Selection of reference genes for gene expression analysis in Liriodendron hybrids' somatic embryogenesis and germinative tissues

Abstract

The differential expression of genes is crucial for plant somatic embryogenesis (SE), and the accurate quantification of gene expression levels relies on choosing appropriate reference genes. To select the most suitable reference genes for SE studies, 10 commonly used reference genes were examined in synchronized somatic embryogenic and subsequent germinative cultures of Liriodendron hybrids by using quantitative real-time reverse transcription PCR. Four popular normalization algorithms: geNorm, NormFinder, Bestkeeper and Delta-Ct were used to select and validate the suitable reference genes. The results showed that elongation factor 1-gamma, histone H1 linker protein, glyceraldehyde-3-phosphate dehydrogenase and α-tubulin were suitable for SE tissues, while elongation factor 1-gamma and actin were best for the germinative organ tissues. Our work will benefit future studies of gene expression and functional analyses of SE in Liriodendron hybrids. It is also serves as a guide of reference gene selection in early embryonic gene expression analyses for other woody plant species.

Figure 1

Somatic embryogenesis and germination groups of Liriodendron hybrids. The somatic embryogenesis group includes PEM and S1–S7, while the germination group includes cotyledon, hypocotyl and radicle. PEM, proembryogenic masses; S1, proembryogenic single cell stage; S2,embryogenic single cell stage; S3, two to four cell proembryo stage; S4, multicell proembryo stage; S5, globular embryo stage; S6, heart/torpedo -shaped embryo stage; S7, cotyledon embryo stage; PL, plantlet developed from somatic embryo. (The images of PEM and S1 to S7 were provided by the Key Laboratory of Forest Genetics and Biotechnology, Ministry of Education of China, Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University).

Figure 2

Expression levels of 10 reference genes in Liriodendron hybrids as determined by the quantification cycle values, also known as the threshold cycle (Ct) values, in the somatic embryogenesis (a) and the germination (b) groups. The boxes indicate the 25th and 75th percentiles, the line across each box represents the median, and whisker caps represent the maximum and minimum Ct values.

Figure 3

Expression stability and rankings of reference genes in Liriodendron hybrids as calculated by geNorm in the somatic embryogenesis (black) and the germination (blue) groups. Genes with the most constitutive expression are indicated on the right side of the graph, with the less stably expressed genes on the left.

Figure 4

Pairwise variation (V) calculated by geNorm to determine the optimal number of reference genes in the somatic embryogenesis (left) and the germination (right) groups of Liriodendron hybrids. A value < 0.15 indicates that the inclusion of an additional reference gene is not required.

Figure 5

ΔCt method for reference gene selection in Liriodendron hybrids. ΔCt variability in reference gene comparisons are shown as medians (lines), 25th percentile to the 75th percentile (boxes) and ranges (whiskers) for all eight samples in the somatic embryogenesis group (a) and three samples in the germination group (b).