Comparison of uridine diphosphate-glycosyltransferase UGT76G1 genes from some varieties of Stevia rebaudiana Bertoni

Stevia leaves contain various components, such as flavonoids, labdanes, chlorophylls, sterols, triterpenoids, mono-disaccharides, organic acids and inorganic salts. Stevia is known to accumulate diterpenoid steviol glycosides, which are approximately 300 times sweeter than regular sugar. Stevioside and rebaudioside A are the main diterpenic glycosides in stevia. Steviol glycosides are the secondary metabolites responsible for the sweetness of stevia. The main objectives of the present study were to determine the concentrations of diterpenic glycosides (stevioside and rebaudioside A) in three stevia varieties (Stevia rebaudiana) via the HPLC-UV technique and to amplify the UGT76G1 gene by PCR using gene-specific primers. The expression levels of the UGT76G1 gene were determined in the three stevia varieties. The PCR products were sequenced and analyzed, and the nucleotide sequences of the UGT76G1 gene were submitted to GenBank and assigned to the following three varieties: Egy1 (MH087463), China1 (MH087464) and Sponti (MH087465). Cluster analysis was used to separate the three varieties into two major clusters based on their phylogenetic relationship. In addition, chemical analysis was carried out to evaluate stevioside and rebaudioside A. The present study concluded that Egy1 and Sponti are closely related varieties as they fall in the same cluster, while China1 forms a separate cluster. Bioprospecting studies could be useful for selection of superior ecotypes of Stevia rebaudiana.

sweet diterpene glycoside extraction. The collected leaves were oven-dried (E. Schulz & Co. Inh. Franz. Skorezewsh KOMEG Technology, China) at 50 °C and then ground to a fine powder according to 34 with some modifications as follows: 5 g of dried leaf powder was extracted with 50 ml of hot methanol using a Soxhlet apparatus for 2 hrs. The extract was filtered using Whatman no. 1 filter paper, and the residue was re-extracted twice with methanol at room temperature. The filtrate was further concentrated in rotary flash evaporator (Type 349, James Jobling and Co. Ltd., England) at 60 °C to 10 ml, and then, 50 ml of distilled water was added to the concentrated extract. A phase separation step was performed to remove plant pigments as follows: 25 ml of diethyl ether was added to the extract in a 500-ml separatory funnel (Sigma Aldrich), and the aqueous phase was collected and extracted with butanol. Finally, the butanol upper layer was collected and refrigerated overnight at 4 °C to allow the purified glycosides to form crystals. Then, the crystals were separated by filtration and analyzed using HPLC 34 . Each stevia sample was extracted twice. preparation of the stevioside standard. Stevioside standard preparation was carried out according to the method described by Nishiyama et al. 35 with minor modifications as follows: dried leaves of S. rebaudiana Bertoni (10 g) collected from Sugar Crops Research Institute (SCRI), Agricultural Research Center (ARC), Ministry of Agriculture, Egypt, were extracted by soaking leaves in 1.0 liter of hot distilled water (85 °C) for 30 minutes. The resulting liquid fractions were filtered using a Buchner filtration system, and the leaves were then washed with an additional volume of hot water (50 ml). The aqueous solution was concentrated to 50 ml in a freeze-drier (Edwards model EF03, England). The extract was defatted by ethyl acetate followed by extraction with isobutyl alcohol (150 ml). The aqueous phase was discarded, and the organic phase was evaporated until dryness by using a rotary evaporator (Type 349, James Jobling and Co. Ltd., England) at 70 °C. The dry pellets were dissolved in hot methanol (100 ml) and allowed to crystallize overnight. The crystals were separated by filtration and dissolved in boiling methanol (50 ml) to obtain a concentrated solution. The solution was clarified with active charcoal (B.D.H. Laboratory Chemicals Division, Poole, England) and left to recrystallize. The procedure was repeated three times until the formation of colorless crystals. The pure solution of the stevioside standard was subjected to HPLC analysis.

