Transcriptome analysis

The total isolated RNAs were used for RNA-Seq on an Illumina HiSeq 2500. The sequencing library is prepared by random fragmentation of the cDNA sample, followed by 5′ and 3′ adapter ligation. Adapter-ligated fragments are then PCR amplified and gel purified. Sequences were screened for primer concatemers, weak signal and poly A/T tails. Low-quality reads were eliminated based on the score value (reads with >30% of bases with quality score (Q value) of <20) and the remaining high quality reads were filtered for short reads below 50 bp. Adaptors were first trimmed, and then reads were further assembled by GS de novo assembler (v2.6)²⁶. Singletons cleaning using Seqclean and lucy with a parameter of minimum length 100 bp Illumina hiseq reads produced in paired-end formats (101 bp) were also assembled using the Trinity software package²⁷. Reads were filtered and trimmed, and then mapped onto ‘Dangshansuli’ (P. bretschneideri Rehd.) coding sequences using the SOAP aligner²⁸.

Clean reads were mapped using bowtie 1.1.2 to generate read alignments for each sample²⁹. The gene expression level was calculated by fragments per kilobase of exon model per million mapped reads³⁰. The RNA-Seq data of unpollinated ovaries were used as the controls. A false discovery rate of <0.001 and an absolute value of |log₂ ratio| > 1 were used as the thresholds for the significance of differentially expressed genes (DEGs). Genes were annotated using the ‘Dangshansuli’ database (http://www.ncbi.nlm.nih.gov/genome/?term=pyrus) as a reference. Three independent biological replications were sequenced and analysed.