ITEM TO CHECK,IMPORTANCE,CHECKLIST
EXPERIMENTAL DESIGN,,
Definition of experimental and control  groups,E,"Experimental groups: 4 groups, tubers with different colors. "
Number within each group,E,The number in each group was 3.
Assay carried out by core lab or investigator's lab?,D,
Acknowledgement of authors' contributions ,D,
SAMPLE,,
Description,E,"Samples were obtained from tubers of Dioscorea cirrhosa L, the colors of these tubers  range from light red to dark red."
     Volume/mass of sample processed,D,"For samples, 50 mg."
    Microdissection or macrodissection,E,Macrodissection
Processing procedure,E,"All samples were stored at -80¡æ  until RNA extraction. After weighing fresh plant tissue, quickly cut 50-100mg into small pieces and place them in a mortar (frozen or liquid nitrogen preserved samples can be directly weighed before taking 25-100mg and placing them in a mortar). Add 1mL of solution A and grind thoroughly. Attention:  the tissue and solution A must be  fully contact to inhibit RNA enzyme activity."
     If frozen - how and how quickly?,E,All the samples were immediately frozen in liquid nitrogen after they were obtained from tubers.
"     If fixed - with what, how quickly?",E,Not fixed
Sample storage conditions and duration (especially for FFPE samples),E,Stored in -80 ¡æ
NUCLEIC ACID EXTRACTION,,
Procedure and/or instrumentation,E,"RNA extraction from samples. Total RNAs were extracted from tuber tissues using Polysaccharide Polyphenol Plant Total RNA extraction Kit (TSP412, Qingke) was added to 50 mg of tissue. Transfer the homogenate into a clean 1.5 mL plastic centrifuge tube. Add 300ul of solution B and 200ul of chloroform to the centrifuge tube, shake for 30 s. Centrifuge at room temperature of 12000 g for 10 mins.  Transfer 700ul (up to 800ul) of the supernatant to another clean 1.5 mL plastic centrifuge tube, with the lower organic phase and middle layer containing proteins and other impurities to avoid contact or absorption. It is best to leave 100ul of supernatant untouched. Add equal volume of solution C, thoroughly invert and mix well. Add the mixture (up to 700ul each time) to a centrifugal adsorption column, centrifuge at room temperature of 12000 g for 1 min, and discard the penetrating solution. Add 700ul of solution D, let stand for 30 s, centrifuge at room temperature of 12000g for 1 min, and discard the penetrating solution. This step can remove residual DNA and proteins. Add 500uL rinsing solution, centrifuge at room temperature of 12000g for 1 min, and discard the penetrating solution. Add 500uL of rinsing solution and repeat. Centrifuge at room temperature of 12000g for 2 mins. This step is very important, otherwise residual ethanol will affect the use of RNA. Transfer the centrifugal adsorption column to an RNase free collection tube, add 50-100 uL of RNase free H2O, and let it for 5 minutes. Centrifuge at room temperature of 12000 g for 2 mins, the solution in the centrifuge tube is the RNA sample,  stored at -80 ¡æ for further use."
     Name of kit and details of any modifications,E,"Goldenstar RT6 cDNA Synthesis Mix (Tsingke,China)
Fast qPCR Mix (SYBR Green I) (Tsingke,China)
We exactly followed the manufacture's protocol.                   "
     Source of additional reagents used ,D,
Details of DNase or RNAse treatment,E,1ug of RNA was treated with 1 ul of gDNA Erase and 2ul 5¡Á gDNA Eraser Buffer ina 20 ul final volume reaction. Digestion of DNA was achieved with 2mins incubation at 42 ¡æ.
Contamination assessment (DNA or RNA),E,"Reverse transcrition controls (without enzyme) were perfomed in order to assess the existence of DNA in the RNA sample. For that purpose, RNA was prosessed as a normal sample in the RT step, except that no reverse transcriptase was added to the reaction mixture. "
Nucleic acid quantification ,E,RNA concentration was assessed using spectrophotometry by measuring the absorbance at 260 nm UV light.
     Instrument and method,E,Spectrophotometry (Eppendorf)
     Purity (A260/A280) ,D,RNA purity wa determined by measuring the absorbance at OD 260 and OD 280
     Yield,D,
RNA integrity method/instrument,E,"RNA integrity was assessed using Agilent's 2100 bioanalyzer (Agilent Technologies,Santa Clara, CA, USA)"
    RIN/RQI or Cq of 3' and 5' transcripts ,E,Not applicable
    Electrophoresis traces,D,
" Inhibition testing (Cq dilutions, spike or other) ",E,The standard curve has been considered sufficient to rule out the presence of inhabitors of reverse-transcription activity or PCR also taking into account the high quality of starting RNAs.
REVERSE TRANSCRIPTION,,
Complete reaction conditions,E,"Reverse transcription was carried out in 20 ul reaction volume. Briefly, 1ul total RNA, 2ul 5¡ÁgDNA Eraser buffer, 1ul gDNA Eraser and nuclease-free water were mixed well and incubated for 2 min at 42 ¡æ to eliminate genomic DNA. For the synthesis of cDNA, 4ul 5¡Á PrimerScript Buffer 2, 1ul PrimeSctipt RT Enzyme Mix 1, 1ul RT Primer Mix and 4 ul nuclease-Free water were then added, and the reaction mixtures were performed using the following conditions: 37¡æ for 15 min, followed by 85¡æ 5s and then held at 4¡æ."
