Reference gene selection for quantitative real-time reverse-transcriptase PCR in orchardgrass subjected to various...

Area of Science:

• Plant molecular biology
• Genomics
• Agricultural science

Background:

• Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is essential for gene expression measurement.
• Accurate qRT-PCR relies on the stability of reference genes.
• Orchardgrass (Dactylis glomerata L.) is a globally important cool-season forage grass.

Purpose of the Study:

• To identify and validate stable reference genes for qRT-PCR in orchardgrass under various abiotic stresses.
• To assess the expression stability of ten candidate reference genes.

Main Methods:

• Selected ten candidate reference genes: ATP-binding (ABC), actin (ACTIN), cyclophilin (CYP2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-amylase 4 (BAM4), zeitlupe (ZTL), MAP Kinase 4 (MPK4), ubiquitin-conjugating enzyme (UBC), S-adenosylmethionine decarboxylase (SAMDC), and translationally controlled tumor protein (TCTP).
• Evaluated gene expression in orchardgrass leaves and roots under drought, salinity, heat, waterlogging, and abscisic acid (ABA) treatments.
• Utilized GeNorm, BestKeeper, NormFinder, and RefFinder for normalization and validation.

Main Results:

• Reference gene expression was found to be stress-dependent.
• For drought stress: ACTIN, CYP2, and ABC were most stable.
• For salt stress: ACTIN, TCTP, and ABC were most stable.
• For heat stress: ACTIN, CYP2, and ABC were suitable.
• For waterlogging: CYP2, MPK4, and ABC were most suitable.
• For ABA treatment: ACTIN, CYP2, and MPK4 were the best.

Conclusions:

• Identified and validated specific reference genes for accurate gene expression quantification in orchardgrass under diverse abiotic stresses.
• The findings provide essential tools for molecular studies in orchardgrass, enhancing stress response research.