Dipteronia Oliver (Aceraceae) is an endangered Chinese endemic genus consisting of two living species, Dipteronia sinensis and Dipteronia dyeriana. However, studies on the population genetics and evolutionary analyses of Dipteronia have been hindered by limited genomic resources and genetic markers. Here, the generation, de novo assembly and annotation of transcriptome datasets, and a large set of microsatellite or simple sequence repeat (SSR) markers derived from Dipteronia have been described. After Illumina pair-end sequencing, approximately 93.2 million reads were generated and assembled to yield a total of 99,358 unigenes. A majority of these unigenes (53%, 52,789) had at least one blast hit against the public protein databases. Further, 12,377 SSR loci were detected and 4179 primer pairs were designed for experimental validation. Of these 4179 primer pairs, 435 primer pairs were randomly selected to test polymorphism. Our results show that products from 132 primer pairs were polymorphic, in which 97 polymorphic SSR markers were further selected to analyze the genetic diversity of 10 natural populations of Dipteronia. The identification of SSR markers during our research will provide the much valuable data for population genetic analyses and evolutionary studies in Dipteronia.

Oaks (Quercus L.) are ideal models to assess patterns of plant diversity. We integrated the sequence data of five chloroplast and two nuclear loci from 50 Chinese oaks to explore the phylogenetic framework, evolution and diversification patterns of the Chinese oak’s lineage. The framework phylogeny strongly supports two subgenera Quercus and Cerris comprising four infrageneric sections Quercus, Cerris, Ilex and Cyclobalanopsis for the Chinese oaks. An evolutionary analysis suggests that the two subgenera probably split during the mid-Eocene, followed by intergroup divergence within the subgenus Cerris around the late Eocene. The initial diversification of sections in the subgenus Cerris was dated between the mid-Oligocene and the Oligocene–Miocene boundary, while a rapid species radiation in section Quercus started in the late Miocene. Diversification simulations indicate a potential evolutionary shift on section Quercus, while several phenotypic shifts likely occur among all sections. We found significant negative correlations between rates of the lineage diversification and phenotypic turnover, suggesting a complex interaction between the species evolution and morphological divergence in Chinese oaks. Our infrageneric phylogeny of Chinese oaks accords with the recently proposed classification of the genus Quercus. The results point to tectonic activity and climatic change during the Tertiary as possible drivers of evolution and diversification in the Chinese oak’s lineage.

Asarum sieboldii, a well-known traditional Chinese medicinal herb, is used for curing inflammation and ache. It contains both the bioactive ingredient asarinin and the toxic compound aristolochic acid. To address further breeding demand, genes involved in the biosynthetic pathways of asarinin and aristolochic acid should be explored. Therefore, the full-length transcriptome of A. sieboldii was sequenced using PacBio Iso-Seq to determine the candidate transcripts that encode the biosynthetic enzymes of asarinin and aristolochic acid. In this study, 63 023 full-length transcripts were generated with an average length of 1371 bp from roots, stems, and leaves, of which 49 593 transcripts (78.69%) were annotated against public databases. Furthermore, 555 alternative splicing (AS) events, 10 869 long noncoding RNAs (lncRNAs) as well as their 11 291 target genes, and 17 909 simple sequence repeats (SSRs) were identified. The data also revealed 97 candidate transcripts related to asarinin metabolism, of which six novel genes that encoded enzymes involved in asarinin biosynthesis were initially reported. In addition, 56 transcripts related to aristolochic acid biosynthesis were also identified, including CYP81B. In summary, these transcriptome data provide a useful resource to study gene function and genetic engineering in A. sieboldii.

Iris (Iridaceae) is one of the most widely admired ornamental plants. It has been used mainly in medicine due to the high concentration of chlorogenic acid (CGA), flavonoids, isoflavones, lignans, and other compounds in its rhizomes. In iris, the gene functions related to CGA and flavonoids biosynthesis are still unclear. In this study, we compared the I. germanica rhizome with a high accumulation level of CGA but a low accumulation level of flavonoids, and the I. pallida rhizome with a low accumulation level of CGA but a high accumulation level of flavonoids at the transcriptome and metabolome levels. A total of 761 metabolites were detected, including 202 flavonoids and 106 phenolic acids based on metabolome profiling. In total, 135 flavonoids were highly accumulated in I. pallida, including three flavanols, 51 flavonoids, 12 flavonoid carbonosides, 31 flavonols, and 21 isoflavones. Based on single-molecule long-read sequencing technology, 94,461 transcripts were identified in iris. Expression analysis indicated that the high accumulation level of C4H and 4CL in I. germanica were essential for CGA accumulation, while CHS, DFR, ANS, ANR, LAR, and 3GT were essential for flavonoids biosynthesis in I. pallida. Many transcription factors such as transcript_83288 (MYB), transcript_57970 (WRKY), and transcript_77465 (WRKY) act as regulators, playing important roles in these biological processes. Our findings provide new insights into the molecular mechanisms associated with the biosynthesis and regulation of flavonoids and CGA in the iris rhizome, and highlight the usefulness of an integrated approach for understanding this process.

Dipteronia Oliv. is an endangered genus found in China with two species, D. sinensis and D. dyeriana. Previous morphological, cytogenetic, and molecular studies have suggested that D. dyeriana is a species related to D. sinensis. However, it is unclear how the two species diverged and whether gene flow exists between these two species. Here, we performed a molecular study at the population level to characterize genetic differentiation and decipher the phylogeographic history for Dipteronia species based on newly sequenced chloroplast DNA (cpDNA) and amplified fragment length polymorphisms (AFLP) date retrieved from our previous studies. No haplotype was shared between the two species in the cpDNA network. However, the phylogenetic analysis suggested that a haplotype found in D. sinensis (H4) showed a closer relationship with haplotypes of D. dyeriana. Based on our estimated time of divergence, these two cpDNA haplotype lineages of Dipteronia diverged at about 31.19 Ma. Furthermore, two genetic clusters with asymmetric gene flow were supported based on the structure analysis, which corresponded with the two Dipteronia species, and we also detected a low level of asymmetric gene flow between these two species according to the MIGRATE analysis using AFLP data. During the Last Glacial Maximum (LGM, c.21 kya BP), the genus’ predicted distribution was more or less similar to that at present, which was also supported by the mismatch analyses that showed no population expansion of the two Dipteronia populations after the LGM. The combined cpDNA and AFLP data revealed significant genetic differentiation between the two Dipteronia species with asymmetric gene flow, which can be explained by the varying phylogeographical histories of these two species.

Dracocephalum moldavica is widely used as an ornamental, medicine, and perfume in industry. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) is widely and accurately utilized for gene expression evaluations. Selecting optimal reference genes is essential for normalizing RT-qPCR results. However, the identification of suitable reference genes in D. moldavica has not been documented. A total of 12 reference genes in D. moldavica were identified by PEG6000 (15%) treatment under hypertonia conditions in different tissues (roots, stem, leaves, flower, seeds and sepal) and during three stages of flower development, then used to validate the expression stability. There were four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper) used to analyze the stability. Finally, the RefFinder program was employed to evaluate the candidate reference genes’ stability. The results showed that ACTIN, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and EF1α (elongation factor-1α) were stable reference genes under the PEG6000 treatment. Heat shock protein 70 (HSP70) was the most stable gene across different flower development stages. ADP-ribosylation factor (ARF) was the most stable gene in different tissues and total samples. This study provides reliable gene expression studies for future research in D. moldavica.