Alkaline dip DNA extraction from skin mucus for high-throughput sexing of sterlets ( <i>Acipenser ruthenus</i> )

Studies on simple DNA extraction methods from fish mucus, which is an ideal resource for noninvasive sampling, are scarce. In the aquaculture of sturgeons such as sterlets (<i>Acipenser ruthenus</i>), a high-throughput genetic sexing method is needed, as only females are reared to maturity for their roe. Here, DNA extraction methods using HotSHOT (hot sodium hydroxide and tris), a novel alkaline dip, alkaline glycol, and water were compared using the skin mucus of sterlets (n = 8) collected with a toothpick. High-throughput sexing of sterlets from six production batches (n = 3953) was also evaluated using HotSHOT or alkaline dip. An alkaline dip with 10-25 mM NaOH was an effective alternative to HotSHOT, eliminating the heating and neutralizing steps. Regarding high-throughput sexing, > 99% (3946) individuals were successfully genotyped on the first PCR trial, and genotype ratios of each batch were close to 1:1, showing applicability of the alkaline dip method for practical sterlet sexing.