Abstract
Jatropha curcas is a promising renewable feedstock for biodiesel and bio-jet fuel production. To study gene expression in Jatropha in different tissues throughout development and under stress conditions, we examined a total of 11 typical candidate reference genes using real-time quantitative polymerase chain reaction (RT-qPCR) analysis, which is widely used for validating transcript levels in gene expression studies. The expression stability of these candidate reference genes was assessed across a total of 20 samples, including various tissues at vegetative and reproductive stages and under desiccation and cold stress treatments. The results obtained using software qBasePLUS showed that the top-ranked reference genes differed across the sample subsets. The combination of actin, GAPDH, and EF1α would be appropriate as a reference panel for normalizing gene expression data across samples at different developmental stages; the combination of actin, GAPDH, and TUB5 should be used as a reference panel for normalizing gene expression data across samples under various abiotic stress treatments. With regard to different developmental stages, we recommend the use of actin and TUB8 for normalization at the vegetative stage and GAPDH and EF1α for normalization at the reproductive stage. For abiotic stress treatments, we recommend the use of TUB5 and TUB8 for normalization under desiccation stress and GAPDH and actin for normalization under cold stress. These results are valuable for future research on gene expression during development or under abiotic stress in Jatropha. To our knowledge, this is the first report on the stability of reference genes in Jatropha.
Highlights
Physic nut (Jatropha curcas, hereafter referred to as Jatropha) is a perennial, oily seed-bearing plant belonging to the family Euphorbiaceae that most likely originated in Central America and is widely distributed in the tropics and subtropics [1,2]
The melting curves for the amplified products of all 11 candidate reference genes showed a single peak, corresponding to a specific melting temperature (Figure S1a), and agarose gel electrophoresis showed a single band at the correct molecular weight for each product (Figure S1b), indicating good specificity of all the primer pairs used in real-time quantitative polymerase chain reaction (RT-qPCR)
The PCR efficiency of each candidate reference gene, which ranged from 90% for UBQ-LIKE to 109% for UBQ10, is shown in Table 1, and these efficiencies were used in the calculations for the subsequent expression data
Summary
Physic nut (Jatropha curcas, hereafter referred to as Jatropha) is a perennial, oily seed-bearing plant belonging to the family Euphorbiaceae that most likely originated in Central America and is widely distributed in the tropics and subtropics [1,2]. Tools for selecting reference genes for RT-qPCR analysis have become available, and several research groups have developed software packages, such as geNorm [38], NormFinder [39] and qBase [40], to identify the most stably expressed genes across a set of samples. These tools are freely available on the web, allowing the identification of the best reference gene for specific experiments. Our results reveal that different reference genes should be selected according to the sample type and that a combination of the most stable reference genes provides a more accurate and reliable method of normalization in RT-qPCR analyses
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