Evolutionary Stasis in Cycad Plastomes and the First Case of Plastome GC-Biased Gene Conversion

GBE Evolutionary Stasis in Cycad Plastomes and the First Case of Plastome GC-Biased Gene Conversion Chung-Shien Wu and Shu-Miaw Chaw* Biodiversity Research Center, Academia Sinica, Taipei, Taiwan *Corresponding author: E-mail: . Data deposition: This project has been deposited at NCBI and DDBJ under the accessions NC_026036, LC049068, LC049070, LC049336, LC049207, LC049069, LC049067, and LC040885. Accepted: June 22, 2015 Abstract Key words: plastome, biased mutation, GC-biased gene conversion, cycad, gymnosperm. Introduction Chloroplasts are photosynthetic plastids (hereafter abbreviated as plastids) and contain their own genomes, called plastomes. During photosynthesis, an excess of solar energy in plastids leads to increased reactive oxygen species (ROS) that can cause DNA damage in plastomes (Kumar et al. 2014). Given that plastomes are constantly exposed to the mutagenic agents of ROS, efficient DNA repair systems are required to prevent plastomes from dysfunction. Moreover, the uniparental, typically maternal, inheritance of plastomes lacks sexual recombination to eliminate deleterious mutations. As a result, plastomes are expected to accumulate mutations over time because of Muller’s ratchet (Muller 1964). In leaf cells, a plastid can contain approximately 1,000 copies of plastomes (Bendich 1987). The nature of multiple plastomes allows for highly efficient gene conversion in asexual genetic systems, correcting mutations and reducing the mutational load of plastomes (Khakhlova and Bock 2006). In plastids, gene conversion takes place in recombination- dependent replications and is able to repairs broken replication forks and maintains plastome stability (Maréchal and Brisson 2010). To date, direct measurements of gene conversion events are only known for the start codons of ycf1 and ycf2 genes in transgenic tobacco plastids, in which AT-biased gene conversion was suggested (Khakhlova and Bock 2006). However, this AT-biased gene conversion in the tobacco plastome was considered exceptional, given that biased gene conversion generally favors incorporation of GC bases in most genomes studied (Smith 2012). Plastomes of seed plants usually contain a pair of inverted repeats (IRs), except for a few lineages, such as legumes and conifers, where one copy of IRs has been completely lost or extremely reduced (e.g., Perry et al. 2002; Cai et al. 2008; Lin et al. 2010; Wu et al. 2011; Guo et al. 2014; Hsu et al. 2014; Wu and Chaw 2014). In angiosperm plastomes, genes located in IRs feature relatively slower substitution rates than those in the single-copy (SC) regions (Wolfe et al. 1987; Maier et al. 1995). These dissimilar substitution rates might result from ß The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact 2000 Genome Biol. Evol. 7(7):2000–2009. doi:10.1093/gbe/evv125 Advance Access publication June 27, 2015 In angiosperms, gene conversion has been known to reduce the mutational load of plastid genomes (the plastomes). Particularly, more frequent gene conversions in inverted repeat (IR) than in single copy (SC) regions result in contrasting substitution rates between these two regions. However, little has been known about the effect of gene conversion in the evolution of gymnosperm plastomes. Cycads (Cycadophyta) are the second largest gymnosperm group. Evolutionary study of their plastomes is limited to the basal cycad genus, Cycas. In this study, we addressed three questions. 1) Do the plastomes of other cycad genera evolve slowly as previously observed in the plastome of Cycas taitungensis? 2) Do substitution rates differ between their SC and IR regions? And 3) Does gene conversion occur in the cycad plastomes? If yes, is it AT-biased or GC-biased? Plastomes of eight species from other eight genera of cycads were sequenced. These plastomes are highly conserved in genome organization. Excluding ginkgo, cycad plastomes have significantly lower synonymous and nonsynonymous substitution rates than other gymnosperms, reflecting their evolutionary stasis in nucleotide mutations. In the IRs of cycad plastomes, the reduced substitution rates and GC-biased mutations are associated with a GC-biased gene conversion (gBGC) mechanism. Further investigations suggest that in cycads, gBGC is able to rectify plastome-wide mutations. Therefore, this study is the first to uncover the plastomic gBGC in seed plants. We also propose a gBGC model to interpret the dissimilar evolutionary patterns as well as the compositionally biased mutations in the SC and IR regions of cycad plastomes. GBE Evolutionary Stasis in Cycad Plastomes genera were also compared. We reasoned that investigating gene conversion by the use of noncoding loci can avoid the effects of selection. Meanwhile, biased mutations of these two regions were evaluated on the basis of the estimated equilibrium GC content (GCeq). This study is the first to uncover the plastomic GC-biased gene conversion (gBGC) in seed plants. In order to explain our novel finding, we proposed a model to link the gBGC mechanism and the evolution of plastomes in cycads. Materials and Methods DNA Extraction and Plastome Sequencing Leaves were harvested from individuals of the eight sampled cycads (supplementary table S1, Supplementary Material online) growing in the greenhouse at Academia Sinica (Taipei). For each sampled species, DNA was extracted using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Extracted DNA was sequenced at Yourgene Bioscience (New Taipei City, Taiwan) using an Illumina GAII sequencer. Sequencing depth was 2.5 GB of 90-bp paired-end reads for each species. Plastome Assembly and Annotation The raw sequencing reads were quality-trimmed and de novoassembled using CLC Genomics Workbench v5.5.1 software (CLC Bio, Aarhus, Denmark). Contigs with length < 1 kb and sequence coverage < 50  were discarded. The remaining contigs were analyzed by a BLAST search against the plastome of Cycas taitungensis (Wu et al. 2007). Contigs that matched the reference plastomic sequences with E-value < 10 10 were designated as plastomic contigs. DNA fragments between plastomic contigs were obtained using the Taq 2X Master Mix Red PCR kits (Ampliqon, Copenhagen, Denmark) with our species-specific primers. Sequences of PCR amplicons were obtained using an ABI 3730 DNA Analyzer (Life Technologies, Taipei, Taiwan). Plastome annotation was performed using DOGMA (Wyman et al. 2004) and tRNAscan-SE 1.21 (Schattner et al. 2005). The annotated genes were confirmed by their alignment with their orthologous genes from published gymnosperm plastomes (...truncated)