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Article

Selection and Validation of Reference Candidate Genes for qRT-PCR Analysis in the Developing Fruit of Phyllanthus emblica L.

by
Tianlei Pu
1,2,3,
Jianmin Yuan
2,3,
Wenlin Qu
2,3,
Chengfei Liao
2,3,
Xiao Lei
2,3,
Kunjian Qian
2,3,
Qiongling Zhao
2,3,
Liangjia Shi
2,3,
Lumin Zhang
2,3,* and
Aizhong Liu
1,*
1
Key Laboratory for Forest Resource Conservation and Utilization in the Southwest Mountains of China (Ministry of Education), College of Forestry, Southwest Forestry University, Kunming 650224, China
2
Tropical Eco-Agriculture Research Institute, Yunnan Academy of Agricultural Sciences, Yuanmou 651300, China
3
National Germplasm Resource Nursery for Characteristic Crops in Dry-Hot Areas, Yuanmou 651300, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(9), 1054; https://doi.org/10.3390/horticulturae11091054
Submission received: 16 July 2025 / Revised: 24 August 2025 / Accepted: 25 August 2025 / Published: 3 September 2025
(This article belongs to the Special Issue From the Field to the Table: Unearthing Wild Fruit Species for Enhanced Prospecting and Utilization—2nd Edition)
Browse Figures
Versions Notes

Abstract

Accurate normalization of target gene expression in qRT-PCR experiments requires the development of stable and efficient housekeeping reference genes, particularly for tissue-specific genes in a given organ. Phyllanthus emblica L., an economically important cash tree, has been applied as medical or functional fruit for years in Asian countries due to its fruit that contains rich and diverse active compounds. Developing housekeeping reference genes is critical to investigate the physiological and molecular regulation of fruit development and ripening for P. emblica genetic improvement in breeding practice. Here, based on the expressional stabilities and efficiencies, expression profiles of 13 candidate genes at various fruit development stages were compared between two accessions using expression levels and multiple statistical methods, including BestKeeper, NormFinder, geNorm, ΔCt, and RefFinder. The validation test was conducted through qRT-PCR analysis of two fruit tissue-specific genes, PeGASA and PeMLP, across the different fruit development stages, combined with the comparison of gene expression consistency between qRT-PCR and transcriptomic data. These analyses identified PeACT and PeUBQ6, two regulators of tissue development and ripening, to be the most suitable housekeeping reference genes. Thus, we recommended PeACT and PeUBQ6 can serve as housekeeping reference genes for conducting qRT-PCR analysis in P. emblica fruit, helpful for investigating gene expression related to fruit development and ripening using the qRT-PCR technique.

