The complete chloroplast genome of Pleione formosana (Orchidaceae)

Abstract

Pleione formosana is an Endangered terrestrial orchid with significant ornamental values. Here, we report the first complete chloroplast genome of P. formosana. The circular genome was 159,843 bp in length and consisted of a pair of inverted repeats (IR 26,879 bp), which were separated by a large single copy region (LSC 87,392 bp) and a small single copy region (SSC 18,693 bp). It contained 135 genes (114 unique), including 87 protein-coding genes, 38 tRNAs, and 8 rRNAs. The maximum likelihood phylogenetic analysis indicated that P. formosana and P. bulbocodioides cluster together are closely related to the genera Bletilla.

Keywords:

• Chloroplast genome
• phylogenetic
• Illumina sequencing
• Pleione formosana

Pleione formosana, Hayata (1911), is a terrestrial orchid, which grows on humus-covered soil or rocks in montane forests and at forest margins, between 600 and 1500 m in southeastern China (1500 and 2500 m in Taiwan island) (Chen et al. 2009). Pleione formosana is a vulnerable species in the Red List (IUCN 2018) and one of the most important parents for a succession of hybrids. Therefore, it is urgent to establish a strategy for the conservation of this important plant. In addition, Pleione formosana belongs to P. bulbocodioides complex (Cribb and Butterfield 1999), which has been thought to be the source of persistent confusion and ambiguity in the taxonomy of Pleione (Gravendeel et al. 2004). In recent years, comparative analysis of the complete chloroplast (cp) genome of different close species has provided a promising method for the study of phylogeny, population dynamics, and species evolution (Shaw et al. 2014). Thus, we aimed to assemble and characterize P. formosana cp genomes to provide a better understanding of the evolution and genetics of genera Pleione.

Total genomic DNA was extracted from fresh leaves (Voucher specimen: 23°32′25.19″N 120°47′57.71″E, AL01A12, FAFU) using a modified CTAB method (Doyle and Doyle 1987) and sequenced by the Illumina Hiseq 2000 sequencing platform (Illumina, CA, USA) at Novogene (Beijing, China). Raw reads were filtered by NGS QC Toolkit (Patel and Jain 2012). The clean reads were firstly aligned to P. bulbocodioides (Genbank Accession No. KY849819) (Shi et al. 2017). Filtered reads were then assembled into contigs in the software CLC Genomics Workbench v8.0 (CLC Bio, Aarhus, Denmark). After assembled, the obtained scaffolds and contigs were assembled into cp genome by Geneious 11.1.15 (Kearse et al. 2012) using the algorithm MUMmer. The genome was automatically annotated using DOGMA (Wyman et al. 2004), then adjusted by Geneious version 11.1.15 (Kearse et al. 2012) and submitted to GenBank with accession number MK361027. The cp genome sequence of P. formosana is 159,843 bp in length, containing a large single copy (LSC) region of 87,392 bp and a small single copy (SSC) region of 18,693 bp, and two inverted repeat (IR) regions of 26,879 bp. The cp genome encoded 135 genes, of which 114 were unique genes (87 protein-coding genes, 38 tRNAs, and 8 rRNAs). Overall, GC content of the whole genome is 37.2%, while the corresponding values of the LSC, SSC, and IR regions are 35.0, 30.3, and 43.1%, respectively.

To further investigate its phylogenetic position, 88 complete cp genomes of Epidendroideae and two species of Orchidoideae were aligned using HomBlocks pipeline (Bi et al. 2018). RAxML-HPC Black-Box version 8.1.24 (Stamatakis et al. 2008) was used to construct a maximum likelihood tree with Ludisia discolor and Goodyera fumata as outgroup. The branch support was computed with 1000 bootstrap replicates. The ML tree analysis indicated that P. formosana and P. bulbocodioides cluster together and are closely related to the genera Bletilla with 100% bootstrap support (Figure 1).

Figure 1. Maximum-likelihood (ML) tree based on 88 complete cp genome in Epidendroideae, with Goodyera fumata and Ludisia discolor (Orchidoideae) as outgroup. The bootstrap value indicated on each node and the position of Pleione formosana is shown in bold.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [31500270], Fujian Agriculture and Forestry University Fund for Distinguished Youth Scholars [XJQ201620], Chiayi Forest District Office, Forestry Bureau [104CRC01], and Innovation of Characteristic Flower Varieties and Industrialization Project of Seedling Breeding Facilities in Fujian Province [2014S1477-7].