DOI: 10.5061/dryad.7sqv9s4xh
title: High quality genomes produced from single MinION flow cells clarify polyploid and demographic histories of critically endangered Fraxinus (ash) species

description: 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.
description: <em>Fraxinus pennsylvanica</em> vouchers and material
 for DNA extraction were collected from Point Gratiot Park in Dunkirk, New
 York, USA. <em>F. americana</em> and <em>F.
 nigra</em> 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 <em>Fraxinus
 pennsylvanica</em> reference assembly as a scaffolding reference.
 All annotations were generated with GeMoMa. 
description: Guppy v5.0.11 Nanoplot
 v1.38.0 Nanostat v1.5.0 Flye
 v2.8.3 Flye v2.9 QUAST
 v5.0.2 BUSCO v5.4.4 GeMoMa
 v1.9 AGAT v1.0.0 Evidential
 Gene RepeatModeler v2.0.1
 RepeatMasker v4.0.1 RagTag
 v2.1.0 MCScan (https://github.com/tanghaibao/jcvi/wiki/MCscan-(Python-version)) HapPy (https://github.com/AntoineHo/HapPy) SAMtools v1.14 Purge Haplotigs v1.1.1 Minimap2 v2.20 bcftools version 1.14 PSMCR (https://github.com/emmanuelparadis/psmcr) Orthofinder v2.5.4 Ksrates v1.1.3
description: # High quality genomes produced from single MinION flow cells clarify
 polyploid and demographic histories of critically
 endangered *Fraxinus* (ash) species --- The following data are genome
 assemblies and annotations generated in this study. In the case of v0.2.1,
 1.4.1, and 1.1, the genomes were previously published and we generated new
 annotations using GeMoMa. ## Description of the data and file structure
 Figure_1_Data.zip #contains data for Figure 1 \|-- Figure_1a #MCScan files
 for generating the karyotype plot in Figure 1a \| |-- *.bck #file
 generated by MCScan for pairwise syneny search \| |-- *.bed #CDS sequence
 coordinates on assembly \| |-- *.cds #CDS sequence fasta file \| |-- *.des
 #file generated by MCScan for pairwise syneny search \| |-- *.prj #file
 generated by MCScan for pairwise syneny search \| |-- *.sds #file
 generated by MCScan for pairwise syneny search \| |-- *.ssp #file
 generated by MCScan for pairwise syneny search \| |-- *.suf #file
 generated by MCScan for pairwise syneny search \| |-- *.tis #file
 generated by MCScan for pairwise syneny search \| |-- *.anchors #seed
 synteny blocks \| |-- *.anchors.new #genes within each synteny block in
 *.anchors.simple file \| |-- *.anchors.simple #more succinct and editable
 form of the .anchors file \| |-- {sp1}.{sp2}.depth.pdf #syntenic gene
 depth between sp1 and sp2 \| |-- .last #raw LAST output \| |--
 .last.filtered #filtered LAST output \| |-- .lifted.anchors #additional
 anchors to form the final synteny blocks \| |-- {sp1}.{sp2}.pdf #syntenic
 dot plot between sp1 and sp2 \| |-- karyotype..pdf #karyotype plot \| |--
 layout. #figure layout file for karyotype plot \| |-- seqids. #sequence
 identification file for karyotype plot \|-- *.xlsx #fractionation bias
 plot source data for each Fraxinus assembly against Vitis vinifera
 Figure_2_Data.zip #RDS data for recreating PSMCR plot (The following
 variables derive from manually set parameters: niters = the number of
 iterations; n = number of time intervals; maxT = the largest possible
 value for time to the most recent common ancestor. The following variables
