Validation of Saccharum SUT Gene Expression

The expression levels of six SUT genes in three tissues (internode 9, 15 and leaf roll in LA-Purple, internode 8,13 and leaf roll in Molokai6081, and internode 6, 9 and leaf roll in SES208) of three Saccharum species were validated by qRT-PCR. Gene-specific primer pairs were designed by using Integrated DNA Technologies (IDT) (http://www.idtdna.com/Primerquest/Home/Index). After treated with DNase I (Tiangen, China), two microgram of RNA was used in reverse transcription with the SuperScript VILO cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s guidelines. The real-time qPCR was performed by using Multicolor Real-Time PCR Detection System (Bio-Rad) with conditions for all reactions were 95 °C for 30s, 40 cycles of 95 °C for 5 s, followed by 60 °C for 30s, and 95 °C for 10s. Melting curve analysis were performed to confirm the PCR specificity. The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and Eukaryotic elongation factor 1a (eEF-1a) were selected as internal standard for normalization [39 (link)], and three replicates were completed for each sample. The relative expression level for each SUT gene in different tissues of three Saccharum species were calculated by using the 2-ΔΔCt method. The correlation coefficient was calculated between the transcript accumulation levels obtained by RNAseq and qRT-PCR using Excel.