1. Introduction

The qRT-PCR (quantitative real-time polymerase chain reaction) technique is commonly used to assess transcript expression, owing to its low cost, high accuracy, speed, specificity, and sensitivity features [1]. The gene expression level is significantly affected by a series of experimental procedures, including primer specificity, the quality of cDNA, the efficiency of reverse transcription and the amount of initial cDNA templates [2,3]. Accurate qRT-PCR results depend on the systematic selection of stable housekeeping genes as internal references for gene expression analysis [4,5]. Usually, ACT (Actin) [6], TUB (Tubulin) [7], UBQ (Ubiquitin) [8], 18S RNA (18S Ribosomal RNA) [9], EF1-α (Elongation Factor 1-α) [10], and RPL (Ribosomal Protein L.) [11] have been widely utilized as reference genes for normalizing qRT-PCR data across various plant tissues.
However, although housekeeping genes are generally considered to be constitutively expressed across different tissues, their transcript levels often exhibit distinct differences under various tissue types [12], experimental conditions [13], and different species [14]. Usually, these commonly used reference genes could not serve as universal and suitable references for specific tissues in a given non-model plant [15]. Thus, selecting appropriate reference genes for specific tissues or developmental stages in a given plant is often crucial [9,16,17]. Choosing suitable reference genes for normalizing gene expression is crucial to obtain accurate and comparable results from qRT-PCR data [18,19]. Several statistical algorithms, including geNorm [20], NormFinder [21], BestKeeper [22], ΔCt [23], and RefFinder [24] have been developed to assess the effectiveness and stability of potential reference genes in various experimental systems. Since the development and regulation of fruit pulp is usually tissue-specific compared with leaves, roots, and shoots, several studies have selected and validated suitable housekeeping reference genes in fruit pulp of species such as pear [25], apple [26], tomato [27], peach [28], and soursop [29]. However, the suitable and stable housekeeping reference genes for qRT-PCR analysis are deficient for investigating fruit development and regulation in Phyllanthus emblica L., an economically important cash tree.
P. emblica L., commonly known as Indian gooseberry or amla, is distributed widely across subtropical and tropical regions of Asia, including India, China, Sri Lanka, Indonesia, Myanmar, and the Malay Peninsula [30]. P. emblica fruit has been applied as medical or functional fruit for many years in India and China because its fruit contains rich and diverse active compounds such as ascorbic acid, flavonoids, tannins, alkaloids, and organic acids [31]. P. emblica fruit has been considered as feedstock for developing new drugs to treat various neurological problems, diabetes, and cardiovascular diseases [30]. Although the genetic diversity of P. emblica wild germplasm is rich, the wild population has largely decreased in recent years due to over-harvesting and habitat destruction [32]. Domesticated cultivars have been planted in many Asian regions for economic return. Investigation of the physiological and molecular regulation of fruit development and ripening is critical for P. emblica genetic improvement in breeding practice. Thus, developing suitable and stable housekeeping reference genes is essential for elucidating these regulatory mechanisms.
In this study, we developed 13 candidate reference genes for P. emblica fruit development from an RNA-Seq-based transcriptome dataset, based on their stable expression profiles in two P. emblica accessions and in fruit pulps at different developmental stages. Integrating five different statistical algorithms (geNorm, NormFinder, BestKeeper, ∆Ct method, and RefFinder), we comprehensively analyzed the data by examining the raw qRT-PCR data of 13 candidate reference genes. Among them, we sorted out the ideal, suitable, and stable housekeeping reference genes, PeACT and PeUBQ6. We assessed the reliability by examining expression profiles of the fruit ripening-related genes PeGASA and PeMLP using qRT-PCR with both the most and least stable reference genes, PeACT, PeUBQ6, and PeRPL2, respectively. This work represents the first report to provide a reliable reference gene for qRT-PCR analysis in developing fruit, facilitating future investigations into the molecular mechanisms in P. emblica fruits.

2. Materials and Methods

2.1. Plant Materials

P. emblica L. germplasms were cultivated in a national germplasm resource nursery located at Yuanmou town (CHN, latitude 25°69′ N, longitude 101°87′ E), Yunnan. Two P. emblica accessions, ‘YGZ400’ (with smaller leaves, smaller fruit size, and thinner sarcocarp) and ‘YGZ16’ (with bigger leaves, bigger fruit size, and thicker sarcocarp), were used as experimental materials [32]. Fruits at the different developing stages of each accession, including 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 weeks after flowering (WAF), were sampled. The stages cover the early, middle, and late stages of fruit development. Samples from three biological replicates were collected, and immediately frozen in liquid nitrogen, stored at  −80 °C until RNA extraction.

2.2. Selection of Reference Candidate Genes

Based on the reference genome data of P. emblica [33], we initially identified multiple common housekeeping reference genes used in other plants. The DNA sequences were extracted from the annotated reference genome data. Further, the transcriptome data from different tissues [33], including leaf, shoot, flower, branch, and fruit, were applied to filter out stable expression candidate genes. After determining the candidate genes, we designed primers in Beacon Designer 8 software (Premier Biosoft International) using the following parameters: the melting temperature (Tm) of the primers was 60 ± 1 °C, with a length of 18–21 bp and an amplification range of 80–150 bp. Primers were synthesized by Tsingke Biotech Co., Ltd. (Kunming, China).

2.3. Total RNA Extraction and cDNA Synthesis

Total RNA was extracted and purified from collected samples of developing fruits for each accession using a modified CTAB method. RNA integrity was tested by 1% agarose gel electrophoresis, and concentration was determined using a full wavelength multifunctional microplate reader (TECAN, Männedorf, Switzerland). Total RNA with an A260/A280 absorbance ratio of 1.9–2.2 and the bright bands of 18S and 28S in agarose gel was considered to be qualified and used for further analysis. cDNA synthesis was performed using the PrimeScriptTM FAST RT reagent Kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturer’s protocol. Briefly, total RNA was treated with 5× gDNA Eraser Premix at 42 °C for 2 min to eliminate potential gDNA contamination. cDNA synthesis was carried out using 1000 ng of total RNA in a 20 μL volume in an Eppendorf tube.

