Abstract
In this study, we constructed and annotated a complete circular chloroplast genome of wild R. glutinosa. The chloroplast genome of wild R. glutinosa is 153,678 bp in length, including two inverted repeat (IR) regions of 25,759 bp, separated by a large single copy (LSC) region of 84,544 bp and a small single copy (SSC) region of 17,616 bp. The genome contains 149 genes, including 104 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Neighbor-joining method phylogenomic analysis showed that wild R. glutinosa formed a monophyletic group, and was sister to other groups of R. glutinosa.
Keywords: Rehmannia glutinosa Wild, chloroplast genome, phylogeny
Wild Rehmannia glutinosa (Gaertn.) DC. is a perennial herb of the Scrophulariaceae family. The wild and cultivated species of R. glutinosa belong to the same plant in classification, and the morphological characteristics are very similar, but the underground root tuber size difference is very obvious (Li et al. 2013). Therefore, different scholars have different nomenclature and classification, and both Chinese Flora and Chinese Pharmacopoeia unify the scientific name of Rehmanniae radix into R. glutinosa from the perspective of large species (Lou et al. 1995). Xie believes that from the viewpoint of paying attention to authentic medicinal materials in the development of traditional Chinese medicine, it is believed that the thickness of underground parts of wild R. glutinosa and cultivated R. glutinosa is different, and the quality of medicinal materials is different. In order to ensure the correct supply of drugs according to the name of drugs, the scientific names of the two are still appropriate to be applied separately (Xie 1990).
In recent years, DNA barcoding has developed into a powerful tool for species identification. Compared with common DNA barcode short fragments, the whole chloroplast genome contains more abundant mutation sites and identification efficiency is high (Jiang et al. 2020). Therefore, in this study, we assembled the complete chloroplast genome of wild R. glutinosa based on next generation sequencing technology, analyzed the basic structure of the chloroplast genome of wild R. glutinosa revealed the phylogenetic relationship between wild and cultivated R. glutinosa by constructing a phylogenetic tree, provided a molecular basis for the classification and nomenclature of wild and cultivated R. glutinosa and laid the foundation for the excavation of excellent genes of wild R. glutinosa.
Fresh leaves of wild R. glutinosa were collected from Wanxianshan (113°61′78.22″E, 35°72′27.10″N) in Xinxiang City, Henan Province, China and stored in the herbarium of Henan University of Traditional Chinese Medicine with voucher specimen number: HZYYHC15. Total genomic DNA was extracted using a Rapid Plant Genomic DNA Isolation extraction kit.
This experiment adopts the Illumina HiSeq PE150 platform, a 300 bp (insertion size) double-ended library was constructed by splicing DNA. First, the quality of the original sequencing data was evaluated by FastQC, then the quality of the sequencing data with low quality was cut by Trimmomatic to obtain relatively accurate and effective data. SPAdes 3.13.1 was used to assemble the filtered sequencing data and GapFiller was used to fill gaps in assembled contigs (Bankevich et al. 2012). PrlnSeS-G was used for sequence correction. Plann is used for initial annotation (Huang and Cronk 2015). The complete annotated chloroplast genomic sequence annotated had been submitted to GenBank under the accession number of MW007380 for wild R. glutinosa.
The structure of chloroplast genome of wild R. glutinosa was circular, and the size was 153,678 bp, including two inverted repeat (IR) regions of 25,759 bp, separated by a large single copy (LSC) region of 84,544 bp and a small single copy (SSC) region of 17,616 bp. The whole chloroplast genome GC content is 37.91%. There are 149 genes in the chloroplast genome of wild R. glutinosa including 104 protein-coding genes, 37 tRNA genes, and eight rRNA genes.
We selected 15 chloroplast genome sequences of R. glutinosa and Scrophularia dentata Royle ex Benth. (Scrophulariaceae) as outgroups and constructed a phylogenetic tree using the ML (maximum likelihood) method (bootstrap: 1000) embedded in MEGA software (Xia et al. 2016), the results showed that R. glutinosa was clustered separately in the mainland of Rehmannia and wild and cultivated R. glutinosa were sister clades. Among them, these samples (KX636157\NC034308) were transplanted from wild to cultivated (Zeng et al. 2017). The phylogenetic tree demonstrated the domestication process from wild to cultivated (Figure 1). Xie’s view is also supported at the chloroplast genome level to classify and name wild and cultivated R. glutinosa.
Figure 1.
The best ML phylogenetic tree (bootstrap: 1000) from 15 Rehmannia complete chloroplast sequences.
Funding Statement
This study was funded by the General Project of the National Natural Science Foundation of China Study on the Pharmacodynamic Material Basis and Quality Markers of Rehmanniae radix with ‘One effect and multiple Sources’ (81973477) and Henan Agricultural Science and Education [2018] No. 14 Technical System of Chinese Medicinal Materials Industry in Henan Province.
Disclosure statement
No potential conflict of interest was reported by the authors.
Data availability statement
The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov/nuccore/MW007380.1/ under the accession no. MW007380. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA682578, SRR13201823, and SAMN16992822, respectively.
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Associated Data
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Data Availability Statement
The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov/nuccore/MW007380.1/ under the accession no. MW007380. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA682578, SRR13201823, and SAMN16992822, respectively.