QTL mapping for the flag leaf-related traits using RILs derived from Trititrigia germplasm line SN304 and wheat cultivar Yannong15 in multiple environments.

BACKGROUND: Developing and enriching genetic resources plays important role in the crop improvement. The flag leaf affects plant architecture and contributes to the grain yield of wheat (Triticum aestivum L.). The genetic improvement of flag leaf traits faces problems such as a limited genetic basis. Among the various genetic resources of wheat, Thinopyrum intermedium has been utilized as a valuable resource in genetic improvement due to its disease resistance, large spikes, large leaves, and multiple flowers. In this study, a recombinant inbred line (RIL) population was derived from common wheat Yannong15 and wheat-Th. intermedium introgression line SN304 was used to identify the quantitative trait loci (QTL) for flag leaf-related traits. RESULTS: QTL mapping was performed for flag leaf length (FLL), flag leaf width (FLW) and flag leaf area (FLA). A total of 77 QTLs were detected, and among these, 51 QTLs with positive alleles were contributed by SN304. Fourteen major QTLs for flag leaf traits were detected on chromosomes 2B, 3B, 4B, and 2D. Additionally, 28 QTLs and 8 QTLs for flag leaf-related traits were detected in low-phosphorus and drought environments, respectively. Based on major QTLs of positive alleles from SN304, we identified a pair of double-ended anchor primers mapped on chromosome 2B and amplified a specific band of Th. intermedium in SN304. Moreover, there was a major colocated QTL on chromosome 2B, called QFll/Flw/Fla-2B, which was delimited to a physical interval of approximately 2.9 Mb and contained 20 candidate genes. Through gene sequence and expression analysis, four candidate genes associated with flag leaf formation and growth in the QTL interval were identified. CONCLUSION: These results promote the fine mapping of QFll/Flw/Fla-2B, which have pleiotropic effects, and will facilitate the identification of candidate genes for flag leaf-related traits. Additionally, this work provides a theoretical basis for the application of Th. intermedium in wheat breeding.

Spatiotemporal and Transcriptional Characterization on Tanshinone Initial Synthesis in Salvia miltiorrhiza Roots.

Tanshinones are the bioactive constituents of Danshen (Salvia miltiorrhiza Bunge), which is used in Traditional Chinese Medicine to treat cardiovascular and other diseases, and they synthesize and accumulate in the root periderm of S. miltiorrhiza. However, there is no relevant report on the initial stage of tanshinone synthesis, as well as the root structure and gene expression characteristics. The present study aims to provide new insights into how these bioactive principles begin to synthesize by characterizing possible differences in their biosynthesis and accumulation during early root development from both spatial and temporal aspects. The morphological characteristics and the content of tanshinones in roots of S. miltiorrhiza were investigated in detail by monitoring the seedlings within 65 days after germination (DAGs). The ONT transcriptome sequencing was applied to investigate gene expression patterns. The periderm of the S. miltiorrhiza storage taproot initially synthesized tanshinone on about 30 DAGs. Three critical stages of tanshinone synthesis were preliminarily determined: preparation, the initial synthesis, and the continuous rapid synthesis. The difference of taproots in the first two stages was the smallest, and the differentially expressed genes (DEGs) were mainly enriched in terpene synthesis. Most genes involved in tanshinone synthesis were up regulated during the gradual formation of the red taproot. Plant hormone signal transduction and ABC transport pathways were widely involved in S. miltiorrhiza taproot development. Five candidate genes that may participate in or regulate tanshinone synthesis were screened according to the co-expression pattern. Moreover, photosynthetic ferredoxin (FD), cytochrome P450 reductase (CPR), and CCAAT binding transcription factor (CBF) were predicted to interact with the known downstream essential enzyme genes directly. The above results provide a necessary basis for analyzing the initial synthesis and regulation mechanism of Tanshinones.

One-Step Regeneration of Hairy Roots to Induce High Tanshinone Plants in Salvia miltiorrhiza.

Salvia miltiorrhiza is a traditional Chinese medicinal plant of Labiatae, which has been widely utilized to treat a variety of cardiovascular and cerebrovascular diseases. However, due to the long growth cycle, low content of active ingredients, and serious quality deterioration of S. miltiorrhiza, the use of biotechnology to improve S. miltiorrhiza to meet the growing demand for clinical applications has become a research hotspot. In this study, a novel one-step hairy root regeneration method was developed, which could rapidly obtain hairy roots and regenerated plants with high tanshinone content. By optimizing the parameters of Agrobacterium rhizogenes transformation in S. miltiorrhiza, it was finally established that the explants were infected in Ar.qual (OD600 = 0.6) for 10 min, co-cultured for 3 days, and then screened on the screening medium containing 7.5 mg/l hygromycin, the maximum transformation frequency can reach 73.85%. GFP and PCR detection yielded a total of 9 positive transgenic hairy root lines and 11 positive transgenic regenerated plants. SmGGPPS1 was successfully overexpressed in positive transgenic regenerated plants, according to the results of qRT-PCR. The content of tanshinone IIA and cryptotanshinone were dramatically enhanced in transgenic regenerated plants and hairy roots by Ultra Performance Liquid Chromatography analysis. Based on the Agrobacterium-mediated transformation of S. miltiorrhiza, this study developed a new method for regenerating plants with transgenic hairy roots. This method provides a foundation for the breeding of S. miltiorrhiza and the sustainable development of medicinal plant resources, as well as provides a useful reference for the application of other species.

Loss of anthocyanidin synthase gene is associated with white flowers of Salvia miltiorrhiza Bge. f. alba, a natural variant of S. miltiorrhiza.

