Abi 3730 genetic analyser

MLPA is a semi-quantitative technique that is designed to detect gene dosage variation based on multiplex PCR method. The SALSA MLPA KIT P245 and P250 (MRC Holland, Amsterdam, Netherlands) were used for MLPA analysis according to the manufacturer’s instructions. PCR amplification products were separated by capillary electrophoresis using ABI 3730 Genetic Analyser (Applied Biosystems, USA). Coffalyser. Net software (MLPA Holland, Amsterdam, Netherlands) was used to analyze and give an interpretation of the raw MLPA data. The detailed information of the probes included in MLPA kits is described in the manufacture’s instructions.

Ten individuals of grass carp and rice were used for SSR validation. For carp, genomic DNA was isolated from tail fin using a salt-extraction method with slight modifications. For rice, genomic DNA was isolated from leaves using the DNeasy Plant mini prep kit (Qiagen, Hilden, Germany). DNA concentration for each sample was determined using NanoDrop (Thermo Scientific, Waltham, MA, USA). Primers were designed using Primer premier 5 software (Lalitha, 2000 ). All the PCR amplifications were carried out in a 10 µL volume containing 1 µL 10× buffer (with Mg²⁺), 100 µM dNTPs, 0.5 µL primer pairs, 1 U Taq DNA polymerase and 20 ng genomic DNA. The reaction program was 5 min at 95 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at annealing temperature 57 °C, 30 s at 72 °C and a final extension at 72 °C for 8 min; then stored at 4 °C. The PCR products were separated by size with Genescan-400 HD size ladders (Applied Biosystems, Foster City, CA, USA), by capillary gel electrophoresis using the ABI 3730 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). The peak heights and fragment sizes were analyzed using GeneMarker software (Holland & Parson, 2011 (link)).

DNA analysis of all samples was conducted at IMR in Bergen during the period 1 May 2017–1 March 2018. DNA was extracted from either fin clips or scales in 96-well plates using the Qiagen DNeasy 96 Blood & Tissue Kit with two negative controls. In total, 31 microsatellite markers were amplified in five PCR multiplexes (microsatellite information (electronic supplementary material, table S1) and amplification protocols (electronic supplementary material, table S2) are found in electronic supplementary material, file 1). PCR products were resolved on an ABI 3730 Genetic Analyser and sized using a 500LIZ size standard (Applied Biosystems). GeneMapper v. 5.0 was used to score alleles manually. A second laboratory technician quality checked each individual sample to ensure scoring accuracy before exporting the data for statistical analysis. Individuals with more than 30% missing alleles were removed from the dataset.

Genetic sex was identified by the presence of the sdY gene (Eisbrenner et al., 2014 (link); Yano et al., 2012 (link)); if the presence of exon 2 and 4 were detected, the individual was designated as male. An ABI Applied Biosystems ABI 3730 Genetic Analyser was used for fragment analysis, the outputs of which were used to call genotypes in GeneMapper (Applied Biosystems, v. 4.0). This assay gives accurate identification of sex, although a very low percentage of fish identified as genetic males are phenotypic females due to carrying an inactive pseudo‐copy of the sdY gene with both exon 2 and 4 (Ayllon et al., 2020 (link)).

MLPA assay and the subsequent results analyses were performed as previously described [12 (link)]. Briefly, The SALSA MLPA KIT P245 (MRC Holland, Amsterdam, Netherlands) were used for MLPA analysis according to the manufacturer’s instructions. PCR amplification products were separated by capillary electrophoresis using ABI 3730 Genetic Analyser (Applied Biosystems, USA). Coffalyser. Net software (MLPA Holland, Amsterdam, Netherlands) was used to analyze and give an interpretation of the raw MLPA data. The detailed information of the probes included in MLPA kits was described in the manufacture’s instructions.

To confirm that the clinical specimens at the two different time points (at initial diagnosis and at day 156) were obtained from the identical patient (patient 1), STR analysis was performed using the AmpFLSTR® Identifiler® Plus kit, according to the manufacturer’s instructions. This assay used 16 STR markers, each with 2 to 28 alleles of different sizes. PCR products were analysed on an ABI 3730 Genetic Analyser (Applied Biosystems, Foster City, CA). Alleles for each STR marker were analysed with an ABI GeneScan analysis program (Applied Biosystems).

DNA was extracted from muscle or branchial tissue with REDExtract-N-AmpTissue PCR Kit (Sigma-Aldrich, St. Louis, MO, USA). A fragment of the mitochondrial Cytochrome Oxidase I (COI) gene was amplified using the universal primers LCO1490 and HCO2198 (Folmer et al., 1994 (link)). Amplifications were performed in a final volume of 20 μL using 10 μL of REDExtract-N-amp PCR reaction mix (Sigma-Aldrich, St. Louis, MO, USA), 0.8 μL of each primer (10 μM), and 2 μL of template DNA. The PCR program consisted of an initial denaturing step at 94 °C for 2 min, 30 amplification cycles (denaturing at 94 °C for 45 s, annealing at 50 °C for 45 s and extension at 72 °C for 50 s), and a final extension at 72 °C for 6 min, on a PCR System 9700 (Applied Biosystems). PCR products were purified using MultiScreen® filter plates (Millipore), labelled using BigDye® Terminator v.3.1 (Applied Biosystems) and sequenced on an ABI 3730 Genetic Analyser (Applied Biosystems) at the Scientific and Technological Centres of the University of Barcelona, Spain (CCiTUB). Other samples were directly sent for purification and sequencing to Macrogen Inc. (Seoul, South Korea). Sequences were edited and aligned using BioEdit® v.7.0.5.3 (Hall, 1999 ).