Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2018 PacBio-based mitochondrial genome assembly of Leucaena trichandra (Leguminosae) and an intrageneric assessment of mitochondrial RNA editing Kovar, Lynsey ; Nageswara-Rao, Madhugiri ; Ortega-Rodriguez, Sealtiel ; Dugas, Diana V ; Straub, Shannon ; Cronn, Richard ; Strickler, Susan R ; Hughes, Colin E ; Hanley, Kathryn A ; Rodriguez, Deyra N ; Langhorst, Bradley W ; Dimalanta, Eileen T ; Bailey, C Donovan Abstract: Reconstructions of vascular plant mitochondrial genomes (mt-genomes) are notoriously compli- cated by rampant recombination that has resulted in comparatively few plant mt-genomes being available. The dearth of plant mitochondrial resources has limited our understanding of mt-genome structural di- versity, complex patterns of RNA editing, and the origins of novel mt-genome elements. Here, we use an efficient long read (PacBio) iterative assembly pipeline to generate mt-genome assemblies for Leucaena trichandra (Leguminosae: Caesalpinioideae: mimosoid clade), providing the first assessment of non- papilionoid legume mt-genome content and structure to date. The efficiency of the assembly approach facilitated the exploration of alternative structures that are common place among plant mitochondrial genomes. A compact version (729 kbp) of the recovered assemblies was used to investigate sources of mt-genome size variation among legumes and mt-genome sequence similarity to the legume associated root holoparasite Lophophytum. The genome and an associated suite of transcriptome data from select species of Leucaena permitted an in-depth exploration of RNA editing in a diverse clade of closely related species that includes hybrid lineages. RNA editing in the allotetraploid, Leucaena leucocephala, is con- sistent with co-option of nearly equal maternal and paternal C-to-U edit components, generating novel combinations of RNA edited sites. A preliminary investigation of L. leucocephala C-to-U edit frequencies identified the potential for a hybrid to generate unique pools of alleles from parental variation through edit frequencies shared with one parental lineage, those intermediate between parents, and transgressive patterns. DOI: https://doi.org/10.1093/gbe/evy179 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-156881 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at: Kovar, Lynsey; Nageswara-Rao, Madhugiri; Ortega-Rodriguez, Sealtiel; Dugas, Diana V; Straub, Shan- non; Cronn, Richard; Strickler, Susan R; Hughes, Colin E; Hanley, Kathryn A; Rodriguez, Deyra N; Langhorst, Bradley W; Dimalanta, Eileen T; Bailey, C Donovan (2018). PacBio-based mitochondrial genome assembly of Leucaena trichandra (Leguminosae) and an intrageneric assessment of mitochondrial RNA editing. Genome Biology and Evolution, 10(9):2501-2517. DOI: https://doi.org/10.1093/gbe/evy179 2 GBE PacBio-Based Mitochondrial Genome Assembly of Leucaena trichandra (Leguminosae) and an Intrageneric Assessment of Mitochondrial RNA Editing Downloaded from https://academic.oup.com/gbe/article-abstract/10/9/2501/5076815 by University of Zurich user on 01 October 2018 Lynsey Kovar1, Madhugiri Nageswara-Rao1, Sealtiel Ortega-Rodriguez1,DianaV.Dugas1, Shannon Straub2, Richard Cronn3, Susan R. Strickler4, Colin E. Hughes5, Kathryn A. Hanley1, Deyra N. Rodriguez6,BradleyW. Langhorst6, Eileen T. Dimalanta6, and C. Donovan Bailey1,* 1Department of Biology, New Mexico State University 2Department of Biology, Hobart and William Smith Colleges, Geneva, New York 3Pacific Northwest Research Station, Corvallis, Oregon 4Boyce Thompson Institute, Ithaca, New York 5Department of Systematic & Evolutionary Botany, University of Zurich, Switzerland 6New England Biolabs, Ipswich, Massachusetts *Corresponding author: E-mail: [email protected]. Accepted: August 17, 2018 Data deposition: This project has been deposited at NCBI SRA under the accession PRJNA379675. Abstract Reconstructions of vascular plant mitochondrial genomes (mt-genomes) are notoriously complicated by rampant recombination that has resulted in comparatively few plant mt-genomes being available. The dearth of plant mitochondrial resources has limited our understanding of mt-genome structural diversity, complex patterns of RNA editing, and the origins of novel mt-genome elements. Here, we use an efficient long read (PacBio) iterative assembly pipeline to generate mt-genome assemblies for Leucaena trichandra (Leguminosae: Caesalpinioideae: mimosoid clade), providing the first