Identification of reference genes for transcript normalization in various tissue types and seedlings subjected to different abiotic stresses of woodland strawberry Fragaria vesca

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Identification of reference genes for transcript normalization in various tissue types and seedlings subjected to different abiotic stresses of woodland strawberry Fragaria vesca Decai Liua, Xiaorong Huanga, Ying Lina, Xiaojing Wanga, Zhiming Yana,b, Quanzhi Wanga,b, Jing Dinga, Tingting Gua,*, Yi Lia,c,*,1 a

State Key Laboratory of Plant Genetics and Germplasm Enhancement and College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, People’s Republic of China State Key Laboratory of Engineering and Technology Center for Modern Horticulture, Jiangsu Vocational College of Agriculture and Forestry, Zhenjiang, Jiangsu 212400, People’s Republic of China c Department of Plant Science and Landscape Architecture, University of Connecticut, Storrs, CT 06269, USA b

ARTICLE INFO

ABSTRACT

Keywords: Reference gene Normalization qRT-PCR Fragaria vesca Abiotic stress Tissue

Quantitative real-time PCR (qRT-PCR) is one of the most popular methods for gene expression studies. However, data on the usage of reference genes in strawberry are limited. In this study, we assayed expression dynamics of 20 candidate reference genes across various organs (root, leaf and flower), different developmental stages of fruits, and in seedlings under abiotic stresses (heat, cold, drought and salt) in woodland strawberry Fragaria vesca. Expression stability of those genes was analyzed by four commonly used statistical algorithms (geNorm, NormFinder, BestKeeper and dalteCt) for comparisons and rankings. Our results indicate that the expression stability of reference genes varies according to tissue types and experimental conditions. Furthermore, two reference genes are sufficient for normalization in tissues and seedlings under the four stress conditions in Fragaria vesca. We recommend FveSAND and FveMT1, FveCHC1 and FveGAPDH as reference genes in organs and fruits, FveCHC1 and FveTIM1, FveSAND and FveTIM1, FveCHP1 and FveAP1, FveCHC1 and FveEF 1a under heat, cold, drought and salt stress conditions, respectively. Our study provides a set of reliable standard reference genes for expression analyses of genes in various tissue types and seedlings under different environment conditions of Fragaria vesca.

1. Introduction Strawberry is one of the most important economic berry crops in the world. Cultivated strawberries (Fragaria × ananassa) have a complex octoploid genome (2n = 8x = 56) which is difficult for genetic studies. The woodland strawberry Fragaria vesca covers a shorter life cycle. The flower structure, flower organ arrangement, early fruit development and other characteristics of woodland strawberry are highly similar to cultivated strawberry. More importantly, Fragaria vesca has a well assembled diploid genome (2n = 14, 240MB), and is identified as an ancestor to the cultivated strawberry genome (Edger et al., 2019). Fragaria vesca is emerging as a model plant for Rosaceae and non-climacteric fruits (Li et al., 2014). Transcriptomic studies in plants have emphasized the key role of

transcriptional regulation in complex metabolism processes (Shinozaki et al., 2018), which is revealed by transcriptomic analyses. However, the functions of the majority of those genes have not been identified. Gene expression profiling is important for identifying genes’ involvements in complex regulatory networks under different conditions (Shinozaki et al., 2003). Quantitative real-time PCR (qRT-PCR) is widely used due to its high sensitivity, good stability, better specificity and relative low cost (Bustin, 2002; Derveaux et al., 2010). The accuracy of qRT-PCR results is dependent on the usage of appropriate reference genes, which act as internal standards for the control of experimental errors between samples (Dheda et al., 2005). Generally speaking, an ideal reference gene should be stably expressed independent of cells and tissues, developmental stages or growth conditions (Radonic et al., 2004; Czechowski et al., 2005a).

Corresponding authors at: State Key Laboratory of Plant Genetics and Germplasm Enhancement and College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, People’s Republic of China. E-mail addresses: [email protected] (T. Gu), [email protected] (Y. Li). 1 Yi Li holds a two month/year visiting professor position at Nanjing Agricultural University. ⁎

https://doi.org/10.1016/j.scienta.2019.108840 Received 27 June 2019; Received in revised form 1 September 2019; Accepted 5 September 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Decai Liu, et al., Scientia Horticulturae, https://doi.org/10.1016/j.scienta.2019.108840

