Bin Li

Many Cypripedium orchids are threatened and legally protected, restricting destructive or large-scale wild sampling and leaving population-scale plastome resources scarce. Using small leaf samples collected with permission and assistance from local reserve staff, we assembled and annotated 62 complete chloroplast genomes from total genomic DNA sequencing data, representing 53 Cypripedium taibaiense and 9 C. flavum individuals. The plastomes were 191,252 to 204,050 bp long and retained the typical quadripartite structure. In the final GenBank annotations, unique intact CDS content was consistent within each species (79 genes in C. taibaiense and 75 in C. flavum), and the four canonical rRNA genes were each present in two copies. A phylogeny based on concatenated nucleotide alignments of protein-coding sequences recovered the two species as reciprocal monophyletic, whereas whole-plastome analyses including non-coding regions captured population-level variation. Across the dataset, we detected 5,574 SNPs and 60,941 indels, mainly in the large and small single-copy regions. This population-scale plastome dataset provides a practical reference for phylogeographic analysis, marker development, and conservation genomic studies in Cypripedium.

The corolla of Rehmannia glutinosa typically exhibits a stable reddish-purple color, but a naturally occurring yellow-flowered variant has recently been identified. To clarify the molecular basis of flower color variant, metabolomics, transcriptomics, and variant analyses were integrated. Metabolomic profiling revealed that the yellow phenotype was associated with lower anthocyanin levels and higher carotenoid levels. Specifically, the decreased cyanidin-3-O-glucoside led to a loss of red, while increased lutein provided the basis for the yellow color. Transcriptomic analysis revealed a downregulation of anthocyanin biosynthetic genes, including CHS, CHI, F3H, DFR, and ANS, in the yellow-flowered variant, and three S6-subgroup R2R3-MYB genes, including the known anthocyanin activator RgMYB41 (gene-DH2020_015992), were downregulated. Variant analysis showed that A12S and G255E in the gene-DH2020_015992 transcription factor were predicted to markedly alter protein conformation and potentially impair regulatory function. Subcellular localization and transcriptional activation assays further supported the functional characterization of gene-DH2020_015992 as a transcription factor. Collectively, these findings suggest that flower color variation in R. glutinosa is driven by MYB-mediated repression of anthocyanin biosynthesis and by carotenoid accumulation. This study provides a comprehensive genetic explanation for flower color variation in R. glutinosa and offers a theoretical foundation for floral pigmentation in plants.

The genus Pourthiaea Decne., a deciduous woody group with high ornamental value, belongs to the family Rosaceae. Here, we reported newly sequenced plastid genome sequences of Pourthiaea beauverdiana (C. K. Schneid.) Hatus., Pourthiaea parvifolia E. Pritz., Pourthiaea villosa (Thunb.) Decne., and Photinia glomerata Rehder & E. H. Wilson. The plastomes of these three Pourthiaea species shared the typical quadripartite structures, ranging in size from 159,903 bp (P. parvifolia) to 160,090 bp (P. beauverdiana). The three Pourthiaea plastomes contained a pair of inverted repeat regions (26,394–26,399 bp), separated by a small single-copy region (19,304–19,322 bp) and a large single-copy region (87,811–87,973 bp). A total of 113 unique genes were predicted for the three Pourthiaea plastomes, including four ribosomal RNA genes, 30 transfer RNA genes, and 79 protein-coding genes. Analyses of inverted repeat/single-copy boundary, mVISTA, nucleotide diversity, and genetic distance showed that the plastomes of 13 Pourthiaea species (including 10 published plastomes) are highly conserved. The number of simple sequence repeats and long repeat sequences is similar among 13 Pourthiaea species. The three non-coding regions (trnT-GGU-psbD, trnR-UCU-atpA, and trnH-GUG-psbA) were the most divergent. Only one plastid protein-coding gene, rbcL, was under positive selection. Phylogenetic analyses based on 78 shared plastid protein-coding sequences and 29 nrDNA sequences strongly supported the monophyly of Pourthiaea. As for the relationship with other genera in our phylogenies, Pourthiaea was sister to Malus in plastome phylogenies, while it was sister to the remaining genera in nrDNA phylogenies. Furthermore, significant cytonuclear discordance likely stems from hybridization events within Pourthiaea, reflecting complex evolutionary dynamics within the genus. Our study provides valuable genetic insights for further phylogenetic, taxonomic, and species delimitation studies in Pourthiaea, as well as essential support for horticultural improvement and conservation of the germplasm resources.

