DOI: 10.5061/dryad.ncjsxksqj
title: Revisiting the origin of octoploid strawberry

description: The cultivated strawberry (Fragaria ×ananassa) is an octoploid, and the
 identity of its four subgenomes has long been a mystery. In their recent
 strawberry genome publication, Edger et al. present a novel hypothesis:
 each subgenome originated from a different extant diploid progenitor, and
 the hexaploid species Fragaria moschata was a direct ancestor. We
 reanalyzed the four octoploid subgenomes in a phylogenomic context and our
 results support only two extant diploids progenitors; we also found no
 support for F. moschata as a direct ancestor. We identified assumptions in
 the Edger et al. tree-searching algorithm that prevent it from accepting
 extinct or unsampled progenitors, and we argue that this is a critical
 weakness of their approach.
description: <b>Phylogenomic Analysis of the Octoploid
 Subgenomes</b> Illumina sequencing libraries were
 prepared from leaf tissue of six diploid <i>Fragaria
 </i>species, and 9-32X genomic coverage was obtained (Supplementary
 Table 1). <i>Fragaria vesca </i>subsp.
 <i>bracteata</i>, <i>F. iinumae </i>and
 <i>F. nipponica</i> were sequenced at the Oregon State
 University Center for Genome Research and Biocomputing Central Services
 Lab with 100 bp single ends on a HiSeq 3000. <i>Fragaria
 mandshurica</i>,<i> F. nilgerrensis</i> and <i>F.
 nipponica </i>were sequenced at Berry Genomics (Hangzhou, China)
 with 150 bp paired ends on a HiSeq 2500. We removed adapters and
 low-quality portions of reads using Trimmomatic (v
 0.35)<sup>1</sup> and settings LEADING:20, TRAILING:20,
 SLIDINGWINDOW:5:20, MINLEN:50. The genome assemblies of octoploid
 <i>Fragaria ananassa</i> ‘Camarosa’, diploid <i>F.
 bucharica </i>(published as <i>F.
 nubicola</i>)<sup>2</sup>, and diploid outgroup
 <i>Potentilla micrantha</i><sup>3</sup> were
 downloaded from the Genome Database for Rosaceae (https://www.rosaceae.org/). The former was subdivided by the Edger et al.<sup>4</sup> subgenome assignment into four sets of seven chromosomes. These assemblies were converted to 20X genomic coverage of random 100 bp sequences using BBTools randomreads.sh (https://jgi.doe.gov/data-and-tools/bbtools/). The above sets of sequence reads were aligned with BWA (v 0.7.12)<sup>5</sup> to the <i>Fragaria vesca </i>v 4.1 genome assembly<sup>6</sup> after masking  repetitive regions of the genome. This was done with BBTools bbmask.sh  (https://jgi.doe.gov/data-and-tools/bbtools/) masking sequences that align to the <i>F. vesca </i>v 4.1 transposable element library downloaded from the Genome Database for Rosaceae (https://www.rosaceae.org/). In total, 4.5% of the genome was masked. This is lower than the 31% transposable element (TE) content reported in supplemental Table 4 of the <i>F. vesca</i> genome publication<sup>6</sup>, but similar to the value inferred from Figure 1 in the same publication. We suggest this discrepancy is due to the inclusion of partial and degenerate TEs in the calculation used in the table, while the figure more accurately represents the fraction of non-unique sequence in the <i>F. vesca </i>reference genome.   The twelve resulting alignments were converted to a variant call format (vcf) file with SAMtools (v 1.9)<sup>7</sup> with the default settings of the mpileup and call options. The vcf file was converted into a multisample variant format (mvf) file using MVFtools (v 0.5.1.4)<sup>8</sup>. Sites with mapping quality below 20 and coverage below 3 were excluded. All heterozygous sites were converted to N, to account for the fact that the octoploid subgenome and outgroup sequences were derived from haploid genome assemblies. MVFtools was used to automate maximum-likelihood (ML) estimates of phylogeny using RAxML (v 8.2.12)<sup>9</sup> with the GTR+Γ model of sequence evolution and 100 bootstrap replicates. A taxon was excluded from the analysis of a 100 kb window if it had &lt;10% of aligned sites. Analyses were conducted for the seven base chromosomes (Fig. 1b, Supplementary Fig. 1) and for 2191 non-overlapping windows of 100 kb across the seven chromosomes (Figs. 1c, 2, Supplementary Table 4). The phylogenetic position of each subgenome relative to diploid species was recorded (see Fig. 2 for details) and summarized for each base chromosome (Supplementary Table 5). Homeologous exchange was inferred when the ‘Camarosa vesca’ subgenome shared a MRCA with <i>F.</i> <i>iinumae</i> or when the other three subgenomes shared a MRCA with the <i>F. vesca </i>clade (<i>F. vesca, F. mandshurica, F. bucharica</i>). <b><i>Fragaria</i></b><b> <i>moschata </i>Linkage Mapping and Phylogenetic Inference<i> </i></b> We used an F<sub>1</sub> cross for linkage mapping of the hexaploid <i>F.