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Studies on reference genes have been reported in several plant species, e.g. Arabidopsis thaliana(Czechowski et al., 2005b), rice (Kim et al., 2003), potato (Nicot et al., 2005), tomato (Exposito-Rodriguez et al., 2008; Lovdal and Lillo, 2009) and soybean (Jian et al., 2008; Libault et al., 2008; Hu et al., 2009). Previous studies on cultivated strawberry Fragaria × ananassa recommended several reference genes for gene expression normalization in strawberry plants (Amil-Ruiz et al., 2013; Galli et al., 2015a; Zhang et al., 2018). However, the reference genes identified in cultivated strawberry may not be appropriate to be directly applied in woodland strawberry. Clancy et al (2013) tested a limited number of candidate reference genes in various organs/tissues under well-controlled conditions in woodland strawberry Fragaria vesca, but no abiotic stress conditions have been assayed (Clancy et al., 2013). Strawberry growth is hindered by multiple abiotic stresses, which cause significant yield reduction of strawberry (Wei et al., 2016). Therefore, we conducted further researches on identification of reference genes in Fragaria vesca. In our study, expression dynamics of 20 candidate reference genes were profiled in various organs (root, leaf and flower), different developmental stages of fruits, and seedlings under abiotic stresses (heat, cold, drought and salt) in Fragaria vesca. The expression stability of those candidate genes were assayed under different growth conditions by ranking analysis using geNorm (Vandesompele et al., 2002), NormFinder (Andersen et al., 2004), BestKeeper (Pfaffl et al., 2004) and deltaCt (Silver et al., 2006). The applicability of recommended reference genes were measured by Geomean of ranking (Cassan-Wang et al., 2012a) and RefFinder (Xie et al., 2012) analyses, which integrated rankings generated by the four methods as introduced above. In conclusion, we recommend to use two reference genes for gene expression normalization under our experiment conditions assayed. Moreover, as shown in our results, tissue types and experimental conditions need to be considered for identification of reference genes.

2.2. RNA extraction and cDNA synthesis Total RNA of organs and seedlings treated with various abiotic stresses was extracted using a TaKaRa MiniBEST Plant RNA Extraction Kit according to the manufacturer’s instructions (TaKaRa, Dalian, China). Total RNA of Fragaria vesca fruits was isolated based on a CTAB method previously described (Chang et al., 1993). The concentration of isolated RNA was determined by the absorbance at 260 nm with Nanodrop ND 1000 spectrophotometer (Nanodrop Technologies, USA). The purity was checked by optical density (OD) absorption ration and on a 1.0% (w/v) agarose gel as well. Subsequently, the first-strand cDNA was synthesized with a 20 μL reaction system using the TaKaRa PrimeScrit RT reagent Kit (Perfect for Real Time) following the manufacturer’s instructions (TaKaRa Biotechnology, Dalian, China). The cDNA was established as ten-fold diluted series (10×, 102×, 103×, 104×, and 105×dilutions) for determining the amplification efficiency (E) and correlation coefficient (R2) analysis, and five-fold diluted for conducting the qPCR analysis of all tissues and treatments. 2.3. PCR primer design Primer pairs were designed using the PrimerQuest Tool online (http://sg.idtdna.com/Primerquest/Home/Index). Each pair of primers was verified by Primer-BLAST on NCBI (https://www.ncbi.nlm.nih. gov/tools/primer-blast/) to confirm the specificity. The primer sequences are listed in Table S1. 2.4. High-throughput quantitative qRT–PCR qRT-PCR runs were performed in MyIQ and iCycler real-time PCR systems (Bio-Rad) using 96-well plates and 20 μL final reaction volume per well. PCR reactions contained 7.6 μL of ddH2O, 10.0 μL of SYBR Green Master Mix (Bio-Rad), 0.4 μL of each specific primer (10 μM), and 1.6 μL of first-strand cDNA template. The following amplification conditions were applied: an initial denaturation step of 95 °C for 30 s; 40 cycles at 95 °C for 5 s; and 60 °C for 30 s. The final dissociation curve was obtained from 65 °C to 95 °C to verify primer specificity. Each assay included three technical and three biological replicates, and a standard curve of five serial dilution points. Mean Cq (quantification cycle) values of the ten-fold dilution series were plotted against the logarithm of the pooled cDNA dilution factors. The Cq values and the following equation were used to determine efficiency (E) of each gene with the slope of a linear regression model: % E = (10[−1/slope] − 1) × 100% (Radonic et al., 2004). Amplification efficiencies were calculated from standard curves with satisfactory linear relationships (R2 > 0.99). All PCR processes displayed efficiency between 90% and 110%.

2. Materials and methods 2.1. Plant growth conditions, stress treatments and material collections Seeds of woodland strawberry Fragaria vesca (Rugen F7-4, kindly provided by Janet Slovin) were sterilized and then planted into MS medium. Seedlings were grown in a growth chamber in which the environment was set at 22℃/20 °C (day/night) and 16 h/8 h (day/night) light conditions. One month-old seedlings were subsequently treated with abiotic stresses. For cold stress treatment, seedlings were treated under 4 ℃ for 1 h, 3 h and 8 h in dark before collection. For heat stress treatment, seedlings were treated under 38℃ for 3 h in light conditions and then recovered at room temperature for 1 h, 3 h and 8 h before collection. For salt and drought treatments, seedlings were treated in 1/2 MS liquid medium supplemented with 150 u M NaCl or 20% PEG on a 100 rpm shaker for 1 h, 3 h and 8 h, respectively. For collection of strawberry fruits, the plants were grown in 15 cm × 15 cm pots in a growth chamber with a 16 h light (22 °C)/8 h dark (20 °C) cycle and 65% relative humidity. Six developmental stages of fruits were collected for qRT-PCR analysis, including small green (SG, 2–4 days post anthesis, DPA), medium green (MG, 8–10DPA), big white (BW, white flesh with green achenes, 20–21 DPA), pre-turning (PT, white flesh with red achenes, 24–25DPA), pink (light pink flesh with red achenes, 26–27 DPA), and red (the flesh is all red, 28–29 DPA) stages. Meanwhile, leaves and roots of seedlings grown in MS medium as well as freshly blooming flowers cultured in growth chamber were collected. All samples were frozen immediately in liquid nitrogen after collection and stored at -80℃ for further research analysis. Three biological replicates were performed for each treatment and tissue/organ.

