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BackgroundPolygonatum kingianumColl. et Hemsl. is an important plant in Traditional Chinese Medicine. The extracts from its tubers are rich in polysaccharides and other metabolites such as saponins. It is a well-known concept that growing medicinal plants in semi-arid (or drought stress) increases their natural compounds concentrations. This study was conducted to explore the morpho-physiological responses ofP. kingianumplants and transcriptomic signatures ofP. kingianumtubers exposed to mild, moderate, and severe drought and rewatering.ResultsThe stress effects on the morpho-physiological parameters were dependent on the intensity of the drought stress. The leaf area, relative water content, chlorophyll content, and shoot fresh weight decreased whereas electrolyte leakage increased with increase in drought stress intensity. A total of 53,081 unigenes were obtained; 59% of which were annotated. We observed that 1352 and 350 core genes were differentially expressed in drought and rewatering, respectively. Drought stress driven differentially expressed genes (DEGs) were enriched in phenylpropanoid biosynthesis, flavonoid biosynthesis, starch and sucrose metabolism, and stilbenoid diarylheptanoid and gingerol biosynthesis, and carotenoid biosynthesis pathways. Pathways such as plant-pathogen interaction and galactose metabolism were differentially regulated between severe drought and rewatering. Drought reduced the expression of lignin, gingerol, and flavonoid biosynthesis related genes and rewatering recovered the tubers from stress by increasing the expression of the genes. Increased expression of carotenoid biosynthesis pathway related genes under drought suggested their important role in stress endurance. An increase in starch and sucrose biosynthesis was evident from transcriptomic changes under drought stress. Rewatering recovered the drought affected tubers as evident from the contrasting expression profiles of genes related to these pathways.P. kingianumtuber experiences an increased biosynthesis of sucrose, starch, and carotenoid under drought stress. Drought decreases the flavonoids, phenylpropanoids, gingerol, and lignin biosynthesis. These changes can be reversed by rewatering theP. kingianumplants.ConclusionsThese results provide a transcriptome resource forP. kingianumand expands the knowledge on the effect of drought and rewatering on important pathways. This study also provides a large number of candidate genes that could be manipulated for drought stress tolerance and managing the polysaccharide and secondary metabolites’ contents inP. kingianum.

Gastrodia elata BI. is an important cultivated medicinal plant in China. To analyze the genetic diversity and evolutionary relationship of the germplasm resources of G. elata, specific Single nucleotide polymorphism (SNP) markers were developed. SLAF analysis was used to compare 28 samples of the same G. elata cultivar. Plants from 4 different varieties or different habitats were collected to explore intraspecific variation and to lay a foundation for resource protection. This will facilitate improved variety breeding in future. In this study, Single nucleotide polymorphism (SNP) genetic variation and differentiation of G. elata f. glauca, G. elata f. viridis, and G. elata f. elata were analyzed using Specific-Locus Amplified Fragment Sequencing (SLAF-seq). A total of 75.95M reads with an average sequencing depth of 19.32 × and a mean Q30 of 91.71% were obtained. Based on the 19,675 polymorphic SLAF tags, 60,238 SNPs were identified and a subset of 22,737 SNPs with minor allele frequency > 0.05 and integrity > 0.5 were selected. A model-based analysis divided the accessions into two groups, wild type G. elata f. glauca and G. elata f. viridis groups. Phylogenetic analysis also clustered the samples into the two major groups. G. elata has high genetic diversity. Population diversity was highest in G. elata f. elata and lowest in G. elata f. viridis. Analysis of molecular variance (AMOVA) revealed significant variations within individuals (92.23%). This study provides new insights into the genetic variation and differentiation of G. elata, which can be exploited to improve existing commercial cultivars.

