Avaii

The primers (LP1 and RP1) for pyrosequencing and PCR‐RFLP were generated by Ruibiotech Company (Beijing, China) using the sequences described by Wilson et al. (2001 (link)) (Table 1). Moreover, optimized primers (LP2 and RP2) were developed to identify PCR‐RFLP results easier (Table 1). Conventional PCR reaction mixtures (20 μl) contained 10 μl PCR SuperMix (Tansgen), 1 μl LP (10 μmol/l), 1 μl RP (10 μmol/l), 1 μl template (100 ng/μl), and 7 μl ddH₂O. The PCR reactions were performed under the following program: 5 min at 94℃, 35 cycles of 30 s at 94℃, 30 s at corresponding annealing temperature (Table 1) and 60 s at 72℃, and 10 min at 72℃ (ABI2720; Applied Biosystems, Waltham, MA, USA). The amplified PCR products were gel purified using the MiniBEST Agarose Gel DNA Extraction Kit (TaKaRa, Dalian, China), and sequenced by Ruibiotech Company (Beijing, China). The purified DNA fragments were digested using AvaII for 4.5 h at 37℃ following the manufacturer's protocol (New England Biolabs, Ipswich, MA, USA).

Double-digest restriction site associated sequencing (ddRAD-Seq) libraries [21 (link)] were prepared for 39 individuals with 8 of these processed in duplicate as technical replicates. For each sample, 300 ng of DNA was digested with two restriction enzymes (AvaII and MspI, New England Biolabs Inc), following manufacturer’s instructions. These two enzymes were selected based on the recommendation of Yang et al [22 (link)], who found that this enzyme pair produced a high number of fragments for many plant species. Adaptors containing sample-specific barcodes and Illumina indices were ligated to each sample. Samples were pooled into three index pools and a size-selection performed on each pool for 300–500 bp fragments using excision from an agarose gel, followed by extraction with a Qiaquick gel extraction kit (Qiagen). Pooled size-selected samples were PCR-amplified to add Illumina indices using Phusion flash high fidelity PCR master mix (Thermo Scientific). Each pooled sample was purified and concentrated with a MinElute kit (Qiagen), quantified with a Qubit dsDNA HS (high sensitivity) assay kit (Thermo Fisher Scientific) and combined in equimolar amounts. A detailed protocol has been deposited in protocols.io (dx.doi.org/10.17504/protocols.io.zgyf3xw). The library was sequenced across a third of a lane using the Illumina HiSeq 2500 to generate 2 × 125 bp reads.

To genotype npc1 mutants, genomic DNA was extracted from either whole embryos/larvae or the caudal fin of adults with DNA extraction buffer (10 mM Tris pH 8.2, 10 mM EDTA, 200 mM NaCl, 0.5% SDS and 200 µg/ml proteinase K). Genomic DNA was further diluted 20-fold and then used as the template for genotyping PCR. For the npc1^y535 allele, genotyping PCR was carried out using a forward primer npc1Ex2F1 (5′-CCAGCACTGTATCTGGTACGG-3′) and a reverse primer npc1Ex3R1 (5′-ACCAGTCTCGGACACAGCTC-3′). The PCR conditions were: 94°C for 2 min; 35 cycles of 94°C for 30 s, 63°C for 30 s, 72°C for 30 s; and 72°C for 5 min. For the npc1^hg37 allele, primers used for genotyping PCR were designed through dCAPS Finder 2.0 online tool (Department of Biology, Washington University, St Louis, MO, USA) to generate an artificial restriction enzyme cutting site on the PCR product. The PCR was carried out using a forward primer Znpc1 Ex7-AvaII-F (5′-TTCTTGACAGCAATCAGCCCCGGTC-3′) and a reverse primer Znpc1 Int7-8-R2 (5′-GAGGGTGTCTGCAGGTTTCACC-3′). The PCR conditions were 94°C for 2 min; 45 cycles of 94°C for 30 s, 63°C for 30 s, 72°C for 30 s; and 72°C for 5 min. PCR products from both npc1^y535 and npc1^hg37 were digested with restriction enzyme AvaII (R0153, New England BioLabs, Ipswich, MA, USA) at 37°C for 8 h. Final digestion products were resolved on 2% agarose gels.

Genotyping for the activin beta A genes was carried out using primers 5′-GGATTATTTTGGACTGGCTCC and 5′-TTCAGATGCAGCATGTTGAGG; these bind to sequences that are identical for both zebrafish and goldfish orthologs and amplify fragments of almost identical size (zebrafish, nucleotides 248–598 in Genbank accession number BC129208; goldfish, nucleotides 134–479 in Genbank accession number AF169032) (Supplementary Fig. 2A). Amplicons were digested with AvaII (New England Biolabs) according to the manufacturer’s instructions and the products resolved on agarose gels. Genotyping for the presence of the gata1:DsRed transgene was carried out with primers 5′-CAAGGAGTTCATGCGCTTCA and 5′-TTCACGCCGATGAACTTCAC (amplicon size 360 bp); genotyping for the presence of the ikaros:eGFP transgene was carried out with primers 5′-TGGTGCCCATCCTGGTCGAG and 5′-GTCCTCGATGTTGTGGCGGA (amplicon size 488 bp).

PCR primers were designed manually on the basis of the SNP/MNP/indel-containing sequences from the VCF2CAPS output file (Table 1). PCR was performed in a total volume of 15 μl containing 75 ng of genomic DNA, 1× PCR buffer (Dongsheng Biotech, Guangzhou, China), 5 mM MgCl₂, 0.25 mM dNTPs, 0.25 μM each primer and 1.1 U of Taq DNA polymerase (Dongsheng Biotech). Temperature cycling was performed as follows: initial denaturation at 94°C for 5 minutes followed by 35 cycles of 92°C for 45 s, 57°C for 45 s and 72°C for 2 minutes, then a final extension at 72°C for 10 minutes.
Twelve microliters of the post-PCR mixture were supplemented with 5 U of a restriction enzyme and incubated at the appropriate temperature for 16 hours. For enzymes AlwI, AvaII, BsiWI, and SspI (New England Biolabs) restriction was carried out at 37°C, while for TaqI (Thermo Fisher Scientific) at 65°C. The resulting restriction fragments were separated in 1% agarose alongside with 5 μl of the 100 bp and 1 kb DNA ladders from Dongsheng Biotech. Prior to gel loading, 2 μl of the 6 × Loading Buffer (Dongsheng Biotech) were added to the post-restriction mixture. DNA electrophoresis was performed in the TBE buffer at a field strength of 4 V/cm for 2 hours. The gels contained ethidium bromide at a concentration of 1.33 μg/ml.