 are automatically calculated and are not meant to be manually
 utilized/accessed: n_free_lambdas = Number of free parameters; logLik =
 log-likelihood; lk = log-likelihood; EMQ = EM-Q scores before and after;
 RI = relative information; Cpi = normalization factor; theta0 = per-site
 mutation rate; rho0 = per-site recombination rate; t_k = time scaled to
 generations; lambda_k = relative population size. RDS files contain 100
 bootstrap replicates) Figure 3_S13_Data.zip #contains data for for Figure
 3 and Figure S13 \|-- Figure_3a_S13a #contains full orthofinder output for
 Figure 3a and Figure S13a \| |-- Citation.txt #citation info \| |--
 Comparative_Genomics_Statistics.tar.gz #run statistics \| |--
 Gene_Duplication_Events.tar.gz #summary of gene duplication events \| |--
 Gene_Trees.tar.gz #original gene trees for each orthogroup \| |-- Log.txt
 #run log \| |-- Orthogroup_Sequences.tar.gz #sequences for the genes in
 each orthogroup \| |-- Orthogroups.tar.gz #info on each orthogroup \| |--
 Orthologues.tar.gz #orthologues for every Sp1 vs Sp2 comparison \| |--
 Phylogenetic_Hierarchical_Orthogroups.tar.gz #genes represented at each
 node of the species/gene duplication tree \| |--
 Phylogenetically_Misplaced_Genes.tar.gz #genes that appear to be out of
 place in the gene tree \| |-- Putative_Xenologs.tar.gz #putative xenologs
 for each species \| |-- Resolved_Gene_Trees.tar.gz #gene trees with nodes
 labelled \| |-- Single_Copy_Orthologue_Sequences.tar.gz #orthogroups that
 contain exactly one gene sequence per species \| |-- Species_Tree.tar.gz
 #txt files for OrthoFinder species tree \|-- Figure_3b #contains Ksrates
 output data for Figure 3b \| |-- config_Fa.txt #configuration file for ks
 rate correction using Fraxinus americana \| |--
 ortholog_distributions.tar.gz #orthologous gene data between each pair of
 species (Fa: Fraxinus_americana.fasta, Fp: Fraxinus_pennsylvanica.fasta,
 Fn: Fraxinus_nigra.fasta, Oe: Olea_europaea.fasta, Of:
 Osmanthus_fragrans.fasta, So: Syringa_oblata.fasta, Fs:
 Forsythia_suspensa.fasta, Js: Jasminum_sambac.fasta, Ca:
 Callicarpa_americana.fasta) \| |-- ortholog_ks_list_db.tsv #file storing
 the ortholog KS value lists between species pairs \| |--
 ortholog_peak_db.tsv #file storing the KS mode estimate between species
 pairs \| |-- paralog_distributions #files generated during the wgd paralog
 KS estimation run for the focal species \| |-- rate_adjustment #collects
 the output files of the substitution rate-adjustment for each species
 relative to the focal species \|-- Figure_13b #single copy orthologue
 sequences tree for Figure 13b \| |-- *.log #ASTRAL log files\ \| |--
 *_cat_t1.tre #ASTRAL tree with quartet branch support values \| |--
 *_cat_t3.tre #ASTRAL tree with local posterior probability branch support
 values \| |-- *_cat.tre #concatenated single copy orthologue sequences
 trees \| |-- *_tree.txt #individual single copy orthologue sequences trees
 Figure_S1_Data.zip #NanoPlot reports for Oxford Nanopore Technologies
 reads \|-- Fraxinus_americana.summaryplots #NanoPlot output for *Fraxinus
 americana* \|-- Fraxinus_nigra.summaryplots #NanoPlot output for *Fraxinus
 nigra* \|-- Fraxinus_pennsylvanica.summaryplots #NanoPlot output for