Tulbaghia violacea Harv. indigenous to southern African countries, is an herbaceous perennial bulbous plant belonging to the family Amaryllidaceae. It is a popular garden plant in China. This attractive plant is traditionally used as medicine and repellent (Kubec et al. 2002; Moodley et al. 2015). In June 2021, T. violacea plants showing typical tospovirus-like symptoms of chlorotic rings patterns, were found at the campus of Yunnan University of Chinese Medicine (Fig.S1). Disease incidence was about 11.0% during the field survey. Total RNA was extracted from symptomatic leaves of T. violacea plants using the TRIzol reagent (ambio, Carlsbad, CA). Reverse transcription (RT)-PCR was conducted to identify the virus using RNA extract as the template. The degenerate primers (dTospo-F2 and dTospo-R2) (Huang et al. 2018) were used to amplify the conserved regions of the orthotospoviral L RNA sequences. No amplification was obtained from extracts of two asymptomatic plants. The amplicons from four symptomatic samples were cloned into the pMD19-T vector (TaKaRa) and sequenced (three clones for each amplicon) by Tsingke (Shanghai, China). The obtained DNA fragments were determined to be 312 bp. The sequences from four symptomatic samples were identical (GenBank acc.no. OK258285) and shared the highest nucleotide identities (98.0%) with a corresponding sequence of segment L of impatiens necrotic spot virus (INSV) isolated (GQ336991) from Phalaenopsis amabilis in Yunnan province, China. To further confirm the INSV infection to T. violacea, the samples were analyzed with the specific primers for the N, NSs and NSm genes of INSV (Table S1), respectively. Amplicons of the expected size, 789 bp, 1344 bp and 912 bp, were produced, respectively. Amplicons were cloned and sequenced. The 789-bp N (ON529554) and 1344-bp NSs (ON529554) gene sequences had 99.1% and 99.3% nucleotide identities with the corresponding region of previously described INSV Phalenopsis isolate (GQ336989), respectively. The 912-bp NSm (ON529553) gene sequence shared 99.5% nucleotide identity with the corresponding region of INSV Phalenopsis isolate (GQ336990). Metavirome and Sanger sequencing were used to complete the genome of INSV from T. violacea. The leaves of the symptomatic sample were used to construct an rRNA-depleted library using Nextera XT reagents (Illumina, San Diego, CA). The library was subjected to RNA-Seq a NovaSeq 6000 platform (Illumina, San Diego, CA). A total of 33,193,233 quality-filtered reads were obtained using BBMAP (https://github.com/BioInfoTools/BBMapBBMap - Bushnell B. - sourceforge.net/projects/bbmap/). Among 161052 reads mapped to virus sequences, 151407 reads (read ratios 94.0%) were mapped to INSV. Three complete segments of INSV genome were determined to 8,778 nt (L segment, Acc. No. ON529552), 4,958 nt (M segment, Acc. No. ON529553), and 2,983 nt (S segment, Acc. No. ON529554) in length. These segments were validated by RT-PCR and Sanger sequencing. Three segments share nucleotide sequence identities of 99.6%, 99.3% and 98.9% with the L (GQ336991), M (GQ336990) and S segments (GQ336989) of INSV Phalenopsis isolate, respectively. The results of sequence comparisons showed no evidence of reassortment between INSV and another orthotospovirus. There was a report of tomato spotted wilt virus infecting T. violacea in Florida, USA (Dey et al. 2019). No other virus infecting T. violacea was reported. INSV has been reported to infect several economically important crops including Phalenopsis, pepper etc. in China (Chen et al. 2016). INSV-infected T. violacea not only losses landscaping value but also plays an important intermedia host role in the spread of INSV. Additional surveys and evaluation will be needed to understand the potential medicinal effect of this virus on this plant. To our knowledge, this is first report of INSV in T. violacea.

The complete genome sequence of a putative novel potyvirus, tentatively named "polygonatum mosaic-associated virus 1" (PMaV1), was sequenced from naturally infected Polygonatum cyrtonema Hua in China. PMaV1 has a typical genome organization of potyviruses with a single large open reading frame (nt 119-9448) that encodes a 3109-aa polyprotein that is predicted to be cleaved into 10 mature proteins by virus-encoded proteases. Pairwise comparisons revealed that PMaV1 shares 71.50% complete genome sequence identity with Polygonatum kingianum virus 4 and 80.00% amino acid sequence identity with Polygonatum kingianum virus 3 of the genus Potyvirus. Phylogenetic analysis indicated that PMaV1 clustered with other potyviruses and that it was most closely related to Polygonatum kingianum virus 3 and Polygonatum kingianum virus 4. These results suggest that PMaV1 is a new member of the genus Potyvirus of the family Potyviridae (Nucleotide sequence data reported are available in the GenBank databases under the accession number OP380926).

The complete genomic sequence of a new potyvirus was determined from Paris polyphylla var. yunnanensis. Its genomic RNA consists of 9571 nucleotides (nt) excluding the 3’ -terminal poly (A) tail, containing the typical open reading frame (ORF) of potyviruses and encoding a putative large polyprotein of 3061 amino acids. The virus shares 54.20-59.60% nt sequence identities and 51.80-57.90% amino acid sequence identities with other potyviruses. Proteolytic cleavage sites and conserved motifs of the potyviruses were identified in the polyprotein and within individual proteins. Phylogenetic analysis indicated that the virus was most closely related to lily yellow mosaic virus. The results suggest that the virus should be classified as a novel species within the genus Potyvirus, which we tentatively name “Paris yunnanensis mosaic chlorotic virus” (PyMNV).11Note: Nucleotide sequence data reported are available in the GenBank databases under the accession number no.ON871824.

The complete genome sequence of a new potyvirus from Paris polyphylla var. yunnanensis was determined. Its genomic RNA consists of 9571 nucleotides (nt), excluding the 3'-terminal poly(A) tail, containing the typical open reading frame (ORF) of potyviruses and encoding a putative large polyprotein of 3061 amino acids. The virus shares 54.20%-59.60% nt sequence identity and 51.80%-57.90% amino acid sequence identity with other potyviruses. Proteolytic cleavage sites and conserved motifs of potyviruses were identified in the polyprotein and within individual proteins. Phylogenetic analysis indicated that the virus was most closely related to lily yellow mosaic virus. The results suggest that the virus should be classified as a member of a novel species within the genus Potyvirus, and we have tentatively named this virus "Paris yunnanensis mosaic chlorotic virus" (PyMCV).

Using the allele-specific diagnostic PCR for the rapid and efficient identification of Chinese herbal medicines and their adulterants. In this paper, we analyze the research object of P. kingianum and its adulterants. The total DNA of the samples to be identified was extracted, and the PCR was amplified by using universal primers. After the homology comparison, the specific sites were found out by using BioEdit software, and the specific PCR primers were designed by Premier primer 5.0 software, and the samples were amplified by the primers. The 331bp bands were amplified by P. kingianum of the specific primers, but the results of their adulterants were not amplified. The results show that the method can provide guidance for the allele-specific diagnostic PCR for quick and easy to select P. kingianum , which is not affected by environmental factors, affecting the growth period of the plant, were identified directly from the molecular level of DNA. This method having important application value in accurate introduction and clinical application can provide a model for other identification.