已知有几种基于 PCR 的方法可用于鸡和其他鸟类的性别鉴定（Çakmak 等人，2017 年；Eiras 等人，2018 年；Gruszczyńska & Grzegrzółka，2021 年；Morinha 等人，2012 年）。虽然一些鸟类性别鉴定方法适用于大规模分析（Chen 等人，2012 年；Clinton 等人，2016 年；He 等人，2019 年；Margulis 和 Danielli，2019 年；Morinha 等人，2013 年；Rosenthal 等人）等，2010)，研究仍在进行中，因为这些方法中的大多数都是昂贵且耗时的。在这里，我们报告了一种新开发的、易于使用的竞争性等位基因特异性 PCR (KASP) 测定法，适用于鸡和其他鸟类的大规模性别鉴定。KASP 测定基于外显子 17 中保守的染色质结构域解旋酶 DNA 结合蛋白 1 ( CHD1 ) 的 W 和 Z 染色体变体之间外显子 17 的 A/G 差异（图 S1）。在CHD1基因中这种变体的上游和下游设计了性别特异性引物，以扩增 46 bp 的产物（图 1；表 S1，图 S1）。扩增子与 PCR 产物重叠，用于 Fridolfsson 和 Ellegren 方法的性别基因分型（1999）（图S1）。此外，使用blast （https://blast.ncbi.nlm.nih.gov/Blast.cgi）从NCBI数据库中获得了鸭、鹅、鹌鹑和火鸡的相似序列，并与鸡序列对齐（图1）。

总共分析了 734 个已知性别的鸡样本（表 S2）。此外，还分析了鸡形目和雁形目其他物种的 55 个样本（表 S2）。如果非鸡样本的性别未知，则通过从 Fridolfsson 和 Ellegren ( 1999 ) 修改的多重 PCR 进行验证（表 S1，图 S1）。样本主要来自广泛的 DNA 收集，该收集是在 AVIANDIV（Lyimo 等人，2014 年）和 SYNBREED（www.synbreed.tum.de）项目的框架内建立的。

每个 KASP 反应包含 20–50 ng 模板 DNA、KASP v. 4.0 2× Master mix 标准 ROX 和 KASP-by-Design 分析混合物 (LGC Genomics)。根据 LGC 协议的标准 KASP 热循环条件在 Eppendorf Mastercycler (Eppendorf) 中进行。扩增后，用 FLUOstar Omega (BMG Labtech) 分析微孔板，对于 FAM 标记的 FRET 盒，使用 485/520 nm 的激发和发射值，对于 HEX 标记的 FRET 盒，530/560 nm 和 584/620 nm ROX 标准。

所有鸡、鸭、鹅、鹌鹑和火鸡都使用新开发的 KASP 测定法正确分配了它们的性别（表 S2）。这种新开发的 KASP 检测方法非常适用于鸡、鸭、鹅、鹌鹑和火鸡，可在更大范围内进行有效的性别测定。

"点击查看英文标题和摘要"

Several PCR-based methods are known for sexing of chickens and other birds (Çakmak et al., 2017; Eiras et al., 2018; Gruszczyńska & Grzegrzółka, 2021; Morinha et al., 2012). While some methods for bird sexing are suitable for large-scale analyses (Chen et al., 2012; Clinton et al., 2016; He et al., 2019; Margulis & Danielli, 2019; Morinha et al., 2013; Rosenthal et al., 2010), research is still ongoing because most of these methods are costly and time consuming. Here we report a newly developed, easy to use competitive allele-specific PCR (KASP) assay that is suitable for large-scale sexing in chickens and other birds. The KASP assay is based on an A/G difference in exon 17 between the W- and Z-chromosomal variants of the conserved chromodomain helicase DNA binding protein 1 (CHD1) in exon 17 (Figure S1). Sex-specific primers were designed up- and downstream of this variant in CHD1 genes to amplify a product of 46 bp (Figure 1; Table S1, Figure S1). The amplicon overlaps with the PCR product for sex genotyping of the method of Fridolfsson and Ellegren (1999) (Figure S1). Furthermore, similar sequences were obtained from NCBI databases using blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for duck, goose, quail and turkey and were aligned with the chicken sequence (Figure 1).

In total, 734 chicken samples with known sex were analysed (Table S2). In addition, 55 samples of other species of the orders Galliformes and Anseriformes were analysed (Table S2). If the sex of non-chicken samples was unknown it was verified by multiplex PCR modified from Fridolfsson and Ellegren (1999) (Table S1, Figure S1). Samples were mainly taken from an extensive DNA collection, which was set up within the framework of the projects AVIANDIV (Lyimo et al., 2014) and SYNBREED (www.synbreed.tum.de).

Each KASP reaction contained 20–50 ng of template DNA, KASP v. 4.0 2× Master mix standard ROX and the KASP-by-Design assay mix (LGC Genomics). The standard KASP thermal cycling conditions according to LGC protocols were performed in an Eppendorf Mastercycler (Eppendorf). After amplification, microplates were analysed with FLUOstar Omega (BMG Labtech) using excitation and emission values of 485/520 nm for the FAM-labelled-FRET cassette, 530/560 nm for the HEX-labelled-FRET cassette and 584/620 nm for the ROX standard.

All chickens, ducks, geese, quails and turkeys were correctly assigned to their sex using the newly developed KASP assay (Table S2). This newly developed KASP assay is well suited to chicken, duck, goose, quail and turkey for efficient sex determination on a larger scale.