Abstract
Due to the lack of effective and stable reference genes, studies on functional genes in Rubus, a genus of economically important small berry crops, have been greatly limited. To select the best internal reference genes of different types, we selected four representative cultivars of blackberry and raspberry (red raspberry, yellow raspberry, and black raspberry) as the research material and used RT-qPCR technology combined with three internal stability analysis software programs (geNorm, NormFinder, and BestKeeper) to analyze 12 candidate reference genes for the stability of their expression. The number of most suitable internal reference genes for different cultivars, tissues, and fruit developmental stages of Rubus was calculated by geNorm software to be two. Based on the results obtained with the three software programs, the most stable genes in the different cultivars were RuEEF1A and Ru18S. Finally, to validate the reliability of selected reference genes, the expression pattern of the RuCYP73A gene was analyzed, and the results highlighted the importance of appropriate reference gene selection. RuEEF1A and Ru18S were screened as reference genes for their relatively stable expression, providing a reference for the further study of key functional genes in blackberry and raspberry and an effective tool for the analysis of differential gene expression.
Highlights
Accepted: 27 September 2021Blackberry and raspberry are important members of the genus Rubus, which belongs to Rosaceae, a moderately large family, with an estimated 85 genera and more than sexual species [1]
The primer annealing temperature was between 60 ◦ C and 61 ◦ C, and the amplification product length was between 72 bp (RuF-box) and 193 bp (RuEEF1B) (Table 1)
The green fruits, immature fruits and mature fruits, leaves, stems, and stem apexes of raspberry and blackberry were used as templates to amplify different candidate reference genes
Summary
Accepted: 27 September 2021Blackberry and raspberry are important members of the genus Rubus, which belongs to Rosaceae, a moderately large family, with an estimated 85 genera and more than sexual species [1]. Blackberry and raspberry fruits are rich in anthocyanins, organic acids, dietary fiber, vitamins, and minerals [1,2]. They have antioxidant, anticancer, and anti-cardiovascular medicinal properties [3]. Quantitative real-time fluorescent polymerase chain reaction (RT-qPCR) is a commonly used method for the determination of gene expression and has been widely used in disease diagnosis and detection, drug development, and scientific research [6,7,8,9]. The commonly used reference genes are usually stable housekeeping genes, Published: 29 September 2021
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