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

<div><p>Physic nut (<i>Jatropha curcas</i> 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 <i>J</i>. <i>curcas</i>. 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, <i>TUB</i> and <i>EF</i> found to be the two most stably expressed reference for staminate flower; while <i>GAPDH1</i> and <i>EF</i> found to be the most stably expressed reference gene for pistillate flowers. Finally, RT-qPCR assays of target gene <i>AGAMOUS</i> using the identified most stable reference genes confirmed the reliability of selected reference genes in different stages of flower development. <i>AGAMOUS</i> 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 <i>J</i>. <i>curcas</i>.</p></div

Most stable and least stable reference genes based on RefFinder analysis.

<p>Most stable and least stable reference genes based on RefFinder analysis.</p

Relative expression of <i>AGAMOUS</i> gene using selected reference genes including most stable and least stable reference genes for normalization in staminate and pistillate flowers.

<p>The error bars represent standard deviation.</p

Expression stability analysis of reference genes assayed by NormFinder software.

<p>Expression stability analysis of reference genes assayed by NormFinder software.</p

Expression stability analysis of reference genes assayed by ΔCt method.

<p>Expression stability analysis of reference genes assayed by ΔCt method.</p

Candidate reference genes and its primer sequences.

<p>Candidate reference genes and its primer sequences.</p

Gene expression stability values (M) and ranking of 10 reference genes as assayed by GeNorm in staminate, pistillate and total samples.

<p>The least stable genes are on the left and the most stable genes on the right.</p

Expression stability analysis of reference genes assayed by BestKeeper software.

<p>Expression stability analysis of reference genes assayed by BestKeeper software.</p

<i>AGAMOUS</i> gene copy number.

<p>The copy number was calculated by standard curve at different developmental stages for staminate and pistillate flower samples of <i>Jatropha curcas</i>.</p

Identification and validation of superior reference gene for gene expression normalization via RT-qPCR in staminate and pistillate flowers of <i>Jatropha curcas</i> – A biodiesel plant - Fig 1

<p><b>A. Primer specificity and amplicon size.</b> Agarose gel electrophoresis (2.0%) indicates amplification of a single PCR product of the expected size for 10 genes (Line 1–10: <i>ACTIN</i>, <i>EF</i>, <i>SLEEPER</i>, <i>GAPDH1</i>, <i>GAPDH2</i>, <i>TUB</i>, <i>UBI</i>, <i>DSK2A</i>, <i>CYC</i>, and <i>PLA</i><b>). B. Melting curve analysis:</b> Melting curves of 10 genes show single peaks.</p