2.4. qRT-PCR Analysis

qRT-PCR was performed on triplicate samples from 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 WAF of YGZ400 and YGZ16 for a total of 66 qRT-PCR reactions, using a Lightcycler 96 device (Roche, Basel, Switzerland) in conjunction with TB Green Premix Ex TaqTM II FAST qPCR (Takara, Dalian, China). The reaction system was prepared in a 20 μL PCR tube, following the manufacturer’s instructions. The PCR conditions consisted of an initial denaturation at 95 °C for 30 s, followed by 50 cycles of 95 °C for 5 s and annealing at 60 °C for 30 s. Melting curve analysis was conducted from 65 to 95 °C with a 0.5 °C increase per second. Three technical replicates were performed for each sample. To confirm primer specificity, melting curve analysis was conducted for each sample’s product. Amplification efficiency (E) and the correlation coefficient (R2) were calculated from standard curves generated using a four-fold dilution series of cDNA, with an initial concentration of 1000 ng/μL.

2.5. Analysis of Gene Stability

Based on the qRT-PCR result, the stability of the 13 candidate reference genes was assessed using geNorm [20], NormFinder [21], RefFinder [24], BestKeeper [22], and ΔCt [23] software packages. Cq values served as input, and data analysis was performed with the web-based qbase+ software. Expression stability measurement (M) was calculated using the geNorm program, which evaluates the average pairwise variations (V) among the tested genes. The NormFinder program assesses both intra- and inter-group variations, with the lowest stability value being assigned the highest rank. The BestKeeper program calculates the CV and SD of Cq values, ranking the genes with the lowest CV and SD as the most stable reference genes. RefFinder is an online tool that evaluates each gene’s ranking from different algorithms, assigns a weight accordingly, and computes the geometric mean of these values as the final ranking score.

2.6. Verification of Stability of Candidate Reference Genes

Gene expression related to fruit development in P. emblica was quantified by qRT-PCR, and the relative expression levels at various developmental stages were determined using the 2−∆∆Ct method [34].

2.7. Data Processing

Statistical analysis was performed with IBM SPSS Statistics 19, and data visualization was performed using Microsoft Excel 2022 and GraphPad Prism 8 software. These tools enable the visualization of experimental results.

3. Results

3.1. Identification of Candidate Reference Genes

We initially referred to commonly used housekeeping reference genes in plants such as ACT, UBQ, EF1α, TUB, TUA, GAPDH, PP2A, RPL, and 18S rRNA (Figure S1). Based on the reference genome data of P. emblica [33], we obtained DNA sequences of these genes. Transcriptome data [34] from leaf, shoot, flower, branch, and fruit tissues were analyzed using the criterion of the mean gene expression level (TPM) > 30 and covariance value (CV) < 0.3 among different tissues [35], resulting in the identification of 13 candidate genes covering one ACT (PeACT), seven UBQs (PeUBQ1, PeUBQ2, PeUBQ3, PeUBQ4, PeUBQ5, PeUBQ6, and PeUBQ7), three RPLs (PeRPL1, PeRPL2, and PeRPL3), and two EF1-αs (PeEF1-α1 and PeEF1-α2) genes for further analyzes. The information of 13 selected candidate genes is presented in Table 1.

3.2. Primer Specificity and Amplification Efficiency

The qRT-PCR experiments were performed using the mixed total RNAs from different development stages with different primer pairs. Primer specificity was verified using agarose gel electrophoresis. The results showed that 13 candidate reference genes exhibited a single band with the anticipated fragment size; there was no primer dimer or other non-specific amplification product detected on agarose gel (Figure S2). The amplification length is within 80 to 140 bp, the amplification efficiency is within 90.27% to 109.54%, and the correlation coefficients (R2) > 0.996. Combining the melting curve showed a single, clear amplification peak for each primer pair, with no primer dimers or secondary bands. We considered these primer pairs to be specific and efficient for further use. Detailed information on 13 selected primer pairs was given in Table 2 and Table S1; their melting curves were shown in Figure 1.

