Mitochondrial RNA Sequencing of CMS Flower Buds

The extracted mitochondrial RNA from the flower buds (3–5 mm size) in CMS line 2074A, its maintainer 2074B and fertile material F₁ (2074A × E5903) were sequenced on an Illumina HiSeq2000 at BGI (Beijing Genomics Institute). Ribosomal RNAs were removed from the extracted mitochondrial RNA using Ribo-Zero (Epicentre, Madison, WI) and the mitochondrial RNA libraries were prepared using Illumina’s TruSeq RNAseq Sample Prep kit. Libraries were sequenced on one lane with 4 Gb clean reads/samples of an average length of 90 nt for paired-end. RNA sequence data quality was checked using FastQC to remove the adapters, low-quality, containing N bases and short sequences with reads Q30 > 85%. The reads were mapped to the assembled mitogenome of CMS line 2074A using bowtie2 [54 (link)] with the following parameters: -D 5 -R 1 -N 0 -L 25 -i S, 1, 2.00. Then, the resulting SAM files from bowtie2 mapping were transformed into BAM files using samtools view program [45 (link)]. The bowtie2 mapping results of these pair-end reads in BAM files were then used to calculate read count for each gene through HTSeq-count program [55 (link)]. Differentially expressed genes that showed up and down regulation between samples were defined based on the standards of cutoff: two-fold change and a p-value of less than 0.05.