 *Fraxinus pennsylvanica* Figure_S2_Data.zip #read coverage histograms for
 primary (v0; a,c,e) and purged (v1; b,d,f) assemblies \|-- * #directories
 for assemblies \| |-- *.aln_stats.plot.png #read coverage histogram figure
 \| |-- *.aln_stats.stats.txt #haploidy stats \| |-- *.aln.sorted.bam.hist
 #histogram source data Figure_S3_Data.zip #synonymous substitution (ks)
 data for primary assemblies (v0) Figure_S4_Data.zip #ks data for purged
 assemblies (v1) and Huff et al. (2022) assemblies (v0.2.1)
 Figure_S5_Data.zip #ks data for *Osmanthus fragrans* (v1.1) and *Olea
 europaea* (v1.1) Figure_S6_Data.zip #MCScan files for generating the
 karyotype plot in Figure S6 \|-- *.bck #file generated by MCScan for
 pairwise syneny search \|-- *.bed #CDS sequence coordinates on assembly
 \|-- *.cds #CDS sequence fasta file \|-- *.des #file generated by MCScan
 for pairwise syneny search \|-- *.prj #file generated by MCScan for
 pairwise syneny search \|-- *.sds #file generated by MCScan for pairwise
 syneny search \|-- *.ssp #file generated by MCScan for pairwise syneny
 search \|-- *.suf #file generated by MCScan for pairwise syneny search
 \|-- *.tis #file generated by MCScan for pairwise syneny search \|--
 *.anchors #seed synteny blocks \|-- *.anchors.new #genes within each
 synteny block in *.anchors.simple file \|-- *.anchors.simple #more
 succinct and editable form of the .anchors file \|-- {sp1}.{sp2}.depth.pdf
 #syntenic gene depth between sp1 and sp2 \|-- .last #raw LAST output \|--
 .last.filtered #filtered LAST output \|-- .lifted.anchors #additional
 anchors to form the final synteny blocks \|-- {sp1}.{sp2}.pdf #syntenic
 dot plot between sp1 and sp2 \|-- karyotype..pdf #karyotype plot \|--
 layout. #figure layout file for karyotype plot \|-- seqids. #sequence
 identification file for karyotype plot Figure_S7_Data.zip #contains data
 for Figure S7 \|-- Figure_S7ad #MCScan files for generating the plots in
 Figure S7a and S7d \| |-- *.bck #file generated by MCScan for pairwise
 syneny search \| |-- *.bed #CDS sequence coordinates on assembly \| |--
 *.cds #CDS sequence fasta file \| |-- *.des #file generated by MCScan for
 pairwise syneny search \| |-- *.prj #file generated by MCScan for pairwise
 syneny search \| |-- *.sds #file generated by MCScan for pairwise syneny
 search \| |-- *.ssp #file generated by MCScan for pairwise syneny search
 \| |-- *.suf #file generated by MCScan for pairwise syneny search \| |--
 *.tis #file generated by MCScan for pairwise syneny search \| |--
 *.anchors #seed synteny blocks \| |-- *.anchors.new #genes within each
 synteny block in *.anchors.simple file \| |-- *.anchors.simple #more
 succinct and editable form of the .anchors file \| |--
 {sp1}.{sp2}.depth.pdf #syntenic gene depth between sp1 and sp2 \| |--
 *.last #raw LAST output \| |-- *.last.filtered #filtered LAST output \|
 |-- .lifted.anchors #additional anchors to form the final synteny blocks
 \| |-- {sp1}.{sp2}.pdf #syntenic dot plot between sp1 and sp2 \| |--
 karyotype..pdf #karyotype plot \|-- *.xlsx #fractionation bias plot source
 data for grape against jasmine Figure_S8_Data.zip #MCScan files for
 generating Figure S8 \|-- *.bck #file generated by MCScan for pairwise
