Deciphering the virome of Chunkung (Cnidium officinale) showing dwarfism-like symptoms via a high-throughput sequencing analysis.

BACKGROUND: Viruses have notable effects on agroecosystems, wherein they can adversely affect plant health and cause problems (e.g., increased biosecurity risks and economic losses). However, our knowledge of their diversity and interactions with specific host plants in ecosystems remains limited. To enhance our understanding of the roles that viruses play in agroecosystems, comprehensive analyses of the viromes of a wide range of plants are essential. High-throughput sequencing (HTS) techniques are useful for conducting impartial and unbiased investigations of plant viromes, ultimately forming a basis for generating further biological and ecological insights. This study was conducted to thoroughly characterize the viral community dynamics in individual plants. RESULTS: An HTS-based virome analysis in conjunction with proximity sampling and a tripartite network analysis were performed to investigate the viral diversity in chunkung (Cnidium officinale) plants. We identified 61 distinct chunkung plant-associated viruses (27 DNA and 34 RNA viruses) from 21 known genera and 6 unclassified genera in 14 known viral families. Notably, 12 persistent viruses (7 DNA and 5 RNA viruses) were exclusive to dwarfed chunkung plants. The detection of viruses from the families Partitiviridae, Picobirnaviridae, and Spinareoviridae only in the dwarfed plants suggested that they may contribute to the observed dwarfism. The co-infection of chunkung by multiple viruses is indicative of a dynamic and interactive viral ecosystem with significant sequence variability and evidence of recombination. CONCLUSIONS: We revealed the viral community involved in chunkung. Our findings suggest that chunkung serves as a significant reservoir for a variety of plant viruses. Moreover, the co-infection rate of individual plants was unexpectedly high. Future research will need to elucidate the mechanisms enabling several dozen viruses to co-exist in chunkung. Nevertheless, the important insights into the chunkung virome generated in this study may be relevant to developing effective plant viral disease management and control strategies.

Effect of explant type and plant growth regulators on callus induction, growth and secondary metabolites production in Cnidium officinale Makino.

This study reports the effect of explant type and plant growth regulators (PGRs) on callus induction in Cnidium officinale. Compared to stem, root explant showed maximum percent callus formation of 75% on Murashige and Skoog (MS) medium containing 2.3 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.2 µM benzyladenine (BA). At 30th day of callus culture on the said medium, callus fresh weight was sevenfold higher than other tested PGRs treatments. It was noted that MS medium supplemented with 27.1 µM 2,4-D showed the highest 0.30 mg g-1 DW of total phenols, while total flavonoids content reached to a maximum of 0.05 mg g-1 DW on the MS medium supplemented with 4.5 µM 2,4-D and 2.2 µM BA. Conversely, maximum (83.9%) DPPH free radical scavenging activity was observed in calli grown on the MS medium supplemented with 2.3 µM 2,4-D and 2.2 µM BA. The high-performance liquid chromatography (HPLC) analysis revealed higher phthalide content in callus than intact roots of in vitro plants. While 3-butylidenephthalide content in callus was comparable to the intact shoots and roots of in vitro grown C. officinale. The concentrations of 2,4-D played a significant role in the production of phthalide and 3-butylidenephthalide. Additional measures are recommended to further enhance their production in vitro.

Metabolic profiling and identification of the genetic varieties and agricultural origin of Cnidium officinale and Ligusticum chuanxiong.

Quality control methods for Cnidium officinale and Ligusticum chuanxiong are lacking because their quality is influenced by multiple factors. Thus, there is a need to develop a multifactorial method for measuring quality that is both standardized and practical. Here, we report a profiling method based on gas chromatography-mass spectrometry (GC-MS) to discriminate among the genetic varieties and agricultural origins of C. officinale and L. chuanxiong. Our metabolome analysis identified 68 metabolites, 13 of which were newly identified in our samples. The S-plot of the OPLS discriminant analysis enabled us to determine significant biomarkers. Using only double-compound biomarkers, the samples were successfully classified into distinct groups defined by genetic variety and cultivation origin. This method will simplify the process of searching for quality control markers that can be used to determine genetic variety and agricultural origin.

Identification and characterization of 12 microsatellite loci in Cnidium dubium (Apiaceae) using next-generation sequencing.

