Strategies for complete plastid genome sequencing

Abstract

Plastid sequencing is an essential tool in the study of plant evolution. This high-copy organelle is one of the most technically accessible regions of the genome, and its sequence conservation makes it a valuable region for comparative genome evolution, phylogenetic analysis and population studies. Here, we discuss recent innovations and approaches for de novo plastid assembly that harness genomic tools. We focus on technical developments including low-cost sequence library preparation approaches for genome skimming, enrichment via hybrid baits and methylation-sensitive capture, sequence platforms with higher read outputs and longer read lengths, and automated tools for assembly. These developments allow for a much more streamlined assembly than via conventional short-range PCR. Although newer methods make complete plastid sequencing possible for any land plant or green alga, there are still challenges for producing finished plastomes particularly from herbarium material or from structurally divergent plastids such as those of parasitic plants.

Keywords: chloroplast; genomic; next-generation sequencing; plant evolution; plastid.

Figure 1

Structural diversity in plastids. (a) Nicotiana has a typical land plant plastid of 156 Kb and tripartite structure, (b) the mycoheterotrophic nonphotosynthetic orchid Epipogium roseum has the smallest plastid genome to date at 19 Kb, and with greatly reduced gene content, (c) green algae, here represented by Chlamydomonas reinhardtii, demonstrate dramatic plastid genome variation and include species with giant plastomes over 500 Kb in length. [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 2

Representation of plastid reads in gDNA sequence libraries of species with different genome sizes. The graph shows the proportion of sequence reads from a phylogenetically diverse range of 27 green plant species that map to a reference database of 100 plant plastomes. Grey shading indicates the 95% confidence interval of the fitted line. Full details are given in Appendix S1 (Supporting information). [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 3

GC by coverage plot of a draft genome assembly. Short reads of the monkey flower Mimulus guttatus (population IM, SRR010318) were assembled with SPAdes genome assembler (v3.7.1) and annotated according to matches to the published Mimulus plastome (Vallejo‐Marín et al. 2016).