The Complete Chloroplast Genome of Colobanthus Apetalus (Labill.) Druce: Genome Organization and Comparison with Related Species
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The complete chloroplast genome of Colobanthus apetalus (Labill.) Druce: genome organization and comparison with related species Piotr Androsiuk1, Jan Paweª Jastrz¦bski1, Łukasz Paukszto1, Adam Okorski2, Agnieszka Pszczółkowska2, Katarzyna Joanna Chwedorzewska3, Justyna Koc1, Ryszard Górecki1 and Irena Gieªwanowska1 1 Department of Plant Physiology, Genetics and Biotechnology, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland 2 Department of Entomology, Phytopathology and Molecular Diagnostics, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland 3 Department of Antarctic Biology, Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warszawa, Poland ABSTRACT Colobanthus apetalus is a member of the genus Colobanthus, one of the 86 genera of the large family Caryophyllaceae which groups annual and perennial herbs (rarely shrubs) that are widely distributed around the globe, mainly in the Holarctic. The genus Colobanthus consists of 25 species, including Colobanthus quitensis, an extremophile plant native to the maritime Antarctic. Complete chloroplast (cp) genomes are useful for phylogenetic studies and species identification. In this study, next-generation sequencing (NGS) was used to identify the cp genome of C. apetalus. The complete cp genome of C. apetalus has the length of 151,228 bp, 36.65% GC content, and a quadripartite structure with a large single copy (LSC) of 83,380 bp and a small single copy (SSC) of 17,206 bp separated by inverted repeats (IRs) of 25,321 bp. The cp genome contains 131 genes, including 112 unique genes and 19 genes which are duplicated in the IRs. The group of 112 unique genes features 73 protein-coding genes, 30 tRNA Submitted 4 January 2018 genes, four rRNA genes and five conserved chloroplast open reading frames (ORFs). Accepted 17 April 2018 Published 23 May 2018 A total of 12 forward repeats, 10 palindromic repeats, five reverse repeats and three complementary repeats were detected. In addition, a simple sequence repeat (SSR) Corresponding author Piotr Androsiuk, analysis revealed 41 (mono-, di-, tri-, tetra-, penta- and hexanucleotide) SSRs, most of [email protected] which were AT-rich. A detailed comparison of C. apetalus and C. quitensis cp genomes Academic editor revealed identical gene content and order. A phylogenetic tree was built based on Ladislav Mucina the sequences of 76 protein-coding genes that are shared by the eleven sequenced Additional Information and representatives of Caryophyllaceae and C. apetalus, and it revealed that C. apetalus and Declarations can be found on C. quitensis form a clade that is closely related to Silene species and Agrostemma githago. page 17 Moreover, the genus Silene appeared as a polymorphic taxon. The results of this study DOI 10.7717/peerj.4723 expand our knowledge about the evolution and molecular biology of Caryophyllaceae. Copyright 2018 Androsiuk et al. Subjects Biodiversity, Bioinformatics, Genetics, Genomics, Plant Science Distributed under Keywords Colobanthus apetalus, Caryophyllaceae, plastid genome, Next-generation sequencing, Creative Commons CC-BY 4.0 Phylogenetics, Genome features OPEN ACCESS How to cite this article Androsiuk et al. (2018), The complete chloroplast genome of Colobanthus apetalus (Labill.) Druce: genome orga- nization and comparison with related species. PeerJ 6:e4723; DOI 10.7717/peerj.4723 INTRODUCTION Chloroplasts are organelles whose main function is the photosynthetic fixation of carbon. They contain the complete enzymatic system for energy production in many metabolic pathways, including the biosynthesis of glucose, amino acids and fatty acids (Neuhaus & Emes, 2000). Chloroplasts are uniparentally inherited: they are inherited maternally in most angiosperms and gymnosperms (Palmer et al., 1988), but are transmitted in the male line in some gymnosperms (Sears, 1980). Their ultrastructure and characteristic genome features indicate that chloroplasts have evolved from free-living cyanobacteria through endosymbiosis (Gray, 1989). In a typical terrestrial plant, the chloroplast (cp) genome is a circular DNA molecule with a conserved quadripartite structure composed of a small single copy (SSC), a large single copy (LSC) and two copies of inverted repeat (IR) regions. The cp genome is the smallest of the plant genomes, and it ranges from 120 kb to 165 kb in most species (Ma et al., 2017). The variations in size can be attributed mostly to the expansion, contraction or even loss of IRs as well as changes in the length of intergenic spacers (Palmer et al., 1988). Due to their compact size, highly conserved status, uniparental inheritance and haploid nature, cp genomes can be effectively used