Selection and validation of reference genes for qRT-PCR normalization in dayflower (Commelina communis) based on the...

Dayflower (Commelina communis), a widely invasive weed, thrives well under a variety of abiotic stresses, including drought and herbicides, and harms the growth of crops such as maize and soybean. Gene expression in dayflower is an important but understudied area due to the lack of reliable reference genes.

Fifteen candidate reference genes, which are common reference genes and homologous to those used in other plants, were selected through RNA-seq datasets of dayflower. The expression stability of these screened reference genes was evaluated under three abiotic stresses (drought, herbicide and copper) and in five organs (roots, stems, leaves, flowers and seeds) using five commonly used software programs (geNorm, NormFinder, BestKeeper, ΔCt and RefFinder). The results showed that API5 and SAND had the highest stability in stems, while SAND, EF1A and API5 had the highest stability in roots. Moreover, SAND, ALDH113 and API5 were most stably expressed under copper stress, and EF1A, SAND and API5 were most stable under drought stress. SAND was consistently the most stably expressed gene in both the organs and all samples. Notably, The SAND gene ranked among the top three in terms of stability in all abiotic treatments and in various organs. This result indicates that the SAND gene is suitable for qRT-PCR experimentation in diverse tissues and under multiple (drought, herbicide and copper) abiotic stress conditions in dayflower.

This study identified the most stably expressed reference genes under three abiotic stresses and in five organs of dayflower, and SAND showed high expression stability under various experimental conditions, making it a reliable reference gene for gene expression analysis experiments under different conditions in dayflower. This study will enhance the precision of the qRT-PCR quantification of candidate genes related to the adaptation significance of dayflower.