     Amount of RNA and reaction volume,E,"Amount of RNA: 3ul, reaction volume: 20ul"
    Priming oligonucleotide (if using GSP) and concentration,E,Oligo dTprimer: 50pmol/20ul reaction system
     Reverse transcriptase and concentration,E,PrimeScrit RT Enzyme Mix 1
     Temperature and time,E,Specified in "Complete reaction conditions"
     Manufacturer of reagents and catalogue numbers,D,
Cqs with and without RT,D*,
Storage conditions of cDNA,D,¡®- 20 ¡æ
qPCR TARGET INFORMATION,,
"If multiplex, efficiency and LOD of each assay.",E,
Sequence accession number,E,NCBI accession number: No.  PRJNA741609
Location of amplicon,D,
     Amplicon length,E,
"     In silico specificity screen (BLAST, etc)",E,Described in text
"     Pseudogenes, retropseudogenes or other homologs?",D,
          Sequence alignment,D,
     Secondary structure analysis of amplicon,D,
Location of each primer by exon or intron (if applicable),E,Not applicable
     What splice variants are targeted?,E,
qPCR OLIGONUCLEOTIDES,,
Primer sequences,E,Table S7 in supplementary materials
RTPrimerDB Identification Number ,D,
Probe sequences,D**,
Location and identity of any modifications,E,No modifications were done
Manufacturer of oligonucleotides,D,
Purification method,D,
qPCR PROTOCOL,,
Complete reaction conditions,E,"qPCR was carried out using SYBR Premix Ex Tag II (Takara: RR820A) in 20ul reaction volumes. In brief, each reaction was comprised of 1ul of the cDNA solution, 10 ul of SYBR Premix Ex Tag (2¡Á), 2 ul of primers, and 7 ul of ddH2O. All qPCR reactions were performed on ABI 7500 Real-Time PCR System (Applied Biosystems, USA), using the following conditions: 95¡æ for 2 mins, followed by 40 cycles at 95¡æ for 15 s, 60¡æ for 15 s, 72 ¡æ for 20 s. "
     Reaction volume and amount of cDNA/DNA,E,  Reaction volume:  20 ul; amount of cDNA/DNA: 1ul.
"     Primer, (probe), Mg++ and dNTP concentrations",E,2 uM Primers; 2 mM MgCl; 0.2 mM dNTP
     Polymerase identity and concentration ,E,TakaRa Ex Taq HS
     Buffer/kit identity and manufacturer ,E,TakaRa 
     Exact chemical constitution of the buffer,D,
"     Additives (SYBR Green I, DMSO, etc.)",E,SYBR Green I
Manufacturer of plates/tubes and catalog number,D,
Complete thermocycling parameters,E,"Initial denaturation: 95¡ãC for 2 minutes,  followed by 40 cycles at 95¡ãC for 15 s , 60¡ãC cycles for 15 s, and 72¡ãC cycles for 20 s"
Reaction setup (manual/robotic),D,Manual
Manufacturer of qPCR instrument,E,ABI 7500 Real-time PCR System (Applied Biosystem. USA)
qPCR VALIDATION,,
Evidence of optimisation (from gradients) ,D,
"Specificity (gel, sequence,  melt, or digest)",E,Specificity was confirmed by melt curve analyses and sequencing analysis (which was provided in Supplementary data). Gene-specific amplification was confirmed by a single band in 2% agarose gel electrophoresis stained with ethidium bromide. 
"For SYBR Green I, Cq of the NTC",E,Not applicable
Standard curves with slope and y-intercept,E,Not applicable
     PCR efficiency calculated from slope,E,No applicable
     Confidence interval for PCR efficiency or standard error,D,
     r2 of standard curve,E,
Linear dynamic range,E,"In average, linear dynamic was considered taking into account the linearity of the standard curves; from 1/10 dilution of cDNA to 1/1000 dilution."
     Cq variation at lower limit,E,Not applicable
     Confidence intervals throughout range,D,
Evidence for limit of detection ,E,Not applicable
"If multiplex, efficiency and LOD of each assay.",E,Not applicable
DATA ANALYSIS,,
"qPCR analysis program (source, version)",E,"Quantification Software version 2.1 (Applied Biosystem, USA)"
     Cq method determination,E,The threshold was defined as the fractional cycle number at which the fluorescence exceeded the given threshold and was calculated using SDS Relative Quantification Software version 2.1 using the automatic baseline setting.
     Outlier identification and disposition,E,Not applicable
Results of NTCs ,E,Not applicable
Justification of number and choice of reference genes,E,Not applicable
Description of normalisation method,E,Described in text
Number and concordance of biological replicates,D,
Number and stage (RT or qPCR) of technical replicates,E,qPCR reactions were perfomed in triplicate.
Repeatability (intra-assay variation),E,Mean standard deviation of triplicates: less than 0.1.
"Reproducibility (inter-assay variation, %CV)",D,
Power analysis,D,
Statistical methods for result significance,E,"T-test, wilcoxon"
"Software (source, version)",E,R software (version 5.0)
Cq or raw data submission using RDML,D,
,,
"Table 1. MIQE checklist for authors, reviewers and editors. All essential information (E) must be submitted with the manuscript.  Desirable",,
"information (D) should be submitted if available. If using primers obtained from RTPrimerDB, information on qPCR target, oligonucleotides,",,
protocols and validation is available from that source.,,
,,
*: Assessing the absence of DNA using a no RT assay is essential when first extracting RNA. Once the sample has been validated as,,
" RDNA-free, inclusion of a no-RT control is desirable, but no longer essential.",,
,,
"**: Disclosure of the probe sequence is highly desirable and strongly encouraged. However, since not all commercial pre-designed assay",,
" vendors provide this information, it cannot be an essential requirement. Use of such assays is advised against.",,