Abstract
Efficient DNA extraction is critical for high-throughput genotyping in aquaculture breeding programs, where cost, speed, and data quality must be balanced. This study compared five rapid extraction methods-HotSHOT, Chelex, Tween, wax-coated dipstick, and bare dipstick-against a conventional cetyltrimethylammonium bromide (CTAB) protocol for use in Lates calcarifer (barramundi) genotyping. DNA yield, purity, and integrity were measured by spectrophotometry, fluorometry, and Agilent TapeStation. Performance was tested in microsatellite genotyping, tyrosinase-related protein 1b (TYRP1b) gene sequencing, and single nucleotide polymorphism (SNP) genotyping on the Tecan Allegro Targeted Resequencing V2 platform. CTAB produced the highest-quality DNA (30.3 ± 17.4 ng/µL; A260/A280 = 1.91 ± 0.26; DIN = 8.78) with reliable results in all assays. Despite lower quality (1.07 ± 0.30 ng/µL; A260/A280 = 1.44 ± 0.18; DIN ≈ 1.6), HotSHOT performed similarly: SNP concordance with CTAB averaged 82.3%, microsatellite amplification reached 93.1% (vs. 99.3% for CTAB), and all HotSHOT samples yielded high-quality TYRP1b sequences. Chelex, Tween, and dipstick methods had lower success, likely due to inadequate yield and purity. HotSHOT was the most effective rapid alternative to CTAB, with significantly higher SNP concordance (82.3% vs. others; p < 0.005). These results show that method choice strongly influences target-capture genotyping success and support HotSHOT as a cost-effective, scalable option for aquaculture genomics.