Transcriptome and Metabolic Analysis of Sorghum Seedlings Exposed to Cadmium Chloride

Based on the physiological results, samples from the 0 and 10 μM CdCl₂ treatments were selected for transcription and metabolic analysis. The roots of the treated and control sorghum seedlings were washed with sterilized distilled water, and were immediately frozen in liquid nitrogen. Total RNA was extracted using the NEBNext^®UltraTM RNA Library Prep Kit for Illumina^®(NEB, USA) following manufacturer’s recommendations. The purity, concentration and integrity of RNA were assessed using a NanoPhotometer^® spectrophotometer (IMPLEN, CA, USA), Qubit^® RNA Assay Kit in Qubit^®2.0 Flurometer (Life Technologies, CA, USA) and RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA), respectively (Ren et al., 2022 (link)). One μg high quality RNA per sample was then used to construct the cDNA library, and was sequenced using an Illumina HiSeq sequencing platform by Metware Biotechnology Co., Ltd. (Wuhan, China). The software fastp v 0.19.3 was used to clean and trim the original data to obtain high-quality clean reads (reads with adapters were filtered out, as were paired reads were the N content exceeded 10% of the base number of the reads, or when the number of low-quality (Q ≤ 20) bases contained in the read exceeds 50% of the bases). Clean data were then mapped to the sorghum reference genome (Sbicolor_454_v3.0.1) using the HISAT v2.1.0 software. The expression abundance of reads was quantified using the fragments per kilobase of transcript per million base pairs (FPKM) value. The differentially expressed genes (DEGs) in the control and treatment groups were screened using DESeq2 v1.22.1 (Ross Ihaka, University of Auckland, New Zealand) with a threshold of |log₂ Fold Change | ≥1 and False Discovery Rate (FDR)< 0.05.