To validate the reliability of differentially expressed genes obtained from Illumina RNA-Seq sequencing, six co-expressed, up-regulated genes from both cultivars: sugar cane_unigene_BMK.40387 (metacaspase-1-like, Q1), sugar cane_unigene_BMK.49302 (ribonuclease 3-like, Q2), sugar cane_unigene_BMK.51436 (pathogenesis-related protein PR-10, Q3), sugar cane_unigene_BMK.57924 (sucrose transporter SUT1, Q4), sugar cane_unigene_BMK.63074 (vacuolar amino acid transporter 1-like, Q5) and sugar cane_unigene_BMK.63784 (heat shock protein-like, Q6) were subjected to RT-qPCR. First-strand cDNAs (10-fold dilution) of sugarcane buds collected from both cultivars 24 h after water inoculation (control) and 24, 48 and 120 h after S. scitamineum inoculation were used as templates, and specific primers were designed according to differential gene sequences [20] (Table S1 in File S1 ). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [21] served as the internal reference gene. SYBR Green staining was applied for RT-qPCR using the ABI 7500 fast real-time PCR system (Applied Biosystems, Foster, CA, USA). The total reaction volume was 25 µL, including 12.5 µL FastStart Universal SYBR Green PCR Master (ROX Medical, Shanghai, China), 0.4 µmol/L primer and 2.0 µL template. Reaction conditions were: 50°C, 2 min; 95°C, 10 min; 95°C, 15 s, 60°C, 1 min, and 40 cycles and three replicates were performed for each. PCR using distilled water as the template was used as a blank control. A 2−ΔΔCt algorithm was applied for quantitative gene expression analysis [22] (link).
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