RNA seq analysis

RNA isolated from tubers at stage 2a was checked for integrity by agarose gel electrophoresis and on the Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). The qualified RNA sample was subjected to library construction using the NEBNextUltra RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s protocols. A cDNA library was constructed and paired-end sequencing was performed on an Illumina HiSeq 2000 platform. After removing reads containing adapter and low-quality reads, about 54.3 million cleaned reads (i.e., 27.15 M paired- end reads, read length greater than 100 bp) were obtained. FASTQ sequence files were deposited at the National Center for Biotechnology Information (NCBI) under BioProject PRJNA591090. The clean reads were assembled as unigenes using Trinity method (Grabherr et al. 2011) with optimized k-mer length of 25 into 118,775 contigs.Analysis of mRNA expression levels
Total RNA was isolated from tuber tissues using CTAB extraction buffer combined with RNA purification columns (Ouyang et al. 2014). Full length RNA transcripts were analyzed by RT-PCR using specific primers for the selected genes (GeSUT4-5-BP and
GeSUT4-3-BP for GeSUT4; GeSUT4-5-BP and GeSUT4D-3-BP for GeSUT4D;
GeSUT3-5-BP and GeSUT3-3-BP for GeSUT3) and 35 cycles of PCR reaction (95 °C for 30 s, 64 °C for 30 s, and 72 °C for 120 s). RNA of G. elata actin 7 (GeAct 7), was also amplified with specific primers (GeAct7-5 and GeAct7-3 ) and used as the internal control for quantification. To quantify the expression, total RNA transcripts were reverse-transcribed and gene-specific primers were used for real-time quantitative PCR as described (Ho et al. 2019). Expression of the GeAct 7 gene was used to determine relative expression levels according to the following equation: 1000 x 2-(Ctgenex-CtGeAct7) (Ct = threshold cycle). All sequences of primers and corresponding targets are shown in Table S1.