3.3. Expression Stability Test of the Candidate Reference Genes

To test the stability and efficiency of 13 primer pairs across fruit development stages, we performed qRT-PCR using cDNAs from 11 stages of fruit development in YGZ400 and YGZ16 accessions, respectively. The 66 cDNA samples, including triplicates for each sample, were used as templates to perform qRT-PCR with each of the 13 selected primer pairs, respectively. The expression stability and efficiency of each primer pair was determined according to its Cq value. All candidate reference genes were expressed in fruits at various developmental stages, showing minimal fluctuation in the expression levels of 13 reference genes. A suitable reference gene should exhibit a moderate expression level (Cq values within 15–30) [36]. The Cq values of 13 tested reference genes varied from 18.07 to 27.17, indicating that all candidates met this basic requirement. PeUBQ3, PeUBQ6, and PeUBQ7 had higher Cq values, reflecting lower expression levels. While PeEF1-α1, PeUBQ2, and PeACT showed lower average Cq values, indicating higher expression levels. Moreover, PeUBQ6 exhibited the narrowest Cq range and could serve as a reliable candidate for target gene normalization, whereas PeRPL2 exhibited the widest range among all candidate reference genes (Figure 2).
Based on the expression levels of each gene at different development stages, the relative expression heatmaps of 13 candidate reference genes between YGZ400 and YGZ16 accessions were obtained, as shown in Figure 3. Optimal reference genes should exhibit consistent and stable expression throughout different development stages. The lower coefficient of variation (CV) value reflects higher stability of the reference gene. Based on Cq value, PeACT showed higher transcript abundance with moderate variation. PeUBQ1 and PeUBQ6 displayed the lower variations in YGZ400, and PeUBQ6 exhibited the least variation in YGZ16 (see Table 3). These results indicated that PeUBQ6 is more stable than other candidate genes.

PCR Stability Test Using Different Statistical Algorithms

Testing PCR stability of reference genes using different statistical algorithms is essential. Here, we used the geNorm, ΔCt method, BestKeeper, and NormFinder algorithms for statistical analyses to further test the PCR stability of 13 candidates. For geNorm analysis, expression stability value (M) negatively reflects PCR stability. Generally, a lower M value means greater stability. We observed that the M values of the 13 candidate reference genes were all below 0.8, with their rankings varying across different accessions. PeACT and PeRPL2 were relatively more stable in YGZ400, and PeUBQ3 and PeRPL1 were more stable in YGZ16, while PeACT and PeUBQ6 were more stable in the combined data (Figure 4A). The NormFinder analysis determines the reference genes stability by calculating the stability value. The lower value indicated the greater stability. Based on NormFinder analysis results, PeACT was the most stable reference candidate gene in YGZ400, YGZ16, and combined samples. (Figure 4B). The BestKeeper analysis determines the stability of reference genes by comparing the standard deviation (SD) between the correlation coefficient and the coefficient of Cq variation (Figure 4C). Since the SD value is inversely related to reference gene stability, PeUBQ6 and PeUBQ1 were found to be more stable in YGZ400, YGZ16, and their combined samples. The ΔCt analysis determines the stability of reference genes by checking the standard deviation of the expression among different tissues, resulting in PeACT being the most stable in the YGZ400, YGZ16, and their combined samples (Figure 4D). Due to the difference in geNorm, NormFinder, BestKeeper, and ΔCt algorithms, the most stable reference gene we obtained was not entirely consistent. Using the comprehensive RefFinder analysis, which calculates the geomean of ranking values obtained from the aforementioned algorithm, we obtained that PeACT was the most stable reference gene in YGZ400, YGZ16, and their combined samples. PeACT and PeUBQ6 were purported as the two most stable reference genes across combined samples, whereas PeRPL2 exhibited the lowest stability (Figure 4E).
To determine the optimal number of reference genes required for normalization, pairwise variations analysis was performed by the geNorm algorithm. According to the threshold value of 0.15 proposed by Vandesompele et al. [20], we found that V2/3 values of YGZ400 and YGZ16 were under this cut-off (Figure 5), indicating that two reference genes were sufficient for qRT-PCR normalization. Combined with these results and the expression stability test, PeACT and PeUBQ6 were selected for further validation.

3.4. Validation of Selected Reference Genes

According to the results obtained from five statistical algorithms, the most stable reference genes, PeACT and PeUBQ6, and the least stable reference gene, PeRPL2, were selected for further experimental validation tests. Since the fruit ripening-associated genes PeMLP (PeChr04G001040.1) and PeGASA (PeSTRG.4700.1.p1) were identified in our previous study from transcriptomic data across four developmental stages of P. emblica fruit (including the 6 WAF, 10 WAF, 18 WAF, and 26 WAF, respectively) [33], we validated their expression patterns in developing fruits by qRT-PCR, using PeACT, PeUBQ6, and PeRPL2 as reference genes. When PeACT and PeUBQ6 were used as reference genes, the expression trends of PeMLP and PeGASA exhibited a small discrepancy across the four stages of YGZ400 and YGZ16. However, when PeRPL2 was used as a reference gene for normalizing, expression trends of target genes exhibited a bigger discrepancy at different stages (Figure 6). Specifically, when PeRPL2 served as the reference gene, the expression levels of PeGASA were discrepant at the 18 WAF stage in YGZ400 and at the 26 WAF stage in YGZ16, while the expression levels of PeMLP were discrepant at the 18 WAF stage in YGZ400 and the 18 WAF stage in YGZ16. Further, we compared the expression profiles of PeGASA and PeMLP at the four development stages based on transcriptomic data (Figure S3). We found that the expressions of PeGASA and PeMLP were highly consistent with the qRT-PCR results when PeACT and PeUBQ6 were used as reference genes. Thus, both PeUBQ6 and PeACT are proposed as optimum housekeeping reference genes for assessing gene-specific expression in developing fruit of P. emblica using the qRT-PCR technique.

