Validation of suitable genes for normalization of diurnal gene expression studies in Chenopodium quinoa

Abstract

Quinoa depicts high nutritional quality and abiotic stress resistance, attracting strong interest in the last years. To unravel the function of candidate genes for agronomically relevant traits, studying their transcriptional activities by RT-qPCR is an important experimental approach. The accuracy of such experiments strongly depends on precise data normalization. To date, validation of potential candidate genes for normalization of diurnal expression studies has not been performed in C. quinoa. We selected eight candidate genes based on transcriptome data and literature survey, including conventionally used reference genes. We used three statistical algorithms (BestKeeper, geNorm and NormFinder) to test their stability and added further validation by a simulation-based strategy. We demonstrated that using different reference genes, including those top ranked by stability, causes significant differences among the resulting diurnal expression patterns. Our results show that isocitrate dehydrogenase enzyme (IDH-A) and polypyrimidine tract-binding protein (PTB) are suitable genes to normalize diurnal expression data of two different quinoa accessions. Moreover, we validated our reference genes by normalizing two known diurnally regulated genes, BTC1 and BBX19. The validated reference genes obtained in this study will improve the accuracy of RT-qPCR data normalization and facilitate gene expression studies in quinoa.

Haplotype variations of major flowering time genes in quinoa unveil their role in the adaptation to different environmental conditions

“…As reference genes, we used the geometric mean of CqIDH ‐ A and CqPTB genes. These genes turned out to be best suited as reference genes because they were stably expressed during different growth stages and different time points of the day (Maldonado‐Taipe, Sarange, Schmöckel, Jung, & Emrani, 2021).…”

“…We mapped Illumina short-read sequences against an improved version of the quinoa reference genome (Jarvis et al, 2017). SNP variants were identified on a population scale using the '-emitRefConfidence' function of the HaplotypeCaller algorithm on each sample, followed by joint genotyping on all samples using GenotypeGVCFs from the Genome Analysis Toolkit (McKenna et al, 2010). The resulting VCF file was further filtered based on minor allele frequency (MAF) ≥ 0.05, minimum mean depth (min-meanDP) ≥ 5.0, and maximum-missing (max-missing) ≤ 0.5 using VCFtools (Danecek et al, 2011) and obtained a high confident SNP data set (HC-SNPs).…”

Screening and validating of endogenous reference genes in Chlorella sp. TLD 6B under abiotic stress

“…Among them, 18S , GTP , IDH and CYP and their combinations were the most stable endogenous reference genes (Table 4 ). Several genes have been shown to be good stable samples, including 18S 53 , 54 , IDH 55 , 56 , and CYP 57 , 58 . In addition, the experimental results showed that GTP can be used as a stable results which is contrary to Zhao’s results 41 ; GAPDH and α-TUB were the least stable genes under various stresses in the present experimental catch, but were the most stable reference in some reported endogenous reference gene studies 59 – 61 , which is an indication that differences in species or experimental conditions lead to uniqueness of genes, therefore, the selection of suitable samples under different stress conditions is necessary to achieve reliable experimental results.…”

Leaf and shoot apical meristem transcriptomes of quinoa (Chenopodium quinoaWilld.) in response to photoperiod and plant development

“…Primers used for gene expression analysis are listed in Supplementary Table 2. CqPTB and CqIDH-A, identified in our previous study as suitable housekeeping genes for RT-qPCR analysis in quinoa, were used as reference genes (Maldonado-Taipe et al ., 2021).…”