Characterization of the complete chloroplast genome sequence of Daphne retusa Hemsl. (Thymelaeaceae), a rare alpine plant species in northwestern China.

Daphne retusa Hemsl. (Thymelaeaceae) is an evergreen shrub plant. First, we characterized the complete nucleotide sequence of chloroplast (cp) genome of D. retusa. The total length of cp genome was found to be 170,553 bp, including a large single copy (LSC) region of 84,886 bp, a small single copy (SSC) region of 2,437 bp, and a pair of inverted repeats (IRs) of 41,617 bp. The cp genome of Daphne retusa Hemsl. contains 134 genes, including 90 protein-coding genes (75 PCG species), 37 transfer RNA genes (29 tRNA species), and 6 rRNA genes (3 RNA species). A total of 13 genes (trnK-UUU, trnS-CGA, atpF, rpoC1, trnL-UAA, trnC-ACA, petD, rpl16, rpl2, ndhB, trnE-UUC, ndhA, and trnA-UGC) contain a single intron, and one gene (ycf3) contains two introns. The GC content in whole cp genome, LSC region, SSC region, and IR region was 36.75%, 34.83%, 28.19%, and 38.96% respectively, like other Thymelaeaceae plants. Phylogenetic analysis suggested that D. retusa has a close relationship with congeneric Daphne tangutica.

Daphne retusa Hemsl. (Thymelaeaceae) is a slow-growing evergreen shrub with a maximum height of 1 m, and it has flexible, off-white stems. This plant often grows on virgin land on hillsides at elevations of 2500–3000 m in the mountains in northwestern China. The dried stem and root bark of D. retusa are known as ‘ZuShima’ which is a common traditional Chinese medicine (Jamila et al. 2018). However, the wild sources of this plant have been nearly used up due to over-exploitation (Geng et al. 2016). In this study, we report the chloroplast genome sequence of D. retusa, and it would be essential to the conservation of this endangered species. The annotated genome sequence has been submitted to GenBank under accession number MW245832.

Fresh leaves of D. retusa were collected from Longnan Mountains (33°38′23″N, 104°50′30″E, 2910 m) in Gansu, China. The total DNA was extracted using the modified CTAB method (Allen et al. 2006) and used for the high-throughput Illumina HiSeq sequencing by Biomarker Technologies, Inc. (Beijing, China). A voucher specimen (ayrx_a) is deposited at the Building B2 of Kechuang 6 Road, Yard 88, Beijing Economic-Technological Development Area (E-mail: lingfeng_zeng@163.com). In total, 47,102,044 raw PE150 reads were obtained and quality-trimmed using the software in the NGSQC Toolkit v2.3.3 (Patel and Jain 2012) with 2% allowed ambiguous bases. The Geneious Prime (Kearse et al. 2012) was used to assemble the complete chloroplast genome. SPAdes v3.11.1 (http://cab.Spbu.Ru/software/SPAdes) was employed to obtain the optimal assembly results using multiple Kmer parameters (107, 117, 127), with the closely related species Daphne tangutica (GenBank: MK455800) serving as the reference. The assembled genome sequence was annotated using CPGAVAS2 (Biomatters Ltd., Auckland, New Zealand) by comparing it to the cpDNA of Daphne tangutica (GenBank: MK455800). Subsequently, a maximum likelihood (ML) tree with 1000 bootstrap replicates was inferred by MEGA7 with default parameters (Kumar et al. 2016). The iTOL (https://itol.embl.de/) was used for tree visualization.

The total length of nucleotide sequence of plastid genome is 170,553 bp, including a large single copy (LSC) region of 84,886 bp, a small single copy (SSC) region of 2,437 bp, and a pair of inverted repeats (IRs) of 41,617 bp (Figure 1). The cp genome of D. retusa contains 134 genes, including 90 protein-coding genes (75 PCG species), 43 transfer RNA genes (37 tRNA species), and 6 rRNA genes (4 RNA species). A total of 13 genes (trnK-UUU, trnS-CGA, atpF, rpoC1, trnL-UAA, trnC-ACA, petD, rpl16, rpl2, ndhB, trnE-UUC, ndhA, and trnA-UGC) contain a single intron, and one gene (ycf3) contains two introns. The GC content in whole cp genome, LSC region, SSC region, and IR region was 36.75%, 34.83%, 28.19%, and 38.96%, respectively, like other Thymelaeaceae plants.

Figure 1.

Maximum likelihood (ML) tree of D. retusa and its related relatives based on the complete chloroplast genome sequences.

To investigate the phylogenetic position of D. retusa, an alignment of Thymelaeaceae chloroplast genome sequences was processed by MAFFT (Katoh and Standley 2013), and a neighbor-joining (NJ) phylogenetic tree (Figure 1) was completed with MAFFT (Katoh and Standley 2013). The results showed close phylogenetic relationships among species in genus Daphne, especially among species Daphne kiusiana, Daphne tangutica, and Daphne Giraldii. The relationships within Daphne can be well distinguished using whole chloroplast genome data. D. retusa has a close relationship with congeneric Daphne tangutica. The complete chloroplast genome sequence of D. retusa facilitates the phylogenetic studies of Thymelaeaceae.