4. Discussion

As mentioned above, P. emblica is a traditional medicine plant and a promising fruit crop as well. Investigation of the physiological and molecular regulation of P. emblica fruit development and ripening is critical for its genetic improvement in breeding practice. Since the qRT-PCR technique has been widely applied in testing gene expression, the development of stable and efficient housekeeping reference genes is critical to ensure experimental accuracy and reproducibility [37]. Generally, reference genes were developed based on their constitutive expression in diverse plants, such as wheat [38], Zizania latifolia [39], and Chrysanthemum [40].
To confirm the suitability of candidate reference genes, validation using a combination of semi-quantitative PCR and qRT-PCR is advisable [41]. Semi-quantitative PCR provides a rapid assessment of transcript detectability and uniformity across different samples, based on single, correctly sized bands with comparable band intensity, thereby identifying primers that yield specific products without apparent bias [42]. qRT-PCR allows precise assessment of gene stability and assay reliability, including verification of primer specificity by melt-curve analysis and agarose gel electrophoresis, calculation of amplification efficiency, linearity, and evaluation of Cq dispersion and statistical algorithms across experimental samples [43]. In this study, semi-quantitative PCR was performed for qualitative analysis to verify the specificity of 13 candidate reference gene primers, and qRT-PCR was conducted for quantitative analysis to assess the expression stability of these reference genes based on the obtained Cq values. By integrating the semi-quantitative PCR with qRT-PCR improves analytical reliability, reduces normalization bias, and provides robust evidence for the suitability of selected reference genes in downstream analyses.
Based on the genome and transcriptome data, we identified 13 continuously expressed genes as housekeeping reference candidate genes to sort out reliable and efficient reference genes for qRT-PCR experiments in this study. The 13 constitutively expressed genes exhibited variable expression in developing fruit of two P. emblica accessions according to their expression stability and efficiency. Similarly, those constitutively expressed genes exhibited variable expressions in different plants, such as strawberries [14], roses [44], and Himalayan onions [45]. Based on the Cq values of each candidate gene, we obtained the relatively suitable candidate PeUBQ6.
Since different statistical algorithms are necessary to test the PCR stability of reference genes, we evaluated the expression of candidate genes using BestKeeper, NormFinder, geNorm, RefFinder, and ΔCt methods. These statistical algorithms depended on different principles, such as geNorm relying on comparing the M value (the value of expression stability) by analyzing the variation coefficients within and between groups for each reference gene across the samples, while the Bestkeeper method was based on a comprehensive criterion, including the SD, CV, and R of the Cq values for each reference gene among samples [15,46]. The RefFinder integrates the results obtained from NormFinder, ΔCt, geNorm, and BestKeeper, providing comprehensive stable values of reference genes. Although the statistical principles were different, the NormFinder, ΔCt, and geNorm algorithms resulted in PeACT being the most stable reference gene. Based on the Bestkeeper method, PeUBQ6 was sorted out as the most stable reference gene. The integrated RefFinder method exhibited that both PeACT and PeUBQ6 were relatively stable and efficient housekeeping reference genes. However, PeRPL2 was identified as the least suitable housekeeping reference gene. Combining the results we obtained from the expression stability test, we recommended that both PeACT and PeUBQ6 could serve as housekeeping reference genes in qRT-PCR experiments to investigate the molecular and physiological mechanisms underlying fruit development and ripening.
Actin, a highly abundant cytoskeletal protein encoded by multiple copies [47], is functionally involved in regulating plant growth and tissue development. Ubiquitin is broadly involved in regulating protein activity and stability by ubiquitination [48]. Although the functions of Actin and Ubiquitin are highly conserved across different tissues and they are commonly developed as housekeeping genes for gene expression normalization in diverse plants, the specific genes encoding Actin and Ubiquitin, that were considered as housekeeping genes are often different due to various genetic backgrounds [49]. Here, we developed PeACT and PeUBQ6 as housekeeping genes in developing fruit of P. emblica, providing new housekeeping gene resources to investigate the expression profiles of genes related to fruit development and ripening (including post-harvest storage) in P. emblica. Further, we compared and tested the expressional reliability of two fruit-specifically expressed genes, PeGASA and PeMLP, during different stages of fruit development using PeACT, PeUBQ6, and PeRPL2 as reference genes. We validated that PeACT and PeUBQ6 could serve as housekeeping reference genes for performing the qRT-PCR experiments in studies related to fruit development and ripening.
Because P. emblica fruits contain rich active compounds, they have been considered a traditional medicine. Investigating the physiological and molecular processes of active compound (secondary metabolite product) biosynthesis is necessary to enhance the content of active compounds in developing fruits by genetic improvement. Accumulations of these secondary metabolite products usually occur at the specific stages of fruit development and ripening [50,51]; thus, the PeACT and PeUBQ6 could be used in testing the expression profiles of genes related to secondary metabolite biosynthesis in given stages of fruit development and ripening in P. emblica. As the current study focused on fruit development and ripening under the normal orchard conditions, it remains uncertain whether the expression stability of PeCT and PeUBQ6 can be applied in response to biotic or abiotic stresses.