2.5. Statistical analyses Four different statistic tools including geNorm, NormFinder, BestKeeper and ΔCt were used to rank the expression stability of the candidate reference genes (Vandesompele et al., 2002; Andersen et al., 2004; Pfaffl et al., 2004; Silver et al., 2006). In addition, we also analyzed the rankings of candidate reference genes by RefFinder online (http://www.leonxie.com/referencegene.php) and Geomean of ranking (GM) to obtain an integration of the rankings generated by the four methods (Xie et al., 2012; Cassan-Wang et al., 2012a). The relative expression profiles of FveNCED2 during fruit development, and FveWRKY34 and FveWRKY48 after salt stress were obtained according to the formula E−ΔΔCT (Pfaffl, 2001).

2

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Fig. 1. Illustration of plant materials applied to the experiment and Cq (quantification cycle) values of the 20 candidate reference genes obtained from qRT-PCR results. a. The schematic diagram for the plant materials. SG-small green, MG-medium green, BW-big white, PT-pre-turning. b. Range of Cq values of the 20 candidate reference genes obtained from the qRT-PCR experiments of 75 Fragaria vesca samples conducted. Comparisons of the variability of Cq values are shown as medians (line in box), 25th percentile to the 75th percentile (boxes) and ranges (whiskers). Bar = 1 cm.

species (Galli et al., 2015b; Guo et al., 2014; Clancy et al., 2013; AmilRuiz et al., 2013), including the most commonly used ACTIN and GAPDH. As shown by qRT-PCR experiment results, among the 20 candidate reference genes, the mean quantification cycle (Cq) values varied between 17.65 (FveRIB413) and 29.70 (FveTUBa), and the standard deviation (SD) were within the range of 0.73–2.76 (Fig. 1b and Table S2). In addition, the correlation coefficient (R²) of those genes ranged from 0.9867 to 0.9999, and the PCR efficiency (E) values of the primers varied from 91.92% to 108.86% (Table S2), which was in line with the experimental requirements of qRT-PCR for primers (Bustin et al., 2009). We could visually find out from the box diagrams that the level of FveTUBa transcripts was the most variable in the 20 candidate genes tested, and that FveEF1a transcripts had the lowest variable Cq values. Other than this, FveRIB413 showed the highest expression level and the mean Cq value was 9.08. (Fig.1b and Table S2). 3.2. Expression stability analysis by geNorm and NormFinder 3.2.1. geNorm analysis The stability of each reference gene in various organs/tissues and seedlings under different treatment conditions was ranked by M value calculated by geNorm analysis. M value is defined as the mean variation of a certain gene within a set of reference genes tested. Lower M values indicate higher expression stability of the candidate reference genes (Vandesompele et al., 2002). An analyzed gene is considered as stably expressed when its M value is below 0.5 (Allen et al., 2008; Gutierrez et al., 2008). As shown in Fig. 2 and Table 1, in terms of expression stability among organs (root, leaf, and flower), the M values of 11 candidate genes were below 0.5, and FveCHC1 and FveENP1 had the lowest M values which indicated their most stable expression. During fruit development, the M values of 10 candidate genes were below 0.5, and FveDBP and FveUBQ1 were the most stably expressed genes. Under the four abiotic stress treatments, all the 20 candidate genes were below 0.5 (Fig. 2). Under heat, cold, salt and drought treatment, the two most stably expressed genes were FveUBP and FveUBQ1, FveENP1 and

Fig. 2. Average expression stability values (M) of the 20 candidate reference genes evaluated by geNorm. Specific M values were calculated under six single experimental conditions tested, as well as by combining all samples together. Refer to Table 1 for the ranking of the 20 genes measured by their expression stability. The lowest M value indicates the most stable expression.

