In Depth Characterization of Repetitive DNA in 23 Plant Genomes Reveals Sources of Genome Size Variation in the Legume Tribe Fabeae

The differential accumulation and elimination of repetitive DNA are key drivers of genome size variation in flowering plants, yet there have been few studies which have analysed how different types of repeats in related species contribute to genome size evolution within a phylogenetic context. This question is addressed here by conducting large-scale comparative analysis of repeats in 23 species from four genera of the monophyletic legume tribe Fabeae, representing a 7.6-fold variation in genome size. Phylogenetic analysis and genome size reconstruction revealed that this diversity arose from genome size expansions and contractions in different lineages during the evolution of Fabeae. Employing a combination of low-pass genome sequencing with novel bioinformatic approaches resulted in identification and quantification of repeats making up 55-83% of the investigated genomes. In turn, this enabled an analysis of how each major repeat type contributed to the genome size variation encountered. Differential accumulation of repetitive DNA was found to account for 85% of the genome size differences between the species, and most (57%) of this variation was found to be driven by a single lineage of Ty3/gypsy LTR-retrotransposons, the Ogre elements. Although the amounts of several other lineages of LTR-retrotransposons and the total amount of satellite DNA were also positively correlated with genome size, their contributions to genome size variation were much smaller (up to 6%). Repeat analysis within a phylogenetic framework also revealed profound differences in the extent of sequence conservation between different repeat types across Fabeae. In addition to these findings, the study has provided a proof of concept for the approach combining recent developments in sequencing and bioinformatics to perform comparative analyses of repetitive DNAs in a large number of non-model species without the need to assemble their genomes

Conserved synteny of genes between chromosome 15 of Bombyx mori and a chromosome of Manduca sexta shown by five-color BAC-FISH

The successful assignment of the existing genetic linkage groups (LGs) to individual chromosomes and the second-generation linkage map obtained by mapping a large number of bacterial artificial chromosome (BAC) contigs in the silkworm, Bombyx mori, together with public nucleotide sequence databases, offer a powerful tool for the study of synteny between karyotypes of B. mori and other lepidopteran species. Conserved synteny of genes between particular chromosomes can be identified by comparatively mapping orthologous genes of the corresponding linkage groups with the help of BAC-FISH (fluorescent in situ hybridization). This technique was established in B. mori for 2 differently labeled BAC probes simultaneously hybridized to pachytene bivalents. To achieve higher-throughput comparative mapping using BAC-FISH in Lepidoptera, we developed a protocol for five-color BAC-FISH, which allowed us to map simultaneously 6 different BAC probes to chromosome 15 in B. mori. We identified orthologs of 6 B. mori LG15 genes (RpP0, RpS8, eIF3, RpL7A, RpS23, and Hsc70) for the tobacco hornworm, Manduca sexta, and selected the ortholog-containing BAC clones from an M. sexta BAC library. All 6 M. sexta BAC clones hybridized to a single M. sexta bivalent in pachytene spermatocytes. Thus, we have confirmed the conserved synteny between the B. mori chromosome 15 and the corresponding M. sexta chromosome (hence provisionally termed chromosome 15)

Genome size evolution and repeat composition of <i>Fabeae</i> species.

<p>(<i>A</i>) Phylogenetic tree of the 23 investigated species with their genome sizes shown by the colours of the terminal branches. Colour gradients within the tree branches indicate inferred genome size changes and species names are represented by the codes given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143424#pone.0143424.t001" target="_blank">Table 1</a>. All nodes except those labelled with "x" were highly supported with posterior probabilities >0.95 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143424#pone.0143424.s001" target="_blank">S1 Fig</a> for details). (<i>B</i>) Graphical representation of the genomic abundances of major types of repetitive sequences. The area of the rectangles are proportional to the total length of individual repeats per monoploid genome size (1Cx) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143424#pone.0143424.s006" target="_blank">S2 Table</a>). For LTR-retrotransposons, the colour of the rectangle indicates the estimated ratio of solo-LTRs to full-length elements (data given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143424#pone.0143424.t003" target="_blank">Table 3</a>). Repeat abbreviations: <i>Chrom</i>, Chromovirus; <i>Max</i>., Maximus/SIRE; <i>DNA</i>, DNA (class II) transposons; <i>SAT</i>, satellite repeats. <i>Copia</i> includes all Ty1/copia lineages except Maximus/SIRE and Angela; <i>LTR</i>, unclassified LTR-retrotransposons, <i>Other 0</i>.<i>01</i> includes remaining repeats with abundance exceeding 0.01% of the genome.</p

Estimated ratios of solo-LTRs to complete elements (<i>Rsf</i>).

<p>Estimated ratios of solo-LTRs to complete elements (<i>Rsf</i>).</p

Estimation of genome size and sequencing of selected <i>Fabeae</i> species.

<p>⁽¹⁾ Seedbank abbreviations: <i>IPK</i>, Leibniz Institute for Plant Genetics and Crop Plant Research; <i>ICARDA</i>, International Center for Agricultural Research in the Dry Areas</p><p>⁽²⁾ In diploids (all species except for the tetraploid <i>V</i>. <i>cracca</i>) 1Cx = 1C. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143424#pone.0143424.s005" target="_blank">S1 Table</a> for details on genome size estimation.</p><p>Estimation of genome size and sequencing of selected <i>Fabeae</i> species.</p

Sequence conservation of repeats between <i>Fabeae</i> species.

<p>The ratio <i>Hs</i>/<i>Ho</i> was calculated for each read within individual groups of repeats, where <i>Hs</i> was the frequency of similarity hits to reads from the same species and <i>Ho</i> was the frequency of hits to reads from all other species. The histograms show the distribution of <i>Hs</i>/<i>Ho</i> ratios for different repeats, with numbers of reads plotted along the y-axis. The <i>Hs</i>/<i>Ho</i> ratios are close to 1 (0 on the log scale) for highly conserved sequences whereas larger values correspond to sequence divergence, resulting in higher frequencies of hits within than between species (for example, a value of 2 on the x-axis corresponds to reads producing 100-fold more intra-specific than inter-specific hits).</p

Southern blot detection of selected Ogre and Angela sequences in the genomes of <i>Fabeae</i> species.

<p>The blots were prepared from equal amounts of genomic DNA of each species digested with <i>Ssp</i>I and hybridized to probes corresponding to sequence variants of Ogre derived from <i>Vicia sylvatica</i> (<i>A</i>), <i>Lathyrus latifolius</i> (<i>B</i>) and <i>Vicia pannonica</i> (<i>C</i>). These variants are evident as narrow parallel paths on a graph representation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143424#pone.0143424.ref031" target="_blank">31</a>] of cluster CL7 (<i>E</i>) where reads from these species are highlighted by blue, red and green colours, respectively (reads of all other species are in grey). For the Angela element cluster CL177, reads of all species (grey dots) generated a narrow linear graph due to their high sequence similarities (<i>F</i>). The corresponding probe detected several conserved bands (arrows) on the Southern blot (<i>D</i>).</p