Selection of stable housekeeping genes for qPCR in biotic and abiotic stressed jute and their application in lignin pathway analysis

Abstract

This is a comprehensive examination of housekeeping gene (HKG) expression in jute (Corchorus sp.), essential for reliable qRT-PCR studies. Jute is the most abundant and vital source of bast (phloem) fibres that have diverse domestic and industrial applications. The functional analysis of genes in jute is crucial for enhancing its commercial potential and value. We specifically focused on 24 HKGs, including nine previously recommended HKGs for jute. We focused the investigation on the only two commercially cultivated species of jute—C. capsularis and C. olitorius. The jute plants were subjected to nine abiotic stresses: osmotic, salinity, low temperature, high temperature, waterlogging, ABA stress hormone, glyphosate herbicide, and heavy metals (copper and cadmium), along with a biotic stress condition—Macrophomina phaseolina (fungal) infection. After rigorous analyses using the ΔCt method, BestKeeper, geNorm, NormFinder, and RefFinder, we identified ubiquitin1 (UBI) and beta-tubulin (BTUB) as the most stable HKGs for both the jute species under various stress conditions. The gene stability of UBI and BTUB was assessed in different jute cultivars, tissues, at different developmental stages, and PCR conditions. As a case study, we investigated eight genes involved in the jute lignin pathway in a low-lignin fibre-producing white jute mutant cultivar—dlpf—compared to its control cultivar, JRC212. Our findings revealed low expression of the three genes- CcCCoAoMT2, CcC4H1, and CcCAD7 in the dlpf cultivar, promising a potential for developing low-lignin jute varieties to meet long-standing industrial demands. The jute HKG expressions under various stress conditions establish a foundation for future research for crop improvement.

Keywords

• Jute
• Housekeeping gene
• Internal control gene
• Reference gene
• Abiotic stress
• Macrophomina phaseolina