3. Results 3.1. Expression profiling of candidate reference genes In order to identify the stably expressed reference genes across various organs, different developmental stages of fruits and in seedlings under abiotic stresses in Fragaria vesca (Fig. 1a), the expression stability of 20 candidate reference genes was evaluated by qRT-PCR assays, and the primer sequences were listed in Table S1. The 20 candidate reference genes were picked from those already reported in other plant 3

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Table 1 The tested 20 candidate reference genes ranked by their expression stability evaluated by geNorm algorithm. Tissues

　

Abiotic Stress

Organs

Fruits

All Samples

Heat

Cold

Drought

Salt

　

Rank

Gene

Ma

Gene

M

Gene

M

Gene

M

Gene

M

Gene

M

Gene

M

1 1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

FveCHC1 FveENP1 FveRIB413 FveTBP FveSAND FveMT1 FveTIP41 FveEF1a FveHDP1 FveTIM1 FveTUBβ FveGAPDH FveCHP1 FveACT-7 FveAP1 FveDBP FveUBQ1 FveBZIP1 FveAATP1 FveTUBa

0.11 0.11 0.23 0.25 0.29 0.32 0.35 0.37 0.39 0.43 0.46 0.51 0.54 0.57 0.61 0.65 0.69 0.74 0.81 2.45

FveDBP FveUBQ1 FveCHC1 FveGAPDH FveEF1a FveSAND FveCHP1 FveACT-7 FveENP1 FveAP1 FveTIM1 FveHDP1 FveAATP1 FveTBP FveTUBβ FveTIP41 FveMT1 FveBZIP1 FveRIB413 FveTUBa

0.05 0.05 0.24 0.26 0.27 0.3 0.35 0.39 0.43 0.46 0.49 0.52 0.55 0.58 0.62 0.67 0.73 0.79 0.85 1.65

FveDBP FveUBQ1 FveMT1 FveTUBa FveCHC1 FveTIM1 FveAATP1 FveTUBβ FveHDP1 FveBZIP1 FveAP1 FveEF1a FveTIP41 FveTBP FveENP1 FveGAPDH FveCHP1 FveRIB413 FveACT-7 FveSAND

0.05 0.05 0.11 0.14 0.17 0.18 0.19 0.2 0.22 0.22 0.23 0.24 0.25 0.26 0.27 0.28 0.29 0.3 0.3 0.31

FveENP1 FveEF1a FveSAND FveTIP41 FveTUBa FveTIM1 FveCHP1 FveAATP1 FveCHC1 FveTUBβ FveGAPDH FveMT1 FveBZIP1 FveAP1 FveTBP FveACT-7 FveRIB413 FveUBQ1 FveHDP1 FveDBP

0.03 0.03 0.04 0.04 0.05 0.07 0.07 0.08 0.1 0.1 0.11 0.13 0.15 0.16 0.17 0.18 0.2 0.23 0.26 0.28

FveTIM1 FveHDP1 FveAP1 FveDBP FveCHP1 FveCHC1 FveTUBβ FveTIP41 FveGAPDH FveENP1 FveMT1 FveUBQ1 FveAATP1 FveSAND FveEF1a FveACT-7 FveBZIP1 FveTBP FveRIB413 FveTUBa

0.04 0.04 0.11 0.11 0.13 0.13 0.15 0.16 0.17 0.19 0.21 0.22 0.23 0.25 0.26 0.27 0.29 0.32 0.37 0.42

FveCHC1 FveEF1a FveSAND FveCHP1 FveTIM1 FveTUBa FveBZIP1 FveTIP41 FveAATP1 FveTUBβ FveMT1 FveTBP FveGAPDH FveDBP FveUBQ1 FveACT-7 FveRIB413 FveENP1 FveHDP1 FveAP1

0.07 0.07 0.1 0.11 0.12 0.14 0.14 0.15 0.16 0.17 0.18 0.19 0.2 0.22 0.23 0.25 0.27 0.28 0.3 0.32

FveCHC1 FveTIM1 FveGAPDH FveACT-7 FveEF1a FveENP1 FveCHP1 FveAP1 FveTUBβ FveMT1 FveHDP1 FveTBP FveDBP FveUBQ1 FveTIP41 FveAATP1 FveBZIP1 FveRIB413 FveSAND FveTUBa

0.39 0.39 0.4 0.42 0.48 0.51 0.54 0.57 0.59 0.61 0.64 0.68 0.72 0.75 0.78 0.82 0.87 0.92 1.61 2.1

a

M: Average expression stability values.

FveEF1a, FveCHC1 and FveEF1a, FveTIM1 and FveHDP1, respectively (Table 1). Furthermore, when all samples (including those from various organs, different developmental stages of fruits, and seedlings under abiotic stresses) were considered, five out of the 20 candidate genes tested had M values smaller than 0.5, and FveCHC1 and FveTIM1 were the most stably expressed genes (Table 1). In some cases, two or more reference genes were required, as one reference gene could not appropriately normalize the gene expression (Vandesompele et al., 2002; Gimenez et al., 2011). For this reason, geNorm was performed to evaluate the optical number of reference genes used for normalization under different conditions. Pairwise variation (Vn/Vn+1) between the sequential ranked normalization factors (NFn and NFn+1, n≥2) was calculated by geNorm (Fig. 3) (Han et al.,

