Abstract
Study on expression of genes for the traits associated with hypoxia tolerance during the germination demands robust choice of reference genes for transcript data normalization and gene validation through real-time quantitative polymerase chain reaction (RT-qPCR). However, reliability and stability of reference genes across different rice germplasms under hypoxic condition have not been accessed yet. Stability performance of reference genes such as eukaryotic elongation factor 1 α (eEF1α), ubiquitin 10 (UBQ10), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18SrRNA), 25S ribosomal RNA (25SrRNA), β-tublin (β-TUB), actin11 (ACT11), ubiquitin C (UBC), eukaryotic elongation factor 4 α (eIF4α), and ubiquitin5 (UBQ5) was accessed through statistical algorithms like geNorm, NormFinder, Comparative ΔCt method, BestKeeper, and RefFinder in three rice germplasms (KHO, RKB, and IR-64) with varied level of tolerance to hypoxic condition during germination. Among all genes used, OsGAPDH was found to be the most suitable reference gene under hypoxic condition. The performance of the highest-ranking reference gene (OsGAPDH) in terms of stability based on statistical algorithms was further validated for its reliability and stability through RT-qPCR with hypoxia-induced target gene OsTTP7. The identified stable housekeeping gene could be used as internal control for gene expression analysis in rice under hypoxia.
Highlights
Devastation due to flooding during sowing and transplanting of rice leads to severe crop loss (Jackson and Ram 2003; Setter et al 1997)
Comparative analyses of all the genes tested revealed a wide range of expression variances of the cycle threshold (Ct) values under all three treatments with the highest variation observed in Os25SRNA and Os18SRNA and lower variation in OsGAPDH, OseEF1α, and OseIF4α
In order to understand the molecular basis of the response of rice to an abiotic stimulus such as hypoxic condition during germination, studies with qPCR have been widely conducted for eukaryotic elongation factor 1 α (eEF1α) glyceraldehyde 3-phosphate dehydrogenase (GAPDH) elF4α β-TUB ubiquitin 10 (UBQ10) ubiquitin C (UBC) 18S ribosomal RNA (18SrRNA) UBQ5 Actin 25S ribosomal RNA (25SrRNA)
Summary
Devastation due to flooding during sowing and transplanting of rice leads to severe crop loss (Jackson and Ram 2003; Setter et al 1997). Several rice genotypes exhibiting a different level of tolerance to hypoxic condition during germination were identified through germplasm screening (Septiningsih et al 2013). A tremendous advancement in breeding for hypoxic germination-tolerant rice varieties supplemented with high throughput tools for genomic studies for quantitative assessment of genes at transcript level provides a greater opportunity to understand the genetic and molecular basis of hypoxia tolerance. The signal cascade emanating from the perception of stimuli led to activation of hypoxia-responsive genes through various transcription factors, either at the transcriptional or posttranscriptional level results in re-programming of adaptive response at metabolic and physiological level (Sasidharan and Mustroph 2011). Changes in signaling and transcriptional regulation are species-specific (Mustroph et al 2010). For these instances, quantitative real-time PCR (RT-qPCR), an analytical
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