Background Polygonatum kingianum Coll. 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 of P. kingianum plants and transcriptomic signatures of P. kingianum tubers exposed to mild, moderate, and severe drought and rewatering. Results The 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. kingianum tuber 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 the P. kingianum plants. Conclusions These results provide a transcriptome resource for P. kingianum and 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 in P. kingianum.

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.

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, speci c 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 intraspeci c 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 Speci c-Locus Ampli ed 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 identi ed 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 signi cant 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.

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.