 syneny search \|-- *.bed #CDS sequence coordinates on assembly \|-- *.cds
 #CDS sequence fasta file \|-- *.des #file generated by MCScan for pairwise
 syneny search \|-- *.prj #file generated by MCScan for pairwise syneny
 search \|-- *.sds #file generated by MCScan for pairwise syneny search
 \|-- *.ssp #file generated by MCScan for pairwise syneny search \|-- *.suf
 #file generated by MCScan for pairwise syneny search \|-- .tis #file
 generated by MCScan for pairwise syneny search \|-- .anchors #seed synteny
 blocks \|-- *.anchors.new #genes within each synteny block in
 *.anchors.simple file \|-- *.anchors.simple #more succinct and editable
 form of the .anchors file \|-- {sp1}.{sp2}.depth.pdf #syntenic gene depth
 between sp1 and sp2 \|-- .last #raw LAST output \|-- .last.filtered
 #filtered LAST output \|-- .lifted.anchors #additional anchors to form the
 final synteny blocks \|-- {sp1}.{sp2}.pdf #syntenic dot plot between sp1
 and sp2 \|-- karyotype..pdf #karyotype plot \|-- layout. #figure layout
 file for karyotype plot \|-- seqids. #sequence identification file for
 karyotype plot Figure_S9_Data.zip #contains data for Figure S9 \|--
 Figure_S9a #MCScan files for generating the plots in Figure S9a \| \|--
 \*.bck #file generated by MCScan for pairwise syneny search \| \|-- \*.bed
 #CDS sequence coordinates on assembly \| \|-- \*.cds #CDS sequence fasta
 file \| \|-- \*.des #file generated by MCScan for pairwise syneny search
 \| \|-- \*.prj #file generated by MCScan for pairwise syneny search \|
 \|-- \*.sds #file generated by MCScan for pairwise syneny search \| \|--
 \*.ssp #file generated by MCScan for pairwise syneny search \| \|-- \*.suf
 #file generated by MCScan for pairwise syneny search \| \|-- \*.tis #file
 generated by MCScan for pairwise syneny search \| \|-- \*.anchors #seed
 synteny blocks \| \|-- {sp1}.{sp2}.depth.pdf #syntenic gene depth between
 sp1 and sp2 \| \|-- \*.last #raw LAST output \| \|-- .last.filtered
 #filtered LAST output \| \|-- .lifted.anchors #additional anchors to form
 the final synteny blocks \| \|-- {sp1}.{sp2}.pdf #syntenic dot plot
 between sp1 and sp2 \|-- \*.xlsx #fractionation bias plot source data for
 *Jasminum sambac* against *Fraxinus americana* Figure_S10_Data.zip #RDS
 data for recreating PSMCR plot (The following variables derive from
 manually set parameters: niters = the number of iterations; n = number of
 time intervals; maxT = the largest possible value for time to the most
 recent common ancestor. The following variables are automatically
 calculated and are not meant to be manually utilized/accessed:
 n_free_lambdas = Number of free parameters; logLik = log-likelihood; lk =
 log-likelihood; EMQ = EM-Q scores before and after; RI = relative
 information; Cpi = normalization factor; theta0 = per-site mutation rate;
 rho0 = per-site recombination rate; t_k = time scaled to generations;
 lambda_k = relative population size. RDS files contain 100 bootstrap
 replicates) Figure_S11_Data.zip #RDS data for recreating PSMCR plot (The
 following variables derive from manually set parameters: niters = the
 number of iterations; n = number of time intervals; maxT = the largest
 possible value for time to the most recent common ancestor. The following