5. Conclusions

After evaluating the expressional stabilities and efficiencies of 13 constitutively expressed genes based on the expression level and different statistical algorithms (including BestKeeper, geNorm, ΔCt method, NormFinder, and RefFinder), we identified PeACT and PeUBQ6 as suitable housekeeping reference genes in qRT-PCR studies focusing on fruit development and ripening of P. emblica. The obtained PeACT and PeUBQ6 reference genes are useful to test the relative expression level of functional genes related to fruit development and ripening, thereby providing insights into the molecular mechanism underlying fruit quality improvement in P. emblica cultivars.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11091054/s1, Table S1. Information about selected primers. Figure S1: Expression heatmap of the selected 13 candidate reference genes and other traditional reference genes in five different tissues (shoot, branch, fruit, leaf, and flower) and four fruits development stages (6WAF, 10WAF, 18WAF, and 26WAF) of YGZ400 and YGZ16. Figure S2: The specific amplification of 13 candidate reference genes by qRT-PCR. Lanes 1–13 are PeUBQ2, PeUBQ5, PeUBQ3, PeUBQ6, PeUBQ4, PeUBQ7, PeRPL3, PeRPL1, PeRPL2, PeEF1-α2, PeEF1-α1, PeACT, and PeUBQ1, respectively. M: DL2000 DNA marker. Figure S3: Relative expression level of PeGASA and PeMLP gene by transcriptome sequencing.

Author Contributions

Resources, Q.Z. and K.Q.; conceptualization, L.Z. and A.L.; methodology, T.P. and X.L.; formal analysis, T.P.; investigation, J.Y. and L.S.; writing—original draft preparation, T.P.; writing—review and editing, A.L., L.Z. and T.P.; project administration, W.Q. and C.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Yunnan Provincial Agricultural Basic Research Joint Project (grant number: 202401BD070001-089 and 202401BD070001-087), National Natural Science Foundation of China (grant number: 32360714), and Yunnan Provincial Science and Technology Plan Basic Research Project (grant number: 202401AU070064).

Data Availability Statement

The sequence data that support the findings of this study are uploaded to NCBI under the accession no. PRJDB18024 and PRJDB18150.

Conflicts of Interest

All authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
qRT-PCRQuantitative Real-Time Polymerase Chain Reaction
GSASGibberellic Acid-Stimulated Arabidopsis
MLPMajor Latex-Like Protein