2012). The cut-off value is 0.15, below which the inclusion of an additional control gene is not necessary for reliable normalization (Cassan-Wang et al., 2012b). As shown in Fig. 3, the inclusion of the third reference gene did not contribute significantly to the variation of the normalization factor (V2/V3 < 0.15) in all tested conditions, indicating that normalization by two best reference genes was sufficient under the conditions assayed. 3.2.2. NormFinder analysis Intra- and intergroup variations used to calculate for normalization factor were taken into account in NormFinder (Andersen et al., 2004). The ranking of the genes and their respective expression stability values (SV) were shown in Table 2. According to the NormFinder analysis, the stability value of most reference genes varied in organs, fruit development and seedlings under stress conditions. In organs, the most stably expressed genes were FveMT1 (SV = 0.01) and FveSAND (SV = 0.01), while FveCHC1 (SV = 0.08) and FveEF1a (SV = 0.1) were the most stably expressed genes in fruit development. Under stress conditions, the most stably expressed genes were FveCHC1 and FveTIM1, FveTIM1 and FveCHP1, FveAP1 and FveDBP, FveCHC1 and FveEF1a, respectively, by heat, cold, drought and salt treatment (Table 2). According to the data from all samples, FveCHC1 (SV = 0.09) and FveTUBβ(SV = 0.09) showed the most stable expression level, and FveRIB413 (SV = 0.3) was the least stable in all samples (Table 2). 3.3. Expression stability analyses by other methods In our study, two additional methods, delta-Ct (ΔCt) comparison and Bestkeeper algorithms, were performed to identify the suitable reference genes. ΔCt method was used to rank the candidate reference genes on the basis of average standard deviation (SD). The relative expression of "pairs of genes" within each sample was compared by this method (Silver et al., 2006). As shown in Table S3, in most cases the most stable candidate reference gene under the different data panels was FveCHC1 or FveSAND, though FveSAND expression was the least stable under heat stress. Bestkeeper Analysis program is used to determine the stability of gene expression based on the SD and coefficient of variation (CV)

Fig. 3. Determination of the optimal number of reference genes for normalization according to geNorm. Pairwise variation (Vn/n+1) analysis was carried out to determine the number of reference genes required for accurate normalization. The cut-off value is 0.15, below which the inclusion of an additional reference gene is not required. *Optimal number of reference genes for normalization. 4

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Table 2 The tested 20 candidate reference genes ranked by their expression stability evaluated by NormFinder. Tissues

　

Abiotic Stress

Organs

Fruits

All Samples

Heat

Cold

Drought

Salt

　

Rank

Gene

SV

Gene

SV

Gene

SV

Gene

SV

Gene

SV

Gene

SV

Gene

SV

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

FveMT1 FveSAND FveTBP FveRIB413 FveCHC1 FveEF1a FveTIP41 FveENP1 FveTIM1 FveHDP1 FveTUBβ FveACT-7 FveCHP1 FveAP1 FveGAPDH FveTUBa FveAATP1 FveDBP FveUBQ1 FveBZIP1

0.01 0.01 0.02 0.07 0.08 0.08 0.08 0.09 0.1 0.12 0.13 0.13 0.14 0.14 0.15 0.23 0.23 0.27 0.38 0.45

FveCHC1 FveEF1a FveGAPDH FveDBP FveAP1 FveUBQ1 FveSAND FveACT-7 FveTUBβ FveHDP1 FveAATP1 FveENP1 FveCHP1 FveTIP41 FveTIM1 FveTBP FveRIB413 FveBZIP1 FveMT1 FveTUBa

0.08 0.1 0.1 0.11 0.12 0.12 0.14 0.14 0.17 0.18 0.19 0.21 0.21 0.23 0.24 0.25 0.3 0.34 0.34 0.44

FveCHC1 FveTIM1 FveGAPDH FveTUBa FveAP1 FveBZIP1 FveHDP1 FveTIP41 FveCHP1 FveTUBβ FveACT-7 FveMT1 FveDBP FveAATP1 FveTBP FveENP1 FveUBQ1 FveRIB413 FveSAND FveEF1a

0.03 0.06 0.07 0.07 0.07 0.08 0.09 0.09 0.1 0.1 0.11 0.11 0.12 0.12 0.13 0.13 0.14 0.15 0.16 0.16

FveTIM1 FveCHP1 FveSAND FveTIP41 FveTUBa FveENP1 FveEF1a FveBZIP1 FveGAPDH FveCHC1 FveTUBβ FveAATP1 FveAP1 FveMT1 FveACT-7 FveTBP FveRIB413 FveUBQ1 FveHDP1 FveDBP

0.01 0.01 0.04 0.05 0.06 0.06 0.06 0.1 0.1 0.1 0.11 0.11 0.12 0.12 0.12 0.13 0.15 0.22 0.24 0.25

FveAP1 FveDBP FveCHP1 FveCHC1 FveHDP1 FveTIM1 FveTUBβ FveMT1 FveTIP41 FveGAPDH FveENP1 FveSAND FveACT-7 FveAATP1 FveUBQ1 FveBZIP1 FveEF1a FveTBP FveRIB413 FveTUBa

