Recalcitrant plant total rna extraction kit

Equal amounts of frozen roots from five excess B-treated or control seedlings (one seedling per pot) of C. grandis or C. sinensis were mixed as a biological replicate. There were three biological replicates for each B treatment. Total RNA was extracted from ca. 300 mg of frozen roots using Recalcitrant Plant Total RNA Extraction Kit (Centrifugal column type, Bioteke Corporation, China). cDNA synthesis and cDNA-AFLP analysis were performed according to Guo et al. (2014 (link)) and Lu et al. (2015 (link)). Database resources of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) were used for the blast analysis of the differentially expressed transcript-derived fragments (TDFs) from excess B-treated C. grandis and C. sinensis roots. TDFs were blasted using BLASTX and BLASTN search engines (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and a TDF was considered to be homologous when it had an E < 0.001 and a similarity ≥50%. Their functional categories were assigned based on the Gene Ontology (http://amigo1.geneontology.org/cgi-bin/amigo/go.cgi) and UniProt (http://www.uniprot.org/) database.

Total RNA was extracted from ~200 mg of frozen leaves mixed equally from four plants (one plant pot⁻¹) using Recalcitrant Plant Total RNA Extraction Kit (Bioteke Corporation, Beijing, China). There were three biological replicates per treatment. A total of six sequencing libraries were constructed according to Guo et al. [37 (link),38 (link)] and sequenced on Illumina HiSeq platform (Illumina Inc., San Diego, CA, USA) at Wuhan MetWare Biotechnology Co., Ltd. (www.metware.cn, accessed on 1 March 2021). The raw transcriptome data have been deposited in NCBI SRA database (accession number: PRJNA702620).

About 300 mg of frozen leaves collected equally from six pots (one plant per pot, one leaf per plant) were mixed as a biological replicate. There were three biological replicates for each treatment. Total RNAs were independently extracted thrice from +Al and control frozen leaves using the Recalcitrant Plant Total RNA Extraction Kit (Centrifugal column type, Bioteke Corporation, Beijing, China) according to the manufacturer’s instructions. Gene-specific primers were designed using Primer Software Version 5.0 (PREMIER Biosoft International, Palo Alto, CA, USA) according to the corresponding sequences of selected proteins in the citrus genome (http://www.phytozome.net/cgi-bin/gbrowse/citrus/). The sequences of the F and R primers used are given in Table S3. qRT-PCR was performed according to Zhou et al. [88 (link)]. Each sample was run in two technical replicates. For the normalization of gene expression, citrus actin (GU911361.1) was used as an internal standard, and the leaves from control plants were used as the reference sample, which was set to 1.