in studies of plant taxonomy and evolution and in species identification (Kress et al., 2005; Chase et al., 2007; Parks, Cronn & Liston, 2009; Yang et al., 2013). The genus Colobanthus, a member of the family Caryophyllaceae, consists of 25 species (The Plant List, 2010) of tufted, mainly cushion-forming perennials from the Pacific region, Australasia to southern South America, sub-Antarctic islands, maritime Antarctic and Hawaiian mountains. These species are related to Spergula and have very small, narrow and dense leaves and solitary, petalless, greenish flowers with four to six, but usually five prominent sepals (Gieªwanowska et al., 2011; Alpine Garden Society, 2017). The available literature is nearly exclusively devoted to only one species of the genus Colobanthus, namely Colobanthus quitensis (Kunth) Bartl. Colobanthus quitensis is a species of special concern as the only representative of Dicotyledoneae in the maritime Antarctic (Skottsberg, 1954). Colobanthus quitensis has been extensively studied to explore the morphological, physiological and biochemical features that constitute the basis of adaptation to extreme Antarctic conditions (Bravo et al., 2007; Gieªwanowska et al., 2011; Gieªwanowska et al., 2014; Bascunan-Godoy et al., 2012; Navarrete-Gallegos et al., 2012; Pastorczyk, Gieªwanowska & Lahuta, 2014; Cuba-Díaz et al., 2017). In contrast, very little is known about the genetic diversity of this species and the genus Colobanthus (Androsiuk et al., 2015; Koc et al., in press). The complete sequence of C. quitensis cp genome has been recently published (Kang et al., 2016), and it paved the way for more sophisticated genomic studies. However, the significance of those studies will be limited without information about the genome composition of other members of the genus Colobanthus. The complete chloroplast genome of eleven Caryophyllaceae species has been sequenced to date (NCBI: the National Center for Biotechnology Information), including eight species of the genus Silene (Silene capitata, S. chalcedonica, S. conica, S. conoidea, S. latifolia, S. noctiflora, S. paradoxa, S. vulgaris), one species of the genus Lychnis (Lychnis wilfordii), one species Androsiuk et al. (2018), PeerJ, DOI 10.7717/peerj.4723 2/23 of the genus Agrostemma (Agrostemma githago) and one species of the genus Colobanthus (Colobanthus quitensis). Colobanthus apetalus (Labill.) forms tufts with soft and grassy leaves (1.5-3 cm in length), stems of up to 3 cm, and small greenish flowers (5 mm in diameter) that are more obvious than in other Colobanthus species. The sepals often have purple borders, and seeds have low rounded papillae. The species has been recorded in New Zealand, south-eastern Australia, including Tasmania (Allan, 1961), and in southern regions of South America (Skottsberg, 1915). In this study, the complete cp genome of Colobanthus apetalus was sequenced with the use of the Illumina MiSeq sequencing technology and compared with other Caryophyllaceae species, in particular with C. quitensis. MATERIALS AND METHODS DNA extraction and chloroplast genome sequencing Fresh leaves of C. apetalus were harvested from greenhouse-grown plants (Department of Plant Physiology, Genetics and Biotechnology, University of Warmia and Mazury in Olsztyn). The seeds of C. apetalus were collected on the south-eastern shore of Lago Roca, near Lapataia Bay, in the Tierra del Fuego National Park in Argentina. Total genomic DNA was extracted from the fresh tissue of a single plant using the Syngen Plant DNA Mini Kit. The quality of DNA was verified on 1 % (w/v) agarose gel and visualized by staining with 0.5 mg/ml ethidium bromide. The amount and purity of DNA samples were assessed spectrophotometrically. Genome libraries were prepared from high-quality genomic DNA using the Nextera XT kit (Illumina Inc., San Diego, CA, USA). The prepared libraries were sequenced on the Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA) with a 150 bp paired-end read, version 3. Annotation and genome analysis The quality of the obtained row reads was checked with the FastQC tool. Row reads were trimmed (5 bp of each read end, regions with more than 5% probability of error per base) and mapped to the reference cp genome of C. quitensis (NC_028080) using Geneious v.R7 software (Kearse et al., 2012) with default medium-low sensitivity settings. The mapped reads were retrieved from the mapping file and used for de novo Velvet preassembly (K-mer—23–41, low coverage cut-off—5, minimum contig length— 300). Preassembled contigs were extended by mapping row reads using customized settings (minimum sequence overlap of 60 bp and 99% overlap identity) with 30 iterations steps. Elongated contigs were de novo assembled after each iteration step to reduce the number and increase the length of sequences and, finally, to create a circular