AbstractBird sex determination is fundamental in various ecological and biological studies, although many avian species cannot be sexed visually due to their monomorphic and/or monochromatic appearance. Thus, reliable laboratory methods for sexing are a prerequisite. Most avian nestlings lack sex-related signs, including the Eurasian pygmy owl (Glaucidium passerinum).

摘要鸟类性别决定是各种生态和生物学研究的基础，尽管许多鸟类由于其单态和/或单色外观而无法通过视觉进行性别鉴定。因此，可靠的性别鉴定实验室方法是先决条件。大多数鸟类雏鸟缺乏与性别有关的体征，包括欧亚侏儒猫头鹰（Glaucidium passerinum）。

We performed laboratory sex determination analysis of this species using blood samples of 242 juveniles and nine adults. It relied on the qPCR of the specific intron from the chromo-helicase DNA-binding protein 1 gene. We tested three primer sets, the P2/P8, 2550F/2718R, and CHD1F/CHD1R, commonly used for bird laboratory sexing.

我们使用242名青少年和9名成年人的血液样本对该物种进行了实验室性别测定分析。它依赖于来自染色体解旋酶DNA结合蛋白1基因的特定内含子的qPCR。我们测试了三个引物组，P2/P8、2550F/2718R和CHD1F/CHD1R，它们通常用于鸟类实验室性别鉴定。

The outcomes were displayed on an agarose gel electrophoresis and a plot from melt curve analysis, which had not been previously conducted in Eurasian pygmy owls. We found that only primer set CHD1F/CHD1R proved reliable, as the only one determined sex with one and two band/s and peak/s on the electrophoresis and the melt curve plot for males and females, respectively.

结果显示在琼脂糖凝胶电泳和熔解曲线分析图上，这是以前在欧亚侏儒猫头鹰中没有进行过的。我们发现，只有引物组CHD1F/CHD1R被证明是可靠的，因为唯一一个在电泳和男性和女性的熔解曲线图上分别具有一个和两个条带/s和峰/s的性别。

The other two primer pairs failed and depicted one band/peak in all specimens regardless of their sex. Therefore, we recommend performing Eurasian pygmy owls’ laboratory sexing by qPCR with CHD1F/CHD1R primers only..

另外两个引物对失败，并且在所有标本中描绘了一个条带/峰，无论其性别如何。因此，我们建议仅使用CHD1F/CHD1R引物通过qPCR进行欧亚侏儒猫头鹰的实验室性别鉴定。。

IntroductionMethods of sex determination based on morphological features are unreliable in monomorphic and monochromatic birds and virtually impossible for juvenile ones1,2,3. This difficulty arises from the minimal sexual dimorphism in many avian species. In birds of prey (owls and diurnal raptors), adult females are usually larger than males and can be sexed during the breeding season because only females incubate and have a brood patch4,5.

引言基于形态特征的性别决定方法在单形和单色鸟类中不可靠，而对于幼鸟则几乎不可能1,2,3。这种困难源于许多鸟类的最小性二态性。在猛禽（猫头鹰和昼夜猛禽）中，成年雌性通常比雄性大，并且可以在繁殖季节进行性别鉴定，因为只有雌性孵化并有一个育雏补丁4,5。

Newly-hatched and half-grown nestlings of birds of prey do not usually show any sex-related physical characteristics and usually cannot be properly sexed based on body size measurements only6,7. However, identifying the sex of newly-hatched chicks holds significant importance in ecological and evolutionary studies, including, for instance, parental sex allocation8,9, sex-specific immune responses10, sex ratio evolution and mating systems11,12,13, research on the evolution of sexual size dimorphism14,15,16, as well as breeding and conservation management17,18,19,20.Nowadays, molecular methods for sex identification are commonly used.

新孵化和半生长的猛禽雏鸟通常不显示任何与性别有关的身体特征，通常只能根据体型测量来正确区分性别6,7。然而，鉴定新孵化雏鸡的性别在生态和进化研究中具有重要意义，包括父母性别分配8,9，性别特异性免疫反应10，性别比进化和交配系统11,12,13，性大小二态性进化研究14,15,16，以及育种和保护管理17,18,19,20。目前，性别鉴定的分子方法是常用的。

The principle of laboratory procedures for bird sexing often hinges on variations in the intron lengths of the conserved chromo-helicase DNA-binding protein 1 (CHD1) gene between the sex chromosomes Z and W. However, methods employing only Z-specific genes have also been developed21. These patterns are applicable to most avian species21,22,23,24.

