Pinellia ternata (Thunb.) Breit. (Abbreviated as P. ternata). It is a commonly prescribed Chinese traditional medicinal herb for the treatment of phlegm, cough, and morning sick. Bulbil reproduction is one of the main reproductive methods of P. ternata. The accurate quantification of gene expression patterns associated with bulbil development might be helpful to explore the molecular mechanism involved in P. ternata reproduction. Quantitative real-time PCR was the most preferred method for expression profile and function analysis of mRNA. However, the reference genes in different tissues of P. ternata in different periods of bulbil development have not been studied in detail. In present study, the expression stability of eight candidate reference genes were determined with programs: geNorm, NormFinder, BestKeeper, and refFinder. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the top- rated reference gene in all samples of P. ternata, while different combinations of reference gene proved to be the most stable depending on development stage and tissue type. Furthermore, the reliability of GAPDH expression was verified by six P. ternata related genes in hormone and nutrient biosynthesis pathways, and the expression profiles of these genes were agreed with the results of RNA-seq digital gene expression analysis. These results can contribute to studies of gene expression patterns and functional analysis of P. ternata involved in bulbil development.

Pinellia ternata (Thunb.) Breit (abbreviated as P. ternata) is a plant with an important medicinal value whose yield is restricted by many factors, such as low reproductive efficiency and continuous cropping obstacles. As an essential breeding material for P. ternata growth and production, the bulbils have significant advantages such as a high survival rate and short breeding cycles. However, the location effect, influencing factors, and molecular mechanism of bulbil occurrence and formation have not been fully explored. In this study, exogenously applied phytohormones were used to induce in vitro petiole of P. ternata to produce bulbil structure. Transcriptome sequencing of mRNA and miRNA were performed in the induced petiole (TCp) and the induced bulbil (TCb). Gene Ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for the identification of key genes and pathways involved in bulbil development. A total of 58,019 differentially expressed genes (DEGs) were identified. The GO and KEGG analysis indicated that DEGs were mainly enriched in plant hormone signal transduction and the starch and sucrose metabolism pathway. The expression profiles of miR167a, miR171a, and miR156a during bulbil induction were verified by qRT-PCR, indicating that these three miRNAs and their target genes may be involved in the process of bulbil induction and play an important role. However, further molecular biological experiments are required to confirm the functions of the identified bulbil development-related miRNAs and targets.

A plant species in a community often grows with some other plant species. While many studies have assessed interspecific interactions between two target plant species, few have considered the impacts of the other plant species (e.g., the third, fourth, and fifth plant species) on these interactions. To assess the impacts, we grew one seedling of each of the five herbaceous plant species that are common in China (Cynodon dactylon, Plantago asiatica, Taraxacum mongolicum, Nepeta cataria, and Leonurus japonicus) alone (no competition) or with one seedling of one, two, three, or four of the other species. The presence of a neighbor plant generally reduced the growth of the target species, suggesting that the interspecific relationships were mostly competitive. The presence of other neighbor species (the third, fourth, and fifth species) could alter the interspecific interactions between two target species, but such effects varied depending on both the identity of the target species and the identity of the other species. Additionally, the effects of the third species depended little on the presence of the fourth and fifth species. We conclude that interspecific interactions between two plant species are commonly regulated by the presence of other species, facilitating species coexistence. However, our findings do not support the idea that the impacts of the fourth and fifth species on interactions among three plant species are common. This study highlights the complex interactions among multiple plant species within a community and also the importance of including these high-order interactions when modelling community dynamics and species coexistence.