RNA-Seq Analysis of Spatiotemporal Gene Expression Patterns During Fruit Development Revealed Reference Genes for Transcript Normalization in Plums
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- Kim, HY., Saha, P., Farcuh, M. et al. Plant Mol Biol Rep (2015) 33: 1634. doi:10.1007/s11105-015-0860-3
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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 (CT) values using BestKeeper, delta (Δ) CT, NormFinder, geNorm, and RefFinder software revealed SAND 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.