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Chloroplast genome structure and phylogenetic analysis of Glycosmis parviflora (Sims) Little 1948, a folk medicinal plant featured in Lingnan Region, China

Abstract Glycosmis parviflora is the most widely spread and the most morphologically varied species of Chinese Glycosmis, and its roots and leaves serve as folk medicines. We sequenced the complete chloroplast (cp) genome of G. parviflora. The cp genome obtained was a circular DNA molecule of 159,825 bp in length, containing one large and one small single copy region (LSC and SSC) of 87,517 and 18,352 bp separated by a pair of 26,978 bp inverted repeat regions (IRs). The overall GC content of the cp genome was 38.40%. The phylogenetic analysis revealed that Glycosmis was strongly supported as a monophyletic group belonging to Clauseneae, and G. parviflora was closely related to G. pentaphylla. The results will provide the basis for the further study of molecular markers and phylogeny of G. parviflora.

Glycosmis is a group of Rutaceaous plants and evergreen glabrous shrub with orange berries (Teja et al. 2021). G. parviflora (Sims) Little 1948 is the most widespread species of the genus in China (Zhang and Hartley 2008). It is a commonly used medicinal plant in Lingnan Region (which is a loosely defined geographic areas south to Nangling Mountains, mainly covering Guangdong Province and Guangxi Zhuang Autonomous Region) of China. In traditional Chinese medicine, the root of G. parviflora is used to eliminate phlegm and relieve cough, and its leaves can dissipate stasis and disperse swelling (Wang 2014). It is also used in other folk medicines (Teja et al. 2021), and the compounds it contains have been highly explored for anticancer activity (Kn€ olker and Reddy 2008). Widely distributed in Lingnan Region, G. parviflora shows great variations in the length of its inflorescences, ranging from 3 cm to 14 cm, and in the morphologies of the leaves, from a simple leaf, unifoliate leaf to a compound leaf with 2-6 leaflets; the leaflets also vary in the shapes, which can be elliptic, oblong or lanceolate (Mou et al. 2012). To identify molecular markers for species and provide information for its phylogenetic position, we sequenced, assembled and annotated the complete chloroplast (cp) genome of G. parviflora.
Fresh leaves of G. parviflora were collected from the medicinal plant garden of Southern Medical University (113 19'43.35"E, 23 11'20.58"N), Guangzhou, China. The collections were in accordance with regulations including our university and regional, national, or international ones. The voucher specimen (Chao Zhi 20200405016) was identified by Professor Zhi Chao and deposited in the herbarium of the School of Traditional Chinese Medicine, Southern Medical University (contact Zhi Chao, chaozhi@smu.edu.cn). The DNA of G. parviflora was extracted by the modified CTAB method (Yang et al. 2014). Sequencing was performed on BGISEQ-500 platform in high output mode with 150 bp paired-end reads at Beijing Genomics Institute (BGI, Shenzhen, China). The cp genome of G. parviflora was assembled by using GetOrganelle v.1.7.1 (Jin et al. 2020) with G. mauritiana and G. pentaphylla as references (Accession No. NC_032686 and NC_032687). The assembled genome was annotated using GeSeq (Annotation of Organellar Genomes) (https://chlorobox.mpimp-golm.mpg.de/geseq.html) (Tillich et al. 2017) and Plastid Genome Annotator (PGA) software (Qu et al. 2019). The annotated sequence has been deposited in GenBank (Accession No. MW714375).
The complete cp genome of G. parviflora showed a typical circular tetramerous structure of 159,825 bp in length. Its quadripartite structure is composed of a large single-copy (LSC) region of 87,517 bp and a small single-copy (SSC) region of 18,352 bp, separated by a pair of inverted repeats (IRs) of 26,978 bp. The complete cp genome encodes 124 genes, including 85 protein-coding genes (PCGs), 30 transfer RNA genes, 8 ribosomal RNA genes and 1 pseudogene(ycf1). Among these genes, 92 genes are single copy, while 8 PCGs, 4 tRNA genes, and 4 rRNA genes in IR regions are duplicated. Moreover, there are ten genes containing one intron, and three genes containing two introns. The GC content of the overall chloroplast genome, IR regions, LSC and SCC are 38.40, 42.9, 36.7, 32.9%, respectively.
In order to gain insight into its phylogenetic position, a phylogenetic analysis was performed using the complete cp genomes of G. parviflora and other 19 species of Rutaceae. The cp genomes of the 19 species were downloaded from GenBank (species names and accession numbers shown in Figure 1). All the cp genomes were aligned with MAFFT (Rozewicki et al. 2019), and then edited manually by MEGA X (Kumar et al. 2018). The maximum likelihood (ML) tree was inferred in RAxML (Stamatakis 2014) with the best fitting substitution model (GTR þ FþR2) determined by the Akaike information criteria (AIC). The bootstrap support was calculated with 1000 replications.
In the phylogenic tree with Phellodendron chinense as an outgroup species, two clades were clearly recognized, which corresponded to Clauseneae and Aurantieae repectively. Glycosmis was a strongly supported monophyletic group (PP ¼ 100) in Clauseneae, and was the sister group to Micromelum. G. parviflora was closely related to G. pentaphylla (Figure 1).

Author contributions
Z C conceived the study, reviewed and revised the drafts of the paper. AM C analyzed data, wrote and revised the manuscript. F L performed the genome assembly and annotation. XN X conducted the molecular experiments, and assisted with data analysis. R H assisted in manuscript revision. EW T collected plant materials and assisted with the molecular experiments. All authors read and approved the final manuscript.

Disclosure statement
No potential conflict of interest was reported by the author(s).