Red walnuts have been widely studied because of their strong antioxidant activity and ornamental value. However, research on the mechanism of anthocyanin biosynthesis in walnuts remains in the initial stage. The regulatory mechanism of TT2-type R2R3-MYB transcription factors in anthocyanin biosynthesis in walnuts is also unclear. Therefore, this study used ‘D2-1’ and ‘Jinghong 1’ walnuts as plant materials. The testa of ‘Jinghong 1’ was red, and its anthocyanin content was significantly higher than that of ‘D2-1’, mainly composed of cyanidin-3-O-arabinoside, cyanidin-3-O-galactoside, and cyanidin-3-O-glucoside. Differentially expressed genes between ‘D2-1’ and ‘Jinghong 1’ testa were enriched in phenylpropanoid biosynthesis and flavonoid biosynthesis. Next, this study identified a TT2-type R2R3-MYB transcription factor JrMYB1L, which was involved in regulating the anthocyanin biosynthesis in the testa of ‘Jinghong 1’. The overexpression of JrMYB1L could promote anthocyanin accumulation in walnut leaves and activate the expression of JrCHS, JrCHI, JrF3H, JrDFR, JrANS, JrUFGT, JrLAR, and JrANR. In addition, yeast two-hybrid results proved that JrMYB1L, JrbHLH42, and JrWD40 proteins could interact with each other. The results of yeast one-hybrid and dual-luciferase assays indicated that JrMYB1L could activate the expression of JrCHS and JrUFGT by binding to their promoters. Based on the above results, this study proposed a possible regulatory mechanism. JrMYB1L activated the expression of JrCHS and JrUFGT in the form of JrMYB1L-JrbHLH42-JrWD40 complex, thereby promoting anthocyanin accumulation in the testa of ‘Jinghong 1’. In summary, this study lays a theoretical foundation for revealing the regulatory mechanism of anthocyanin biosynthesis in red walnut and contributes to the breeding of new varieties of red walnuts with more edible and ornamental value.

Walnut is one of four major nuts in the world. China has abundant walnut germplasm resources, but there are still shortcomings in the identification of germplasm resources. This study used different walnut varieties as materials and developed 14 high-quality SSR molecular markers from 60 pairs of primers based on genome re-sequencing results. This study analyzed the genetic diversity of Chinese walnut genetic resources using 14 selected SSR markers. A total of 64 alleles were detected in 47 walnut resources, with an average of 4.571 alleles per locus. The variation range of polymorphism information content was 0.096~0.711, with an average value of 0.422. Cluster analysis, population genetic structure, and principal coordinate analysis divided 47 walnut resources into ordinary walnuts, Juglans hopeiensis, and Liaoyi 1. In addition, core SSR markers (Jr45, Jr40, Jr29, Jr35, and Jr11) were selected from 14 SSR markers, which were sufficient to distinguish 47 walnut resources. At the same time, 47 unique molecular fingerprints of walnuts were constructed using these core SSR markers. This study provides strong scientific support for rapid and efficient identification, germplasm innovation, and a variety of property protection of walnut germplasm.

Secondary flowering is the phenomenon in which a tree blooms twice or more times a year. Along with the development of blueberry (Vaccinium corymbosum L.) fruits in spring, a large number of secondary flowers on the strong upright spring shoots were noticed in blueberries planted in the greenhouse. To reveal the cause and possible regulatory mechanism of the phenomenon, we clarified the phenological characteristics of flower bud differentiation and development on the spring shoots by combining phenological phenotype with anatomical observation. Furthermore, the changes in carbohydrates, trehalose-6-phosphate (Tre6P), and the relationship among the key enzyme regulatory genes for Tre6P metabolism and the key regulatory genes for flower formation during the differentiation process of apical buds and axillary buds were investigated. The results showed that the process of flower bud differentiation and flowering of apical and axillary buds was consistent, accompanied by a large amount of carbohydrate consumption. This process was positively correlated with the expression trends of VcTPS1/2, VcSnRK1, VcFT, VcLFY2, VcSPL43, VcAP1, and VcDAM in general, and negatively correlated with that of VcTPP. In addition, there is a certain difference in the differentiation progress of flower buds between the apical and axillary buds. Compared with axillary buds, apical buds had higher contents of sucrose, fructose, glucose, Tre6P, and higher expression levels of VcTPS2, VcFT, VcSPL43, and VcAP1. Moreover, VcTPS1 and VcTPS2 were more closely related to the physiological substances (sucrose and Tre6P) in axillary bud and apical bud differentiation, respectively. It was suggested that sucrose and trehalose-6-phosphate play a crucial role in promoting flower bud differentiation in strong upright spring shoots, and VcTPS1 and VcTPS2 might play a central role in these activities. Our study provided substantial sight for further study on the mechanism of multiple flowering of blueberries and laid a foundation for the regulation and utilization of the phenomenon of multiple flowering in a growing season of perennial woody plants.