ارزیابی بیان ژن‌های مرتبط با پاسخ دفاعی در رقم حساس سیب "پریما" در مواجهه با بیماری شانکر اروپایی با رویکرد توالی‌یابی رونوشت‌ها

Objective
European canker is one the most destructive diseases affecting apple trees in temperate growing regions and is caused by the fungal pathogen Neonectria ditissima. Due to the pathogen’s ability to persist year-round, effective disease control remains challenging. This study aimed to identify key genes and signaling pathways associated with susceptibility to N. ditissima. Elucidating the expression patterns and functional roles of these genes provides insight into the molecular mechanisms underlying disease development and may contribute to the development of more targeted and sustainable disease management strategies.
Materials and methods
The susceptible apple cultivar ‘Prima’ was inoculated by applying a fungal spore suspension to selected buds. Both inoculated and control samples (n = 18) were collected at 5, 15, and 30 days post-inoculation, with three biological replicates per time point, and used for total RNA extraction. RNA quality and quantity were evaluated prior to whole-transcriptome sequencing. Sequencing was performed in paired-end mode on the Illumina HiSeq 2000 platform. Raw read quality was assessed using FastQC, and high-quality reads were aligned to the apple reference genome using TopHat2. Differentially expressed genes (DEGs) were identified after normalization with DESeq2, and functional pathway enrichment analysis was conducted using the KEGG database.
Results
RNA-Seq analysis showed that the majority of transcriptional changes in the susceptible cultivar ‘Prima’ occurred at 15 and 30 days following inoculation with N. ditissima. A total of 6,996 differentially expressed genes (DEGs; FDR < 0.05) were identified, with more than 55% being up-regulated and approximately 45% down-regulated during the intermediate and late stages of infection. In contrast, no significant changes in gene expression were detected at 5 days post-inoculation. Notably, genes associated with cell wall biosynthesis and reinforcement, including CAD and LAC1, were down-regulated, while MLO6, a gene previously linked to enhanced disease susceptibility, showed strong up-regulation. Collectively, these expression patterns point to a disrupted coordination between structural and chemical defence responses in the susceptible cultivar, indicating an overall ineffective defence strategy against the pathogen.
Conclusions
The results suggest that in the susceptible apple cultivar, defence responses are mainly triggered at later stages of N. ditissima infection and are not sufficiently activated during the early phase of pathogen establishment. The gene expression patterns observed indicate a disruption in key defence-related pathways, along with altered regulation of genes associated with susceptibility. Taken together, genes that exhibited consistent and significant expression changes in this study represent potential candidates for molecular markers in apple breeding programmes, although their utility will require validation in independent populations and further functional characterization.