Meiosis is a specialized type of cell division occurring in sexually reproducing organisms to generate haploid cells known as gametes. In flowering plants, male gametes are produced in anthers, being encased in pollen grains. Understanding the genetic regulation of meiosis key events such as chromosome recognition and pairing, synapsis and recombination, is needed to manipulate chromosome associations for breeding purposes, particularly in important cereal crops like wheat. Reverse transcription-quantitative PCR (RT-qPCR) is widely used to analyse gene expression and to validate the results obtained by other transcriptomic analyses, like RNA-seq. Selection and validation of appropriate reference genes for RT-qPCR normalization is essential to obtain reproducible and accurate expression data. In this work, twelve candidate reference genes were evaluated using the mainstream algorithms geNorm, Normfinder, BestKeeper and ΔCt, then ranked from most to least suitable for normalization with RefFinder. Different sets of reference genes were recommended to normalize gene expression data in anther meiosis of bread and durum wheat, their corresponding genotypes in the absence of the Ph1 locus and for comparative studies among wheat genotypes. Comparisons between meiotic (anthers) and somatic (leaves and roots) wheat tissues were also carried out. To the best of our knowledge, our study provides the first comprehensive list of reference genes for robust RT-qPCR normalization to study differentially expressed genes during male meiosis in wheat in a breeding framework.

减数分裂是有性生殖生物体中发生的一种特殊类型的细胞分裂，以产生称为配子的单倍体细胞。在开花植物中，雄配子在花药中产生，被包裹在花粉粒中。需要了解减数分裂关键事件（例如染色体识别和配对、突触和重组）的遗传调控，以操纵染色体关联以实现育种目的，特别是在小麦等重要谷类作物中。逆转录定量 PCR (RT-qPCR) 广泛用于分析基因表达并验证其他转录组分析（如 RNA-seq）获得的结果。选择和验证用于 RT-qPCR 标准化的适当参考基因对于获得可重复且准确的表达数据至关重要。在这项工作中，使用主流算法 geNorm、Normfinder、BestKeeper 和 ΔCt 评估了 12 个候选参考基因，然后使用 RefFinder 从最适合到最不适合标准化进行排序。建议使用不同组的参考基因来标准化面包和硬粒小麦花药减数分裂中的基因表达数据，以及在缺少Ph1位点的情况下相应的基因型，并用于小麦基因型之间的比较研究。还对减数分裂（花药）和体细胞（叶和根）小麦组织进行了比较。据我们所知，我们的研究提供了第一个全面的参考基因列表，用于稳健的 RT-qPCR 标准化，以在育种框架中研究小麦雄性减数分裂期间的差异表达基因。