Genes encoding membrane proteins showed stable expression in rice under aerobic condition: novel set of reference genes for expression studies
- 47 Downloads
A set of reference genes expressing stably under aerobic and anaerobic conditions in rice is essential to execute omics studies relating to aerobic adaptations. Stability of expression of ten rice reference genes, viz. Actin, eEF-1a, eIF-5C, Exp1, Exp2, Memp, SKP1A, TF-SUI1, TPH, and UBQ5 was validated across six experimental sets in shoot and root tissues at seedling, tillering, and panicle initiation stages. Comprehensively, Memp (Membrane protein), TPH (Tumor protein homolog), and Exp1 (Expressed protein) were revealed as the most stable set with acceptable M and V value according to the gold standards of qRT-PCR using various algorithms/tools. The identified set of reference genes was validated using root trait genes, which showed concurrence with the functional expression patterns in the aerobic and anaerobic adapted cultivars. The Memp (Membrane protein), TPH (Tumor protein homolog), and Exp1 (Expressed protein) genes are the most stable reference genes across the root and shoot at various developmental stages under aerobic and anaerobic conditions in rice. This is the first study for accurate and reliable relative gene expression analysis in rice grown in aerobic and anaerobic conditions.
KeywordsAerobic Anaerobic Gene expression qRT-PCR Reference gene Rice
Quantitative real-time polymerase chain reaction
Coefficient of variation
Days after germination
Deeper rooting 1
- PI stage
Panicle initiation stage
Tumor protein homolog
The authors are thankful to Director, ICAR-IIRR (Institute Research Council Project Codes ABR/CI/BT/12 and ABR/CI/BT/15) and Director, Institute of Biotechnology, PJTSAU for providing necessary facilities for research. Amol S. Phule acknowledges the University Grant Commission (UGC), New Delhi, India for providing National Fellowship for furnishing the doctoral programme.
PAK, MSM, and KMB conceived, planned, and designed study. ASP, KMB, PS, and MBB conducted experiments. KMB and ASP analyzed the data and wrote the manuscript. PAK and MSM critically edited manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Andersen C, Jensen J, Orntoft T (2004) Normalization of real-time quantitative reverse transcription PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245. https://doi.org/10.1158/0008 CrossRefPubMedGoogle Scholar
- Bevitori R, Oliveir MB, Grossi-de-Sá MF, Lanna AC, da Silveira RD, Petrofeza S (2014) Selection of optimized candidate reference genes for qRT-PCR normalization in rice (Oryza sativa L.) during Magnaporthe oryzae infection and drought. Genet Mol Res 13(4):9795–9805. https://doi.org/10.4238/2014.November.27.7 CrossRefPubMedGoogle Scholar
- Pabuayon IM, Yamamoto N, Trinidad JL, Longkumer T, Raorane ML, Kohli A (2016) Reference genes for accurate gene expression analyses across different tissues, developmental stages and genotypes in rice for drought tolerance. Rice 9(1):32. https://doi.org/10.1186/s12284-016-0104-7 CrossRefPubMedPubMedCentralGoogle Scholar
- Patel DP, Das A, Munda GC, Ghosh PK, Sandhya J, Kumar M (2010) Evaluation of yield and physiological attributes of high-yielding rice varieties under aerobic and flood-irrigated management practices in mid-hills ecosystem. Agric Water Manag 97(9):1269–1276. https://doi.org/10.1016/j.agwat.2010.02.018 CrossRefGoogle Scholar
- Pathak H, Tewari AN, Sankhyan S, Dubey DS, Mina U, Singh VK, Bhatia A (2011) Direct-seeded rice: potential, performance and problems—a review. Curr Adv Agric Sci 3(2):77–88Google Scholar
- Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations. Biotechnol Lett 26(6):509–515. https://doi.org/10.1023/B:BILE.0000019559.84305.47 CrossRefPubMedGoogle Scholar
- Singh A, Kumar P, Gautam V, Rengasamy B, Adhikari B, Udayakumar M, Sarkar AK (2016) Root transcriptome of two contrasting indica rice cultivars uncovers regulators of root development and physiological responses. Sci Rep 6:39266. https://doi.org/10.1038/srep39266 CrossRefPubMedPubMedCentralGoogle Scholar
- Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):RESEARCH0034. https://doi.org/10.1186/gb-2002-3-7-research0034 CrossRefPubMedPubMedCentralGoogle Scholar