Introduction

Every year, 1.6 billion tons of food goes to waste. This is about one-third of the food that is produced for human consumption1. Unpredictable ripening of fruit is one of the main causes of loss after harvest. This is particularly true of climacteric fruits, which are characterized by a spike in ethylene biosynthesis, known as system 2 (S2) ethylene production, and a concomitant burst in respiration at the onset of ripening. The ethylene receptor antagonist 1-methylcyclopropene (1-MCP) is used to impart a longer shelf life by limiting the ethylene perception and activation of downstream ripening responses2,3,4.

Uniquely, in European pear fruit (Pyrus communis), 1-MCP treatment may irreversibly inhibit endogenous or system 2 ethylene production and the respiratory climacteric5,6. Furthermore, exogenous ethylene application does little to affect the capacity of 1-MCP-treated pears to ripen7,59.

Functional annotation

The master transcriptome fasta produced from the Illumina assembly was imported into OmicsBox 1.1.135 (BioBam Bioinformatics S.L., Valencia, Spain) for functional annotation of expressed contigs. Contig sequences were identified by a blastx alignment against the NCBI ‘Viridiplantae’ database with and e-value specification of 10.0E-3. GO annotation was assigned using the ‘Map**’ and ‘Annotation’ features using default parameters to generate a functionally annotated master assembly60.

Differential expression analysis

Temporally differentially expressed genes were identified using the time course, multi-series differential expression feature in the OmicsBox suite, which employs the maSigPro R package61. The FDR cutoff value was set to 0.05. The statistical analysis ensured that genes that did not meet the assumption of equal variances were eliminated from the analysis, which was particularly important given that the three experiments were performed at different times throughout the 2018 season. The DEGs and expression values were matched with their corresponding functional annotations (Supplementary File 5).

GO enrichment analysis

Gene ontology (GO) enrichment analysis was conducted to determine over and underrepresented biological processes, molecular functions, and cellular components among the differentially expressed sequences using the OmicsBox Enrichment Analysis (Fisher’s Exact Test) function60 (Supplementary File 8). The annotated master transcriptome was used as the reference dataset, and the set of genes identified as differentially expressed over time in the treatment group versus the control group was used as the test dataset.

qRT-PCR validation

Primers for qRT-PCR targeting seven differentially expressed genes in the ripening-related pathways discussed previously were designed using the NCBI Primer-BLAST tool62. 200 ng RNA for each sample was used to generate 1st strand cDNA using the Invitrogen VILO kit (Life Technologies, Carlsbad, CA USA). cDNA preparations were then diluted to 20 ng/uL. Final library concentrations were quantified using a Qubit fluorometer (Carlsbad, CA). qRT-PCR technical replicate reactions were prepared for each of the gene targets using the iTAQ Universal SYBR Green Supermix with ROX reference dye (BioRad, Hercules, CA) per the manufacturer’s protocols with 20 ng of template cDNA. In a Stratagene MX3005P, the following thermocycler profile was used: 95 °C initial disassociation for 2:30 minutes followed by 50 amplification cycles (95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s) and a final, single-cycle phase to generate a dissociation curve (95 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s). The LinRegPCR tool was used to calculate the Cq values for each reaction63,64 (Supplementary File 10). Cq values, which were calculated from efficiency scores below 1.80 or above 2.20 were considered sufficiently low in confidence and were deemed unacceptable and were omitted from the analysis.