Chestnut (Castanea mollissima) is a deciduous tree species with major economic and ecological value that is widely used in the study of floral development in woody plants due its monoecious and out-of-proportion characteristics. Squamosa promoter-binding protein-like (SPL) is a plant-specific transcription factor that plays an important role in floral development. In this study, a total of 18 SPL genes were identified in the chestnut genome, of which 10 SPL genes have complementary regions of CmmiR156. An analysis of the phylogenetic tree of the squamosa promoter-binding protein (SBP) domains of the SPL genes of Arabidopsis thaliana, Populus trichocarpa, and C. mollissima divided these SPL genes into eight groups. The evolutionary relationship between poplar and chestnut in the same group was similar. A structural analysis of the protein-coding regions (CDSs) showed that the domains have the main function of SBP domains and that other domains also play an important role in determining gene function. The expression patterns of CmmiR156 and CmSPLs in different floral organs of chestnut were analyzed by real-time quantitative PCR. Some CmSPLs with similar structural patterns showed similar expression patterns, indicating that the gene structures determine the synergy of the gene functions. The application of gibberellin (GA) and its inhibitor (Paclobutrazol, PP₃₃₃) to chestnut trees revealed that these exert a significant effect on the number and length of the male and female chestnut flowers. GA treatment significantly increased CmmiR156 expression and thus significantly decreased the expression of its target gene, CmSPL6/CmSPL9/CmSPL16, during floral bud development. This finding indicates that GA might indirectly affect the expression of some of the SPL target genes through miR156. In addition, RNA ligase-mediated rapid amplification of the 5′ cDNA ends (RLM-RACE) experiments revealed that CmmiR156 cleaves CmSPL9 and CmSPL16 at the 10th and 12th bases of the complementary region. These results laid an important foundation for further study of the biological function of CmSPLs in the floral development of C. mollissima.

Chinese chestnut (Castanea mollissima Blume) is native to China and distributes widely in arid and semi-arid mountain area with barren soil. As a perennial crop, chestnut is an alternative food source and acts as an important commercial nut tree in China. Starch is the major metabolite in nuts, accounting for 46 ~ 64% of the chestnut dry weight. The accumulation of total starch and amylopectin showed a similar increasing trend during the development of nut. Amylopectin contributed up to 76% of the total starch content at 80 days after pollination (DAP). The increase of total starch mainly results from amylopectin synthesis. Among genes associated with starch biosynthesis, CmSBEs (starch branching enzyme) showed significant increase during nut development. Two starch branching enzyme isoforms, CmSBE I and CmSBE II, were identified from chestnut cotyledon using zymogram analysis. CmSBE I and CmSBE II showed similar patterns of expression during nut development. The accumulations of CmSBE transcripts and proteins in developing cotyledons were characterized. The expressions of two CmSBE genes increased from 64 DAP and reached the highest levels at 77 DAP, and SBE activity reached its peak at 74 DAP. These results suggested that the CmSBE enzymes mainly contributed to amylopectin synthesis and influenced the amylopectin content in the developing cotyledon, which would be beneficial to chestnut germplasm selection and breeding.

