Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.)

Abstract

Background: Perennial ryegrass (Lolium perenne L.) is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use.

Results: Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2) were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L.) samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h), a moderately, but stably expressed eEF1A (s), and combined expression of multigene eEF1A (m). NormFinder identified eEF1A (s) and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field.

Conclusions: This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples, helping pave the way to conduct gene expression studies in perennial biomass crops under field-conditions. From our study several stably expressed reference genes have been validated. This provides useful candidates for reference gene selection in perennial ryegrass under conditions other than those tested here.

Figure 1

Crossing point (Cp) value variability in candidate reference gene comparisons for the 442 perennial ryegrass samples. Variability is displayed as medians (lines), 25^th percentile to the 75^th percentile (boxes) and ranges (whiskers).

Figure 2

Expression stability and ranking of candidate reference genes as calculated by geNorm. (A) all 442 perennial ryegrass tissue samples, (B) 422 field-grown samples harvested following different defoliation management, (C) 13 laboratory-grown samples, (D) 218 perennial ryegrass stubble samples, (E) 220 perennial ryegrass leaf samples, (F) four perennial ryegrass callus, inflorescence and root samples, (G) five perennial ryegrass etiolated seedlings of different cultivars, (H) four field-grown samples harvested at the peak of each season, (I) three laboratory-grown samples to evaluate water stress and (J) four laboratory-grown samples to evaluate cold stress. A lower value of average expression stability (M) indicates more stable gene expression.

Figure 3

Pairwise variation (V) to determine the optimal number of reference genes for accurate normalisation. (A) all 442 perennial ryegrass tissue samples, (B) 422 field-grown samples harvested following different defoliation management, (C) 13 laboratory-grown samples, (D) 218 perennial ryegrass stubble samples, (E) 220 perennial ryegrass leaf samples, (F) four perennial ryegrass callus, inflorescence and root samples, (G) five perennial ryegrass etiolated seedlings of different cultivars, (H) four field-grown samples harvested at the peak of each season, (I) three laboratory-grown samples to evaluate water stress and (J) four laboratory-grown samples to evaluate cold stress.

Figure 4

Relative quantification of the target gene EF-Tu in perennial ryegrass leaf tissue throughout regrowth. Normalisation was carried out using the four most stable reference genes defined by geNorm, the two most stable reference genes defined by NormFinder or the least stable reference gene, eEF1A (h).