Assembly and Annotation of Red Spruce (Picea rubens) Chloroplast Genome, Identification of Simple Sequence Repeats, and Phylogenetic Analysis in Picea

Abstract

We have sequenced the chloroplast genome of red spruce (Picea rubens) for the first time using the single-end, short-reads (44 bp) Illumina sequences, assembled and functionally annotated it, and identified simple sequence repeats (SSRs). The contigs were assembled using SOAPdenovo2 following the retrieval of chloroplast genome sequences using the black spruce (Picea mariana) chloroplast genome as the reference. The assembled genome length was 122,115 bp (gaps included). Comparatively, the P. rubens chloroplast genome reported here may be considered a near-complete draft. Global genome alignment and phylogenetic analysis based on the whole chloroplast genome sequences of Picea rubens and 10 other Picea species revealed high sequence synteny and conservation among 11 Picea species and phylogenetic relationships consistent with their known classical interrelationships and published molecular phylogeny. The P. rubens chloroplast genome sequence showed the highest similarity with that of P. mariana and the lowest with that of P. sitchensis. We have annotated 107 genes including 69 protein-coding genes, 28 tRNAs, 4 rRNAs, few pseudogenes, identified 42 SSRs, and successfully designed primers for 26 SSRs. Mononucleotide A/T repeats were the most common followed by dinucleotide AT repeats. A similar pattern of microsatellite repeats occurrence was found in the chloroplast genomes of 11 Picea species.

Keywords: Pinaceae; comparative genome analysis; conifers; genome assembly and annotation; genome sequences; microsatellites; organellar genome; phylogeny; plastid genome.

Figure 1

Near-complete chloroplast genome of Picea rubens, annotated using GeSeq v1.79 and organized using OGDRAW version 1.3.1 [(*) asterisk represents intron-containing genes in organelle genomes]. The ndh genes in the figure are truncated pseudogenes.

Figure 2

mVISTA-based visual representation of the aligned genomes of the 11 Picea species using annotation of the P. sitchensis chloroplast genome as the reference.

Figure 3

(a) Patterns of the number of microsatellites repeats in the chloroplast genomes of Picea sitchensis, P. engelmannii, P. glauca, P. chihuahuana, P. neoveitchii, P. abies, P. asperata, P. crassifolia, P. jezoensis, P. mariana, and P. rubens. (b) Repeat motif distribution in the chloroplast genomes of Picea sitchensis, P. engelmannii, P. glauca, P. chihuahuana, P. neoveitchii, P. abies, P. asperata, P. crassifolia, P. jezoensis, P. mariana, and P. rubens.

Figure 3

(a) Patterns of the number of microsatellites repeats in the chloroplast genomes of Picea sitchensis, P. engelmannii, P. glauca, P. chihuahuana, P. neoveitchii, P. abies, P. asperata, P. crassifolia, P. jezoensis, P. mariana, and P. rubens. (b) Repeat motif distribution in the chloroplast genomes of Picea sitchensis, P. engelmannii, P. glauca, P. chihuahuana, P. neoveitchii, P. abies, P. asperata, P. crassifolia, P. jezoensis, P. mariana, and P. rubens.

Figure 4

A rooted neighbor-joining phylogenetic tree of 11 Picea species using Pinus thunbergii as the outgroup, based on their whole chloroplast genome sequences. The numbers on the nodes are the percent support from 1000 bootstraps.