Plant Breeding Department, Institute for Sustainable Agriculture, Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC), Alameda del Obispo s/n, Apartado 4084, 14080, Córdoba, Spain.
Área de Fisiología Vegetal. Universidad de Córdoba. Campus de Rabanales, edif. C4, 3ª planta, Córdoba, Spain.
Sci Rep. 2020 Feb 17;10(1):2726. doi: 10.1038/s41598-020-59580-5.
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.
减数分裂是一种在有性繁殖生物中发生的特殊类型的细胞分裂，用于产生称为配子的单倍体细胞。在开花植物中，雄性配子在花药中产生，被花粉粒包裹。为了进行育种目的操纵染色体的关联，需要理解减数分裂关键事件的遗传调控，如染色体识别和配对、联会和重组。在重要的谷类作物如小麦中，这一点尤其重要。逆转录定量 PCR（RT-qPCR）广泛用于分析基因表达，并验证 RNA-seq 等其他转录组分析获得的结果。选择和验证 RT-qPCR 标准化的合适参考基因对于获得可重复和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 评估了十二个候选参考基因，然后使用 RefFinder 从最适合到最不适合的顺序进行排序。推荐了不同的参考基因集，用于标准化面包和硬质小麦花药减数分裂、缺少 Ph1 基因座的基因型以及小麦基因型之间的比较研究中的基因表达数据。还对减数分裂（花药）和体细胞（叶片和根系）小麦组织之间进行了比较。据我们所知，我们的研究提供了第一个用于在小麦雄性减数分裂中研究差异表达基因的稳健 RT-qPCR 标准化的综合参考基因列表，这是在育种框架下进行的。