MAIN CONCLUSION: SmANS deletion leads to white flower mutation in Salvia miltiorrhiza. SmANS deletion leads to white flower mutation in Salvia miltiorrhiza. Abstract Salvia miltiorrhiza is an essential traditional Chinese medicine (TCM) with purple flowers, and S. miltiorrhiza Bge. f. alba is a unique intraspecific variation with white flowers. The molecular mechanism of flower color formation in S. miltiorrhiza will provide vital information for the variation and evolution. Here, we performed HPLC, transcriptomic, and re-sequencing analyses of purple-flowered S. miltiorrhiza line 'Zihua105' (ZH105) and white-flowered S. miltiorrhiza Bge. f. alba line 'Baihua18' (BH18). Delphinidin was the most anthocyanidin in ZH105, which become the main different between ZH105 vs. BH18 flowers. Transcriptome analysis revealed 299 differentially expressed genes (DEGs). SmANS, the anthocyanidin synthase gene in the down-stream anthocyanin biosynthesis pathway, was significantly expressed in ZH105 corollas, suggesting it might play a key role in white petal formation. Whole-genome re-sequencing revealed that a 6.75 kb segment located on chromosome 5, which contains the complete sequence of the SmANS genes, was lost in BH18 and another S. miltiorrhiza Bge. f. alba line. In contrast, the other five purple-flowered S. miltiorrhiza lines both possessed this segment. Further molecular marker identification also confirmed that wild S. miltiorrhiza Bge. f. alba lines lost regions that contained a complete or important part of SmANS sequences. Subsequently, the research showed that the deletion mutant of SmANS genes resulted in the natural white flower color variant of S. miltiorrhiza.

SmGDB: genome database of Salvia miltiorrhiza, an important TCM Plant.

BACKGROUND: Salvia miltiorrhiza is an important traditional Chinese medicinal (TCM) plant and a model plant in the genetic study of TCM. A series of omics related to Danshen have been published. Integrating, managing, storing, and sharing data has become an urgent problem to be solved in S. miltiorrhiza genetic studies. OBJECTIVES: The genome database is the link for the exchange, acquisition, and use of different omics data between data producers and users, maximizing value and utilization of data. METHODS: The genome database included DSS3 genome and five RNA-Seq data. The back-end performs data search and retrieval through the LAMP (Linux, Apache, MySQL, PHP) framework. RESULTS: Here, we present SmGDB (S. miltiorrhiza genome database; http://8.140.162.85/ ), which houses the latest version of genome sequence and annotation data for S. miltiorrhiza, combining three unpublished RNA-Seq data from our group and two released RNA-Seq data. We also identified a novel gene cluster including seven CYP71D genes involved in the tanshinone synthesis pathway based on genome sequences and expression data. Besides, SmGDB provides user-friendly web interfaces for querying and browsing gene annotation, structure, location, and expression profiles for concerned genes. Popular bioinformatics tools such as 'BLAST', 'Search', 'Heatmap', 'JBrowse', etc., were also provided in SmGDB. CONCLUSIONS: SmGDB will provide utility for characterizing the structure of the S. miltiorrhiza genome and better understanding gene functions and biological processes underlying complex secondary metabolism in Danshen.

A high-quality reference genome sequence of Salvia miltiorrhiza provides insights into tanshinone synthesis in its red rhizomes.

Salvia miltiorrhiza Bunge, also known as red sage or Danshen, is an important traditional Chinese medicine (TCM) that has been used for thousands of years to treat cardiovascular and other diseases. It is also considered an important model TCM plant. Here, a high-quality reference genome of S. miltiorrhiza was generated by combining PacBio long-read sequencing and chromatin interaction mapping (Hi-C) technologies, resulting in the chromosome-scale assembly of a 594.75-Mb genome sequence with a contig N50 of 2.70 Mb. This assembly shows the highest level of continuity for a Danshen genome generated thus far. The S. miltiorrhiza genome contained 32,483 protein-coding genes, with a repetitive DNA content of approximately 64.84%. The high percentage of young LTRs suggests that multiple TE transposition bursts occurred recently in S. miltiorrhiza. Genes unique to secondary metabolism pathways were expanded in the S. miltiorrhiza genome. A new CYP450 gene cluster was identified in the phloem of red roots where active components were synthesized. This reference genome sequence will facilitate future studies aimed at the elucidation of the secondary metabolism synthesis pathway and the genetic improvement of S. miltiorrhiza.

Comparative RNA-Sequence Transcriptome Analysis of Phenolic Acid Metabolism in Salvia miltiorrhiza, a Traditional Chinese Medicine Model Plant.

Salvia miltiorrhiza Bunge is an important traditional Chinese medicine (TCM). In this study, two S. miltiorrhiza genotypes (BH18 and ZH23) with different phenolic acid concentrations were used for de novo RNA sequencing (RNA-seq). A total of 170,787 transcripts and 56,216 unigenes were obtained. There were 670 differentially expressed genes (DEGs) identified between BH18 and ZH23, 250 of which were upregulated in ZH23, with genes involved in the phenylpropanoid biosynthesis pathway being the most upregulated genes. Nine genes involved in the lignin biosynthesis pathway were upregulated in BH18 and thus result in higher lignin content in BH18. However, expression profiles of most genes involved in the core common upstream phenylpropanoid biosynthesis pathway were higher in ZH23 than that in BH18. These results indicated that genes involved in the core common upstream phenylpropanoid biosynthesis pathway might play an important role in downstream secondary metabolism and demonstrated that lignin biosynthesis was a putative partially competing pathway with phenolic acid biosynthesis. The results of this study expanded our understanding of the regulation of phenolic acid biosynthesis in S. miltiorrhiza.