Chestnut blight, one of the most serious branch diseases in Castanea caused by Cryphonectria parasitica, which has ravaged across American chestnut and most of European chestnut since the early twentieth century. Interestingly, the Chinese chestnut is strongly resistant to chestnut blight, shedding light on restoring the ecological status of Castanea plants severely affected by chestnut blight. To better explore the early defense of Chinese chestnut elicited in response to C. parasitica, the early stage of infection process of C. parasitica was observed and RNA sequencing-based transcriptomic profiling of responses of the chestnut blight-resistant wild resource ‘HBY-1’ at 0, 3 and 9 h after C. parasitica inoculation was performed. First, we found that 9 h was a critical period for Chinese chestnut infected by C. parasitica, which was the basis of further study on transcriptional activation of Chinese chestnut in response to chestnut blight in the early stage. In the transcriptome analysis, a total of 283 differentially expressed genes were identified between T9 h and Mock9 h, and these DEGs were mainly divided into two clusters, one of which was metabolism-related pathways including biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, amino sugar and nucleotide sugar metabolism, and photosynthesis; the other was related to plant-pathogen interaction and MAPK signal transduction. Meanwhile, the two clusters of pathways could be connected through junction among phosphatidylinositol signaling system, phytohormone signaling pathway and α-Linolenic acid metabolism pathway. It is worth noting that genes associated with JA biosynthesis and metabolic pathway were significantly up-regulated, revealing that the entire JA metabolic pathway was activated in Chinese chestnut at the early stage of chestnut blight infection. We identified the important infection nodes of C. parasitica and observed the morphological changes of Chinese chestnut wounds at the early stage of infection. In response to chestnut blight, the plant hormone and MAPK signal transduction pathways, plant-pathogen interaction pathways and metabolism-related pathways were activated at the early stage. JA biosynthesis and metabolic pathway may be particularly involved in the Chinese chestnut resistance to chestnut blight. These results contributes to verifying the key genes involved in the resistance of Chinese chestnut to C. parasitica.

Chinese chestnut (Castanea mollissima), a species with recalcitrant seeds, is an important source of nuts and forest ecosystem services. The germination rate of recalcitrant seeds is low in natural habitats and decreases under conditions of desiccation and low temperature. The germination rate of cultivated Chinese chestnut seeds is significantly higher than that of wild seeds. To explore the reasons for the higher germination rate of cultivated seeds in Chinese chestnut, 113,524 structural variants (SVs) between the wild and cultivated Chinese chestnut genomes were detected through genome comparison. Genotyping these SVs in 60 Chinese chestnut accessions identified allele frequency changes during Chinese chestnut domestication, and some SVs are overlapping genes for controlling seed germination. Transcriptome analysis revealed downregulation of the abscisic acid synthesis genes and upregulation of the beta-amylase synthesis genes in strongly selected genes of cultivated seeds. On the other hand, hormone and enzyme activity assays indicated a decrease in endogenous ABA level and an increase in beta-amylase activity in cultivated seeds. These results shed light on the higher germination rate of cultivated seeds. Moreover, phosphatidic acid synthesis genes are highly expressed in seed germination stages of wild Chinese chestnut and may play a role in recalcitrant seed germination. These findings provide new insight into the regulation of wild seed germination and promote natural regeneration and succession in forest ecosystems.

This study investigates the taxonomic status, phylogenetic relationships, and genetic diversity of Japanese chestnut (Castanea crenata Siebold & Zucc.) in Liaodong, China, and across East Asia. Additionally, it evaluates core germplasm resources through cluster and population structure analyses using simple sequence repeat (SSR) marker data from 13 Castanea henryi, 18 Castanea seguinii, and 27 Castanea mollissima, and 142 Japanese chestnut resources. The results show that the East Asian Castanea genus forms a monophyletic group with distinct interspecific boundaries. Japanese chestnut and two varieties/lines of C. seguinii (187 and 170) form a sister clade, indicating a close phylogenetic relationship. All Japanese chestnut resources are divided into two branches, with considerable admixture. The genetic diversity analysis revealed that the 142 Japanese chestnut varieties/lines collectively possessed 141 allelic loci, with genetic distances (GDs) ranging from 0.429 to 0.880 with an average of 0.740. Based on unique characteristics, seven resources with distinctive features were selected as mandatory. A total of 41 core germplasm resources were finally determined using the simulated annealing method. The comparative analysis revealed that, aside from a notable difference in polymorphic information loci, the core germplasm and original germplasm showed no significant differences in other genetic diversity parameters. This indicates that the 41 core germplasm resources effectively preserve the genetic diversity of the original germplasm and have been influenced by artificial selection. This study provides a scientific basis for conserving and using C. crenata germplasm resources.