Dnase 1

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.

Total RNA was extracted from each sample using the RNAprepPure Plant Kit DP441 (Tiangen Biotech CO., LTD, Beijing, China) according to the manufacturer’s instructions, followed by DNase I (Tiangen Biotech CO., LTD, Beijing, China) treatment to eliminate DNA contamination. The integrity of RNA was determined by 1.5% agarose gel electrophoresis. The purity and concentration of total RNA was determined using NanoDrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA, US). RNA samples with a concentration higher than 60 ng/μL and a ratio of A260/A280 between 1.8 and 2.0 were required for cDNA preparation. Synthesis of cDNA was conducted in the PrimerScript™ RT cDNA Synthesis Kit (TaKaRa Bio Inc., Dalian, China) using 1.0 μg RNA solution. The obtained cDNA was then diluted with 10 times nuclease-free water to prepare RT-qPCR.

Total RNA was extracted using TRIzol reagent (Invitrogen) and was then treated with DNase I (Tiangen Biotech, Beijing, China). The cDNA for the qRT-PCR was synthesised with the PrimeScript RT reagent kit and the gDNA Eraser (TaKaRa). The PCR amplification procedure for GAPDH was as follows: 94°C for 3 min; 30 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 30 s; and a terminal elongation at 72°C for 5 min. The amplification of LIN28B was performed as follows: 95°C for 2 min; 30 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s; and a final step at 72°C for 5 min. The qRT-PCR assays were performed using the SYBR PrimeScript RT-PCR kit (TaKaRa) for GAPDH and LIN28B.

Frozen samples were ground to a fine power in liquid nitrogen using a mortar and pestle sterilized at 180°C for 8 h. Total RNA was extracted from the collected tissues following Li [68 ]. To eliminate any traces of genomic DNA contamination after RNA extraction, DNase I (Tiangen, Beijing, China) was used as recommended by the manufacturer. The integrity of the RNA was assessed on a 1% (w/v) agarose (Invitrogen, CA, USA) gel. RNA concentration and the 260/280 as well as 260/230 absorbance ratios were determined using an Infinite^® 200 PRO (Tecan, Männedorf, Switzerland). First-strand cDNA was synthesized from 1 μg of DNase I-treated RNA using anchored-oligo (dT)s primers according to the manufacturer’s instructions (Promega, Madison, USA). Before each qRT-PCR stage, cDNA products were diluted five-fold prior to use.

Total RNA was isolated from samples of jejunum, ileum, and colon with TRIzol regent (Tiangen Biotech, Beijing) and then treated with DNase I (Tiangen Biotech, Beijing) according to the manufacturer's instructions. Subsequently, we tested the integrity, purity, and concentration of RNA. Fastking cDNA first-strand synthesis Kit, Super real premix plus, 2 × Taq PCR mastermix, and 5 × RNA loading buffer were purchased from Beijing Tiangen Biochemical Technology Co., Ltd. The PCR reaction process was as follows: 95°C for 2 min, followed by amplification in 40 cycles of 95°C for 5 s, 15 s at 60°C, and 20 s at 72°C, and then 65°C and 95°C for 5 s, using the C1000 Touch^TM Thermal Cycler Real-Time System (Bio-Rad). Referring to the data of the National Center for Biotechnology Information (NCBI) database, fluorescent quantitative-specific primers were designed by primer 5.0 software. All primers are synthesized by Thermo Fisher Technology Co., Ltd. and are presented in Table 2. GAPDH was used as an internal control to normalize the expression of target gene transcripts.

We used WT, cry1 mutant, CRY1-ovx, CCT1, and CNT1 seedlings for real-time quantitative PCR. Seedlings were germinated on half-strength MS plates plus 1% sucrose and placed at 4°C for 3 days and then transferred to white light for 12 h before all seedlings placed in darkness for another 4 days. Then half of the seedlings were exposure to 30 μmol/m²/s blue light for 1 h, and another half of the seedlings were continue grown in dark. Total RNAs were isolated with RNAprep Plant kit (TIANGEN) followed by DNase I (TIANGEN) treatment. Then 500 ng sample of total RNA were used to reverse-transcribed to 10 μl cDNA using iScriptcDNA Synthesis kit (Bio-Rad). To validate our expression profile data, we selected genes that showed significant expression changements according to our different aim and performed real-time quantitative PCR. qRT-PCR was described previously (Zhang et al., 2014 (link); He et al., 2015 (link)) and ACT2 was used as internal control for qRT-PCR. The used primers are listed in Table S4.

Total RNA was isolated using a RNAprep Pure Plant Kit and treated with DNase I (Tiangen, Beijing, China) according to manufacturer’s instructions. RNA concentrations and quality were determined by a Thermo 2000 Bioanalyzer with a RNA NanoDrop (Thermo Scientific, USA). Library preparation for RNA-Seq was conducted using a TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, Cat. RS-122-2101, USA) according to manufacturer’s protocol. And the final libraries were quantified by QuantiFluor dsDNA System (Promega, USA) and sequenced on an Illumina HiSeq 2500 platform (Illumina Inc. USA) at Berry Genomics Corporation, Beijing, China.