Scaffolding using optical map, genetic map and synteny with closely related fish genomes produced chromosome-level assembly of the Asian seabass genome.

<p>(A) Comparison of <i>L</i>. <i>calcarifer</i> to two closely related fish species (<i>G</i>. <i>aculeatus</i>, and <i>D</i>. <i>labrax</i>) at the genome-wide level. Colours used for depicting assembled chromosomes are random for each of the three genomes. Different colours in a single <i>L</i>. <i>calcarifer</i> linkage group are used to represent the inter-chromosomal rearrangements. Black arcs show collinear blocks that are intra-chromosomally rearranged between the species. (B) Genome assembly (middle panel) shown anchored to two (LG15 and LG18) of the twenty four <i>L</i>. <i>calcarifer</i> linkage groups while the right panel represents the scaffolded assembly (regions in grey depict the additional contigs brought together by scaffolding).</p

Annotation statistics of the Asian seabass genome.

<p>Annotation statistics of the Asian seabass genome.</p

<i>Lates calcarifer</i> has the best metrics from among the assembled fish genomes till date.

<p>The <i>L</i>. <i>calcarifer</i> genome contig N50 and scaffold N50 values were compared to the following fish genomes: <i>Anguilla japonica</i>, <i>Astatotilapia burtoni</i>, <i>Astyanax mexicanus</i>, <i>Boleophthalmus pectinirostris</i>, <i>Ctenopharyngodon idellus</i>, <i>Cynoglossus semilaevis</i>, <i>Cyprinus carpio</i>, <i>Danio rerio</i>, <i>Dicentrarchus labrax</i>, <i>Electrophorus electricus</i>, <i>Esox lucius</i>, <i>Gadus morhua</i>, <i>Gasterosteus aculeatus</i>, <i>Larimichthys crocea</i>, <i>Latimeria chalumnae</i>, <i>Metriaclima zebra</i>, <i>Neolamprologus brichardi</i>, <i>Notothenia coriiceps</i>, <i>Oncorhynchus mykiss</i>, <i>Oreochromis niloticus</i>, <i>Oryzias latipes</i>, <i>Pundamilia nyererei</i>, <i>Periophthalmodon schlosseri</i>, <i>Periophthalmus magnuspinnatus</i>, <i>Salmo salar</i>, <i>Scartelaos histophorus</i>, <i>Takifugu flavidus</i>, <i>Takifugu rubripes</i>, <i>Tetraodon nigroviridis</i>, <i>Thunnus orientalis</i>, and <i>Xiphophorus maculatus</i> (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005954#pgen.1005954.s002" target="_blank">S1 Table</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005954#pgen.1005954.s003" target="_blank">S2 Fig</a> for more details).</p

Survey of the <i>L</i>. <i>calcarifer</i> genome assembly identified long stretches of TRs lacking in the short read-based assembly and a continuous assembled telomeric region identified at the end of LG3.

<p>(A) Stretches of TRs were virtually missing from the <i>L</i>. <i>calcarifer</i> short read assembly (SRA) generated using 80X Illumina reads scaffolded with ~11,000 BAC ends (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005954#pgen.1005954.s016" target="_blank">S1 Table</a>) whereas the long read assembly (LRA) had a good representation of TRs (upper panel) and the different repeats were more fragmented in the SRA <i>vis-à-vis</i> the LRA (lower panel). (B) Arrangement of telomere monomer sequence (TTAGGG) on a single assembled contig, (unitig_1659; ~0.5 Mb) placed at the terminal end of LG3 (region indicated in orange). Every occurrence of the monomer is indicated by green bars. A highly dense region of (TTAGGG)n was observed between 455.5–466.9 kb, containing the monomer repeated in tandem 1,655 times. The region upstream to this dense region had short dispersed stretches of (TTAGGG)n and contained eight predicted genes (indicated by blue boxes).</p

Analysis of re-sequenced genomes supported the existence of <i>L</i>. <i>calcarifer</i> species complex and its separation into two species (and a third variety).

<p>(A) PCA analysis of <i>L</i>. <i>calcarifer</i> populations using SNPs, (B) PCA analyses using truss morphometric data for representative fishes from the Indian region (red), SE Asia (green) and Australia (blue), (C) Genome-wide nucleotide diversity (Pi) plot representing the three representative species/sub-species of <i>L</i>. <i>calcarifer</i>. Circos was used to plot nucleotide diversity representing <i>L</i>. <i>calcarifer</i> from the Indian region (red), Australia/Papua New Guinea (blue), and SE Asia/Philippines (green) based on SNPs in 50 kb non-overlapping windows. The outer scale is 1 Mb. LG refers to the 24 linkage groups of <i>L</i>. <i>calcarifer</i> with the discrepancies identified in the process of genome scaffolding reflected in the altered IDs for certain linkage groups (LG7 split into two—LG7_1 and LG7_2; LG16 and LG 22 combined- LG16_LG22) and (D) Admixture analyses showing iterations from K = 2,3,4,6,7,8. Each individual is represented by a vertical bar. Abbreviations: I-EC (India-Eastern coast), I-WC (India-Western coast), KH (Cambodia), TH-EC (Thailand-Eastern Coast), VN (Vietnam), SG (Singapore), PH (Philippines), ID-SJ (Indonesia-South Jakarta), ID-SW (Indonesia-Sulawesi), ID-K (Indonesia-Kalimantan), AU-D (Australia-Darwin), PG (Papua New Guinea), AU-QLD (Australia-Queensland).</p

Fluorescence <i>in situ</i> hybridization revealed the localization of tandem repeats in centromeric/pericentromeric regions of the Asian seabass genome and characterization of B chromosomes.

<p>Labeled painting B chromosomes and tandem repeat probes were hybridized to metaphase chromosomes. The chromosomal position of three tandem repeats (green): (A) Sat_LM- centromeres; (B) Lca_217 and Lca_38 (C) pericentromeric region. (D) B chromosome-derived probes, ChB5 (green) and ChB6 (red), reveal the presence of a B chromosome in the <i>L</i>. <i>calcarifer</i> karyotype, as indicated by arrowhead. Chromosomes were counterstained with DAPI (blue). Bar is 10 μm for all images. (E) Association of B chromosomes with the linkage groups. Each linkage group is represented in coloured blocks, and the shadings delineate the genome superscaffolds (after optical mapping) that were assigned to the given linkage group. Rearrangements of portions from the four linkage groups, namely LG5, LG9, LG17 and LG19, together with regions without linkage group assignment (U) comprised the B chromosome.</p

Phylogenetic analyses from 24 species (including 21 ray-finned fishes) depicting the relationship of Asian seab<i>ass</i> with the other percomorphs.

<p>Maximum Likelihood (ML) tree based on a genome-wide set of 313 strict one-to-one orthologs from 24 species. The concatenated and trimmed alignment spans 127,424 amino acid positions. The scale bar represents 0.05 substitutions per site.</p