0.01 0.01 0.03 0.03 0.03 0.04 0.04 0.06 0.07 0.08 0.08 0.09 0.09 0.1 0.1 0.11 0.11 0.13 0.25 0.37

FveCHC1 FveEF1a FveSAND FveTIM1 FveCHP1 FveBZIP1 FveTUBa FveTIP41 FveGAPDH FveDBP FveMT1 FveAATP1 FveTBP FveUBQ1 FveTUBβ FveENP1 FveRIB413 FveAP1 FveACT-7 FveHDP1

0.02 0.02 0.05 0.06 0.07 0.08 0.08 0.1 0.11 0.11 0.11 0.11 0.13 0.13 0.14 0.16 0.18 0.19 0.21 0.22

FveCHC1 FveTUBβ FveAP1 FveCHP1 FveTIM1 FveEF1a FveACT-7 FveGAPDH FveENP1 FveMT1 FveHDP1 FveAATP1 FveTUBa FveTBP FveTIP41 FveSAND FveDBP FveUBQ1 FveBZIP1 FveRIB413

0.09 0.09 0.1 0.1 0.1 0.11 0.12 0.12 0.13 0.15 0.16 0.16 0.16 0.16 0.19 0.19 0.21 0.23 0.29 0.3

SV, stability value. Table 3 Comprehensive ranking of the tested 20 candidate reference genes by the GM methoda. Tissues

　

Abiotic Stress

Organs

Fruits

All Samples

Heat

Cold

Drought

Salt

Rank

Gene

GMb

Gene

GM

Gene

GM

Gene

GM

Gene

GM

Gene

GM

Gene

GM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

FveMT1 FveTBP FveSAND FveCHC1 FveTIP41 FveRIB413 FveEF1a FveENP1 FveHDP1 FveTIM1 FveTUBβ FveCHP1 FveDBP FveUBQ1 FveACT-7 FveGAPDH FveAP1 FveBZIP1 FveAATP1 FveTUBa

3.5 4 4.25 5 6 6.5 6.75 7.25 8 10.8 11.8 12.5 13 13.5 14 14.8 15 15.8 18.5 19

FveCHC1 FveEF1a FveGAPDH FveDBP FveUBQ1 FveSAND FveACT-7 FveTUBβ FveENP1 FveAP1 FveCHP1 FveAATP1 FveTIM1 FveHDP1 FveTBP FveBZIP1 FveMT1 FveTIP41 FveRIB413 FveTUBa

2.5 3 3 4.25 4.75 7 9.5 9.5 10 10.5 10.5 11.8 12.8 13.8 13.8 14.5 14.5 16.8 18.5 19

FveCHC1 FveTIM1 FveAP1 FveTUBa FveBZIP1 FveGAPDH FveTIP41 FveTUBβ FveAATP1 FveHDP1 FveCHP1 FveDBP FveUBQ1 FveACT-7 FveMT1 FveTBP FveEF1a FveENP1 FveSAND FveRIB413

3.25 4.25 6.75 7 7.75 8 8.5 8.75 9.5 9.5 10.8 11 12.3 12.5 12.5 13 14.8 15.3 16 18.5

FveSAND FveTIM1 FveCHP1 FveENP1 FveEF1a FveTIP41 FveAATP1 FveCHC1 FveTUBa FveBZIP1 FveTUBβ FveGAPDH FveACT-7 FveMT1 FveTBP FveAP1 FveUBQ1 FveRIB413 FveDBP FveHDP1

3 3 4 4.75 5.25 5.5 7.5 8.5 8.5 10.5 10.5 11.5 12.3 14 14 14.5 16.8 17.8 18.8 19.3

FveAP1 FveDBP FveCHP1 FveCHC1 FveTIM1 FveHDP1 FveTUBβ FveTIP41 FveGAPDH FveENP1 FveMT1 FveSAND FveTBP FveUBQ1 FveAATP1 FveEF1a FveRIB413 FveTUBa FveACT-7 FveBZIP1

3.25 4.5 5 5.5 5.5 6.25 6.25 7.75 9.25 10.5 11.3 13.3 13.5 13.5 14.5 15.3 15.5 15.5 16 17.8

FveCHC1 FveEF1a FveBZIP1 FveSAND FveTUBa FveTIM1 FveCHP1 FveTIP41 FveGAPDH FveMT1 FveAATP1 FveTUBβ FveTBP FveDBP FveACT-7 FveUBQ1 FveRIB413 FveENP1 FveHDP1 FveAP1

3 3 5 5.25 6 7.5 7.75 8.5 9 9 9.75 10.3 12.3 12.8 14.8 15.3 16.3 17.3 17.8 19.5

FveCHC1 FveTIM1 FveEF1a FveTUBβ FveCHP1 FveGAPDH FveACT-7 FveENP1 FveAP1 FveTBP FveMT1 FveDBP FveHDP1 FveUBQ1 FveTIP41 FveSAND FveAATP1 FveBZIP1 FveRIB413 FveTUBa

