Screening Of Internal Reference Genes Based On De Novo Transcriptome Data Of Cranberry Fruit

The selection of the reference gene is critical for the results of gene expression analysis using real-time quantitative PCR(qRT-PCR).In this study,Referring to the traditional reference genes in plants and combining transcriptome data of cranberry fruit,ten candidate internal reference genes(ACTIN,cyclophilin,elongation factor 1α,F-box protein family,glyceraldehyde-3-phosphate dehydrogenase,beta tubulin,sand family protein,18 S ribosomal RNA,protein phosphate Enzyme 2A regulatory subunit,RH 8)were selected for qRT-PCR,and three statistical softwares(geNorm,NormFinder and Bestkeeper)were used for data analysis.The candidate internal reference genes were evaluated for their stability of expression under the influence of different experimental factors(cranberries of different cultivars;different tissues of cranberries;cranberries treated with salt stress;cranberries treated with alkali stress;simulated drought stress treatment of cranberries).The main results are:1.Among the cranberries of different cultivars,PP2 A is the best choice for a single internal reference gene,and PP2A+SAND is the best choice for the combination of internal reference genes;18S rRNA is the candidate internal reference gene with the most unstable expression level in this sample set.2.In different tissues of cranberry,SAND is the best choice for a single internal reference gene,and PP2A+SAND is the best combination of internal reference genes;ACTIN is the candidate internal reference gene with the most unstable expression level in this sample set.3.When cranberry is subjected to abiotic stress,both PP2 A and CYP 2 can be used as the best choice for a single internal reference gene in cranberry treated with salt stress;PP2A is the best choice as a single internal reference gene in cranberry treated with alkali stress;In PEG simulated drought-treated cranberry and abiotic stress-treated cranberry,SAND are the best choice for a single internal reference gene;PP2A + SAND is the best combination of internal reference genes for cranberries when subjected to the any of above abiotic stress conditions;when cranberry is subjected to abiotic stress,the expression level of GAPDH cannot be maintained in a stable state.When all the test materials are combined as a sample set for data analysis,the stable expression of a single internal reference gene or a combination of internal reference genes could not be accurately screened.In summary,it is recommended to select appropriate internal reference genes or combinations of internal reference gene for specific experimental conditions to standardize data for more reliable results of gene expression analysis.This study is an initial attempt to screen out the appropriate internal reference genes in cranberries under different experimental conditions,combining the transcriptome data.This study lay the foundation for the study of gene expression analysis of cranberry under specific experimental conditions,revealing the biological characteristics and genetics and breeding of cranberry.