A cultured stock of masculinized rainbow trout was diagnosed with Y‐linked markers (sdY and OmyY1) aiming to detect neomales before their use at the production level. To achieve a reliable diagnosis, the following steps were considered: (1) PCR amplification of the housekeeping β-actin gene to determine the DNA quality of samples, (2) validation of the Y‐linked markers by their PCR amplification in male and female samples with known sex, and (3) molecular sexing of the masculinized juveniles based on male-specific (XY genotype) and neomale-specific (XX genotype) PCR product band patterns visualized on agarose gel. The validity and concordance of the markers were assessed. The housekeeping gene identified samples with negative PCR amplification revealing a poor DNA quality. The OmyY1 marker presented a more distinctive PCR product band pattern between males and females than the sdY marker and identified a higher proportion of true males (sensitivity = 1.0 and 0.91, respectively). The OmyY1 marker accurately identified 105 neomales of the 198 masculinized individuals on account their consistent and distinctive PCR product band pattern. Among both markers, there was a medium high positive concordance (γ index = 0.7). It is concluded that the OmyY1 marker shows the best performance to reliably detect neomales, a step that is essential to have certified breeders for the production of all-female progenies in fish farming.

对雄性化虹鳟鱼的养殖种群进行 Y 连锁标记（sdY和OmyY1）诊断，旨在在用于生产水平之前检测新雄性。为了实现可靠的诊断，考虑了以下步骤：（1）管家β-肌动蛋白基因的PCR扩增以确定样本的DNA质量，（2）通过在男性和女性中PCR扩增来验证Y连锁标记已知性别的样本，以及（3）基于在琼脂糖凝胶上可视化的雄性特异性（XY基因型）和新雄性特异性（XX基因型）PCR产物条带图案对雄性化幼体进行分子性别鉴定。评估了标记的有效性和一致性。看家基因鉴定出 PCR 扩增呈阴性的样本，表明 DNA 质量较差。与sdY 标记相比，OmyY1标记在男性和女性之间呈现出更独特的 PCR 产物条带模式，并识别出更高比例的真正男性（灵敏度分别为 1.0 和 0.91）。由于其一致且独特的 PCR 产物带模式，OmyY1标记准确地识别了 198 个男性化个体中的 105 个新雄性。在这两个标记中，存在中高阳性一致性（γ 指数 = 0.7）。结论是，OmyY1标记显示出可靠检测新雄性的最佳性能，这是在养鱼业中拥有经过认证的育种者生产全雌性后代的关键步骤。