 variables are automatically calculated and are not meant to be manually
 utilized/accessed: n_free_lambdas = Number of free parameters; logLik =
 log-likelihood; lk = log-likelihood; EMQ = EM-Q scores before and after;
 RI = relative information; Cpi = normalization factor; theta0 = per-site
 mutation rate; rho0 = per-site recombination rate; t_k = time scaled to
 generations; lambda_k = relative population size. RDS files contain 100
 bootstrap replicates) Figure_S12_Data.zip #RDS data for recreating PSMCR
 plot (The following variables derive from manually set parameters: niters
 = the number of iterations; n = number of time intervals; maxT = the
 largest possible value for time to the most recent common ancestor. The
 following variables are automatically calculated and are not meant to be
 manually utilized/accessed: n_free_lambdas = Number of free parameters;
 logLik = log-likelihood; lk = log-likelihood; EMQ = EM-Q scores before and
 after; RI = relative information; Cpi = normalization factor; theta0 =
 per-site mutation rate; rho0 = per-site recombination rate; t_k = time
 scaled to generations; lambda_k = relative population size. RDS files
 contain 100 bootstrap replicates) Figure_S14_Data.zip #orthologous ks data
 between each *Fraxinus* assembly (v1) and *Vitis vinifera*
 Figure_S15_Data.zip #orthologous ks data between each *Fraxinus* assembly
 (v1) and the *F. pennsylvanica* reference assembly (v1.4.1)
 Figure_S16_Data.zip #contains data for Figure S16 \|-- \*.gencov #source
 data for read depth histogram plots \|-- \*.png #read depth histograms
 generated from Purge Haplotigs for each primary *Fraxinus* assembly (v0)
 Fraxinus_americana_v0.fasta.gz Fraxinus_americana_v0.gff.gz
 Fraxinus_americana_v0.proteins.fasta.gz
 Fraxinus_americana_v0.repeats.gff.gz Fraxinus_nigra_v0.fasta.gz
 Fraxinus_nigra_v0.gff.gz Fraxinus_nigra_v0.protein.fasta.gz
 Fraxinus_pennsylvanica_v0.fasta.gz Fraxinus_pennsylvanica_v0.gff.gz
 Fraxinus_pennsylvanica_v0.protein.fasta.gz
 Fraxinus_pennsylvanica_v0.repeats.gff.gz Fraxinus_americana_v1.fasta.gz
 Fraxinus_americana_v1.gff.gz Fraxinus_americana_v1.protein.fasta.gz
 Fraxinus_americana_v1.repeats.gff.gz Fraxinus_nigra_v1.fasta.gz
 Fraxinus_nigra_v1.gff.gz Fraxinus_nigra_v1.protein.fasta.gz
 Fraxinus_nigra_v1.repeats.gff.gz Fraxinus_nigra_v0.repeats.gff.gz
 Fraxinus_pennsylvanica_v1.fasta.gz Fraxinus_pennsylvanica_v1.gff.gz
 Fraxinus_pennsylvanica_v1.protein.fasta.gz
 Fraxinus_pennsylvanica_v1.repeats.gff.gz
 Fraxinus_americana_v1Ragtag.cds.fasta.gz
 Fraxinus_americana_v1Ragtag.fasta.gz Fraxinus_americana_v1Ragtag.gff.gz
 Fraxinus_americana_v1Ragtag.longest_iso.gff.gz
 Fraxinus_americana_v1Ragtag.longest_iso.protein.fasta.gz
 Fraxinus_americana_v1Ragtag.protein.fasta.gz
 Fraxinus_nigra_v1Ragtag.cds.fasta.gz Fraxinus_nigra_v1Ragtag.fasta.gz
 Fraxinus_nigra_v1Ragtag.gff.gz Fraxinus_nigra_v1Ragtag.longest_iso.gff.gz
 Fraxinus_nigra_v1Ragtag.longest_iso.protein.fasta.gz
 Fraxinus_nigra_v1Ragtag.protein.fasta.gz
 Fraxinus_pennsylvanica_v1Ragtag.cds.fasta.gz
 Fraxinus_pennsylvanica_v1Ragtag.fasta.gz
 Fraxinus_pennsylvanica_v1Ragtag.gff.gz
 Fraxinus_pennsylvanica_v1Ragtag.longest_iso.gff.gz
 Fraxinus_pennsylvanica_v1Ragtag.longest_iso.protein.fasta.gz
