The chloroplast genome sequence of the ornamental plant Petunia hybrida

Abstract

Petunia hybrida is a popular ornamental plant and a model for chloroplast research. A complete P. hybrida chloroplast genome sequence would advance phylogenetic studies in the Solanaceae sub-family Petunioideae and is essential for developing synthetic biology tools to study and manipulate chloroplast function for fundamental and industrial applications. Here we present the 156,575 bp P. hybrida chloroplast genome sequence. It is comprised of 87,083 bp large single copy and 18,630 bp small single copy regions separated by 25,431 bp inverted repeats. Seventy-nine proteins, 30 transfer-RNAs and four ribosomal RNA species are encoded by 113 unique genes. The results of phylogenetic analysis using complete Solanaceous chloroplast genomes are consistent with other data that supports an early split for the P. hybrida lineage away from the branch giving rise to groups containing major crops such as tobacco (Nicotianoideae) and potato (Solanoideae).

Keywords:

• Chloroplast biotechnology
• chloroplast genome
• Petunioideae
• Petunia hybrida
• Solanaceae

Petunia hybrida is the world’s most popular bedding plant (Van der Krol and Immink 2016) and a model for genetic research (Gerats and Vandenbussche 2005). P. hybrida belongs to the Solanaceae, a family with major crops including tobacco (Nicotiana tabacum), tomato (Solanum lycopersicon) and potato (Solanum tuberosum). P. hybrida has a rich history of chloroplast research (Bovenberg et al. 1981; Palmer and Thompson 1982; Cornu and Dulieu 1988). Recently, P. hybrida chloroplast transformants (Zubko et al. 2004) provided the first experimental verification of enhanced containment of chloroplast transgenes in the field (Horn et al. 2017). The P. hybrida sequence reported here represents the first complete chloroplast genome (cp-genome) sequence for a member of the Petunioideae. This facilitates phylogenetic studies of this sub-family (Ando et al. 2005) as well as advancing chloroplast transformation technologies in P. hybrida (Avila and Day 2014).

P. hybrida var. Pink Wave was obtained from Thompson and Morgan, Ipswich, UK (52°02′44.5″N, 1°05′50.0″E) and stored in the Plant Science Laboratories, Manchester University (53°27′51.9″N 2°13′46.0″W). DNA extracted from Percoll-gradient-purified chloroplasts (Primavesi et al. 2008) was sequenced using 300 bp paired end reads (Nextera DNA kit libraries) on the Illumina MiSeq platform (San Diego, USA). De novo assembly of the 156,575 bp cp genome (GenBank Accession MF459662) used a rbcL seed sequence (Aldrich et al. 1986) and NOVOPlasty v 2.5.9 (Dierckxsens et al. 2017). Average coverage was over 10,000 reads (Geneious R11, Biomatters Ltd, Auckland). Annotation used GeSeq (Tillich et al. 2017), CpGAVAS (Liu et al. 2012) followed by manual curation with Vector NTI advance v9 (Invitrogen, Carlsbad).

The 156,575 bp P. hybrida cp genome has the typical inverted repeat (IR) organisation of land plants (Palmer and Thompson 1982) and coding content of Solanaceae cp-genomes (Amiryousefi et al. 2018). Two 25,431 bp IRs separate an 87,083 bp large single copy region from a 18,630 bp small single copy region. It contains 113 unique genes encoding 79 proteins, 30 transfer-RNAs and 4 ribosomal RNA (rRNA) genes. The rps12 gene is trans-spliced and the psbL and ndhD genes contain ACG start codons consistent with RNA editing.

Complete cp-genomes were aligned using MAFFT (Katoh and Standley 2013) in Geneious R11 after removing one copy of the large inverted repeat. Geneious R11 tree builder used neighbour joining (Tamura and Nei 1993) and Davidia involucrata as the outgroup. The resulting tree (Figure 1) shows the Petunioideae branching away from the group that contains the Nicotianodeae and Solanoideae. This is consistent with the Petunia lineage splitting from the branch giving rise to the Nicotianoideae and Solanoideae about 30 million years ago (Bombarely et al. 2016). High conservation of P. hybrida and N. tabacum cp-genomes (98% base identity) is consistent with successful transplantation of P. hybrida chloroplasts into N. tabacum (Glimelius and Bonnett 1986) and the use of N. tabacum chloroplast sequences to target integration of transgenes in P. hybrida chloroplasts (Zubko et al. 2004). The P. hybrida cp-genome sequence provides a template for editing the P. hybrida cp-genome by homologous recombination (Avila et al. 2016).

Figure 1. Neighbour joining consensus tree of indicated chloroplast genomes. Bootstrap values (1000 replicates) are indicated at nodes. Distances shown for truncated branches. The position of P. hybrida MF459662 (this work) is arrowed. Sequences retrieved from GenBank. Scale bar: substitutions per site.

Acknowledgements

We thank the Genomics Technologies Next Generation Sequence Facility and Richard Leong in research IT for their support, and Genbank for sequences used in the Phylogenetic Tree.

Disclosure statement

The authors alone are responsible for the content and writing of the article. None of the authors report any conflict of interest.

Additional information

Funding

The work was supported by The University of Manchester.