assessment of non-papilionoid legume mt-genome content and structure to date. The efficiency of the assembly approach facilitated the exploration of alternative structures that are common place among plant mitochondrial genomes. A compact version (729 kbp) of the recovered assemblies was used to investigate sources of mt-genome size variation among legumes and mt-genome sequence similarity to the legume associated root holoparasite Lophophytum. The genome and an associated suite of transcriptome data from select species of Leucaena permitted an in-depth exploration of RNA editing in a diverse clade of closely related species that includes hybrid lineages. RNA editing in the allotetraploid, Leucaena leucocephala, is consistent with co-option of nearly equal maternal and paternal C-to-U edit components, generating novel combinations of RNA edited sites. A preliminary investigation of L. leucoce- phala C-to-U edit frequencies identified the potential for a hybrid to generate unique pools of alleles from parental variation through edit frequencies shared with one parental lineage, those intermediate between parents, and transgressive patterns. Key words: Caesalpinoideae, mimosoid clade, iterative assembler, PacBio, transgressive, hybrid. Introduction the sizes of mt-genomes have been shown to vary over Mitochondrial genomes (mt-genomes) are known to be 100-fold across eukaryotes (Wu, Cuthbert, et al. 2015), highly reduced in terms of genic content when compared wherein the land plants display some of the largest and with their alphaproteobacterial ancestors. The transfer of mi- most variable assemblies (Gualberto and Newton 2017). tochondrial genes to the nucleus is well-known across eukar- Major differences in mt-genome size are attributed to non- yotes, where gene content can vary slightly even among coding sequences, as variation in the total number of genes closely related taxa (KuboandNewton2008). In addition, and their combined lengths account for a limited amount of ß The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/by-nc/4.0/),whichpermitsnon- commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Genome Biol. Evol. 10(9):2501–2517. doi:10.1093/gbe/evy179 Advance Access publication August 20, 2018 2501 Kovar et al. GBE the observed inter genomic variation. Acquisition of noncod- species of angiosperms are needed to better understand the ing sequences is due in part to horizontal DNA transfer be- degree of variation in RNA editing between recently diverged tween other organelles and exogenous sources (Plitmann lineages as well as the impact of interspecific hybridization on 1993; Bergthorsson et al. 2003; Moweretal.2010; Warren patterns of editing. Such variation has been posited to provide et al. 2016), but a large portion of noncoding sequences are potentially important variation contributing to adaptation and not conserved between closely related species. Thus, their population-level divergence (Gommans et al. 2009). origins often remain unclear. The combination of land plant mt-genome structural var- Downloaded from https://academic.oup.com/gbe/article-abstract/10/9/2501/5076815 by University of Zurich user on 01 October 2018 Variation in plant mt-genome structure is also widespread, iation and their RNA-editing features illustrate several impor- even among members of the same species. This is mostly due tant features associated with the complex nature of these to the presence of dispersed repeats that contribute to exten- genomes and their transcriptomes. However, these dynamic sive homologous recombination (Stern and Palmer 1984; genomes remain among the least well surveyed eukaryotic Alverson et al. 2010; Gualberto and Newton 2017). During organellar genomic systems (e.g., Richardson et al. 2013). the early days of mt-genome analysis, plants were believed to Within the economically and ecologically important legume have circular mt-genomes like their metazoan counterparts. plant family there are currently seven mt-genomes available Continued work involving microscopy and related techniques for comparative studies (table 1), but these are all repre- in plant mt-DNAs recovered mostly linear molecules of varying sentatives of subfamily Papilionoideae. Even within this size, with most all much larger than the standard 16 kbp phylogenetically restricted set of representatives, there is metazoan mitochondrial genomes that had been character- considerable mt-genome variation, including a lack of ized at the time (Ward et al. 1981).