鸟类性别鉴定的实验室程序的原理通常取决于性染色体Z和W之间保守的染色体解旋酶DNA结合蛋白1（CHD1）基因内含子长度的变化。然而，仅使用Z特异性基因的方法也已被开发21。这些模式适用于大多数鸟类21,22,23,24。

Different protocols are applied to the ratites (e.g., ostriches—Struthioniformes), in which the Z and W chromosomes are essentially homomorphic, i.e., identical in size21,25,26. Primer sets specific to the CHD1 gene have been independently developed, with the ones most frequently utilised being P2/P827, 2550F/2718R28, and CHD1F/CHD1R29.

不同的方案适用于比率（例如，鸵鸟Struthioniformes），其中Z和W染色体基本上是同态的，即大小相同21,25,26。CHD1基因特异的引物组已经独立开发，最常用的是P2/P827、2550F/2718R28和CHD1F/CHD1R29。

In addition, advanced te.

此外，高级te。

Table 1 The names and sequences of the individual primers utilised within this study.Full size tableSix samples (three males and three females) were sequenced using all three primer pairs. If only one band was present in the gel, the amplicon was directly purified by Monarch® PCR & DNA Cleanup Kit. In the case of female samples with two bands in the gel, each DNA fragment was initially extracted from the gel, followed by DNA purification using the Monarch® DNA Gel Extraction Kit.

表1本研究中使用的单个引物的名称和序列。使用所有三对引物对全尺寸tableSix样品（三只雄性和三只雌性）进行测序。如果凝胶中只有一条带，则通过Monarch®PCR＆DNA Cleanup Kit直接纯化扩增子。对于凝胶中有两条带的雌性样品，首先从凝胶中提取每个DNA片段，然后使用Monarch®DNA凝胶提取试剂盒进行DNA纯化。

SEQme Ltd., Czechia, sequenced the purified products. The obtained sequences were identified by the Basic Local Alignment Search Tool (hereafter BLAST) in the NCBI.ResultsMelt curve analysis of qPCR products of CHD1F/CHD1R primers depicted one peak at temperature 83 °C for males and two peaks at temperatures 83 °C and 86 °C for females (Fig. 1).

捷克SEQme有限公司对纯化产物进行了测序。通过NCBI中的基本局部比对搜索工具（以下简称BLAST）鉴定获得的序列。结果CHD1F/CHD1R引物的qPCR产物的熔解曲线分析显示，雄性在83°C温度下有一个峰，雌性在83°C和86°C温度下有两个峰（图1）。

Likewise, qPCR products of the same primer set displayed on 3% agarose gel electrophoresis both the one band for males (CHD1Z fragment long ∼ 500 bp—base pair) and two bands for females (CHD1Z fragment and CHD1W fragment long ∼ 900 bp) (Fig. 2A; Supplementary Fig. 1). In the case of the P2/P8, melt curve analysis of qPCR products depicted one peak at the temperature 81.5 °C for all specimens only (Supplementary Fig. 2).

同样，相同引物组的qPCR产物在3%琼脂糖凝胶电泳上显示出雄性的一条带（CHD1Z片段长约500 bp碱基对）和雌性的两条带（CHD1Z片段和CHD1W片段长约900 bp）（图2A；补充图1）。在P2/P8的情况下，qPCR产物的熔融曲线分析仅描绘了所有样品在81.5°C温度下的一个峰（补充图2）。

Furthermore, a single band was displayed on the 3% agarose gel electrophoresis only, showing a fragment of ∼ 350 bp for all specimens—both males and females (Fig. 2B; Supplementary Fig. 3). Primer set 2550F/2718R yielded similar results as the previous primer, except that melt curve analysis depicted one peak at the temperature of 83 °C for both males and females (Supplementary Fig. 4) and a single band on the electrophoresis showed a fragment of ∼ 630 bp (Fig.

