Pinus uliginosa (Neumann) is one of the most controversial, mysterious and threatened taxa from an aggregate of European mountain pines—the Pinus mugo complex (Christensen 1987). The origin of this taxa is not accurately resolved and taxonomical position is still unclear and thus widely discussed in the scientific literature (Hamerník and Musil 2007). Decreasing population size, lack of natural regeneration and ongoing hybridization with the other pines altogether make this taxa threatened by extinction (Wachowiak et al. 2005; Lewandowski and Dering 2006). Highly conservative genome organization, very close relations among the taxa in the P. mugo complex, and lack of any diagnostic markers or taxa determinants for P. uliginosa were all demonstrated previously using morphological and anatomical traits, polymorphism of allozymes, as well as molecular cytogenetics and flow cytometry. On the other hand, recently reported chemotaxonomic markers for P. uliginosa cannot be directly applied for monitoring of gene flow in natural populations or for hybrids identification (Bonikowski et al. 2015; Celiński et al. 2015). Whole-plastid-based barcodes have been shown to have a great potential in species discrimination, especially for closely related taxa where none of single barcodes or their different combinations has sufficient resolution (Li et al. 2015).

Here, we report a complete chloroplast genome sequence of the P. uliginosa, determined using the next-generation sequencing method. The assembled and annotated complete nucleotide sequence of P. uliginosa chloroplast genome has been deposited in GenBank under accesion number KX833097.

Fresh pine needles were collected from a single tree of P. uliginosa (accession no. 06_0526_0008) grown in the Dendrological Garden, Poznań University of Life Sciences (52°25′37″N, 16°53′48″E). Intact chloroplasts fraction was isolated from needles according to Vieira et al. (2014). Plastome DNA was extracted using CTAB protocol (Doyle and Doyle 1990). The chloroplast DNA of P. uliginosa was fragmented using Covaris E210 (Covaris, Woburn, MA, USA). The library was prepared with a NEBNext DNA Library Prep Kit (New England Biolabs Inc., Ipswich, MA, USA) using dual-indexing TruSeq HT (Illumina Inc., San Diego, CA, USA). Paired-end (PE, 2 × 250nt) sequencing of a library was performed using Illumina MiSeq (MiSeq Reagent kit v2) at the Genomed sequencing facility (Warsaw, Poland) following manufacture’s protocols (Illumina, Inc., San Diego, CA, USA). The automatic primary analysis and the de-multiplexing of the raw reads were performed on MiSeq with the use of MiSeq Reporter (MSR) v2.4 (BaseSpace). In total, 1,355,084 paired-end reads (2 × 250 bp) were obtained. The obtained reads were trimmed using Cutadapt 1.4 and assembled with CLC Genomics Workbench 7.0.4 in reference based assembly mode (with D17510.1.1—Pinus thunbergii chloroplast DNA as a reference). As a result, 27,419 paired-end reads with the average length 239,22 bp were assembled into 45 contigs with mean coverage of 54,40×. Gaps and ambiguous sites were PCR-amplified and re-sequenced using Sanger method, using 3130x Genetic Analyzer (Applied Biosystems). Protein-coding genes and genes for non-coding RNAs were annotated with DOGMA (Dual Organellar GenoMe Annotator; Wyman et al. 2004) and cpGAVAS (Chloroplast Genome Annotation, Visualization, Analysis, and GenBank Submission Server; Liu et al. 2012). Annotated sequence was aligned to reference genome using MAFFT (Katoh et al. 2002) and all features were manually inspected to cross-validate the predictions. A circular map of the chloroplast genome was drawn with the OGDRAW software (Lohse et al. 2013). The overall GC content was analyzed using Geneious 9.1.2 (Kearse et al. 2012). REPuter (Kurtz et al. 2001) was used to visualize the large (>1000 bp) duplicated sequences in P. uliginosa cp genome by forward versus reverse complement (palindromic) alignment.

The complete chloroplast (cp) genome of P. uliginosa is 119,877 bp in length (Fig. 1). The overall G + C content of the P. uliginosa chloroplast genome is 38.5%, which is similar to the other reported for Pinaceae cp genomes (Duan et al. 2016; Fang et al. 2016). The cp genome of P. uliginosa contains a total of 112 predicted functional genes including 73 protein-coding genes, 4 ribosomal RNA (rRNA) and 35 transfer RNA (tRNA) genes (Table 1). Five tRNA genes were duplicated from two (trnF-GAA, trnH-GUG, trnS-GCU and trnV-GAC) to four times (trnM-CAU). Twelve genes (petB, petD, atpF, rpl2, rpl16, rpoC1) and six tRNA genes contain one intron, while one gene (ycf3) contains two introns. There were no large inverted repeats (IRs) in the P. uliginosa plastome.

Fig. 1
figure 1

Chloroplast genome map of P. uliginosa. Genes drawn inside the circle are transcribed clockwise, and those outside are counterclockwise. Genes belonging to different functional groups are color-coded. The darker gray in the inner circle corresponds to GC content, while the lighter gray corresponds to AT content

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Table 1 List of genes present in P. uliginosa chloroplast genome
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Here reported chloroplast genome of P. uliginosa provides useful genomic resources for further studies on determination of taxa-diagnostic markers in the P. mugo complex, analysis of hybridization and introgression processes in this complex, characterization of genetic diversity of P. uliginosa and more effective protection of its threatened gene pools.