Date Published: February 1, 2018
Publisher: Public Library of Science
Author(s): Izabela Ruduś, Jan Kępczyński, Gayle E. Woloschak.
Molecular studies of primary and secondary dormancy in Avena fatua L., a serious weed of cereal and other crops, are intended to reveal the species-specific details of underlying molecular mechanisms which in turn may be useable in weed management. Among others, quantitative real-time PCR (RT-qPCR) data of comparative gene expression analysis may give some insight into the involvement of particular wild oat genes in dormancy release, maintenance or induction by unfavorable conditions. To assure obtaining biologically significant results using this method, the expression stability of selected candidate reference genes in different data subsets was evaluated using four statistical algorithms i.e. geNorm, NormFinder, Best Keeper and ΔCt method. Although some discrepancies in their ranking outputs were noticed, evidently two ubiquitin-conjugating enzyme homologs, AfUBC1 and AfUBC2, as well as one homolog of glyceraldehyde 3-phosphate dehydrogenase AfGAPDH1 and TATA-binding protein AfTBP2 appeared as more stably expressed than AfEF1a (translation elongation factor 1α), AfGAPDH2 or the least stable α-tubulin homolog AfTUA1 in caryopses and seedlings of A. fatua. Gene expression analysis of a dormancy-related wild oat transcription factor VIVIPAROUS1 (AfVP1) allowed for a validation of candidate reference genes performance. Based on the obtained results it can be recommended that the normalization factor calculated as a geometric mean of Cq values of AfUBC1, AfUBC2 and AfGAPDH1 would be optimal for RT-qPCR results normalization in the experiments comprising A. fatua caryopses of different dormancy status.
Seed dormancy is an innate seed property responsible for the temporal suppression of germination despite suitable conditions [1, 2]. Still on the mother plant, seeds acquire a state of primary dormancy which after shedding, depending on external factors (e.g. temperature, light, different chemicals), decreases gradually or is abruptly lost to allow seed germination . Seeds of many plant species require variable periods of dry after-ripening or moist chilling to become non-dormant. Either in primary dormant seeds or seeds that fully or partially lost dormancy after dispersal or harvest, secondary (induced) dormancy may be imposed by unfavorable environmental cues [3, 4]. The physiological phenomenon of seed dormancy and its alterations are widely recognized to have both ecological and agricultural significance [5, 6].
Comparative studies of the dormancy-related gene expression in A. fatua caryopses of different dormancy status, employing a powerful method of RT-qPCR, may provide insights into molecular mechanisms of the dormancy induction, maintenance and release as well as germination process per se. Therefore, several experiments were planned and performed to obtain reliable results which would facilitate further work on the expression profiles of the genes of interest involved in those physiological phenomena in A. fatua. Although there are several publications dedicated to reference genes used in seed biology studies [40, 47, 48, 58, 59, 60], to our knowledge, this is the first report concerning reference gene selection and validation for that specific developmental stage of A. fatua life cycle comprising dormant and germinating caryopses.
From our findings it can be concluded that the normalization factor calculated as a geometric mean of Cq values of two UBC homologs accompanied by AfGAPDH1 would be optimal for RT-qPCR results normalization in the experiments comprising A. fatua caryopses of different dormancy status. This conclusion remains in agreement with the approach proposed by Vandesompele et al.  and widely considered as the most appropriate in gene expression studies. Based on the obtained results it would be advisable to use AfUBC1, AfUBC2 and AfGAPDH1 as reference genes also in further studies on molecular basis of seed dormancy induction, maintenance, and release in A. fatua caryopses on condition of prior validation.