此外，仅在3%琼脂糖凝胶电泳上显示一条带，显示所有男性和女性标本的约350 bp片段（图2B；补充图3）。引物组2550F/2718R产生了与先前引物相似的结果，除了熔解曲线分析显示雄性和雌性在83°C的温度下都有一个峰（补充图4），电泳上的一条带显示了约630 bp的片段（图4）。

2C; Supplementary Fig. 3). In total, 242 blood samples of nestlings were sexe.

2C；补充图3）。总共有242份雏鸟的血液样本是性别的。

Table 2 Identification of different quantitative polymerase chain reaction (qPCR) products after Sanger sequencing by the Basic Local Alignment Search Tool (BLAST) using the National Centre for Biotechnology Information (NCBI) database. The table presents the lengths of the DNA fragments CHD1Z and CHD1W on the chromo-helicase DNA-binding protein 1 (CHD1) gene from Z and W chromosomes produced by three sets of primers for males (M) and females (F).

表2使用国家生物技术信息中心（NCBI）数据库，通过基本局部比对搜索工具（BLAST）对桑格测序后的不同定量聚合酶链反应（qPCR）产物进行鉴定。该表显示了来自Z和W染色体的染色体解旋酶DNA结合蛋白1（CHD1）基因上的DNA片段CHD1Z和CHD1W的长度，该基因由三组引物产生于雄性（M）和雌性（F）。

The fragment lengths were obtained from sequencing after quality filtering and are stated in base pairs (bp). Further, the owl species and the accession number of the identified sequences with the highest similarity percentages (per cent identity) of the CHD1 gene using the BLAST in the NCBI database are represented.

片段长度是通过质量过滤后的测序获得的，并以碱基对（bp）表示。此外，还表示了使用NCBI数据库中的BLAST对CHD1基因具有最高相似百分比（同一性百分比）的已鉴定序列的猫头鹰种类和登录号。

All sequences identified as CHD gene from different owl species belong to the family of Strigidae.Full size tableDiscussionOur molecular analyses showed that out of the three primer sets employed, only the CHD1F/CHD1R primers provided reliable results. The primers CHD1F/CHD1R depicted one peak for males and two for females using melt curve analysis and showed one band for males and two bands for females on agarose gel electrophoresis.

从不同猫头鹰物种中鉴定为CHD基因的所有序列都属于Strigidae家族。全尺寸表讨论我们的分子分析表明，在所使用的三个引物组中，只有CHD1F/CHD1R引物提供了可靠的结果。引物CHD1F/CHD1R使用熔解曲线分析描绘了雄性的一个峰和雌性的两个峰，并且在琼脂糖凝胶电泳上显示了雄性的一条带和雌性的两条带。

Samples of adults of known sex confirmed the accuracy of the primers. The other two tested primer sets, the P2/P8 and 2550F/2718R, displayed only one peak/band in all specimens, regardless of the tested individual’s sex.Although the identification of PO sex using primers P2/P8 and 2550F/2718R remained unachievable, many other studies commonly used these primers and even described them as offering the broadest possible coverage for various Neognathae species35,37,38,42.

已知性别的成年人样本证实了引物的准确性。另外两个测试的引物组P2/P8和2550F/2718R在所有样本中仅显示一个峰/带，而与测试个体的性别无关。尽管使用引物P2/P8和2550F/2718R鉴定PO性别仍然无法实现，但许多其他研究通常使用这些引物，甚至将其描述为为为各种Neognathae物种提供尽可能广泛的覆盖范围35,37,38,42。

Our sequencing results showed that only CHD1Z was amplified employing 2550F/2718R in both PO males and females. We s.

我们的测序结果表明，在PO雄性和雌性中，仅使用2550F/2718R扩增了CHD1Z。我们s。

Data availability

数据可用性

The raw data supporting the conclusions of this article will be made available by the authors without undue reservation. The sequences obtained in this investigation have been deposited into the NCBI GenBank database under accession numbers PP391271 and PP391272.

