Efficient hybrid strategies for assembling the plastome, mitochondriome, and large nuclear genome of diploid Ranunculus cassubicifolius (Ranunculaceae)

Currently, we lack efficient strategies - in terms of laboratory work and cost, as well as assembly quality - to reliably reconstruct large and complex genomes from non-model plants. This often impairs the study of evolutionary intricate groups characterized by hybridization, polyploidy, and/or apomixis. The large Ranunculus auricomus polyploid complex is an angiosperm model system for apomixis, evolution, and biogeography research. However, neither plastid, mitochondrial, nor high-quality nuclear genomes are available, and a genus-wide draft genome has yet to be assembled, limiting phylogenomic, ecological, and taxonomic analyses thus far. We tested different Illumina (short, highly accurate reads), Oxford Nanopore Technology (ONT; long, less accurate reads), and Illumina-ONT-hybrid assembly strategies using the diploid species R. cassubicifolius , a sexual diploid progenitor of the R. auricomus complex, and selected the best assemblies in terms of completeness, contiguity, and BUSCO quality scores. We first assembled the entire plastome (156 Mbp; 78 protein-coding genes, 30 tRNAs, and 4 rRNAs) and mitochondriome (1,260 Mbp; 39 protein-coding genes, 13 tRNAs, 3 rRNAs), using an Illumina- and hybrid-based strategy, respectively. The best out of 11 strategies used raw Nanopore reads assembled and polished by filtered Nanopore and Illumina reads accompanied by genome annotation yielded a 3.45 Gbp high-quality genome with (>99% completeness), 97% single-copy and duplicated complete BUSCO genes found, median contig length (N50) of 104 Mbp, and 39,544 predicted genes. The genomic information presented herein helps to improve phylogenomic analyses in this species complex, and will enable improved functional, biogeographic, and population genomic analyses for the R. auricomus complex, the genus, and beyond in Ranunculaceae in the future. Significance Statement The Ranunculus auricomus complex is a model system in flowering plant apomixis, evolution, and biogeography research, but thus far no genus-wide complete genome exists. Using Illumina and/or Nanopore data, we assembled the plastome, mitochondriome, and high-quality diploid genome. We created efficient assembly strategies, which are applicable to other non-model systems and useful for improving phylogenomic analyses, enabling better functional (e.g., apomixis), biogeographic, and population genomic analyses at the complex, but also at the genus and family level. ### Competing Interest Statement The authors have declared no competing interest.