Introduction: Stephania longa, a medicinal plant renowned for producing cepharanthine, has gained significance due to the compound's notable antiviral properties against SARS-CoV-2. However, a comprehensive genetic understanding of S. longa has been lacking. This study aimed to develop a high-quality, chromosome-level genome assembly to uncover the genetic intricacies and evolutionary narrative of this species. By integrating genomic data with metabolomic and transcriptomic analyses, we sought to identify key genes involved in cepharanthine biosynthesis.Methods: We employed a multi-faceted approach comprising genome assembly, phylogenetic analysis, gene family dynamics investigation, metabolomic profiling, and gene expression analysis across various tissues of S. longa. This integrated strategy enabled the identification of key genes involved in cepharanthine biosynthesis and elucidated the species’ evolutionary history.Results: Our phylogenetic analysis clarified the placement of the genus Stephania within the Ranunculales order and revealed its notably high mutation rate. We identified gene family expansions and signs of positive selection likely contributing to Stephania’s unique metabolic capabilities. Metabolomic profiling uncovered complex regulatory mechanisms orchestrating the biosynthesis and distribution of cepharanthine and related metabolites. Through the integration of genomic, transcriptomic, and metabolomic data, we identified genes with expression patterns and evolutionary trajectories suggesting pivotal roles in cepharanthine biosynthesis, including those involved in crucial biosynthetic steps.Discussion: This comprehensive study, integrating genomic, metabolomic, and transcriptomic approaches, provides valuable insights into S. longa's biosynthetic potential. It not only enhances our understanding of the species but also establishes a foundation for future investigations into the biosynthesis and therapeutic exploitation of cepharanthine and related alkaloids.

The genus Rorippa belongs to the family Brassicaceae, and its members usually have high medicinal value. The genus consists of approximately 75 species and mainly grows in the Northern Hemisphere, occurring in every continent except Antarctica. The taxonomy and phylogenetic relationships of Rorippa are still unsettled, largely due to complex morphological variations in Rorippa, which were caused by frequent hybridization events. Here, we sequenced four complete plastid genomes of Rorippa species by Illumina paired-end sequencing. The four new plastid genomes of Rorippa ranged in total size from 154,671 bp for R. palustris to 154,894 bp for R. sylvestris. There are 130 genes in the four plastomes, embodying 8 rRNA, 37 tRNA, and 85 protein-coding genes. Combining with six published plastid genomes, we carried on comparative and phylogenetic analyses. We found that the ten Rorippa plastid genomes were conservative in gene number and order, total size, genomic structure, codon usage, long repeat sequence, and SSR. Fourteen mutational hotspot regions could be selected as candidate DNA barcoding to distinguish Rorippa plants. The phylogenetic trees clearly identified that ten Rorippa species displayed monophyletic relationships within the tribe Cardamineae based on plastomes and nrDNA ITS sequences. However, there are significant cytonuclear discordances in the interspecific relationships within Rorippa, as well as the intergeneric relationships between Rorippa and its related genera. We inferred that the cytonuclear discordance is most likely a result of interspecific hybridization within Rorippa, as well as intergeneric hybridization with its related genera. These plastid genomes can offer precious information for studies of species authentication, evolutionary history, and the phylogeny of Rorippa.

The size of leaves is a vital factor in the development and overall biomass of a plant, serving as a key indicator of how a plant adapts to its environment. Rhamnus heterophylla, a species known for its heteromorphic leaves of varying sizes, presents an intriguing case for studying leaf development at the molecular level. To gain insights for further studies on the underlying mechanisms, we constructed a comprehensive reference transcriptome database using both SMART sequencing and Illumina RNA-seq technologies. Our analysis of the transcriptome data identified 88,546 isoforms, featuring an N50 size of 2386 base pairs. Furthermore, we identified 2932 transcription factors from 55 gene families, along with 14,947 unigenes that underwent alternative splicing. By comparing the gene expression patterns between large and small leaves, we pinpointed 982 differentially expressed genes (DEGs). Among these DEGs, 116 genes exhibit significantly greater activity in small leaves, while 866 genes display significantly greater activity in large leaves. Functional enrichment analyses revealed the significant involvement of these DEGs in various hormone signaling pathways. Notably, we detected a significant decrease in the expression of several genes associated with auxin synthesis, such as ARFs, GRF8, and IAA27, in small leaves. This finding sheds light on their potential role in leaf size regulation in R. heterophylla, providing valuable insights into the genes underlying this mechanism.