Quantitative Analysis of Citrus Root Transcripts

Root tips of six plants from different pots were mixed as a biological replicate. Equal amounts of root tips were collected from each plant. There were three biological replicates for each treatment. Total RNAs were independently extracted three times from the frozen roots of Al-toxic and control plants using Recalcirtant Plant Total RNA Extraction Kit (Centrifugal column type, Bioteke Corporation, China) according to manufacturer’s instructions. Gene-specific primers were designed using Primer Software Version 5.0 (PREMIER Biosoft International, CA, USA) according to the corresponding sequences of selected proteins in Citrus genome (http://www.phytozome.net/cgi-bin/gbrowse/citrus/). The sequences of the F and R primers used are given in Additional file 3. qRT-PCR analysis was performed according to Zhou et al. [70 (link)]. For the normalization of gene expression, β-tubulin (JN580571) gene was used as an internal standard and the roots from control plants were used as reference sample, which was set to 1.