Identification and validation of stable reference genes for RT-qPCR analyses of Kobresia littledalei seedlings

• Published in: BMC plant biology 2024-05, Vol.24 (1), p.389-389, Article 389
• Main Authors: Sun, Haoyang, Li, Chunping, Li, Siyu, Ma, Jiaxin, Li, Shuo, Li, Xin, Gao, Cai, Yang, Rongchen, Ma, Nan, Yang, Jing, Yang, Peizhi, He, Xueqing, Hu, Tianming
• Format: Article
• Language: English
• Subjects: Abscisic acid; Abscisic Acid - metabolism; Actin; Algorithms; Biological products industry; Botanical research; Cold; Cyperaceae; Cyperaceae - genetics; Dehydrogenases; Dominant species; Drought; Droughts; Efficiency; Gene expression; Gene Expression Profiling - methods; Gene Expression Regulation, Plant; Genes; Genes, Plant; Genetic aspects; Genetic research; Genetic transcription; Genomes; Genomics; Gibberellins; Gibberellins - metabolism; Glyceraldehyde-3-phosphate dehydrogenase; Identification and classification; Kinases; Kobresia; Kobresia littledalei; Leaves; Meadows; Methods; Molecular biology; Muscle proteins; Normalization; Phosphates; Physiological aspects; Plant genetics; Plants (botany); Polymerase chain reaction; Proteins; Real-Time Polymerase Chain Reaction; Reference gene; Reference Standards; Reproducibility of Results; Reverse transcriptase; Reverse transcription; Roots; RT-qPCR; Salts; Seedlings; Seedlings - genetics; Seeds; Stability; Stems; Stress, Physiological - genetics; Superoxide; Superoxide dismutase; Testing
• ISSN: 1471-2229; 1471-2229
• DOI: 10.1186/s12870-024-04924-w

Kobreisa littledalei, belonging to the Cyperaceae family is the first Kobresia species with a reference genome and the most dominant species in Qinghai-Tibet Plateau alpine meadows. It has several resistance genes which could be used to breed improved crop varieties. Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) is a popular and accurate gene expression analysis method. Its reliability depends on the expression levels of reference genes, which vary by species, tissues and environments. However, K.littledalei lacks a stable and normalized reference gene for RT-qPCR analysis. The stability of 13 potential reference genes was tested and the stable reference genes were selected for RT-qPCR normalization for the expression analysis in the different tissues of K. littledalei under two abiotic stresses (salt and drought) and two hormonal treatments (abscisic acid (ABA) and gibberellin (GA)). Five algorithms were used to assess the stability of putative reference genes. The results showed a variation amongst the methods, and the same reference genes showed tissue expression differences under the same conditions. The stability of combining two reference genes was better than a single one. The expression levels of ACTIN were stable in leaves and stems under normal conditions, in leaves under drought stress and in roots under ABA treatment. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was stable in the roots under the control conditions and salt stress and in stems exposed to drought stress. Expression levels of superoxide dismutase (SOD) were stable in stems of ABA-treated plants and in the roots under drought stress. Moreover, RPL6 expression was stable in the leaves and stems under salt stress and in the stems of the GA-treated plants. EF1-alpha expression was stable in leaves under ABA and GA treatments. The expression levels of 28 S were stable in the roots under GA treatment. In general, ACTIN and GAPDH could be employed as housekeeping genes for K. littledalei under different treatments. This study identified the best RT-qPCR reference genes for different K. littledalei tissues under five experimental conditions. ACTIN and GAPDH genes can be employed as the ideal housekeeping genes for expression analysis under different conditions. This is the first study to investigate the stable reference genes for normalized gene expression analysis of K. littledalei under different conditions. The results