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Figure 1. Melting curves of 13 candidate reference genes, including (A) PeUBQ1, (B) PeUBQ2, (C) PeUBQ3, (D) PeUBQ4, (E) PeUBQ5, (F) PeUBQ6, (G) PeUBQ7, (H) PeRPL1, (I) PeRPL2, (J) PeRPL3, (K) PeEF1-α1, (L) PeEF1-α2, (M) PeACT, respectively. The different color lines represent the melting curves of different P. emblica accessions across different developmental stages.
Figure 1. Melting curves of 13 candidate reference genes, including (A) PeUBQ1, (B) PeUBQ2, (C) PeUBQ3, (D) PeUBQ4, (E) PeUBQ5, (F) PeUBQ6, (G) PeUBQ7, (H) PeRPL1, (I) PeRPL2, (J) PeRPL3, (K) PeEF1-α1, (L) PeEF1-α2, (M) PeACT, respectively. The different color lines represent the melting curves of different P. emblica accessions across different developmental stages.
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Figure 2. Distribution of Cq values of 13 candidate reference genes acquired by qRT-PCR in P. emblica. The Cq distribution boxes indicate 75th and 25th percentiles, lines across the box indicate the median Cq values, and the dots illustrate their distribution.
Figure 2. Distribution of Cq values of 13 candidate reference genes acquired by qRT-PCR in P. emblica. The Cq distribution boxes indicate 75th and 25th percentiles, lines across the box indicate the median Cq values, and the dots illustrate their distribution.
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Figure 3. Expression heatmap of 13 candidate reference genes tested by qRT-PCR, (A,B) shows the Cq values of YGZ400 and YGZ16 in different fruits development stages, including 6WAF, 8WAF, 10WAF, 12WAF, 14WAF, 16WAF, 18WAFAF, 20WAF, 22WAF, 24WAF, and 26WAF, respectively. The red color indicates low Cq values, reflecting high gene expression levels, while the blue color represents high Cq values, indicating low gene expression levels.
Figure 3. Expression heatmap of 13 candidate reference genes tested by qRT-PCR, (A,B) shows the Cq values of YGZ400 and YGZ16 in different fruits development stages, including 6WAF, 8WAF, 10WAF, 12WAF, 14WAF, 16WAF, 18WAFAF, 20WAF, 22WAF, 24WAF, and 26WAF, respectively. The red color indicates low Cq values, reflecting high gene expression levels, while the blue color represents high Cq values, indicating low gene expression levels.
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Figure 4. Stability analysis of 13 candidate reference genes in YGZ400, YGZ16, and combined samples were conducted using five distinct statistical algorithms. Stability values of 13 reference genes obtained through (A) geNorm analysis, (B) NormFinder analysis, (C) BestKeeper analysis, (D) ΔCt analysis, and (E) RefFinder analysis. The lower stability value indicated the higher stability.
Figure 4. Stability analysis of 13 candidate reference genes in YGZ400, YGZ16, and combined samples were conducted using five distinct statistical algorithms. Stability values of 13 reference genes obtained through (A) geNorm analysis, (B) NormFinder analysis, (C) BestKeeper analysis, (D) ΔCt analysis, and (E) RefFinder analysis. The lower stability value indicated the higher stability.
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Figure 5. Pairwise variation (V) values derived from geNorm to demonstrate the optimal number of reference genes for normalization in different fruit development stages.
Figure 5. Pairwise variation (V) values derived from geNorm to demonstrate the optimal number of reference genes for normalization in different fruit development stages.
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Figure 6. Relative expression level of PeGASA and PeMLP in different accessions, normalized using selected reference genes. (A) Relative expression level of PeGASA in YGZ400. (B) The expression level of PeGASA in YGZ16. (C) The expression level of PeMLP in YGZ400. (D) The expression level of PeMLP in YGZ16. The expression levels were normalized by PeACT, PeUBQ6, and PeRPL2, with three technical repetitions, different lowercase letters indicate statistical significance at level p < 0.05 during each stages. One-way ANOVA was used to evaluate statistical significance.
Figure 6. Relative expression level of PeGASA and PeMLP in different accessions, normalized using selected reference genes. (A) Relative expression level of PeGASA in YGZ400. (B) The expression level of PeGASA in YGZ16. (C) The expression level of PeMLP in YGZ400. (D) The expression level of PeMLP in YGZ16. The expression levels were normalized by PeACT, PeUBQ6, and PeRPL2, with three technical repetitions, different lowercase letters indicate statistical significance at level p < 0.05 during each stages. One-way ANOVA was used to evaluate statistical significance.
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Table 1. Information on 13 candidate reference genes for P. emblica.
Table 1. Information on 13 candidate reference genes for P. emblica.
Gene NameGene IDArabidopsis
Homolog		CDS Length (bp)Mean TPMCV
PeUBQ1 (Non-canonical ubiquitin conjugating enzyme 1)PeChr11G010870.1At3g1700090953.160.29
PeUBQ2 (Ubiquitin-conjugating enzyme E2)PeChr09G004080.1At3g5256044151.440.24
PeUBQ3 (E3 ubiquitin ligase)PeChr22G009860.1At1g6504096642.950.29
PeUBQ4 (Ubiquitin-protein ligases)PeSTRG.11463.1.p1At2g0276045973.410.29
PeUBQ5 (Ubiquitin-like protein 5)PeChr02G004390.1At5g42300222114.920.28
PeUBQ6 (Ubiquitin and ubiquitin-like proteins)PeChr26G003160.2At1g3134046536.210.25
PeUBQ7 (Ubiquitin-like protein)PeSTRG.21462.1.p1At2g17200170143.270.20
PeRPL1 (Ribosomal protein RPL1)PeChr26G007230.1At3g096301227135.420.29
PeRPL2 (Ribosomal protein L2)PeSTRG.17299.1.p1At4g36130783160.570.29
PeRPL3 (Ribosomal protein L10)PeChr02G001910.3At1g26910798192.730.30
PeEF1-α1 (Translation elongation factor 1 alpha)PeSTRG.12624.1.p1At5g603901350148.270.24
PeEF1-α2 (Translation elongation factor 1 alpha)PeSTRG.1197.1.p1At5g603901350217.270.23
PeACT (Actin depolymerizing factor)PeSTRG.26181.1.p1At3g46010420141.440.26
Table 2. Information about selected primers.
Table 2. Information about selected primers.
Gene NameAmplification Length/bpR2Efficiency/%
PeUBQ11280.999190.27%
PeUBQ2800.9983100.07%
PeUBQ3880.9994102.00%
PeUBQ41050.996697.74%
PeUBQ5890.9988101.64%
PeUBQ61310.9983109.54%
PeUBQ71140.999297.67%
PeRPL1960.999797.17%
PeRPL2860.999795.24%
PeRPL31230.9974104.69%
PeEF1-α11230.9999101.14%
PeEF1-α21400.9962102.15%
PeACT1060.999497.28%
Table 3. The Cq value analysis of the candidate reference genes.
Table 3. The Cq value analysis of the candidate reference genes.
Gene NameYGZ400YGZ16Combined
MeanSDCV/%MeanSDCV/%MeanSDCV/%
PeUBQ122.720.642.8123.200.783.3722.960.753.27
PeUBQ220.490.874.2620.970.693.3020.730.823.95
PeUBQ324.080.783.2524.690.903.6724.380.893.67
PeUBQ421.150.733.4621.900.693.1421.530.803.72
PeUBQ520.890.874.1421.731.175.3821.311.105.18
PeUBQ622.990.662.8723.610.572.4123.300.692.95
PeUBQ723.250.883.7824.190.873.6023.720.994.17
PeRPL121.530.914.2421.940.944.2821.740.944.33
PeRPL221.810.773.5423.681.134.7622.741.345.91
PeRPL321.000.773.6521.390.683.1721.190.753.52
PeEF1-α119.600.773.9220.000.803.9919.800.804.05
PeEF1-α222.400.914.0622.271.205.3622.341.064.72
PeACT20.890.773.6721.490.813.7621.190.843.95
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Pu, T.; Yuan, J.; Qu, W.; Liao, C.; Lei, X.; Qian, K.; Zhao, Q.; Shi, L.; Zhang, L.; Liu, A. Selection and Validation of Reference Candidate Genes for qRT-PCR Analysis in the Developing Fruit of Phyllanthus emblica L. Horticulturae 2025, 11, 1054. https://doi.org/10.3390/horticulturae11091054

