The complete plastome sequence of the endangered orchid Kuhlhasseltia nakaiana (Orchidaceae)

Abstract In this study, we report the complete plastome sequence of Kuhlhasseltia nakaiana (F.Maek.) Ormerod (Orchidaceae) (NCBI acc. no. KY354041), an endangered plant species protected by the national law of Korea. The gene order and number in the K. nakaiana plastome were similar to a typical orchid plastome. The complete plastome was 147,614 bp in length and consisted of a large single copy region of 81,617 bp and a small single copy region of 13,673 bp, separated by two inverted repeats of 26,162 bp. The plastome contained 103 genes, of which 69 were protein-coding genes, 30 were tRNA genes, and four were rRNA genes. Fourteen genes contained one intron and two genes (clpP and ycf3) had two introns. The AT content of the plastome was 60.5%. A total of 74 simple sequence repeat regions were identified from the plastome. Phylogenetic analysis determined that K. nakaiana was a member of the tribe Cranichideae and revealed the sister group relationship between K. nakaiana and Ludisia discolor within the tribe Cranichideae.

Kuhlhasseltia nakaiana (F.Maek.) Ormerod, a terrestrial orchid in the genus Kuhlhasseltia, is native to Korea, Japan, Taiwan, and the Philippines (Saeki et al. 2014). The genus Kuhlhasseltia is composed of five species (Lee 2011). In nature, K. nakaiana is very rare and only three populations were recorded in Korea. This species has, therefore, been designated an endangered and protected plant species in Korea. It is a tiny plant of less than 10 cm in height with only a few small leaves along a single erect stem. K. nakaiana belongs to the subfamily Orchidoideae of the family Orchidaceae (APG IV 2016). To develop genetic markers for K. nakaiana for use in conservation studies, we sequenced and analysed the plastome of this species.
K. nakaiana plant material was collected from its natural habitat of Jeju Island, Korea, under a collection permit from the environmental protection authority of the Korean government. As this species is seriously threatened species, we were permitted to collect only one K. nakaiana individual. Therefore, a representative specimen was not deposited in herbarium. But, the extracted DNAs were deposited in the Plant DNA Bank in Korea (PDBK 2016-0443). Fresh leaves were ground into powder in liquid nitrogen and total DNA was extracted using a G-spin TM II Plant Genomic DNA extraction kit (iNtRON). The complete plastome sequence was generated using Illumina MiSeq (San Diego, CA) and assembled by Geneious 6.1.8 (Kearse et al. 2012). An average sequence coverage of 400 times the plastome size was obtained.
Annotations were performed using the National Center for Biotechnology Information (NCBI) BLAST and tRNAscan-SE programs (Lowe and Eddy 1997). The complete plastome sequence was submitted to the NCBI database under the accession number KY354041.
The gene order and number in K. nakaiana were similar to those of a typical angiosperm such as Panax, Nicotiana, and Sesamum (Shinozaki et al. 1986;Kim and Lee 2004;Yi and Kim 2012), with the exception of the ndh genes. A typical plant plastome contains 11 ndh family genes; the K. nakaiana plastome, however, contained only the ndhE gene, with the other 10 ndh genes pseudogenized or lost. These ndh gene losses are not unique to the K. nakaiana plastome and occur commonly in the plastomes of Orchidaceae plants (Chang et al. 2006;Wu et al. 2010;Lin et al. 2015). In total, the plastome of K. nakaiana contained 103 unique genes including 69 protein-coding genes, 30 tRNA genes, and four rRNA genes. Fourteen genes had a single intron while the clpP and ycf3 genes had two introns. The length of the complete plastome of K. nakaiana was 147,614 bp; this was composed of a large single copy (LSC) region of 81,617 bp, a small single copy (SSC) region of 13,673 of bp, and two inverted repeats (IRs) of 26,162 bp. The average AT content of the plastome was 60.5%. We identified a total of 74 simple sequence repeat (SSR) loci including 61 mono-SSRs, 10 di-SSRs, and three tri-SSRs. Some of these plastome SSRs may useful for the development of genetic markers among K. nakaiana populations.
Phylogenetic analyses were performed on a dataset that included 78 protein-coding genes (excluding ycf1) and four rRNA genes extracted from 38 taxa in the NCBI database and Cymbidium macrorhizon (KY354040) and K. nakaiana (KY354041). Fritillaria hupehensis and F. taipaiensis, representing the sister order Liliales, were used as outgroups. The gaps for lost genes were treated as missing bases. The 82 gene sequences were aligned with MUSCLE in Geneious 6.1.8; the aligned data matrix consisted of a total of 70,620 bp. This alignment is used for phylogenetic analysis using RAxML v. 7.7.1 (Stamatakis et al. 2008). An ML tree was obtained with an ML estimation value of -302818.486357. The sister group relationship between K. nakaiana and Ludisia discolor was confirmed with 100% bootstrap value support (Figure 1). Both species occurred in the tribe Cranichideae within Orchidoideae.