Abstract
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.
Highlights
Bromus sterilis is an annual weedy grass, causing high yield losses in winter cereals
With the development of next-generation sequencing technologies, there is a need to validate the expression of a greater number of genes involved in abiotic stresses7,8. qRT-polymerase chain reaction (PCR) is widely used for such comparative gene expression studies
The A260/A280 values ranged from 1.90 to 2.05. These samples were further used to synthesise complementary DNA (cDNA), which was used for the quantitative real-time polymerase chain reaction (qRT-PCR) experiments
Summary
Bromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. The most commonly used references genes for normalisation of plant gene expression studies are ubiquitin (UBQ), β-tubulin (β-TUB), ribosomal RNA genes (18S rRNA and 25S rRNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic elongation factor (eEF), eukaryotic translation initiation factor 1 (eIF1), actin (ACT), acetyl-CoA carboxylase (ACCase) etc[9,18] These genes are known to have a stable expression in any given condition, several studies documented variability in their expression level between species of plants or different stress conditions or developmental stages[19,20,21]. We had selected eight common candidate reference genes (UBQ, ACT,GAPDH, 18S rRNA, 25S rRNA, ACCase, β-TUB and eEF) identified in B. sterilis and evaluated the stability of their gene expression in three developmental stages (two-leaves, three-leaves and four-leaves), two different plant organs (shoots and leaves) and one abiotic stress (drought stress). Global warming has resulted in an increase of air temperature and evapotranspiration, leading to agricultural droughts, affecting both crops and weeds
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