High quality genomes produced from single MinION flow cells clarify polyploid and demographic histories of critically endangered Fraxinus (ash) species

With populations of threatened and endangered species declining worldwide, efforts are being made to generate high-quality genomic records of these species before they are lost forever. Here, we demonstrate that data from single Oxford Nanopore Technologies (ONT) MinION flow cells can, even in the absence of highly accurate short DNA-read polishing, produce high-quality de novo plant genome assemblies adequate for downstream analyses, such as synteny and ploidy evaluations, paleodemographic analyses, and phylogenomics. This study focuses on three North American ash tree species in the genus Fraxinus (Oleaceae) that were recently added to the International Union for Conservation of Nature (IUCN) Red List as critically endangered. Our results support a whole genome triplication at the base of the Oleaceae as well as a subsequent whole genome duplication shared by Syringa, Osmanthus, Olea, and Fraxinus. Finally, we demonstrate the use of ONT long-read sequencing data to reveal patterns in demographic history.

Methods
Fraxinus pennsylvanica vouchers and material for DNA extraction were collected from Point Gratiot Park in Dunkirk, New York, USA. F. americana and F. nigra vouchers and material for DNA extraction were collected from the College Lodge Forest in Brocton, New York, USA. Samples were sequenced on an Oxford Nanopore Technologies’ GridION instrument utilizing MinION flowcells (version R9.4). Genome assemblies were generated using Flye and reduced to haploid assemblies using Purge Haplotigs. Genomes were additionally scaffolded using RagTag and the recent Fraxinus pennsylvanica reference assembly as a scaffolding reference. All annotations were generated with GeMoMa.