Hindawi BioMed Research International Volume 2020, Article ID 3536761, 12 pages https://doi.org/10.1155/2020/3536761 Research Article Characteristic and Phylogenetic Analysis of the Complete Chloroplast Genomes of Three Medicinal Plants of Schisandraceae Dachuan Zhang,1 Jiahao Wang,1 Liang Xu ,1,2 Yanping Xing,1,2 Tingting Zhang,1 Shengnan Li,1 Yanyun Yang,1 Guihua Bao,3 Wuliji Ao,3 and Tingguo Kang 1,2 1School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China 2Liaoning Quality Monitoring and Technology Service Center for Chinese Materia Medica Raw Materials, Dalian 116600, China 3School of Mongol Medicine, Inner Mongolia University for Nationalities, Tongliao 028000, China Correspondence should be addressed to Liang Xu; [email protected] and Tingguo Kang; [email protected] Received 4 July 2020; Accepted 5 October 2020; Published 17 October 2020 Academic Editor: Yong-Pyo Lim Copyright © 2020 Dachuan Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Schisandra chinensis, which has a high development value, has long been used as medicine. Its mature fruits (called Wuweizi in Chinese) have long been used in the famous traditional Chinese medicine (TCM) recorded in the “Chinese Pharmacopoeia.” Chloroplasts (CP) are the highly conserved primitive organelles in plants, which can serve as the foundation for plant classification and identification. This study introduced the structures of the CP genomes of three Schisandraceae species and analyzed their phylogenetic relationships. Comparative analyses on the three complete chloroplast genomes can provide us with useful knowledge to identify the three plants. In this study, approximately 5 g fresh leaves were harvested for chloroplast DNA isolation according to the improved extraction method. A total of three chloroplast DNAs were extracted. Afterwards, the chloroplast genomes were reconstructed using denovo combined with reference-guided assemblies. General characteristics of the chloroplast genome and genome comparison with three Schisandraceae species was analyzed by corresponding software. The total sizes of complete chloroplast genomes of S. chinensis, S. sphenanthera, and Kadsura coccinea were 146875 bp, 146842 bp, and 145399 bp, respectively. Altogether, 124 genes were annotated, including 82 protein-coding genes, 34 tRNAs, and 8 rRNAs of all 3 species. In SSR analysis, only S. chinensis was annotated to hexanucleotides. Moreover, comparative analysis of chloroplast Schisandraceae genome sequences revealed that the gene order and gene content were slightly different among Schisandraceae species. Finally, phylogenetic trees were reconstructed, based on the genome-wide SNPs of 38 species. The method can be used to identify and differentially analyze Schisandraceae plants and offer useful information for phylogenetics as well as further studies on traditional medicinal plants. 1. Introduction China Pharmacopoeia. Wuweizi is often used as a diarrhea antispasmodic agent in clinical practice, and it has astringent Wuweizi (Schisandra chinensis), first recorded in “Shen- solidifying, Qi and fluid replenishing, as well as kidney and nong’s Herbal Classic of Materia Medica,” has long been used heart tonifying effects [1]. K. coccinea is known as the “magic in traditional Chinese medicine (TCM) as a top grade medi- longevity fruit,” which can be eaten as a fruit, and its root and cine; specifically, it has been utilized as a tonic medicine for root skin can be used for medicinal purposes, as recorded in about 2000 years [1]. In many Asian countries like Japan “Gui Medicine.” Besides, it can promote qi and activate blood and South Korea, Schisandra is also listed as a pharmaco- circulation, reduce swelling, and relieve pain [4]. poeia variety [2, 3]. Wuweizi (Schisandra chinensis), Nanwu- Chloroplasts are important organelles for photosynthesis weizi (Schisandra sphenanthera), and Heilaohu (Kadsura in plants, which have distinct physiological characteristics, coccinea) have displayed medicinal value. Nanwuweizi (Schi- such as relatively conservative and single parental inheritance sandra sphenanthera) is the same as Wuweizi in historical [5, 6]. Chloroplasts can be used for plant species identification, medicine; both of them were listed separately in the 2000 hybridization, and phylogenetic analysis. In 1986, Japanese 2 BioMed Research International scientists had first obtained the complete chloroplast genome Various plants of Schisandraceae have exhibited high sequence of plants in tobacco [7]. With the rapid development economic and medicinal value. This paper is aimed at analyz- of high-throughput sequencing in recent years, this emerging ing the complete chloroplast genomes