2.5 3.25 4.75 5.5 6 7 7.25 7.5 9 10 11.8 12 12 12.8 14.3 14.5 16.3 17.5 18 18

a b

Calculated by the integration of rankings produced by geNorm, NormFinder, Δ Ct and BestKeeper methods. GM：Geomean of ranking values.

values obtained by the Cq values of the candidate reference genes (Pfaffl et al., 2004). SD value is negatively correlated with gene stability, which means that more stable genes have lower SD values. FveUBQ1 (SD = 0.15) and FveTUBβ (SD = 0.66) were identified as the most stably expressed gene in organs and fruit development, respectively. In the four abiotic stress treatments, the most stable reference gene identified was different, in that FveACT-7 (SD = 0.25), FveAATP1 (SD = 0.29), FveTBP (SD = 0.34) and FveBZIP1 (SD = 0.15) had the lowest SD under heat, cold, drought and salt treatment, respectively (Table S4). Furthermore, when all samples were considered, FveTUBβ (SD = 0.69) was the most stable reference gene, followed by FveEF1a (SD = 0.72).

3.4. Comprehensive stability analysis of candidate reference genes Ranking results of the 20 candidate reference genes obtained from the four individual methods showed substantial differences, due to different algorithms used in the software programs. Thus, we decided to integrate the results from the four methods to obtain a comprehensive ranking for the 20 candidate reference genes by Geomean of ranking (GM) values firstly. In this method, the gene with the lowest GM value was considered to be the most stable one. As shown in Table 3, we found that FveCHC1 exhibited relatively low GM value in fruit development, under heat and salt stresses and for all samples. On the other hand, FveRIB413 or FveTUBa was not recommended as an internal 5

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Fig. 4. Relative expression profiles of FveNCED2 and WRKY genes in Fragaria vesca (a–c) Expression profiles of FveNCED2 in fruit ripening from the small green stage to the red stage. SG small green, MG medium green, BW big white, PT pre-turning. Expression profiles of FveNCED2 are normalized by the top-ranked reference gene FveCHC1 (a), the combination of two recommended genes FveCHC1 and FveGAPDH (b), or the less recommended reference gene FveRBI413 (c) as the reference genes. Results are presented as a percentage of the value in SG which was arbitrarily set to 100%. (d–i) Expression profiles of FveWRKY34 and FveWRKY48 genes under salt stress conditions. Expression profiles of FveWRKY34 and FveWRKY48 are normalized by the top-ranked reference gene FveCHC1 (d and g), the combination of two recommended genes FveCHC1 and FveEF1a (e and h), or the less recommended reference gene FveAP1 (f and i) as the reference genes. Results are presented as a percentage of the value under 0 h salt stress conditions which was arbitrarily set to 100%. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

control for its poor expression stability displayed in the comprehensive analysis. Finally, RefFinder was applied to mine the appropriate reference genes, which was based on both Cq and geomean of ranking values (Xie et al., 2012). According to the results of RefFinder, FveCHC1 was the most stably expressed gene when all samples were considered, which is consistent with the results of GM (Table S5). In tissues, FveSAND and FveGAPDH were recommended as the stably expressed reference gene in organs and fruits, respectively. In addition, under the four stress

conditions, the stably expressed genes identified by RefFinder also showed high expression stability in GM method, as FveCHC1, FveTIM1, FveCHP1 and FveCHC1 were recommended under heat, cold, drought and salt treatment, respectively. 3.5. Expression profiling of FveNCED2, FveWRKY34 and FveWRKY48 To assess the influence of reference genes on the normalized expression profile of a gene of interest, the transcript level of genes 6

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Table 4 The top two reference genes for each method and two reference genes we ultimately recommend. Tissues

Abiotic Stress

All Samples

method

Rank

Organs Gene

Fruits Gene

Heat Gene

Cold Gene

Drought Gene

Salt Gene

Gene

geNorm

1 1 1 2 1 2 1 2 1 2 1 2 1 2

FveCHC1 FveENP1 FveMT1 FveSAND FveSAND FveMT1 FveUBQ1 FveDBP FveMT1 FveTBP FveSAND FveEF1a FveMT1 FveSAND

FveDBP FveUBQ1 FveCHC1 FveEF1a FveCHC1 FveGAPDH FveTUBβ FveEF1a FveCHC1 FveEF1a FveGAPDH FveCHC1 FveCHC1 FveGAPDH

FveDBP FveUBQ1 FveCHC1 FveTIM1 FveCHC1 FveTIM1 FveACT-7 FveGAPDH FveCHC1 FveTIM1 FveCHC1 FveGAPDH FveCHC1 FveTIM1

FveENP1 FveEF1a FveTIM1 FveCHP1 FveSAND FveTIP41 FveAATP1 FveTIM1 FveSAND FveTIM1 FveTIM1 FveAATP1 FveSAND FveTIM1

FveTIM1 FveHDP1 FveAP1 FveDBP FveCHC1 FveHDP1 FveTBP FveTUBa FveAP1 FveDBP FveCHP1 FveAP1 FveAP1 FveCHP1

