Deep sequencing of Ptilidium (Ptilidiaceae) suggests evolutionary stasis in liverwort plastid genome structure

摘要

Background and aims - Organellar genome sampling is patchy for non-vascular groups, with the earliest land plants poorly represented; currently only two liverworts, two mosses and one hornwort have sequenced, annotated plastid genomes. This is in partdue to methodological difficulties that have hampered attempts to generate plastid genome data from liverworts. In this paper we present a method that overcomes some of the inherent difficulties by circumventing the need for plastid enrichment, but thatalso provides other valuable information from nuclear and mitochondrial regions including sequences from loci that may be phylogenetically useful, and potential population-level markers such as single nucleotide polymorphisms and microsatellites. Methods - A shotgun library developed from total genomic liverwort DNA was subjected to high-throughput pyrosequencing using the Roche 454 platform. Plastid reads were bioinformatically identified, assembled and annotated. To maximize usage of the vast numberof reads generated using 454 sequencing technology, combined nuclear, mitochondrial and plastid contigs were also screened for microsatellite markers, and presumed nuclear contigs were scanned for protein domains. Key Results - This is the first plastidgenome to be assembled for a leafy liverwort (i.e. Ptilidium) and also the first such genome to be sequenced using next generation technology for any bryophyte. The 119,007 base long plastid genome of Ptilidium pulcherrimum contains 88 protein-coding genes, four rRNAs and thirty tRNAs. The Inverted Repeat occurs between trnV-GAC and traN-GUU. Functional copies of the two plastid-encoded sulphate import protein-coding genes (cysA and cysT) are absent, although pseudogenes are present in the same positionthat the functional genes occupy in Marchantia. Microsatellites: 197 novel potential primer pairs for P. pulcherrimum were found. Presumed nuclear Ptilidium contigs gave multiple hits to Class I transposable elements. Conclusions - The arrangement of genes is identical to the plastid of the complex thalloid liverwort Marchantia, suggesting that structural rearrangements are rare in hepatics. This dataset represents a valuable resource for novel phylogenetic and population level marker design in hepatics.