title: RNA-seq of Gymnosporangium yamadae inoculated apple leaves (30 days after inoculation)
design: In early spring, juniper twigs with mature G. yamadae telia were collected and placed in a sterile plastic container with sterile water to overnight to hydrate telia. Germinated telia with high concentration of basidiospores were mixed with sterile water and divided into several sterile vials for inoculation. Thirty two-year healthy apple (Malus domestica cv Fuji) seedlings were divided into two groups, one was sprayed with G. yamadae basidiospores, the other was treated with sterile water as control. After inoculation, these seedlings were covered with transparent plastic bags and placed in greenhouse at same conditions (moist level > 95% and temperature at 20 for two days). About 10 days after inoculation (10 dpi), globoid and blackish spermogonia appeared on the upper surface of apple leaves. We removed the bags on the seedlings and transferred the flowerpots outdoors. Tubular aecia extruded from the lower surface of apple leaves at 30 days after inoculation (30 dpi). Three biological replicates of 50mg of apple leaves (5  5 cm2) inoculated apple leaves with typical rust symptoms and mock inoculated apple leaves were harvested at two time points, respectively. Total RNA for each replicate was isolated and sequenced using Illumina Hiseq platform (Supplementary file: Figure S2). After transcriptome sequencing and quality control process, 264 and 269 million paired clean reads were obtained for infected apples leaves, and 194 and 151 million clean reads from healthy leaves, respectively at 10dpi and 30dpi. Paired-end reads were aligned to the M. domestica reference genome using Tophat v.2.0.12 and the mapped rates are relatively lower in infected leaves than in healthy leaves and lowest at 30dpi (24.68% for average), which reflected the development of rust fungi inside infected leaves (Table 1). Pearson correlation coefficients (PCCs) (r2) were calculated between samples to assess overall similarity between samples and visualized using the function pheatmap package in R . The results showed that the correlation coefficients of the sample between biological replicates are greater than the sample outside the biological replicate, in other words, most of the variation in gene expression stem from the infection process and life-cycle stage change. Furthermore, principal components analysis (PCA) indicated that the transcriptomes of inoculated and mock inoculated at two time points are quite independent from each other, and the replicates from one condition is relatively centralized.