Polygonati Rhizoma and Polygonati odorati Rhizoma, known as “Huangjing” and “Yuzhu” in China, are medicinal Polygonatum species resources with top-grade medical and edible properties. The chloroplast (cp) genome has been used to study species diversity, evolution, and breeding of species for applications in genetic engineering. Codon usage bias (CUB), a common and complex natural phenomenon, is essential for studies of codon optimization of exogenous genes, genetic engineering, and molecular evolution. However, the CUB of medicinal Polygonatum species chloroplast genomes has not been systematically studied. In our study, a detailed analysis of CUB was performed in the medicinal Polygonatum species chloroplast genomes. We investigated the codon bias of 204 plastid protein-coding genes (PCGs) in 4 medicinal Polygonatum species using CodonW and CUSP online software. Through the analysis of the codon bias index, we found that the medicinal Polygonatum species chloroplast genomes had weak codon usage bias. In addition, our results also showed a high preference for AT bases in medicinal Polygonatum species chloroplast genomes, and the preference to use AT-ending codons was observed in these species chloroplast genomes. The neutrality plot, ENC plot, PR2-Bias plot, and correspondence analysis showed that compared with mutation pressure, natural selection was the most important factor of CUB. Based on the comparative analysis of high-frequency codons and high expression codons, we also determined the 10-11 optimal codons of investigative medicinal Polygonatum species. Furthermore, the result of RSCU-based cluster analysis showed that the genetic relationship between different medicinal Polygonatum species could be well reflected. This study provided an essential understanding of CUB and evolution in the medicinal Polygonatum species chloroplast genomes.

Background: Polygonatum kingianum has been widely used as a traditional Chinese medicine as well as a healthy food. Because of its highly variable morphology, this medicinal plant is often difficult to distinguish from other related verticillate leaf types of the Polygonatum species. The contaminants in P. kingianum products not only decrease the products’ quality but also threaten consumer safety, seriously inhibiting the industrial application of P. kingianum.Methods: Nine complete chloroplast (cp) genomes of six verticillate leaf types of the Polygonatum species were de novo assembled and systematically analyzed.Results: The total lengths of newly sequenced cp genomes ranged from 155,437 to 155,977 bp, including 86/87 protein-coding, 38 tRNA, and 8 rRNA genes, which all exhibited well-conserved genomic structures and gene orders. The differences in the IR/SC (inverted repeats/single-copy) boundary regions and simple sequence repeats were detected among the verticillate leaf types of the Polygonatum cp genomes. Comparative cp genomes analyses revealed that a higher similarity was conserved in the IR regions than in the SC regions. In addition, 11 divergent hotspot regions were selected, providing potential molecular markers for the identification of the Polygonatum species with verticillate leaf types. Phylogenetic analysis indicated that, as a super barcode, plastids realized a fast and efficient identification that clearly characterized the relationships within the verticillate leaf types of the Polygonatum species. In brief, our results not only enrich the data on the cp genomes of the genus Polygonatum but also provide references for the P. kingianum germplasm resource protection, herbal cultivation, and drug production.Conclusion: This study not only accurately identifies P. kingianum species, but also provides valuable information for the development of molecular markers and phylogenetic analyses of the Polygonatum species with verticillate leaf types.

Group Ⅲ WRKY transcription factors (TFs) play pivotal roles in responding to the diverse abiotic stress and secondary metabolism of plants. However, the evolution and function of WRKY66 remains unclear. Here, WRKY66 homologs were traced back to the origin of terrestrial plants and found to have been subjected to both motifs’ gain and loss, and purifying selection. A phylogenetic analysis showed that 145 WRKY66 genes could be divided into three main clades (Clade A–C). The substitution rate tests indicated that the WRKY66 lineage was significantly different from others. A sequence analysis displayed that the WRKY66 homologs had conserved WRKY and C2HC motifs with higher proportions of crucial amino acid residues in the average abundance. The AtWRKY66 is a nuclear protein, salt- and ABA- inducible transcription activator. Simultaneously, under salt stress and ABA treatments, the superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities, as well as the seed germination rates of Atwrky66-knockdown plants generated by the clustered, regularly interspaced, short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system, were all lower than those of wild type (WT) plants, but the relative electrolyte leakage (REL) was higher, indicating the increased sensitivities of the knockdown plants to the salt stress and ABA treatments. Moreover, RNA-seq and qRT-PCR analyses revealed that several regulatory genes in the ABA-mediated signaling pathway involved in stress response of the knockdown plants were significantly regulated, being evidenced by the more moderate expressions of the genes. Therefore, the AtWRKY66 likely acts as a positive regulator in the salt stress response, which may be involved in an ABA-mediated signaling pathway.

Background Dendrobium catenatum is a valuable traditional medicinal herb with high commercial value. D. catenatum stems contain abundant polysaccharides which are one of the main bioactive components. However, although some genes related to the synthesis of the polysaccharides have been reported, more key genes need to be further elucidated. Results In this study, the contents of polysaccharides and mannose in D. catenatum stems at four developmental stages were compared, and the stems’ transcriptomes were analyzed to explore the synthesis mechanism of the polysaccharides. Many genes involved in starch and sucrose metabolisms were identified by KEGG pathway analysis. Further analysis found that sucrose synthase (SUS; EC 2.4.1.13) gene maybe participated in the polysaccharide synthesis. Hence, we further investigated the genomic characteristics and evolution relationships of the SUS family in plants. The result suggested that the SUS gene of D. catenatum (DcSUS) had undergone the expansion characterized by tandem duplication which might be related to the enrichment of the polysaccharides in D. catenatum stems. Moreover, expression analyses of the DcSUS displayed significant divergent patterns in different tissues and could be divided into two main groups in the stems with four developmental stages. Conclusion In general, our results revealed that DcSUS is likely involved in the metabolic process of the stem polysaccharides, providing crucial clues for exploiting the key genes associated with the polysaccharide synthesis.