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

Abstract

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 hexaploidy event 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.

Fig. 1. Polyploidy events in jasmine and ash since their divergence from grapevine.

a Macrosynteny on a chromosome-scale between grapevine (Vitis vinifera) and jasmine (Jasminum sambac) and black ash (Fraxinus nigra). Numbers below chromosomes indicate chromosome number. Syntenic blocks between Vitis chromosome 3, Jasminum, and Fraxinus are connected by light blue lines. Syntenic blocks between Vitis chromosome 16, Jasminum, and Fraxinus are connected by red lines. Fractionation bias between syntenic blocks of Vitis chromosome 19 and F. americana (b), F. americana (c), and F. pennsylvanica (d) reveals 3 pairs of subgenomes (for six subgenomes total), one pair of which retaining far fewer syntenic genes since the ancient hexaploidy at the base of Oleaceae. The Y-axis represents the percent of gene retention of each Fraxinus syntenic block compared with windows of genes on Vitis chromosome 19. The X-axis represents 100-gene windows along Vitis chromosome 19. Line color corresponds with Fraxinus chromosome numbers in the key to the right of (d).

Fig. 2. Pairwise Sequentially Markovian Coalescent with R (PSMCR) paleodemographic analyses for our long read Fraxinus species and the short read F. pennsylvanica reference assembly.

a PSMCR for our three long-read Fraxinus assemblies. b PSMCR for our long-read F. pennsylvanica assembly and the short read F. pennsylvanica assembly. Demographic curve colors represent the following genome assemblies: blue: F. americana; green: F. nigra; pink: F. pennsylvanica; gold: short-read F. pennsylvanica. PSMCR plots generated by mapping F. americana, F. nigra, and F. pennsylvanica ONT long-reads against their own ONT long-read assemblies. All curves assume a generation time of 15 years. Mutation rates for F. americana and F. nigra curves have been adjusted in order to correct for differences in segregating sites and genome size. Mutation rates used were 6.76 × 10⁻⁹, 4.60 × 10⁻⁹, 7.77 × 10⁻⁹, and 2.51 × 10⁻⁹ nucleotide generation^-1 for F. americana, F. nigra, the long-read F. pennsylvanica, and the short-read F. pennsylvanica respectively. The bcftools multiallelic and rare-variant calling model was used and maxt was set to 10 for PSMCR. The thin black arrow points to a pronounced Ne hump in the long-read F. pennsylvanica’s demographic curve.

Fig. 3. Polyploidy events for Oleaceae and relatives.

a OrthoFinder species tree with inferred gene duplications and associated polyploidy events. Values at nodes and terminal branches are the numbers of gene duplication events with at least 50% of the descendant species maintaining both copies of the duplicated genes. Branch lengths represent average substitutions per site among orthogroup input trees. Red-orange triangles represent known or proposed whole genome “triplication” (hexaploidy) events, with the triangle shared by all members of the tree being the ancient gamma hexaploidy event. Purple squares represent known or proposed whole genome duplication (WGD) events. The positioning of hexaploidy and WGD events along branches is arbitrary and does not represent the relative timing of those events. The Lamiales order, Oleaceae family, and Oleeae tribe are highlighted and labeled. Branch support values are displayed in Fig. S13a; these are derived from the proportion of species trees derived from single-locus gene trees supporting each bipartition. b Positioning of Oleaceae species splits relative to polyploidy events in Fraxinus americana. Syntenic (anchor) Ks parolog pair distribution for F. americana is shown in gray. Lognormal mixture model clustering of the median Ks values of the syntenic paralog pairs indicate two putative polyploidy events represented with blue and red curves. Vertical dashed lines labeled a and b denote the modes of these curves and represent the timing of these events. Vertical lines labeled with 1–6 are rate-adjusted modal estimates of one-to-one ortholog Ks distributions between F. americana and other species, representing speciation events. Phylogenetic tree branch lengths represent Ks distances estimated from ortholog Ks distributions, with short branches unlabeled. Dashed branches have lengths that cannot be computed by the software. Manually superimposed labels match with the Ks plot and represent F. americana polyploidy events and divergence with other lineages on the tree. OrthoFinder tip labels are as follows: (Lamiales, Oleaceae) F. pennsylvanica: Fraxinus pennsylvanica; F. pennsylvanica (v1.4.1): F. pennsylvanica (Huff et al.); F. americana: Fraxinus americana; F. americana (v0.2.1): Fraxinus americana (Huff et al.); F. nigra: Fraxinus nigra; F. nigra (v0.2.1): Fraxinus nigra (Huff et al.); O. fragrans: Osmanthus fragrans; O. europaea: Olea europaea; S. oblata: Syringia oblata; F. suspensa: Forsythia suspensa; J. sambac: Jasminum sambac; (other Lamiales) C. americana: Callicarpa americana; T. grandis: Tectona grandis; (Gentianales) C. humblotiana: Coffea humblotiana; O. pumila: Ophiorrhiza pumila; (Rosids) T. cacao: Theobroma cacao; P. tomentosa: Populus tomentosa; P. avium: Prunus avium; B. rapa: Brassica rapa; A. thaliana: Arabidopsis thaliana; V. vinifera: Vitis vinifera.