Combined analysis of the chloroplast genome and transcriptome of the Antarctic vascular plant Deschampsia antarctica Desv

Abstract

Background: Antarctic hairgrass (Deschampsia antarctica Desv.) is the only natural grass species in the maritime Antarctic. It has been researched as an important ecological marker and as an extremophile plant for studies on stress tolerance. Despite its importance, little genomic information is available for D. antarctica. Here, we report the complete chloroplast genome, transcriptome profiles of the coding/noncoding genes, and the posttranscriptional processing by RNA editing in the chloroplast system.

Results: The complete chloroplast genome of D. antarctica is 135,362 bp in length with a typical quadripartite structure, including the large (LSC: 79,881 bp) and small (SSC: 12,519 bp) single-copy regions, separated by a pair of identical inverted repeats (IR: 21,481 bp). It contains 114 unique genes, including 81 unique protein-coding genes, 29 tRNA genes, and 4 rRNA genes. Sequence divergence analysis with other plastomes from the BEP clade of the grass family suggests a sister relationship between D. antarctica, Festuca arundinacea and Lolium perenne of the Poeae tribe, based on the whole plastome. In addition, we conducted high-resolution mapping of the chloroplast-derived transcripts. Thus, we created an expression profile for 81 protein-coding genes and identified ndhC, psbJ, rps19, psaJ, and psbA as the most highly expressed chloroplast genes. Small RNA-seq analysis identified 27 small noncoding RNAs of chloroplast origin that were preferentially located near the 5'- or 3'-ends of genes. We also found >30 RNA-editing sites in the D. antarctica chloroplast genome, with a dominance of C-to-U conversions.

Conclusions: We assembled and characterized the complete chloroplast genome sequence of D. antarctica and investigated the features of the plastid transcriptome. These data may contribute to a better understanding of the evolution of D. antarctica within the Poaceae family for use in molecular phylogenetic studies and may also help researchers understand the characteristics of the chloroplast transcriptome.

Figure 1. Map of the chloroplast genome of Deschampsia antarctica.

Genes lying outside of the outer circle are transcribed clockwise, while those inside the circle are transcribed counterclockwise. Genes belonging to different functional groups are color coded. The innermost darker gray corresponds to GC, while the lighter gray corresponds to AT content. IR, inverted repeat; LSC, large single copy region; SSC, small single copy region.

Figure 2. Sequence alignment of eight Poaceae chloroplast genomes.

The top line shows genes in order (transcriptional direction indicated by arrows). The sequence similarity of the aligned regions between Deschampsia antarctica and the other seven species is shown as horizontal bars indicating the average percent identity between 50% and 100% (shown on the y-axis of the graph). The x-axis represents the coordinate in the chloroplast genome. Genome regions are color coded as protein-coding (exon), tRNA or rRNA, and conserved noncoding sequences (CNS).

Figure 3. Comparison of the rbcL-psaI region among eight Poaceae species.

The genes and intergenic regions between rbcL and psaI are indicated by boxes, with the length presented in bp. (Lp: Lolium perenne, Fa: Festuca arundinacea, As: Agrostis stolonifera, Hv: Hordeum vulgare, Ta: Triticum aestivum, Bd: Brachypodium distachyon, Os: Oryza sativa subsp. japonica).

Figure 4. Maximum parsimony analysis of nine Poaceae species based on the whole plastome sequence.

The plastome sequences of Oryza sativa and Bambusa oldhamii were included as outgroup species. The phylogenetic tree was drawn using MEGA5, and bootstrap support was achieved using 1,000 replicates.

Figure 5. Repeat analysis in the Deschampsia antarctica chloroplast genome.

Repeat sequences are compared among eight chloroplast genomes in the Poaceae family. To identify repeat sequences, the REPuter program was used. Repeats with length >20 bp and sequence identity e-value <10⁻³ were selected and categorized to four types based on their orientations (F: forward, P: palindromic, R: reverse).

Figure 6. Distribution of plastid small RNAs in the Deschampsia antarctica chloroplast genome.

The reads from small RNA-seq were divided into two groups according to the length (20–24 nt and >30 nt) and aligned to the D. antarctica chloroplast genome with 100% identity. The distributions of reads were compared between the two groups. In total, 12,753,636 reads were distributed unevenly in the chloroplast genome with high density in the coding regions of psbA and rbcL, intergenic regions, and inverted repeat regions in which most of the rRNA genes exist. The 27 loci enriched with 20–24 nt RNAs are indicated in red, along with the number of reads. The y-axis shows the number of reads (from 0 to 1000).

Figure 7. Relative locations of small RNAs in the Deschampsia antarctica chloroplast genome.

a Relative locations of plastid small RNAs according to the gene structure; b examples of small RNAs located proximal to the 5′ ends of the coding genes; c examples of small RNAs located proximal to the 3′ end of the coding genes.

Figure 8. Sequence conservation among orthologs of plastid small RNAs.

To determine if the identified sRNAs are evolutionarily conserved, Deschampsia antarctica sRNAs were compared with the plastid sRNAs identified in Arabidopsis, rice, or barley , . The sequence aligments of sRNAs which have >90% sequence homology are shown. The multiple sequence alignments were performed with ClustalW2 algorithm (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and visualized with Jalview program . The consensus sequences between ortholog sRNAs were shown at the bottom of each alignment.