Superscript 2 kit

Total RNA was extracted from two sweetpotato lines SR at 60, 90, 120, and 150 DAP using the RNApure Plant Kit (DNase I) (CWBIO, Beijing, China). cDNA was reverse-transcribed using the SuperScript II Kit (TaKaRa, Beijing, China) according to the manufacturer’s instructions. Four selected DEGs from the RNA-Seq were validated using quantitative real-time PCR (qRT-PCR) with the one-step real-time PCR System (Applied Biosystems, Foster, USA). The qRT-PCR of each reaction (total volume 20 μL) contained 10 μL of SYBR Master Mix (2×, (TaKaRa, Dalian, China), 1.0 μL of primers, 1.0 μL of the cDNA template, and 7 μL of RNase-free water. The 2^−ΔΔCT method was used to calculate the relative expression levels of genes [24 (link)]. The sweetpotato tublin gene was used as a reference. The PCR procedure was: 95 °C for 60 s, 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, and elongation at 72 °C for 20 s, then a melting curve was generated and analyzed. All the primers used for the qRT-PCR validation are listed in Table S3. Three biological replicates were used in statistical analysis and the values in figures were means ± SD (standard deviation). Statistically significant differences at p < 0.05 and p < 0.01 were indicated by asterisks * and **, respectively.

For quantitative real-time PCR (qRT-PCR) analysis, total RNA was extracted from young leaves of ygl7, 810S and ygl7-NIL using an RNA Prep Pure Plant kit (Tiangen Co., Beijing, China). RNA was reverse transcribed using a SuperScript II kit (TaKaRa). Real-time PCR was performed using a SYBR Premix Ex Taq™ kit (TaKaRa) on an ABI prism 7900 Real-Time PCR System. We selected two types of genes for analysis. The first type are associated with chlorophyll biosynthesis and includes ChlD (YGL7), ChlI, ChlH, YGL1, HEMA1, and PoPA [32] (link). The second type are associated with photosynthesis, including the genes Cab1R, Cab2R, PsaA, PsbA, and RbcL [32] (link). qRT-PCR primers come from Zhou KN's paper [32] (link) (primers were DXJCHLD to DXJrbcL, Table S2).

For expression analysis of Zm00001d023265 and Zm00001d023267, total RNA was extracted using an RNAprep Pure Plant kit (Tiangen) from ear leaves of Jing2416K and Jing2416 at three time points: 48 Days After Sowing (DAS) (before the appearance of visible lesions in ear leaves of Jing2416), 55 DAS (lesions just visible) and 66 DAS (lesions clearly visible). First-strand cDNA was synthesized using a SuperScript II kit (Takara) following the manufacturer’s recommendations. Primer pairs were designed using GenScript (https://www.genscript.com/tools/real-time-pcr-taqman-primer-design-tool) and are listed in Supplementary Table S3. Quantitative real-time PCR amplifications were carried out using a SYBR Premix Ex Taq kit (Takara) as described previously (Wang et al., 2017 (link)), with the maize Actin gene (Zm00001d012277) used as an internal reference. Relative expression levels of the two genes were determined by the 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)).