作者将毫无保留地提供支持本文结论的原始数据。在这项研究中获得的序列已保存在NCBI GenBank数据库中，登录号为PP391271和PP391272。

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Download referencesAcknowledgementsWe thank Rauno Varjonen, Chiara Morosinotto and Jorma Nurmi for assisting in collecting blood samples of Eurasian pygmy owls in the Kauhava study area. We further thank Petr Kouba for his help with the graphic design of the figures. The research project was supported by two grants provided to MK by the Regional Fund of the South Ostrobothnia of the Finnish Cultural Foundation (Business ID 0116947-3 and 10201775).Author informationAuthors and AffiliationsDepartment of Ethology and Companion Animal Science, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 129, Praha 6 – Suchdol, 165 00, CzechiaSimona Stehlíková Sovadinová & Marek KoubaLaboratory of Anaerobic Microbiology, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Vídeňská 1083, Praha 4 – Krč, 142 20, CzechiaChahrazed Mekadim & Jakub MrázekSection of Ecology, Department of Biology, University of Turku, Turku, 20014, FinlandErkki KorpimäkiDepartment of Game Management and Wildlife Biology, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences Prague, Kámycká 129, Praha 6 – Suchdol, 165 00, CzechiaMarek KoubaAuthorsSimona Stehlíková SovadinováView author publicationsYou can also search for this author in.

下载参考文献致谢我们感谢Rauno Varjonen，Chiara Morosinotto和Jorma Nurmi在考哈瓦研究区协助收集欧亚侏儒猫头鹰的血液样本。我们进一步感谢Petr Kouba在图形设计方面的帮助。该研究项目得到了芬兰文化基金会南奥斯特罗博尼亚区域基金会向MK提供的两项资助（商业ID 0116947-3和10201775）。作者信息作者和附属机构捷克生命科学大学布拉格农业生物学、食品和自然资源学院行为学和伴生动物科学系，卡姆切卡129，普拉哈6–苏赫多尔，165 00，捷克科学院动物生理学和遗传学研究所无氧微生物学实验室，捷克科学院动物生理学和遗传学研究所，维德切斯克1083，普拉哈4–Krč，142 20，CzechiaChahrazed Mekadim&Jakub Mrázekssection of Ecology，Turku大学生物系，20014年，捷克生命科学大学布拉格林业与木材科学学院芬兰德尔基·科尔皮姆·基德（FinlandErkki KorpimäkiDepartment of Game Management and Wildlife Biology），卡拉格（Kámycká129），普拉哈（Praha 6–Suchdol），165 00，捷克共和国库巴（CzechiaMarek KoubauthorsSimona StehlíkováSovadinováView）作者出版物你也可以在中搜索这位作者。

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PubMed Google ScholarContributionsS.S.S. wrote the main manuscript text; M.K., C.M., and E.K. participated in the writing and editing of the text. S.S.S. performed laboratory analyses (isolation DNA, qPCR, sequencing, etc.) with training and essential assistance from C.M. and supervision by J.M.

PubMed谷歌学术贡献。S、 美国写了主要的手稿文本；M、 K.，C.M。和E.K.参与了文本的撰写和编辑。S、 S.S.在C.M.的培训和必要协助以及J.M.的监督下进行了实验室分析（分离DNA，qPCR，测序等）。

E.K. collected blood samples within the study area. M.K. created the graphic design of the figures. All authors reviewed the manuscript and approved the submitted version.Corresponding authorCorrespondence to.

E、 K.在研究区域内收集血液样本。M、 K.创造了人物的平面设计。所有作者都审阅了稿件并批准了提交的版本。对应作者对应。

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Reprints and permissionsAbout this articleCite this articleStehlíková Sovadinová, S., Mekadim, C., Korpimäki, E. et al. Comparison three primer pairs for molecular sex determination in Eurasian pygmy owls (Glaucidium passerinum).

转载和许可本文引用本文StehlíkováSovadinová，S.，Mekadim，C.，Korpimäki，E。等人比较了三对引物对欧亚侏儒猫头鹰（Glaucidium passerinum）的分子性别决定。

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Keywords2550F/2718RBirds of preyCHD1F/CHD1RLaboratory sexingP2/P8Sequencing

preyCHD1F/CHD1Laboratory sexingP2/P8测序的关键词2550F/2718RBirds

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