Quercus (oak) is an important economic and ecological tree species in the world, and it is the necessary feed for oak silkworm feeding. Chloroplasts play an important role in green plants but the codon usage of oak chloroplast genomes is not fully studied. We examined the codon usage of the oak chloroplast genomes in detail to facilitate the understanding of their biology and evolution. We downloaded all the protein coding genes of 26 non-redundant chloroplast reference genomes, removed short ones and those containing internal stop codons, and finally retained 50 genes shared by all genomes for comparative analyses. The base composition, codon bias, and codon preference are not significantly different between genomes but are significantly different among genes within these genomes. Oak chloroplast genomes prefer T/A-ending codons and avoid C/G-ending codons, and the psbA gene has the same preference except for the codons encoding amino acid Phe. Complex factors such as context-dependent mutations are the major factors affecting codon usage in these genomes, while selection plays an important role on the psbA gene. Our study provided an important understanding of codon usage in the oak chloroplast genomes and found that the psbA gene has nearly the same codon usage preference as other genes in the oak chloroplasts.

Quercus dentata is a deciduous oak species widely distributed in northern China, with short petioles and dense grayish brown stellate tomentose on the abaxial surface (Lyu et al., 2018). Q. dentata is cold-tolerant (Du et al., 2022), and its broad leaves are used for tussah silkworm rearing, traditional Chinese medicine, kashiwa mochi in Japan, and Manchu delicacy in Northeast China (Wang et al., 2023). In June 2020, a single Q. dentata plant with brown leaf spots was observed in the Oak Germplasm Resources Nursery (N41°82', E123°56') in SYAU, Shenyang, China. From 2021 through 2022, other two nearby Q. dentata plants (six trees in all) became diseased with similar brown spots on their leaves. The small brown lesions with subcircular or irregular shape gradually expanded, and then the entire leaf turned brown. Under magnification, the diseased leaves contain many conidia. To identify the pathogen, diseased tissues were surface sterilized in 2% sodium hypochlorite for 1 min, and washed in sterile distilled water. Lesion margins were plated onto potato dextrose agar and incubated at 28°C in darkness. The aerial mycelium changed color, from white to dark gray, and dark olive green pigmentation was observed on the medium reverse side after 5 days of incubation. The emerging fungal isolates were repurified by the single-spore method. The mean length and width of spores were 20.32 ± 1.90 × 5.2 ± 0.52 μm (n=50). These morphological characteristics resembled the description of Botryosphaeria dothidea (Slippers et al., 2014). For molecular identification, internal transcribed spacer (ITS) region, translation elongation factor1 alpha (tef1-α), and beta-tubulin (tub) were amplified. These new sequences GenBank accession nos. are OQ383627.1, OQ387861.1 and OQ387862.1. Blastn searches showed 100% homology with ITS sequence of B. dothidea strain P31B (KF293892.1) and 98 to 99% similarity with tef and tub sequences of B. dothidea isolate ZJXC2 (KP183219.1) and B. dothidea isolate SHSJ2-1 (KP183133.1). The sequences were also concatenated for phylogenetic analysis (maximum likelihood). Result support isolate SY1 in the same clade as B. dothidea. Based on the multi-gene phylogeny and morphology, the isolated fungus associated with brown leaf spot on Q. dentata was identified as B. dothidea. Pathogenicity tests were performed on five-year-old potted plants. Conidial suspensions (106 conidial/mL) were applied on punctured leaves using a sterile needle and non-punctured leaves. Non-inoculated plants spayed with sterile water served as control. Plants were placed in a growth chamber at 25°C on a 12h fluorescent light/dark regime. Symptoms similar to those from natural infections were observed after 7 to 9 days (non-punctured also infected). No symptoms were found on non-inoculated plants. The pathogenicity test was repeated three times. Fungi re-isolated from inoculated leaves were comfirmed as B. dothidea on the basis of morphological and molecular characterization as described above, fulfilling Koch’s postulates. B. dothidea was previously reported as a pathogen causing branch diebacks and twig dieback on sycamore, red oak (Quercus rubra), and English oak (Quercus robur) in Italy (Turco et al., 2006). It has also been reported to cause leaf spot on Celtis sinensis, Camellia oleifera and Kadsura coccinea in China (Wang et al., 2021; Hao et al., 2022; Su et al., 2021). To our knowledge, this is the first report of B. dothidea inducing leaf spot on Q. dentata in China.

The Chinese oak silkworm is commonly used in pupal diapause research. In this study, a long photoperiod was used to trigger pupal diapause termination. Genes encoding three hormones, namely prothoracicotropic hormone (PTTH), ecdysis triggering hormone (ETH), and eclosion hormone (EH), were studied. Additionally, ecdysteroids (mainly 20-hydroxyecdysone, 20E) were quantified by HPLC. Pupal diapause stage was determined by measuring respiratory intensity. The pupae enter a low metabolic rate, which starts approximately 1 month after pupal emergence. ApPTTH expression showed a small increase at 14 days and then a larger increase from 35 days under the long photoperiod treatment. A similar pattern was observed for the titer of 20E in the hemolymph. However, ApETH expression later increased under the long photoperiod treatment (42 days) just before eclosion. Moreover, ApEH expression increased from 21 to 35 days, and then decreased before ecdysis. These results suggest that hormone-related gene expression is closely related to pupal development. Our study lays a foundation for future diapause studies in A. pernyi.

Drought is a significant environmental limiting factor that restricts the growth of Quercus wutaishanica Mayr. The MYB transcription factor plays a wide role in controlling the growth of plants. In this study, the QwMYB108 gene was cloned and the bioinformatics was analyzed, and we examined how QwMYB108 responded to various gradient drought stresses. The results demonstrated that QwMYB108 encoded 275 amino acids using an 828 bp open reading frame. Subcellular localization indicated that the gene was located in the nucleus. Phylogenetic analysis showed that QwMYB108 was close to Q. robur, and that the highest level of expression was found in leaves, which was significantly different from other tissues. The expression of QwMYB108 increased as the stress degree rose when drought stress was present, and there was a significant difference between severe drought stress and other gradient stress. In this study, the function of QwMYB108 in drought stress response was investigated, and the drought response function gene of Q. wutaishanica was further explored to provide a theoretical basis.