Identification and validation of superior reference gene for gene expression normalization via RT-qPCR in staminate and pistillate flowers of Jatropha curcas - A biodiesel plant

PLoS One. 2017 Feb 24;12(2):e0172460. doi: 10.1371/journal.pone.0172460. eCollection 2017.


Physic nut (Jatropha curcas L) seed oil is a natural resource for the alternative production of fossil fuel. Seed oil production is mainly depended on seed yield, which was restricted by the low ratio of staminate flowers to pistillate flowers. Further, the mechanism of physic nut flower sex differentiation has not been fully understood yet. Quantitative Real Time-Polymerase Chain Reaction is a reliable and widely used technique to quantify the gene expression pattern in biological samples. However, for accuracy of qRT-PCR, appropriate reference gene is highly desirable to quantify the target gene level. Hence, the present study was aimed to identify the stable reference genes in staminate and pistillate flowers of J. curcas. In this study, 10 candidate reference genes were selected and evaluated for their expression stability in staminate and pistillate flowers, and their stability was validated by five different algorithms (ΔCt, BestKeeper, NormFinder, GeNorm and RefFinder). Resulting, TUB and EF found to be the two most stably expressed reference for staminate flower; while GAPDH1 and EF found to be the most stably expressed reference gene for pistillate flowers. Finally, RT-qPCR assays of target gene AGAMOUS using the identified most stable reference genes confirmed the reliability of selected reference genes in different stages of flower development. AGAMOUS gene expression levels at different stages were further proved by gene copy number analysis. Therefore, the present study provides guidance for selecting appropriate reference genes for analyzing the expression pattern of floral developmental genes in staminate and pistillate flowers of J. curcas.

Publication types

  • Validation Study

MeSH terms

  • Algorithms
  • Biofuels
  • Flowers / genetics*
  • Flowers / growth & development
  • Gene Expression Regulation, Plant
  • Genes, Essential*
  • Genes, Plant*
  • Genomic Instability
  • Jatropha / genetics*
  • Jatropha / growth & development
  • MADS Domain Proteins / genetics
  • Plant Oils / isolation & purification
  • Real-Time Polymerase Chain Reaction / standards
  • Seeds / chemistry
  • Seeds / genetics
  • Seeds / growth & development
  • Sex Differentiation / genetics


  • Biofuels
  • MADS Domain Proteins
  • Plant Oils

Grant support

This research was supported by the National Natural Science Foundation of China (Grand Number: 31270359). Fund received by JW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.