</i> <i>moschata</i>. Our F<sub>1</sub> mapping population was derived from two parental plants collected from Slovenia (46.6827°N, 16.2951°W). Following our previous protocols<sup>10</sup>, seeds of the experimental cross (<i>N</i> = 192) were planted in a custom soil mixture (2 : 1, Fafard 4 : sand top-dressed with Sunshine Redi-earth Plug & Seedling; Sun Gro Horticulture, Agawam, MA, USA), and grown under 16 °C /21 °C night/day temperatures and a 14-h photoperiod in a growth chamber at the University of Pittsburgh for 11 wk. We selected a random subset (<i>N</i> = 46) of the F<sub>1</sub> progeny for targeted sequence capture. Targeted sequence capture was performed using previously developed <i>Fragaria</i> baits (v 2.0)<sup>10,11</sup>. These 20,000 capture baits of 100 bp each are relatively randomly distributed across the seven base chromosomes (1–7). DNA was isolated from silica-dried leaf tissue of the 46 progeny and two parents at Ag-Biotech (Monterey, CA). We constructed individually indexed genomic libraries using the NEBNext Ultra DNA Library Prep Kit (New England BioLabs, Ipswich, MA, USA), which were then target enriched<sup>10</sup> and sequenced using a 1/3 lane of 150 bp paired ends on a HiSeq 3000 at the Oregon State University Center for Genome Research and Biocomputing Central Services Lab. The hexaploid linkage mapping involved four steps: quality filtering of paired-end capture reads, mapping reads to the above-mentioned diploid <i>F</i>. <i>vesca</i> v 4.1 genome assembly, genotype calling in polyploids using POLiMAPS (v 1.1)<sup>12</sup>, and linkage mapping using OneMap<sup>13</sup>. First, we removed adapters and low-quality portions of paired-end reads using Trimmomatic (v 0.35)<sup>1</sup> as described above, and merged the paired-end reads using PEAR (v 0.9.6)<sup>14</sup> with a minimum overlap size of 20 bp. Second, we mapped both the merged and un-merged paired-end reads to the <i>F</i>. <i>vesca</i> v 4.1 reference using BWA (v 0.7.12)<sup>5</sup>. The sorted BAM files were generated with SAMtools (v 1.9)<sup>7</sup> and then were used to create the mpileup file. Third, with the mpileup file, we conducted polyploid genotype calling using POLiMAPS  (v 1.1)<sup>12</sup>, in which heterozygous and homozygous loci were identified with the default parameters, except for the depth of ≥32X per progeny for the hexaploid. Lastly, to construct maternal and paternal linkage groups (LGs), we assigned SNPs to the most likely LGs based on a logarithm of odds (LOD) threshold of 5 using OneMap<sup>13</sup> in R (v 3.3.3)<sup>15</sup>. LGs with at least 20 SNPs were used for subsequent analyses. To infer the phylogenetic placement of <i>F.</i> <i>moschata</i> in relation to the octoploid strawberry and other diploid <i>Fragaria</i>, we first extracted the quality-filtered reads that contained the above LG SNPs, and mapped these reads to the <i>F</i>. <i>vesca</i> v 4.1 reference to generate consensus LG sequences using POLiMAPS  (v 1.1). We next used POLiMAPS  (v 1.1) and the variant call format file described in the above Phylogenomic Analysis of Octoploid Subgenomes section to generate multiple sequence alignments among <i>F. moschata</i> LG sequences, the four octoploid reference subgenomes, diploid <i>Fragaria </i>genomes and the outgroup <i>Potentilla</i>. Phylogenetic inference was conducted for each of chromosomes 1–7 and for maternal and paternal LGs separately, using the ML method with the GTR+Γ model and 100 bootstrap replicates in RAxML (v 8.0.26)<sup>9</sup>.  
description: Alignment files (phylip format) and resulting phylogenetic trees
 (nexus format). A. Analysis of Fragaria moschata
 subgenomes:
 Fragaria_moschata.subgenome.alignments.tar.gz<br>
 Fragaria_moschata.subgenome.trees B. Analysis of
 Fragaria x ananassa 'Camarosa' subgenomes. The nexus trees with
 branch length and bootstrap values are in column 4 of the tree-results.txt
 files. See the first line header for other fields.
 octoploid.subgenome.phylogeny.2191-windows.alignments.tar.gz<br> octoploid.subgenome.phylogeny.2191-windows.tree-results.txt<br> octoploid.subgenome.phylogeny.7-chromosomes.alignments.tar.gz<br> octoploid.subgenome.phylogeny.7-chromosomes.tree-results.txt Key to codes used in octoploid subgenome alignment files:<br> s0       vesca<br> s1       Camarosa_iinumae<br> s2       Camarosa_nipponica<br> s3       Camarosa_vesca<br> s4       Camarosa_viridis<br> s5       bucharica<br> s6       potentilla<br> s7       bracteata<br> s8       iinumae<br> s9       nipponica<br> s10      mandshurica<br> s11      nilgerrensis<br> s12      viridis