AMA Style

Pu T, Yuan J, Qu W, Liao C, Lei X, Qian K, Zhao Q, Shi L, Zhang L, Liu A. Selection and Validation of Reference Candidate Genes for qRT-PCR Analysis in the Developing Fruit of Phyllanthus emblica L. Horticulturae. 2025; 11(9):1054. https://doi.org/10.3390/horticulturae11091054

Chicago/Turabian Style

Pu, Tianlei, Jianmin Yuan, Wenlin Qu, Chengfei Liao, Xiao Lei, Kunjian Qian, Qiongling Zhao, Liangjia Shi, Lumin Zhang, and Aizhong Liu. 2025. "Selection and Validation of Reference Candidate Genes for qRT-PCR Analysis in the Developing Fruit of Phyllanthus emblica L." Horticulturae 11, no. 9: 1054. https://doi.org/10.3390/horticulturae11091054

APA Style

Pu, T., Yuan, J., Qu, W., Liao, C., Lei, X., Qian, K., Zhao, Q., Shi, L., Zhang, L., & Liu, A. (2025). Selection and Validation of Reference Candidate Genes for qRT-PCR Analysis in the Developing Fruit of Phyllanthus emblica L. Horticulturae, 11(9), 1054. https://doi.org/10.3390/horticulturae11091054

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