Prediction of candidate reference genes in VBT transcriptomics and design of primers
The fruit transcriptome includes green fruits, red fruits, and blue fruits, which represent the progressive stages of fruit maturity. 27 Based on previous studies and transcriptomic analysis, we have identified ten reference genes. There were five actin-related genes (actin-related protein 2/3 complex subunit 2A (actin 1), actin-7 (actin 2), actin depolymerization factor 2 (actin 3), actin-97 (actin 4) and actin-related protein 3 (actin 5)), 50S ribosomal protein L27 (RP), ubiquitin-conjugating enzyme E25 (UBE), protein containing the domain of l ubiquitin DSK2b (UBQ), NADH dehydrogenase (NADH), and adenylate kinase 4 (ADK), all of which have been widely used for gene validation in plants and fruits (Table S1). Simultaneously, RNA from green, red and blue fruits was extracted and pure total RNA was quantified. Additionally, primers for RT–qPCR were designed (Table S2), and then these primers were confirmed using melting curves. The results showed that only one peak for each primer pair was present, indicating exceptionally high primer specificity (Fig. 1).
Reference candidate gene expression levels
To assess expression levels of candidate reference genes, cycle threshold (Ct) values of ten candidate reference genes in green, red and blue fruits were acquired with three replicates. Expression levels of these ten reference genes varied widely, with Ct values ranging from 17.61 to 29.73 cycles. According to the Ct values, the expression levels of actin 2 in the three fruit samples were the highest and actin 5 was the lowest (Fig. 2). In green fruits, all gene expression levels ranged from actin2 > actin3 > NADH > actin4 > UBE > ADK > UBQ > actin1 > RP > actin5 (Table S3). In red and blue fruits, the arrangement changed in the order actin2 > actin3 > NADH > actin4 > ADK > UBE > UBQ > actin1 > RP > actin5 (Table S3). Based on these data, we also found that the expression levels of actin3, NADH, ADKand EBU showed narrow variances across the three stages of fruit development, which convincingly suggested that their expression levels were stable in all three fruits (Fig. 2). These data demonstrated that these genes could be used as reference genes.
Expression stability of reference candidate genes
The average expression stability (M value) of the ten genes was calculated by geNorm (version 3.5). The M values of the ten green, red and blue fruit genes were below the threshold of 1.5, meaning that all selected genes could be used as reference genes (Table 1). However, the M value of these genes showed great variability; the M value of the RP gene was the highest, while those of the NADH and ADK genes were the smallest, suggesting that they had the highest expression stability (Table 1). These results showed that NADH and ADK are the best reference genes, followed by actin2. To better assess the optimal number of reference genes, the pairwise variation (Vnot/Vn+1) was analyzed using geNorm, and 0.15 was used as a cut-off value according to a previous study. The results showed that all Vnot/Vn+1 values were less than 0.15. Moreover, the pairwise variation value (V2/V3) was significantly less than 0.15 (Fig. 3), suggesting that only two reference genes were needed for the genetic profile; thus, we assumed that no additional reference genes were needed.
In addition to geNorm analysis, NormFinder analysis is also a necessary method to confirm the best reference gene. The gene with the smallest S value was the most suitable reference gene. According to NormFinder analysis, the S value of all candidate genes was less than 1.0, indicating that all reference genes we evaluated could be used for RT-qPCR analysis. Among them, the S value of the actin1 gene (0.03) was the smallest and that of RP (0.785) was the largest (Table 1), indicating that the actin1 gene is the most stable reference gene and the results were not not consistent with geNorm analysis.
Finally, BestKeeper was used to assess the stability of the reference genes through the standard deviation (SD) and the coefficient of variance (CV) of the Ct values. If the SD value of the Ct values was > 1, the gene could not not be defined as a reference gene. According to BestKeeper’s analysis, both Ct values of all genes were less than 1.0, indicating that all genes we selected met BestKeeper’s standard. Among them, however, actin3, UBE and NADH are the three genes with the lowest SD values. Like the SD value, these three genes had smaller CV values (Table 1). However, UBQ, actin5 and RP both had higher SD values and CV values (Table 1). So, while these results demonstrate that the ten genes we selected could be used as reference genes, the actin3, UBE, and NADH genes are the best choices overall.
Full ranking recommended
Since the analysis by the three methods is not uniform, we compared and ranked the reference genes by RefFinder, which is a comprehensive web tool combined with geNorm, Normfiner, BestKeeper and Ct values. The overall final ranking based on the ranking of each program is shown in Table 2. These results showed that actin2ADK, actin1and NADH were the four genes with smaller values, and the overall ranking values (actin2, 2.59; ADK, 2.83; actin1, 3.15; NADH, 3.20) were very close (Table 2). However, the partial Ct values of actin1 in soft fruits were greater than 26, suggesting that its expression levels are slightly low and may not be suitable for RT-qPCR analysis of genes at all stages of fruit development. Considering all of these results, we conclude that the three reference genes, actin2ADK and NADH are the best reference genes for RT–qPCR analysis at all stages of fruit development.
Identification of reference genes using marker genes in fruit development
Since ACO and PG genes are generally up-regulated in fruit ripening programs, assessing their expression levels should be the best way to confirm the reliability of reference genes. Additionally, the ranking scores of the top three reference genes (actin2, ADK, and NADH) is close, meaning any are suitable for gene expression analysis. To assess the reference gene, expression levels of ACO1, ACO2, and PG were calculated using NADH, ADK, NADH+ADK, RP, and actin5. We found that the transcription levels of RP-normalized ACO genes were not consistent with their expression characteristics (Fig. 4). Moreover, actin5-normalized PG transcript levels in berries were not consistent with PG expression characteristic and transcriptomic analysis (Fig. 4). However, the results calculated by NADH and ADK reflect the expression characteristics of these genes and the transcriptomic analysis (Fig. 4). Moreover, NADH + ADK as a coreference gene is not consistent with NADH or ADK alone (Fig. 4). Taken together, NADH or ADK are the most suitable reference genes for RT–qPCR analysis in VBT fruit development.
Differential expressed genes from validation of transcriptomic analysis using NADH
To further assess the reliability of the reference genes, the expression levels of 15 genes related to flavonoid synthesis were normalized to the expression level of NADH. These genes code for shikimate O-hydroxycinnamoyltransferase (SHT), flavonol synthase (FLS), flavonoid 3′-monooxygenase (F3MO), flavonoid 3′,5′-hydroxylase 2 (F35H2), dihydroflavonol 4-reductase ( DFR), leucoanthocyanidin dioxygenase (LDOX), leucoanthocyanidin reductase (LAR), anthocyanidin reductase (ANR), UDP-glycosyltransferase 74B1 (UGT), caffeoyl-CoA O-methyltransferase (CcoA), probable caffeoyl-CoA O-methyltransferase (At4g) , hydroxypalmitate O-feruloyl transferase (HOFT), transcription factor MYB113 (MYB113), acetylajmalan esterase (Ace), and 2-oxoglutarate-dependent dioxygenase (ODD) (Table S4). The results showed that the Pearson correlation coefficients of the 12 genes are greater than 0.9 (Fig. 5, Table S5), indicating that their RT–qPCR results are highly consistent with the FPKM values in the transcriptome. Moreover, the Pearson correlation coefficients of FLS and LAR2and ANR2 arranged from 0.81 to 0.89 (Table S5), indicating a weaker correlation. However, their expression profiles were also very consistent with the transcriptomic data. Taken together, the NADH reference gene is very reliable for RT-qPCR analysis at the fruit ripening stage.