Leaf RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and RNA sequencing was performed in Novogene (Tianjin, China). A total of 18 libraries (2 cultivars × 3 treatments × 3 biological replicates) were sequenced on the Illumina platform (HiSeq 6000), which produced paired-end reads of 150-nucleotide. The adapter sequences and low-quality bases were removed, and the clean data was de novo assembled using the Trinity platform (Trinity) [50 (link)].
The Expectation Maximization (RSEM) was used to map the transcripts to reference unigenes via RNA-seq. The read count numbers were calculated and translated to FPKM (fragments per kilobase of transcript per million mapped reads) gene values [51 (link)]. DESeq was used for analysing differentially expressed genes (DEGs) [52 (link)]. We used FDR ≤ 0.05 and |log2Fold > Change| ≥ 1 as threshold for screening DEGs. All identified DEGs were mapped to gene ontology (GO) and Tokyo Encyclopedia of Genes and Genomes (KEGG) databases. The significantly enriched biochemical pathways were obtained using KOBAS with corrected P-value ≤0.05.
The Expectation Maximization (RSEM) was used to map the transcripts to reference unigenes via RNA-seq. The read count numbers were calculated and translated to FPKM (fragments per kilobase of transcript per million mapped reads) gene values [51 (link)]. DESeq was used for analysing differentially expressed genes (DEGs) [52 (link)]. We used FDR ≤ 0.05 and |log2Fold > Change| ≥ 1 as threshold for screening DEGs. All identified DEGs were mapped to gene ontology (GO) and Tokyo Encyclopedia of Genes and Genomes (KEGG) databases. The significantly enriched biochemical pathways were obtained using KOBAS with corrected P-value ≤0.05.
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