PREMISE OF THE STUDY: We present a set of 12 polymorphic nuclear microsatellite loci, identified for the first time within the herbaceous plant species Cnidium dubium (Schkuhr) Thell. using next-generation sequencing. METHODS AND RESULTS: To characterize these loci, 40 plants were analyzed genetically. The number of alleles ranged from 2 to 23, with an average of 8.3 alleles per locus. Mean observed and expected heterozygosities ranged from 0.25 to 0.95 and 0.30 to 0.94, respectively. CONCLUSIONS: All 12 nuclear microsatellite loci showed scorable and polymorphic fragments after polymerase chain reaction. The new marker set can be used for future studies of genetic diversity and differentiation as well as estimation of gene flow and spatial genetic structures.

Molecular identification of "Chuanxiong" by nucleotide sequence and multiplex single base extension analysis on chloroplast trnK gene.

Chloroplast trnK gene sequences of Cnidium officinale and Ligusticum chuanxiong were determined to establish an effective method for identifying Japanese Senkyu and Chinese Chuanxiong, the two which have the same drug name in Chinese characters, similar external feature, but different botanical origins. Three sites of nucleotide differences were found between these 2 species at positions 767,924 and 964 from upstream in trnK gene sequence, allowing molecular identification of the two plants and crude drugs. Further, three kinds of specific primers of 14 mer, 23 mer and 30 mer long were designed to detect these 3 sites of marker nucleotides. By using multiplex single base extension (MSBE) analysis with the 3 specific primers, C. officinale and L. chuanxiong could be distinguished clearly by the electrophoretograms, where 3 peaks with different color of ddTMP, ddCMP and ddTMP were observed in case of C. officinale and those of ddGMP, ddAMP and ddGMP in L. chuanxiong. Moreover, trnK gene sequence of "Dongxiong," a kind of Chuanxiong cultivated in Northeast China, suggested that its botanical origin was C. officinale.

[Analysis on intraspecific variation of Cnidium monnieri by RAPD].

OBJECTIVE: To study the genetic variation of Cnidium monnieri from different regions. METHODS: Random amplified polymophic DNA technique was used to analyze genetic polymorphy of Cnidium monnieri from 9 different regions, and dendrogram was constructed by UPGMA. RESULTS: 20 random primers were used for polymorphic selection. A total of 75 bands were amplified, 64 of which were polymorphism. CONCLUSION: The smaller the geographic distances between two Cnidium monnieri, the smaller genetic differences. However, it should not be overlooked that natural environment, especially the local ecological conditions may also show some influences on the genetic variations among different C. monnieri samples.

[matK and its nucleotide sequencing of crude drug chuanxiong and phylogenetic relationship between their species from China and Japan].

AIM: To provide more molecular evidences for species relationship between Chuanxiong (Ligusticum chuanxiong Hort.) from China and Japanese Chuanxiong (Senkyu in Japanese) (Cnidium officinale Makino). METHODS: To sequence such two genes as internal transcribed spacer (ITS) from nuclear rDNA and maturase for lysine (matK) in tRNA(lys) (UUU) intron from chloroplast DNA of both Ligusticum chuanxiong and Cnidium officinale using PCR direct sequencing and to analyze the sequence variation of two genes between these two species. RESULTS: The matK gene sequence of Ligusticum chuanxiong and Cnidium officinale is 1268 bp in length, coding 422 amino acids of maturase protein. ITS gene sequence 699 bp, consisting of 54 bp of 18S rRNA-3', 215 bp of ITS1, 162 bp of 5.8S rRNA, 222 bp of ITS2, 46 bp of 26S rRNA-5'. Multiple sequence alignment shows that the sequence of two genes between dried crude drug and fresh voucher material of Ligusticum chuanxiong and Cnidium officinale, there is 1 variable site (T-->C) in matK (upstream at 595 nt) and ITS (ITS1 at 54 nt) between Ligusticum chuanxiong and Cnidium officinale. CONCLUSION: Based on homology analysis of two genes plastid matK and nuclear ITS, the origin of Chuanxiong from China and Japan ought to be identical, the scientific name Cnidium officinale of Japanese Chuanxiong should be changed to Ligusticum chuanxiong.