which is crucial for blueberry breeding. The V. carlesii mt genomes were sequenced using Illumina and Nanopore, which total length was 636,904 bp with 37 protein coding genes, 20 tRNA genes, and three rRNA genes. We found four pairs of long repeat fragments homologous recombination mediated the generation of substructures in the V. carlesii mt genome. We predicted 383 RNA editing sites, all converting cytosine (C) to uracil (U). According to the phylogenetic analysis, V. carlesii and V. macrocarpon of the Ericaceae exhibited the closest genetic relationship. This study provides a theoretical basis for understanding the evolution of higher plants, species classification and identification, and will also be useful for further utilization of Vaccinium germplasm resources.