Identification and validation of reference genes for qRT-PCR based studies in horse gram (Macrotyloma uniflorum)

The quantitative real-time polymerase chain reaction (qRT-PCR) is the most sensitive and commonly used technique for gene expression studies in biological systems. However, the reliability of qRT-PCR results depends on the selection of reference gene(s) for data normalization. Horse gram (Macrotyloma uniflorum) is an important legume crop on which several molecular studies have been reported. However, the stability of reference genes has not been evaluated. In the present study, nine candidate reference genes were identified from horse gram RNA-seq data and evaluated in two horse gram genotypes, HPK4 and HPKM317 under six abiotic stresses viz. cold, drought, salinity, heat, abscisic acid and methyl viologen-induced oxidative stress. The results were evaluated using geNorm, Bestkeeper, Normfinder and delta-delta Ct methods and comprehensive ranking was assigned using RefFinder and RankAggreg software. The overall result showed that TCTP was one of the most stable genes in all samples and in genotype HPK4, while in HPKM317 profilin was most stably expressed. However, PSMA5 was identified as least stable in all the experimental conditions. Expression of target genes dehydrin and early response to dehydration 6 under drought stress was also validated using TCTP and profilin for data normalization, either alone or in combination, which confirmed their suitability for qRT-PCR data normalization. Thus, TCTP and profilin genes may be used for qRT-PCR data normalization for molecular and genomic studies in horse gram.