Oryzias latipes

Fertilized eggs of medaka and zebrafish were routinely collected as described previously [11] (link), [14] (link), [17] (link). The embryos were incubated at 28°C until 6 days (zebrafish) or 7 days (for medaka) post fertilization. Except for the Heino strain, all the strains and transgenic lines of both medaka and zebrafish embryos were incubated with 5×PTU to prevent pigmentation. The embryos were fixed with 4%PFA at 4°C overnight. For fluorescent immunostaining of either cryosecctions or whole mount embryos, fixed embryos were dechorionated (for medaka) and equilibrated in 1×PTw (1×PBS at pH 7.3, 0.1% Tween), followed by appropriate steps including the heating method for either cyrosection or whole mount immunostaining (see below for details). Prior to perform combined whole mount in situ hybridization and immunostaining, dechorionated medaka embryos were washed with 1×PTw for 5 min several times, and then stored in 100% MeOH at −20°C at least two days. Those embryos were subjected to fluorescent whole mount in situ hybridization including the heating method and immunostaining (see below for details).

For in vivo linearization of the donor plasmids, sgRNA-1 target site (T1) was cloned into the Golden GATEway cloning system [24 (link)] via oligo annealing (T1_F 5’-GATCAGGCCTGCAGCTGGGCGAGGGCGATGCCACCTACGGCTCGAGCTCGTAC-3’, T1_R 5’-GAGCTCGAGCCGTAGGTGGCATCGCCCTCGCCCAGCTGCAGGCCT-3’).
Homology flanks were selected according to integration sites and PCR amplified with primers extended with BamHI (forward primer) or KpnI (reverse primer) restriction sites via Q5 polymerase (NEB) from wildtype medaka genomic DNA (actb 5’ homology flank: F 5’-GGGGATCCCAGCAACGACTTCGCACAAA-3’, R 5’-GGGGTACCGGCAATGTCATCATCCATGGC-3’; rx2 5’ homology flank: F 5’-GCCGGATCCAAGCATGTCAAAACGTAGAAGCG-3’, R 5’-GCCGGTACCCATTTGGCTGTGGACTTGCC-3’). eGFP^var was generated via fusion PCR (fragment 1 eGFP_F 5’-GCCGGATCCGGAGTGAGCAAGGGCGAGGAGCT-3’, eGFPvar_R 5’-GTACGTCGCGTCACCTTCACCCTCGCCGGAC-3’; fragment 2 eGFPvar_F 5’-TGAAGGTGACGCGACGTACGGCAAGCTGACCCTG-3’, eGFP_R 5’-GCCGGTACCTCCCTTGTACAGCTCGTCCATGCC-3’) with Q5 polymerase (NEB) on an eGFP template. eGFP forward and reverse primers were extended with BamHI or KpnI restriction sites, respectively for cloning via Golden GATEway.

Four datasets were used for synteny analyses: 1) the reference Esox lucius transcriptome (see ‘RNA-seq and assembly’ section of Methods); 2) stickleback protein sequences from the Feb. 2006 Broad/gasAcu1 release; 3) zebrafish protein sequences from the Jul. 2010 Zv9/danRer7 release and; 4) medaka protein sequences from the Oct. 2005 NIG/UT MEDAKA1/oryLat 2 release. Stickleback, zebrafish and medaka sequences and their associated genomic location information were obtained from the UCSC Genome Browser [92] (link). Scaffold locations for northern pike transcripts were obtained through mapping using GMAP [93] (link); linkage group assignments followed if the host scaffold had been previously mapped to a group in the genetic map. Using the BLASTX and TBLASTN programs, BLAST alignments (E-value ≤1e-5) were obtained between the northern pike transcripts and the proteins of each other fish species. Orthology between northern pike transcripts and other fish protein sequences was determined using the reciprocal best hit (RBH) paradigm requiring at least 50% of each sequence was covered in non-overlapping BLAST alignments (HSPs) from the other. Synteny between two species (Figure 6) and scaffold continuity (Figure S2) were examined by plotting the genomic locations of each sequence in a relevant orthologue pair.
The analysis of synteny between northern pike and Atlantic salmon (Table 2) was performed by obtaining the flanking sequence of chromosome-associated SNPs in Atlantic salmon and identifying the strongest BLASTN hits (E-value ≤1e-10) between these sequences and northern pike scaffolds with a known linkage group.

Repetitive elements were identified de novo using RepeatModeler v2.0.1 (Flynn et al. 2020 (link)) with the “LTRStruct” option. RepeatMasker v4.1.1 (Tempel 2012 (link)) was used to screen known repetitive elements with two inputs: (1) the RepeatModeler output and (2) the vertebrata library of Dfam v3.3 (Storer et al. 2021 (link)). The resulting output files were validated and merged before redundancy was removed using GenomeTools v1.6.1 (Gremme et al. 2013 (link)). To identify and annotate candidate gene models, BRAKER v2.1.6 (Brůna et al. 2021 (link)) was used with mRNA and protein evidence. For annotation with BRAKER, the chromosome sequences were soft masked using the maskfasta function of BEDTools v2.30.0 (Quinlan 2014 (link)) with the “soft” option. Protein evidence consisted of protein records from UniProtKB/Swiss-Prot (UniProt Consortium 2021 (link)) as of 2021 January 11 (563,972 sequences) as well as selected fish proteomes from the NCBI database (A. ocellaris: 48,668, Danio rerio: 88,631, Acanthochromis polyacanthus: 36,648, Oreochromis niloticus: 63,760, Oryzias latipes: 47,623, Poecilia reticulata: 45,692, Stegastes partitus: 31,760, Takifugu rubripes: 49,529, and Salmo salar: 112,302). Transcriptomic reads from 13 tissues were used as mRNA evidence. These Illumina short reads were trimmed with Trimmomatic v0.39 (Bolger et al. 2014 (link)) as described above and mapped to the chromosome sequences with HISAT2 v2.2.1 (Kim et al. 2019 (link)). The resulting SAM files were converted to BAM format with SAMtools v1.10 (Li et al. 2009 (link)) and used as input for BRAKER. Of the resulting gene models, only those with supporting evidence (mRNA or protein hints) or with homology to the Swiss-Prot protein database (UniProt Consortium 2021 (link)) or Pfam domains (Mistry et al. 2021 (link)) were selected as final gene models. Homology to Swiss-Prot protein database and Pfam domains was identified using Diamond v2.0.9 (Buchfink et al. 2015 (link)) or InterProScan v5.48.83.0 (Zdobnov and Apweiler 2001 (link)), respectively. Functional annotation of the final gene models was completed using NCBI BLAST v2.10.0 (Altschul et al. 1990 (link)) with the NCBI non-redundant (nr) protein database. Gene Ontology (GO) terms were assigned to A. clarkii genes using the BLAST output and the “gene2go” and “gene2accession” files from the NCBI ftp site (https://ftp.ncbi.nlm.nih.gov/gene/DATA/). Completeness of the gene annotation was assessed with BUSCO v4.1.4 (actinopterygii_odb10) (Simão et al. 2015 (link)).