Evolutionary and biotechnology implications of plastid genome variation in the inverted-repeat-lacking clade of legumes Jamal Sabir 1 , Erika Schwarz 2 , Nicholas Ellison 3 , Jin Zhang 2 , Nabih A. Baeshen 1 , Muhammed Mutwakil 1 , Robert Jansen 1,2 and Tracey Ruhlman 2, * 1 Department of Biological Sciences, Faculty of Science, King Abdulaziz University (KAU), Jeddah, Saudi Arabia 2 Department of Integrative Biology, University of Texas at Austin, Austin, TX, USA 3 Grasslands Research Centre, Palmerston North, New Zealand Received 11 November 2013; revised 22 January 2014; accepted 24 January 2014. *Correspondence (fax 512-232-9592; email truhlman@austin.utexas.edu) Keywords: Lens culinaris, Vicia faba, gene transfers, acetyl-CoA, carboxylase, licorice. Summary Land plant plastid genomes (plastomes) provide a tractable model for evolutionary study in that they are relatively compact and gene dense. Among the groups that display an appropriate level of variation for structural features, the inverted-repeat-lacking clade (IRLC) of papilionoid legumes presents the potential to advance general understanding of the mechanisms of genomic evolution. Here, are presented six complete plastome sequences from economically important species of the IRLC, a lineage previously represented by only five completed plastomes. A number of characters are compared across the IRLC including gene retention and divergence, synteny, repeat structure and functional gene transfer to the nucleus. The loss of clpP intron 2 was identified in one newly sequenced member of IRLC, Glycyrrhiza glabra. Using deeply sequenced nuclear transcriptomes from two species helped clarify the nature of the functional transfer of accD to the nucleus in Trifolium, which likely occurred in the lineage leading to subgenus Trifolium. Legumes are second only to cereal crops in agricultural importance based on area harvested and total production. Genetic improvement via plastid transformation of IRLC crop species is an appealing proposition. Comparative analyses of intergenic spacer regions emphasize the need for complete genome sequences for developing transformation vectors for plastid genetic engineering of legume crops. Introduction Understanding the evolutionary processes that act on genomes is fundamental to the elucidation of the mechanisms that underlie all of eukaryotic evolution. The study of the plastid genome (plastome) provides a tractable model in that they are relatively compact and gene dense, having been pared down to a limited set of core coding regions through endosymbiotic horizontal transfer (Timmis et al., 2004). As the availability of complete plastome sequences has increased, the notion that plastomes across the plant kingdom lack diversity has diminished. In addition to variation at the nucleotide level, structural variability can provide informative characters for increased phylogenetic resolu- tion at low taxonomic levels and among basal lineages (Graham et al., 2000; Kelchner, 2000). However, microstructural changes (i.e. small inversions in noncoding regions) have been found to be recurrent in angiosperms limiting their power for phylogeny (Kelchner and Wendel, 1996; Kim and Lee, 2005). The frequency of more profound plastome rearrangements such as large inversions, gene and intron gains and losses, expansion, contrac- tion and loss of the inverted repeat (IR) varies across groups allowing a comparative evaluation of genomic evolutionary history. A number of mechanisms may promote gene order changes (reviewed in Wicke et al., 2011; Jansen and Ruhlman, 2012; Ruhlman and Jansen, 2014). Inversions may occur through homologous recombination between members of repeat families. Transfer RNA genes share some sequence similarity and thus may serve as foci for recombination events (e.g. Triticum, Ogihara et al., 1988). In a few cases, sequence motifs associated with repeat elements have been identified at inversion endpoints. While it remains that most plastomes examined have no notable changes in gene order, several lineages have multiple rearrange- ments, including conifers (e.g. Pinus, Wakasugi et al., 1994; Wu et al., 2011) and several angiosperm lineages (Campanulaceae, Haberle et al., 2008; Fabaceae, Cai et al., 2008; Magee et al., 2010; Saski et al., 2005; Geraniaceae, Blazier et al., 2011; Chumley et al., 2006; Guisinger et al., 2011; Weng et al., 2013; Lobeliaceae, Knox and Palmer, 1999; Silene, Ingvarsson et al., 2003; Sloan et al., 2012). Examination of plastome structural rearrangements can sim- plify comparative genomic evaluation relative to nuclear genom- ics. Nonetheless, inferences regarding mechanism will be obscured by the many events that have occurred throughout the history of individual lineages facilitating the need to employ model systems. Among the groups that display an appropriate level of variation for structural features, the Fabaceae (legumes), in particular within the subfamily Papilionoideae (papilionoids), presents the potential to advance general understanding of the Please cite this article as: Sabir J., Schwarz E., Ellison N., Zhang J., Baeshen N.A., Mutwakil M., Jansen R. and Ruhlman T. (2014) Evolutionary and biotechnology implications of plastid genome variation in the inverted-repeat-lacking clade of legumes. Plant Biotechnol. J., doi: 10.1111/pbi.12179 ª 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd 1 Plant Biotechnology Journal (2014), pp. 1–12 doi: 10.1111/pbi.12179