Characterization of the complete chloroplast genome sequence of Pinus pumila (Pinaceae)

Abstract

Pinus pumila is a rare and endangered conifer plant in the family Pinaceae. Here, we determined the complete chloroplast (cp) genome sequence of P. pumila. The cpDNA was 117,398 bp in length, containing a pair of 475 bp inverted repeat regions (IR), which was divided into large single copy region (LSC) and small single copy region (SSC) with 64,606 and 51,842 bp in length, respectively. The GC content in cpDNA, LSC region, SSC region and IR region were 38.8%, 38.0%, 39.8% and 37.9%, respectively. The cpDNA contained 114 genes, including 74 protein-coding genes (CDS), 36 transfer RNA genes (tRNA) and 4 ribosomal RNA genes (rRNA). A phylogenetic analysis revealed that P. pumila is more closely related with congeneric P. fenzeliana var. dabeshanensis and P. sibirica.

KEYWORDS:

• Pinus pumila
• chloroplast genome
• phylogenetic analysis

Pinus pumila is a rare and endangered plant in the family Pinaceae, which distributed in Northeast China, Japan and Eastern Siberia. Previous studies have focused on the genetic structure, paleoclimatic implications, vegetation dynamics, photosynthesis and respiration for this species (Okitsu and Ito 1984; Kajimoto 1990; Watano et al. 2004; Anderson et al. 2010; Chen et al. 2016). In this study, we sequenced and assembled the complete chloroplast genome (cpDNA) of P. pumila. The annotated cpDNA of P. pumila has been deposited into the GenBank with the accession number number MH046871.

Fresh leaf tissues were collected in the Greater Hinggan mountains, Heilongjiang Province, China (N: 51.62°; E: 124.00°; H: 627m). The total genomic DNA was extracted using the cetyltrimethyl ammonium bromide (CTAB) method (Doyle and Doyle 1987). DNA sample and voucher specimen (No. PPLZH2016137) of P. pumila were deposited in the Northwest University Museum (NUM). Meanwhile, we constructed a pair-end (PE) library with 350 bp insert size fragments using TruSeq DNA sample preparation kits (Sangon, Shanghai, China). Thereafter, we sequenced at least 1.2 GB of clean data for P. pumila. The detailed next-generation sequencing was conducted on the Illumina Hiseq 2500 platform by Sangon Biotech (Shanghai, China). Then, we trimmed the low-quality reads by NGSQCToolkit v2.3.3 (Patel and Jain 2012). After removing the low-quality sequences, the clean reads were assembled using MIRA v4.0.2 (Chevreux et al. 2004) and MITObim v1.8 (Hahn et al. 2013) with the cp genome of Pinus armandii (KP412541), as reference. A total of 208,396 bp reads generated with average coverage of 266.7. Then, we used DOGMA (http://dogma.ccbb.utexas.edu/) (Wyman et al. 2004) and Geneious v8.0.2 (Kearse et al. 2012) to annotate the chloroplast genome. Finally, we acquired the P. pumila complete chloroplast genome sequences.

The complete chloroplast (cp) genome sequence of P. pumila was 117,398 bp in length, containing a pair of 475 bp inverted repeat regions (IR), one large single copy region (LSC) with 64,606 bp in length and one small single copy region (SSC) of 51,842 bp in length. The GC content in cpDNA, LSC region, SSC region and IR region were 38.8%, 38.0%, 39.8% and 37.9%, respectively. The cpDNA contained 114 genes, including 74 protein-coding genes (CDS), 36 transfer RNA genes (tRNA) and 4 ribosomal RNA genes (rRNA).

Twenty-five available complete chloroplast genomes, 23 Pinus species with Cupressus chengiana (KY392754) and Cupressus gigantean (KT315754) as outgroups, all of the 26 complete chloroplast sequences were aligned using MAFFT (Katoh and Standley 2013) with the default parameters. We constructed a maximum likelihood phylogenetic tree based on these complete plastomes using MAGA7 (Kumar et al. 2016) with 1000 bootstrap replicates under the GTRGAMMA model, which was determined using the Modeltest v3.7 (Posada and Crandall 1998). The phylogenetic analysis showed that the most of monophyletic clade with high bootstrap value (Figure 1). P. pumila is more closely related with P. fenzeliana var. dabeshanensis and P. sibirica. Meanwhile, the P. pumila chloroplast genome will provide information for the conservation, utilization and management.

Figure 1. Maximum likelihood tree based on the complete chloroplast genome sequences of 26 species. The numbers on the branches are bootstrap values.

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

This work was co-supported by the Shaanxi Provincial Key Laboratory Project of Department of Education [grant no. 17JS135].