Identification and testing of reference genes for Sesame gene expression analysis by quantitative real-time PCR

Abstract

Sesame (Sesamum indicum L.) is an ancient and important oilseed crop. However, few sesame reference genes have been selected for quantitative real-time PCR until now. Screening and validating reference genes is a requisite for gene expression normalization in sesame functional genomics research. In this study, ten candidate reference genes, i.e., SiACT, SiUBQ6, SiTUB, Si18S rRNA, SiEF1α, SiCYP, SiHistone, SiDNAJ, SiAPT and SiGAPDH, were chosen and examined systematically in 32 sesame samples. Three qRT-PCR analysis methods, i.e., geNorm, NormFinder and BestKeeper, were evaluated systematically. Results indicated that all ten candidate reference genes could be used as reference genes in sesame. SiUBQ6 and SiAPT were the optimal reference genes for sesame plant development; SiTUB was suitable for sesame vegetative tissue development, SiDNAJ for pathogen treatment, SiHistone for abiotic stress, SiUBQ6 for bud development and SiACT for seed germination. As for hormone treatment and seed development, SiHistone, SiCYP, SiDNAJ or SiUBQ6, as well as SiACT, SiDNAJ, SiTUB or SiAPT, could be used as reference gene, respectively. To illustrate the suitability of these reference genes, we analyzed the expression variation of three functional sesame genes of SiSS, SiLEA and SiGH in different organs using the optimal qRT-PCR system for the first time. The stability levels of optimal and worst reference genes screened for seed development, anther sterility and plant development were validated in the qRT-PCR normalization. Our results provided a reference gene application guideline for sesame gene expression characterization using qRT-PCR system.

Fig. 1

Expression levels of ten candidate reference genes tested in 32 sesame samples. a C _t values of ten candidate reference genes with three replicates. b The mean C _t values of ten candidate reference genes in all sesame samples. The boxes represent mean C _t values. The bars indicate the maximum and minimum values

Fig. 2

Expression stability values (M) of 10 genes in eight sample groups (a–h) by geNorm software. a All sesame samples (S1–S32), b vegetative tissues in different developing stages (S1–S6), c biotic stress treatment (S7–S9) and normal control (S1–S3), d abiotic stress treatment (S10–S18) and normal control (S1–S3), e ABA treatment (S19–S21) and normal control (S1–S3), f developing buds (S22–S24), g developing seeds (S25–S28), h germinating seeds (S29–S31)

Fig. 3

Pairwise variation (V) analysis of 10 sesame candidate reference genes in eight sample groups. Asterisk indicates the optimal number of reference genes for a–h sample groups. a–h Sample groups are the same as in Fig. 2

Fig. 4

Expression stability analysis of ten reference genes in eight sample groups by BestKeeper. a–h Sample groups were the same as in Fig. 2. SD standard deviation. A lower average expression stability value indicates more stable expression

Fig. 5

The expression level of the SiSS in different plant organs. SiUBQ6 (i), SiAPT (ii) and SiCYP (iii) were used as recommended internal controls defined by NormFinder, geNorm and BestKeeper. SiGAPDH (iv) and Si18S RNA (v) were used as the worst internal controls accordingly

Fig. 6

The expression level of the SiLEA in different plant organs. SiUBQ6 (i), SiAPT (ii) and SiCYP (iii) were used as recommended internal controls defined by NormFinder, geNorm and BestKeeper. SiGAPDH (iv) and Si18S RNA (v) were used as the worst internal controls accordingly

Fig. 7

The expression level of the SiLEA in developing seed (5–35 DAF) organs. SiTUB (i), SiACT (ii), SiAPT (iii) and SiDNAJ (iv) were used as recommended internal controls defined by NormFinder, geNorm and BestKeeper. Si18S RNA (v) was used as the worst internal controls accordingly

Fig. 8

The expression level of the SiGH in fertile anthers (FA) and sterile anthers (SA) of different sizes (early anther with 2.1–4.0 mm length; late anther with 4.1–7.0 mm length). SiUBQ6 (i), SiHistone (ii) and Si18S RNA (iii) were used as recommended internal controls defined by NormFinder, geNorm and BestKeeper. SiGAPDH (iv) and SiTUB (v) were used as the worst internal controls accordingly