Plant Molecular Biology Reporter

, Volume 33, Issue 6, pp 1634–1649 | Cite as

RNA-Seq Analysis of Spatiotemporal Gene Expression Patterns During Fruit Development Revealed Reference Genes for Transcript Normalization in Plums

  • Ho-Youn Kim
  • Prasenjit Saha
  • Macarena Farcuh
  • Bosheng Li
  • Avi Sadka
  • Eduardo Blumwald
Original Paper


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.


Fruit development Gene expression Plum Quantitative real-time reverse transcription PCR Reference gene(s) 



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.

Conflict of Interest

The authors declared that they have no conflict of interest.

Supplementary material

11105_2015_860_Fig5_ESM.gif (32 kb)
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)

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High resolution image (TIFF 2,741 kb)
11105_2015_860_Fig6_ESM.gif (137 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)

11105_2015_860_MOESM2_ESM.tif (8.1 mb)
High resolution image (TIFF 8,285 kb)
11105_2015_860_Fig7_ESM.gif (261 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)

11105_2015_860_MOESM3_ESM.tif (9.8 mb)
High resolution image (TIFF 10,073 kb)
11105_2015_860_Fig8_ESM.gif (46 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)

11105_2015_860_MOESM4_ESM.tif (813 kb)
High resolution image (TIFF 813 kb)
11105_2015_860_MOESM5_ESM.xlsx (1.1 mb)
Supplementary data 1 (XLSX 1,118 kb)
11105_2015_860_MOESM6_ESM.xlsx (17 kb)
Supplementary data 2 (XLSX 17 kb)
11105_2015_860_MOESM7_ESM.docx (17 kb)
Table S1 (DOCX 17 kb)
11105_2015_860_MOESM8_ESM.docx (35 kb)
Table S2 (DOCX 35 kb)
11105_2015_860_MOESM9_ESM.docx (22 kb)
Table S3 (DOCX 21 kb)
11105_2015_860_MOESM10_ESM.docx (19 kb)
Table S4 (DOCX 18 kb)
11105_2015_860_MOESM11_ESM.docx (28 kb)
Table S5 (DOCX 28 kb)


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Ho-Youn Kim
    • 1
  • Prasenjit Saha
    • 1
  • Macarena Farcuh
    • 1
  • Bosheng Li
    • 1
  • Avi Sadka
    • 2
  • Eduardo Blumwald
    • 1
  1. 1.Department of Plant SciencesUniversity of California DavisDavisUSA
  2. 2.Department of Fruit Tree Sciences, Institute of Plant SciencesA.R.O. Volcani CenterBet DaganIsrael

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