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Identification of reference genes for real-time PCR gene expression studies during seed development and under abiotic stresses in Cyamopsis tetragonoloba (L.) Taub

Guar (Cyamopsis tetragonoloba) is an important industrial crop. The knowledge about genes of guar involved in various processes can help in developing improved varieties of this crop. qRT-PCR is a preferred technique for accurate quantification of gene expression data. This technique requires the use of appropriate reference genes from the crop to be studied. Such genes have not been yet identified in guar. The expression stabilities of the 10 candidate reference genes, viz., CYP, ACT 11, EF-1α, TUA, TUB, ACT 7, UBQ 10, UBC 2, GAPDH and 18S rRNA were evaluated in various tissues of guar under normal and abiotic stress conditions. Four different algorithms, geNorm, NormFinder, BestKeeper and ∆Ct approach, were used to assess the expression stabilities and the results obtained were integrated into comprehensive stability rankings. The most stable reference genes were found to be CYP and ACT 11(tissues), ACT 11, UBC 2 and ACT 7 (seed development), ACT 7 and TUB (drought stress), TUA, UBC 2 and CYP (nitrogen stress), TUA and UBC 2 (cold stress), GAPDH and ACT 7 (heat stress) and GAPDH and EF-1a (salt stress). These results indicated the necessity of identifying a suitable reference gene for each experimental condition. Four selected reference genes were validated by normalizing the expression of CtMT1 gene. To the best of our knowledge this is the first report on the identification of reference genes in guar. These findings are likely to provide a boost to the gene expression studies in this important crop.