A Quantitative Polymerase Chain Reaction Protocol for Sex Identification of Zebra Finch Embryos Using Blood Samples

Investigating sex differences in avian developmental biology research is increasingly relevant for clinical and ecology-based studies. Songbirds like zebra finches (Taeniopygia guttata), like most birds, are not sexually dimorphic at hatching. Their plumage is indistinguishable as embryos, complicating experiments where sex is not yet visually evident, but biological differences between sexes are expected (e.g., gene expression studies). The current methods for zebra finch embryo genetic sex identification where only a single W locus in analyzed are prone to false identification because of genetic variation or technical failure of amplification. As a result, any further validation steps of the amplified product such as gel electrophoresis adds additional steps, only to receive inaccurate results. Thus, there is a need for an efficient and accurate method for time-sensitive developmental and behavioral studies. Here, we developed an approach that targets two different W loci, using SYBR-based quantitative PCR to analyze amplification curves. We applied this method to determine sex of 30 zebra finch embryos (embryonic day 13) and subsequently confirmed genetic sex in all cases by brain transcriptome sequencing. Using this multi-marker approach, we also identified primer sets that are effective for sex determination in chickens. Overall, our findings contribute to the field of avian development biology by offering an efficient and accurate method for genetic sex determination. The CSV files attached have (1) qPCR DNA Absorbance (A260/280 ratios) and DNA Yield (ng/µL) data from E13 zebra finch embryo's trunk blood, and (2) raw data from the qPCR quantification amplification and melt curve analyses.

在鸟类发育生物学研究中探究性别差异，对临床研究与基于生态学的研究而言愈发具有重要意义。斑胸草雀（Taeniopygia guttata）这类鸣禽，与多数鸟类一样，孵化时并无性别二态性。其胚胎阶段的羽毛无法区分性别，这使得在性别尚无法通过视觉辨别、但已预期存在两性生物学差异（如基因表达研究）的实验中，研究流程变得复杂。
当前用于斑胸草雀胚胎遗传性别鉴定的方法多仅分析单个W位点，由于遗传变异或扩增技术失败，这类方法极易出现鉴定错误。为此，研究人员往往需额外增设凝胶电泳等扩增产物验证步骤，却仍难以获得准确结果。因此，针对时效性较强的发育与行为研究，亟需一种高效且准确的鉴定方法。
本研究开发了一种靶向两个不同W位点的方法，采用基于SYBR的定量PCR（qPCR）分析扩增曲线。我们运用该方法对30枚胚胎发育第13天的斑胸草雀胚胎进行了性别鉴定，并通过大脑转录组测序验证了所有样本的遗传性别。
借助这种多标记物方法，我们还筛选出了可有效用于鸡性别鉴定的引物套装。总体而言，本研究提供了一种高效准确的遗传性别鉴定方法，为鸟类发育生物学领域的研究作出了贡献。
附件中的CSV文件包含两类数据：(1) 胚胎发育第13天斑胸草雀躯干血液的qPCR DNA吸光度（A260/280比值）与DNA产量（ng/µL）数据；(2) qPCR定量扩增与熔解曲线分析的原始数据。