of S. chinensis, S. sphe- technology has been applied to investigation in various fields. nanthera, and K. coccinea, so as to explore the basis for For instance, Li et al. had investigated the structural character- identification and genetic relationships among these three. istics and genetic evolution of Magnoliaceae plants through Notably, S. chinensis and S. sphenanthera have not been chloroplast genome-wide analysis of Magnolia grandiflora in accurately distinguished in the history of medication use, 2013, and had verified the negative correlation of the IR region and mixed phenomena can be observed. Therefore, exploring length in some Magnoliaceae plants with the pseudogene the differences at the gene level between these two is not only length ψycf1 [8]. In 2013, Yang et al. had carried out chloro- conducive to identifying these two traditional Chinese medi- plast genome-wide structural analysis to identify and analyze cines, but it also lays the vital genetic foundation for the various plants of genus Cymbidium, and examined their future cultivation of S. chinensis and S. sphenanthera. This genetic relationships [9]. Their results proved that the correla- study is aimed at exploring the intrinsic relationship and dif- tion between species based on organelle gene sequencing was ference in the chloroplast gene structure of S. chinensis reliable. through internal analysis of S. chinensis and S. sphenanthera, Magnoliaceae has always been a research hotspot. So far, as well as K. coccinea. Besides, phylogenetic analysis of Schi- the chloroplast genomes of about 20 plant species have been sandraceae with other families was also carried out to obtain sequenced [10]. Typically, Magnolia is also a controversial the position of Schisandra during phylogenetic evolution. group in botany, which is mainly attributed to the classifica- tion of botany. In the APG IV system, Schisandraceae 2. Materials and Methods includes Schisandra and Illicium. In 2007, Hansen et al. ana- lyzed the chloroplast gene structures of four plants, including 2.1. Plant Materials. The fresh S. chinensis leaves were col- medicinal anise [11]. In 2017, Guo et al. examined the chlo- lected from Dalian, China (E121°52′34.96″,N39°03′43.96″). roplast genes of S. chinensis and determined that the Illicium The fresh leaves of S. sphenanthera were collected from Shang- genus was a sister branch of Schisandra [12]. In 2018, Li and luo, China (E110°02′27.55″,N33°55′34.10″). The fresh leaves Zheng analyzed the chloroplast genomes of K. coccinea and of K. coccinea were obtained from the channel Xufeng Chinese obtained the evolutionary structure of K. coccinea by means Herbal Medicine Cooperative (Huaihua, Hunan Province, of phylogenetic tree analysis [13]. Modern methods have China (E110°05′03.05″,N27°28′30.10″). These three species been utilized to analyze chloroplasts, such as simple sequence were identified by Dr. Tingguo Kang from the Liaoning Uni- repeat (SSR) analysis, IR boundary analysis, and phylogenetic versity of Traditional Chinese Medicine in Shenyang, China. analysis. Of them, SSR is frequently used to identify species Permission for using plant fruit samples in biological experi- and analyze the genetic difference. Previous phylogenetic ments had been granted. Plant samples were deposited in the research focusing on the phylogenetic tree of consensus pro- herbarium of Liaoning University of Traditional Chinese tein clustering has only taken into consideration the coding Medicine and the genomic DNA was stored in the Key Labo- region variation, which has certain limitations. Therefore, ratory of Traditional Chinese Medicine in the University this paper had been carried out with the aim of analyzing (Dalian, China, 116600). the phylogenetic tree of genome-wide single-nucleotide poly- morphisms (SNPs), with the consideration of variations in 2.2. Chloroplast DNA Extraction and Sequencing. Approxi- the coding region and the noncoding region. Notably, our mately 5 g fresh leaves were harvested for chloroplast DNA analysis was more comprehensive, and our results were more isolation according to an improved extraction method [25]. accurate and reliable. After DNA isolation, 1 μg purified DNA was fragmented, In the 2015 China Pharmacopoeia, all the three medicinal which was then utilized to construct the short-insert libraries plants examined in this study are classified into Magnoliaceae; (insert size of 430 bp) in accordance with the manufacturer’s however, in the latest APG IV classification system [14], Schi- protocol (Illumina), followed by sequencing on the Illumina sandraceae is classified into a separate family, which includes HiSeq 4000 [26]. Illicium