 Fraxinus_pennsylvanica_v1Ragtag.protein.fasta.gz
 Fraxinus_americana_v0.2.1.cds.fasta.gz Fraxinus_americana_v0.2.1.fasta.gz
 Fraxinus_americana_v0.2.1.gff.gz
 Fraxinus_americana_v0.2.1.longest_iso.gff.gz
 Fraxinus_americana_v0.2.1.longest_iso.protein.fasta.gz
 Fraxinus_americana_v0.2.1.protein.fasta.gz
 Fraxinus_americana_v0.2.1.repeats.gff.gz
 Fraxinus_nigra_v0.2.1.cds.fasta.gz Fraxinus_nigra_v0.2.1.fasta.gz
 Fraxinus_nigra_v0.2.1.gff.gz Fraxinus_nigra_v0.2.1.longest_iso.gff.gz
 Fraxinus_nigra_v0.2.1.longest_iso.protein.fasta.gz
 Fraxinus_nigra_v0.2.1.protein.fasta.gz
 Fraxinus_nigra_v0.2.1.repeats.gff.gz
 Fraxinus_pennsylvanica_v1.4.1.cds.fasta.gz
 Fraxinus_pennsylvanica_v1.4.1.fasta.gz
 Fraxinus_pennsylvanica_v1.4.1.gff.gz
 Fraxinus_pennsylvanica_v1.4.1.longest_iso.gff.gz
 Fraxinus_pennsylvanica_v1.4.1.longest_iso.protein.fasta.gz
 Fraxinus_pennsylvanica_v1.4.1.protein.fasta.gz
 Fraxinus_pennsylvanica_v1.4.1.repeats.gff.gz
 Forsythia_suspensa_v1.1.cds.fasta.gz Forsythia_suspensa_v1.1.fasta.gz
 Forsythia_suspensa_v1.1.gff.gz Forsythia_suspensa_v1.1.longest_iso.gff.gz
 Forsythia_suspensa_v1.1.longest_iso.protein.fasta.gz
 Forsythia_suspensa_v1.1.protein.fasta.gz
 Osmanthus_fragrans_v1.1.cds.fasta.gz Osmanthus_fragrans_v1.1.fasta.gz
 Osmanthus_fragrans_v1.1.gff.gz Osmanthus_fragrans_v1.1.longest_iso.gff.gz
 Osmanthus_fragrans_v1.1.longest_iso.protein.fasta.gz
 Osmanthus_fragrans_v1.1.protein.fasta.gz Genome assemblies and annotations
 with version v0, v1, and v1Ragtag were generated in this study. Assemblies
 and annotations with v0 represent primary Flye assemblies. Assemblies and
 annotations with v1 represent haploid Flye assemblies generated using
 Purge Haplotigs. Assemblies and annotations with v1Ragtag represent the v1
 assemblies scaffolded to chromosome-level using RagTag and the Huff et al.
 2021 Fraxinus pennsylvanica assembly as a reference. Genome assemblies
 with versions 0.2.1 and 1.4.1 were not generated in this study. The
 annotations were publicly available, but in a format that couldn't be
 used in our analyses. We annotated these genomes using GeMoMa. Genome
 assemblies with version 1.1 were not generated in this study. New
 annotations were generated using GeMoMa due to the originals not being
 publicly available at the time the analyses were done. ## Sharing/Access
 information Links to other publicly accessible locations of the data: *
 Data was derived from the
 following sources: * Huff, M., Seaman, J., Wu, D., Zhebentyayeva, T.,
 Kelly, L.J., Faridi, N., Nelson, C.D., Cooper, E., Best, T., Steiner, K.
 and Koch, J., 2022. A highquality reference genome for Fraxinus
 pennsylvanica for ash species restoration and research. Molecular ecology
 resources, 22(4), pp.1284-1302. * Li, L.F., Cushman, S.A., He, Y.X. and
 Li, Y., 2020. Genome sequencing and population genomics modeling provide
 insights into the local adaptation of weeping forsythia. Horticulture
 research, 7. * Yang, X., Yue, Y., Li, H., Ding, W., Chen, G., Shi, T.,
 Chen, J., Park, M.S., Chen, F. and Wang, L., 2018. The chromosome-level
 quality genome provides insights into the evolution of the biosynthesis
 genes for aroma compounds of Osmanthus fragrans. Horticulture research, 5.