Screening of Internal Reference Genes Analysis in Alfalfa under Different Abiotic Stress Conditions

Alfalfa is a kind of high-quality legume grass with high crude protein content, good palatability, high grass yield and good nitrogen fixation capacity. Abiotic stresses have become the key factors restricting the yield and quality in alfalfa and other crops, such as drought, extreme changes in ambient temperature, floods, land salinization and acidification, while under the different abiotic stress conditions, gene expression levels will also change accordingly. The selection and using of appropriate reference genes are crucial for the accuracy of gene expression quantification.

We chose the candidate internal reference genes of alfalfa from transcriptome sequence datasets (162 RNA-seq sequencing data) through comparative analysis. Finally, 10 candidate reference genes were selected. These candidate reference gene expressions were determined by RT-qPCR under three common abiotic stresses in production, such as alkali, drought and low temperature. The stability index of these candidate genes was calculated and evaluated correspondently using specific softwares and different algorithms, such as GeNorm, Normfinder, Bestkeeper, ΔCt method and an online analysis tool RefFinder.

The results showed that all 10 pair candidate reference genes could be used in gene expression quantification except for GAPDH and Ms.33066 under low-temperature stress based on screening criteria. Under alkaline stress, the optimal reference gene is UBL-2a and the optimal combination of reference genes is GAPDH and UBL-2a; Under drought stress, the optimal reference gene is Rer1 and the optimal combination of reference genes is Ms.33066 (some candidate reference genes haven't been annotated yet, using gene ID abbreviation number of Medicago sativa L. instead) and Actin; Under low-temperature stress, the optimal reference gene is Ms.99505 and the optimal combination of reference genes is Ms.65463, UBL-2a, Ms.99505 and Actin. Among all the samples, the optimal reference gene is MS.99505 and the optimal combination of reference genes is MS.073307, Rer1, MS.99505 and UBL-2a. GAPDH and Actin aren't the most appropriate reference genes of alfalfa under different abiotic stresses, the optimal reference gene and the optimal combination of reference genes under other abiotic stress need further validation. This paper provides scientific evidence for quantitative analysis of the genes of alfalfa.