Department of Agroecology and Crop Production, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 1176, 165 00, Prague 6, Suchdol, Czech Republic.
Faculty of Forestry and Wood Sciences, EXTEMIT-K and EVA.4.0 Unit, Czech University of Life Sciences, Kamýcká 1176, 165 00, Prague 6, Suchdol, Czech Republic.
Sci Rep. 2021 Jun 28;11(1):13393. doi: 10.1038/s41598-021-92780-1.
Bromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.
硬雀麦是一年生杂草，会导致冬小麦严重减产。这种物种频繁使用除草剂导致了抗除草剂性的出现。必须揭示硬雀麦抗除草剂的机制，因为这个问题正在成为全球性威胁。qRT-PCR 和下一代测序技术可以阐明抗除草剂的机制。尽管 qRT-PCR 可以计算出精确的倍数变化，但它的精确度取决于参考基因的表达。没有任何基因可以作为通用的参考基因，无论在任何特定条件下表达是否稳定。因此，有必要为每个物种识别合适的参考基因。据我们所知，目前还没有关于任何雀麦物种合适的参考基因的报道。因此，在本文中，我们通过 qRT-PCR 实验评估了 8 个基因的稳定性，然后通过最常用的 5 种软件对参考基因选择进行表达稳定性排名，以评估其稳定性。我们的研究结果表明，在硬雀麦中，可以使用 18S rRNA 和 ACCase 组合来归一化 qRT-PCR 数据。此外，还建议针对不同的实验条件选择参考基因。本研究结果将有助于未来在硬雀麦和其他相关草种中的分子研究。
