Identification and validation of reference genes in vetiver (Chrysopogon zizanioides) root transcriptome

Vetiver [Vetiveria zizanioides (L.) Roberty] is a perennial C-4 grass traditionally valued for its aromatic roots/root essential oil. Owing to its deep penetrating web-forming roots, the grass is now widely used across the globe for phytoremediation and the conservation of soil and water. This study has used the transcriptome data of vetiver roots in its two distinct geographic morphotypes (North Indian type A and South Indian type B) for reference gene(s) identification. Further, validation of reference genes using various abiotic stresses such as heat, cold, salt, and drought was carried out. The de novo assembly based on differential genes analysis gave 1,36,824 genes (PRJNA292937). Statistical tests like RefFinder, NormFinder, BestKeeper, geNorm, and Delta-Ct software were applied on 346 selected contigs. Eleven selected genes viz., GAPs, UBE2W, RP, OSCam2, MUB, RPS, Core histone 1, Core histone 2, SAMS, GRCWSP, PLDCP along with Actin were used for qRT-PCR analysis. Finally, the study identified the five best reference genes GAPs, OsCam2, MUB, Core histone 1, and SAMS along with Actin. The two optimal reference genes SAMS and Core histone 1 were identified with the help of qbase + software. The findings of the present analyses have value in the identification of suitable reference gene(s) in transcriptomic and molecular data analysis concerning various phenotypes related to abiotic stress and developmental aspects, as well as a quality control measure in gene expression experiments. Identifying reference genes in vetiver appears important as it allows for accurate normalization of gene expression data in qRT-PCR experiments.