FveCHC1 FveEF1a FveCHC1 FveEF1a FveCHC1 FveEF1a FveBZIP1 FveTUBβ FveCHC1 FveEF1a FveCHC1 FveEF1a FveCHC1 FveEF1a

FveCHC1 FveTIM1 FveCHC1 FveTUBβ FveCHC1 FveTIM1 FveTUBβ FveEF1a FveCHC1 FveTIM1 FveCHC1 FveTUBβ FveCHC1 FveTUBβ

NormFinder Δ Ct BestKeeper GM RefFinder recommend

FveNCED2, FveWRKY34 and FveWRKY48 with known expression patterns were analyzed. FveNCED2 was reported to be essential in ABA biosynthesis during strawberry fruit ripening (Wang et al., 2017). Thus, FveNCED2 was analyzed in strawberry fruits at six ripening stages during ripening. Three ways of normalization were performed, including normalization by using two reference genes (the most recommended FveCHC1 and FveGAPDH), and by FveCHC1 or FveRIB413 representing the best and less recommended reference gene during fruit development and ripening (Fig. 4a-c). The relative expression profiles of FveNCED2 obtained by using reference gene combinations by FveCHC1 and FveGAPDH, or by FveCHC1 along, exhibited an increasing trend during ripening, which was consistent with expectation. In contrast, the relative expression level normalized by FveRIB413 was distinct, especially at the medium green (MG), big white (BW), pre-turning (PT) and Pink stages. The results were not unexpected, as FveRIB413 ranked poorly in all statistical methods tested. WRKY family proteins are involved in various biotic or abiotic stress responses in plants (Chen et al., 2012; Banerjee and Roychoudhury, 2015). FveWRKY34 and FveWRKY48 were revealed to be activated by salt stress in strawberry (Wei et al., 2016). Thus, we profiled the transcript levels of FveWRKY34 and FveWRKY48 under salt stress conditions to testify the performance of different ways of normalization. The results showed that by normalized to two reference genes (the most recommended FveCHC1 and FveEF1a) or the best recommended reference gene FveCHC1, the relative expression of FveWRKY34 and FveWRKY48 genes were elevated after salt stress (Fig. 4d–e and g–h), which was consistent with previous reports (Wei et al., 2016). However, the expression level of FveWRKY34 and FveWRKY48 normalized to the less recommended reference gene FveAP1 showed an irregular expression pattern (Fig. 4f and i). Overall, the reference genes identified in our analysis are appropriate for normalization of gene expressions in Fragaria vesca.

the developmental stages and experimental conditions. Two reference genes are sufficient for normalization under the experimental conditions tested. We recommend FveSAND and FveMT1, FveCHC1 and FveGAPDH as reference genes in organs and fruits, FveCHC1 and FveTIM1, FveSAND and FveTIM1, FveCHP1 and FveAP1, FveCHC1 and FveEF1a under heat, cold, drought, salt stress conditions, respectively (Table 4). If one reference gene is to be used for normalization, FveCHC1 could be considered as it performs as one of the most stable internal controls in most conditions. Identification of reference genes in cultivated strawberry has been conducted previously (Amil-Ruiz et al., 2013; Zhang et al., 2018). Our results suggest that although there is substantial similarity between cultivated and woodland strawberry, the reference genes recommended for gene expression normalization in cultivated strawberry are not necessary suitable in woodland strawberry. For example, FaRIB413 was identified as a suitable internal control in Fragaria × ananassa in previous studies (Amil-Ruiz et al., 2013). While in the sets of samples investigated in our study, the expression of FveRIB413 is relatively unstable (Table 2 and 3 and Table S3–S5). 5. Conclusion In summary, our study represents a comprehensive analysis of 20 reference genes as internal controls in qRT-PCR analysis in various tissues and seedlings under abiotic stresses in Fragaria vesca. Our data suggest that two reference genes are sufficient for normalization under our experiment conditions. Furthermore, FveCHC1 could be considered for normalization if one reference gene is to be used. Our results provide useful tools for reliable expression normalization of the genes of interest in future research in woodland strawberry. Declaration of Competing Interest The authors have no conflicts of interest to declare.

4. Discussion

Acknowledgements

qRT-PCR has been widely used for gene expression analysis in highthroughput transcriptomic studies (Wong and Medrano, 2005). A reliable internal control should show minimal changes through experiment. Determination of the expression stability of reference genes prior to their use for normalization of expression levels of genes of interest is critical for qRT-PCR data interpretation.In this study, the expression stabilities of 20 candidate reference genes were identified in organs (root, leaf and flower), fruit development and seedlings under four abiotic stress conditions (heat, cold, drought and salt). Overall, our results suggest that reference genes should be validated according to

This work was supported by the National Natural Science Foundation of China [31672123 to T.G.] and the Fundamental Research Funds for the Central Universities[KYZ201605 to T.G.]. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.scienta.2019.108840. 7

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