RNA sequencing and analyses
The RNAprep Pure Plant Plus Kit was used to extract total RNA from the
roots, stems, and leaves of rosemary samples according to the
manufacturer’s instructions. The RNA-seq library was constructed on the
Illumina HiSeq X Ten platform and sequenced to 150-bp paired reads, and
then de novo assembled. The quality control of the assembled sequence
included low-quality filtering, removal of N-containing bases, and
removal of 3’ and 5’ end sequencing adapters. Raw data were filtered
using SOAPnuke software
(www.bgitechsolutions.com) with
the following filtering parameters: -n 0.01 -l 20 -q 0.4 -A0.25
–cutAdaptor -Q 2 -G –polyX50 –minLen 150. After the raw data
had been filtered, FastQC
(https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to
monitor data quality. Three sets of biological replicates were made for
each of the three parts of the roots, stems and leaves, and 9 sample RNA
sequences were obtained for subsequent transcriptome analyses.
The ‘WGCNA’ package of R (v4.0) was used to separate all genes into
modules of color blocks, calculate the PCCs between gene expression and
the content of differential metabolites, and construct a weighted gene
co-expression network based on PCCs. In addition, genes were regarded as
highly corelated to candidate metabolites if PCCs were >
0.6 and p-values < 0.05.