Abstract
Transcriptional analysis that uncovers fruit ripening-related gene regulatory networks is increasingly important to maximize quality and minimize losses of economically important fruits such as plums. RNA sequencing (RNA-Seq) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) are important tools to perform high-throughput transcriptomics. The success of transcriptomics depends on the high-quality transcripts from polyphenolic- and polysaccharide-enriched plum fruits, whereas reliability of quantification data relies on accurate normalization using suitable reference gene(s). We optimized a procedure for high-quality RNA isolation from vegetative and reproductive tissues of climacteric and non-climacteric plum cultivars and conducted high-throughput transcriptomics. We identified 20 candidate reference genes from significantly non-differentially expressed transcripts of RNA-Seq data and verified their expression stability using qRT-PCR on a total of 141 plum samples which included flesh, peel, and leaf tissues of several cultivars collected from three locations over a 3-year period. Stability analyses of threshold cycle (C T) values using BestKeeper, delta (Δ) CT, NormFinder, geNorm, and RefFinder software revealed S AND protein-related trafficking protein (MON), elongation factor 1 alpha (EF1α), and initiation factor 5A (IF5A) as the best reference genes for precise transcript normalization across different tissue samples. We monitored spatiotemporal expression patterns of differentially expressed transcripts during the developmental process after accurate normalization of qRT-PCR data using combination of two best reference genes. This study also offers a guideline to select best reference genes for future gene expression studies in other plum cultivars.
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Acknowledgments
This research was supported by the Will W. Lester Endowment of the University of California to E.B.. M.F. is a recipient of a fellowship from the Programa Formacion de Capital Humano Avanzado CONICYT, Chile. The authors are thankful to Dr. Ellen Tumimbang for technical support.
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The authors declared that they have no conflict of interest.
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Ho-Youn Kim and Prasenjit Saha contributed equally to this work.
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Fig. S1
Illustration of plum fruit developmental changes and tissue samples used to evaluate reference genes. Fruits from developmental stages, except S1 (early stage), Stage 2 (S2, pit hardening), Stage 3 (S3, 2nd exponential growth phase) and Stage 4 (S4, ripe stage) of cultivars BB (Burbank), BG (Burgundy), DL (Dolly), EH (Elephant Heart), MT (Methley), SK (Simka), SR (Santa Rosa), SM (Sweet Miriam), QA (Queen Ann) were included in the study. See Table S1 for detailed sample description. (GIF 32 kb)
Fig. S2
Non-differential expression patterns of 20 potential reference genes from RNA-Seq analyses. Data showed log-transformed values of total RNA-Seq reads from immature stage (IS, S2) and ripe stage (RS, S4) of a climacteric cultivar (SR) and a non- climacteric cultivar (SM) in three biological replicates (R1, R2, and R3) collected during 2011. Green-yellow-red color scale depicts low-medium-high expression levels of each gene. See Table 3 for detail characteristic of candidate reference genes and Fig. S1 for developmental stages. (GIF 137 kb)
Fig. S3
Dissociation curve analyses for the conformation of specific qRT-PCR amplification from each primer pair. Melt curves showing the single peak generated after qRT-PCR using gene specific primer pair from each sample. Arrow head represents no template controls (NTC). (GIF 260 kb)
Fig. S4
Conformation of specificity of primer pairs for precise amplification of reference gene after qRT-PCR. Agarose gel showing the expected amplicon size from each primer pair after qRT-PCR of cDNAs pooled form all samples. Lane name corresponds to each reference gene used for qRT-PCR. M1 and M2 represent 50 base pair (bp) and 100 bp DNA size marker, respectively. (GIF 46 kb)
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Table S2
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Kim, HY., Saha, P., Farcuh, M. et al. RNA-Seq Analysis of Spatiotemporal Gene Expression Patterns During Fruit Development Revealed Reference Genes for Transcript Normalization in Plums. Plant Mol Biol Rep 33, 1634–1649 (2015). https://doi.org/10.1007/s11105-015-0860-3
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DOI: https://doi.org/10.1007/s11105-015-0860-3