Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.).
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.
Lee
Julia M
JM
DairyNZ Ltd, Private Bag 3221, Hamilton 3240, New Zealand. julia.lee@dairynz.co.nz
Roche
John R
JR
Donaghy
Danny J
DJ
Thrush
Anthony
A
Sathish
Puthigae
P
D020869:Gene Expression Profiling / Q000592:standards*; D017343:Genes, Plant; D006657:Histones / Q000032:analysis / Q000235:genetics; D008129:Lolium / Q000235:genetics*; D020648:Peptide Elongation Factor 1 / Q000032:analysis / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000032:analysis / Q000235:genetics; D016601:RNA-Binding Proteins / Q000032:analysis / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D044763:Ubiquitin-Conjugating Enzymes / Q000032:analysis / Q000235:genetics
D006657:Histones; D020648:Peptide Elongation Factor 1; D016601:RNA-Binding Proteins; D044763:Ubiquitin-Conjugating Enzymes; D046988:Proteasome Endopeptidase Complex