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

Abstract

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.

Stability evaluation of reference genes for gene expression analysis by RT-qPCR in soybean under different conditions

“…Real-time quantitative reverse transcription PCR (RT-qPCR) is one of the most commonly used techniques to examine transcript levels due to its sensitivity, specificity, wide dynamic range, and high throughput capacity [ 1 – 3 ]. As a valuable tool for basic research, RT-qPCR is available in many fields, such as diagnostics, biotechnology and microbiology [ 4 – 9 ].…”

“…During the past few years, there have been lots of reports published with the aim of identifying and evaluating suitable reference genes for expression analysis in different plant species under abiotic stress [ 11 , 13 , 16 , 19 , 23 , 24 ], at different developmental stages or tissues [ 1 , 3 , 12 , 21 , 22 ], after infection with fungi, virus or bacteria [ 3 , 13 , 17 , 18 , 20 , 24 , 25 ]. However, there is a lack of validated reference genes under soybean mosaic virus (SMV) infection and nitrogen stress.…”

Evaluation of reference genes for reverse transcription quantitative real-time PCR (RT-qPCR) studies in Silene vulgaris considering the method of cDNA preparation

“…The RefFinder tool [ 59 ] which compilates four algorithms: geNorm [ 60 ], NormFinder [ 61 ], BestKeeper [ 62 ] and the comparative delta-Ct method [ 63 ] did not evaluate candidate genes congruently with standalone geNorm and NormFinder software. This discrepancy among various analytical tools has been described previously [ 23 , 65 , 66 , 67 , 68 ]. However, despite modest inconsistencies, Sv_ACT and Sv_GAPDH 1 primer pairs were consistently ranked among the first three most stable reference genes, no matter which analytical tool was applied.…”

“…Both GAPDH [ 5 , 23 , 48 , 49 , 50 , 65 ] and ACT [ 38 , 39 , 40 ] are often used as reference genes for gene expression quantitation in angiosperms, including the genus Silene [ 4 ]. The ACT gene was reported to be affected by stress conditions [ 69 ], but our sample sets did not include stressed individuals.…”

“…Although the majority of angiosperms are hermaphroditic, some species produce female, male or hermaphroditic flowers on the same individual (monoecy), or on distinct individuals (dioecy). In these species, gender-specific expression patterns, in addition to organ- and tissue-specificity, has to be considered, when evaluating reference genes [ 4 , 23 , 24 ].…”