Genemapper v6

Parentage analysis was performed to confirm that the sampled fish corresponded to the correct family, as described in [57 ]. Briefly, DNA was extracted from fin clips from parents and offspring, amplified at 12 microsatellite loci [58 (link)], and visualized on an AB3500 automated DNA sequencer. GeneMapper V6 (Applied Biosystem) was used to determine allele lengths, which were imported into both Cervus v. 3.0.7 [59 (link)] and COLONY v. 2.0.6 [60 (link)]. Parentage analysis was performed using COLONY's full likelihood approach and Cervus's 90% confidence likelihood approach. Parentage assignment was considered successful when both programs identified the same set of parents. If the identified parents were not crossed in the breeding design, the next most probable parentage assignment was used. If both programs did not suggest the same pair, the most probable pair between the possible crosses was assigned. Inability to assign both parents to an individual resulted in a failed assignation.

All PCR experiments involved four independent biological replicates. For conventional end-point PCR, primers in exons flanking the alternative splicing event were used to amplify 1 μL of cDNA with the Taq DNA polymerase (New England Biolabs) for 35 cycles in a 25 μL total volume. RT-PCR products were resolved by gel electrophoresis in 2% agarose gels and bands were quantified using the Image Lab 6.0.1 software (Bio-Rad). The identity of the amplified products was verified by Sanger sequencing. For semi-quantitative analysis of fluorescently marked PCR products, the same protocol was applied except that FAM-labeled forward primers were used and the cycling conditions were as follows: 94 °C for 3 min followed by 26 cycles at 94 °C for 30 s, 59 °C for 30 s and 72 °C for 45 s and a final extension step at 72 °C for 20 min. Fluorescently labeled PCR fragments were separated by size on an Applied Biosystems^® 3500 Dx Genetic Analyzer and analyzed with the GeneMapper v6.0 software (Applied Biosystems). Ratios of splicing isoforms were determined as the peak area of one specific isoform divided by the total peak areas for the other detected isoforms. Primer sequences for RT-PCR are provided in the Supplementary Table 1.