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.