RESUMEN
Studies of gene expression are very important for the identification of genes that participate in different biological processes. Currently, reverse transcription quantitative real-time PCR (RT-qPCR) is a high-throughput, sensitive and widely used method for gene expression analysis. Nevertheless, RT-qPCR requires precise normalisation of data to avoid the misinterpretation of experimental data. In this sense, the selection of reference genes is critical for gene expression analysis. At this time, several studies focus on the selection of reference genes in several species. However, the identification and validation of reference genes for the normalisation of RT-qPCR have not been described in amaranth. A set of seven housekeeping genes were analysed using RT-qPCR, to determine the most stable reference genes in amaranth for normalisation of gene expression analysis. Transcript stability and gene expression level of candidate reference genes were analysed in different tissues, at different developmental stages and under different types of stress. The data were compared using the geNorm, NormFinder and Bestkeeper statistical methods. The reference genes optimum for normalisation of data varied with respect to treatment. The results indicate that AhyMDH, AhyGAPDH, AhyEF-1α and AhyACT would be optimum for accurate normalisation of experimental data, when all treatment are analysed in the same experiment. This study presents the most stable reference genes for normalisation of gene expression analysis in amaranth, which will contribute significantly to future gene studies of this species.
Asunto(s)
Amaranthus/genética , Proteínas de Plantas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Algoritmos , Cartilla de ADN , Regulación de la Expresión Génica de las Plantas , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , TranscriptomaRESUMEN
Agricultural application of wastewater sludge has become the most widespread method of disposal, but the environmental effects on soil, air, and crops must be considered. The effect of wastewater sludge or urea on sunflower's (Helianthus annuus L.) growth and yield, the soil properties, and the resulting CO(2) and N(2)O emissions are still unknown. The objectives of this study were to investigate: i) the effect on soil properties of organic or inorganic fertilizer added to agricultural soil cultivated with sunflower, ii) how urea or wastewater sludge increases CO(2) and N(2)O emissions from agricultural soil over short time periods, and iii) the effect on plant characteristics and yield of urea or wastewater sludge added to agricultural soil cultivated with sunflower. The sunflower was fertilized with wastewater sludge or urea or grown in unamended soil under greenhouse conditions while plant and soil characteristics, yield, and greenhouse gas emissions were monitored. Sludge and urea modified some soil characteristics at the onset of the experiment and during the first two months but not thereafter. Some plant characteristics were improved by sludge. Urea and sludge treatments increased the yield at similar rates, while sludge-amended soil significantly increased N(2)O emissions but not CO(2) emissions compared to the other amended or unamended soils. This implies that wastewater sludge increased the biomass and/or the yield; however, from a holistic point of view, using wastewater sludge as fertilizer should be viewed with concern.