Analysis of sGs by HpLC.
High-performance liquid chromatography (HPLC) technology can be used to directly measure the levels of steviol glycosides (rebaudioside A and stevioside) in Stevia rebaudiana Bertoni 36 . The levels of stevia sweetener compounds were estimated at the Central Laboratory, Faculty of Science, Alexandria University. Leaf extracts were separated and identified by HPLC according to 37 as follows: the stevioside solution was filtered through a Millipore membrane (13 mm diameter, 0.5 μm pore size) and analyzed using HPLC with a stevioside standard as an internal standard (10 mg/ml). Different extracts of stevia leaves were injected into an HPLC instrument (Shimadzu, Tokyo, Japan; model SPD-6AV) equipped with an LC-GA UV-vis detector and an Alex C-R 4 A recorder. The separation was carried out on a Zorbax NH2 column (25 cm × 0.4 mm I.D.; Dupont, Wilmington, DE, USA) with acetonitrile (HPLC grade, Fisons Co., England) as the mobile phase (acetonitrile: water (80: 20 v/v), adjusted to pH 5 with H 3 PO 4 ). The flow rate was 2 ml/min; the UV detection wavelength was 210 nm; the recorder chart speed was 20 nm/min; and the analysis was performed at ambient temperature (25 °C). Two samples per variety were analyzed, and the quantities of stevioside and rebaudioside A were calculated from the area under each peak.
PCR amplification of the stevia UGT76G1 gene. Fresh leaf samples of three stevia varieties (three months old), namely, China1, Egy1 and Sponti, were used to extract total genomic DNA using the DNA Mini-Prep Kit (BIO BASIC, Canada). The stevia UGT76G1 gene was amplified by using gene-specific primers UGT76G1 FP (5′ AACGTCAGTCAAACCCAATG3′) and UGT76G1 RP (5′ CTCACATAACCAACAACCATCC3′) according to 33 . The PCR was performed in a 25-µl reaction mixture containing 1 µl of 100 ng/µl DNA, 1 µl of each primer at a concentration of 0.01 nmol/µl, 12.5 µl of master mix (2.5 µl of 10 × PCR buffer, 1.5 µl of 25 mM MgCl 2 , 2.0 µl of 2.5 mM dNTP, and 0.125 µl of Taq DNA polymerase) (BIOLINE, UK) and 9.5 µl of water (H 2 O). The PCR www.nature.com/scientificreports www.nature.com/scientificreports/ program was as follows: denaturation for 5 min at 95 °C; 35 cycles of 40 s at 94 °C, 40 s at 55 °C and 2 min at 72 °C; and 72 °C for 10 min. Then, 2% agarose gel electrophoresis with ethidium bromide was used to separate the PCR product. The image was recorded using a gel documentation system (Alpha Image, USA). Clearly separated DNA bands of PCR products (approximately 1500 bp) were cut from the gel and purified using the GF-1 AmbiClean Kit (PCR & Gel, Vivantis, WE CARE, Malaysia). Then, the purified PCR products were submitted for sequencing at Macrogene (Korea). The sequences were analyzed and compared by NCBI BLAST (http://blast.ncbi.nlm. nih.gov); the sequences were aligned to generate a phylogenetic tree by using Molecular Evolutionary Genetics Analysis (MEGA5) software. The sequences were submitted to GenBank (gb-admin@nbci.nlm.nih.gov), and each sequence was assigned a GenBank accession number.  Table  (1). The stevia actin gene was used as an internal control for data normalization. For relative quantification of gene expression, qRT-PCR was conducted in an Eppendorf Master Cycler ep realplex using the following PCR cycling conditions: 2 min at 95 °C, followed by 40 cycles of 5 s at 95 °C, 10 s at 60 °C and 5 s at 72 °C; then, melting curve analysis was performed according to 33 . Expression of the UGT76G1 gene was determined by quantitative qRT-PCR on a Thermo Scientific PikoReal 96 real-time PCR system (www.thermoscientific.com/pikoreal) with the SYBR Green SensiFAST TM SYBR ® No-ROX Kit (BIOLINE). Relative quantification by real-time PCR was performed in a 10 µl volume containing 1 μl of cDNA, 5 µl of 2 × SensiFAST SYPBR ® No-ROX mix, 0.5 µl of each primer, and 3 µl of H2O. For quantitative real-time PCR data analysis, relative expression of UGT76G1 was calculated based on the threshold cycle using the 2 −ΔΔCq method 38 . The expression levels of target genes were normalized using the stevia actin gene as an internal control, and the relative transcript levels were calculated as follows according to 39 .

Quantitative real-time PCR analysis (qRT-PCR). Primer design for the UGT76G1 gene is recorded in
where Cq expression 2 nCq 39 statistical analysis. Data analysis was performed by using the Excel software program; gene expression was examined for three biological replicates of each variety. A t-test at p < 0.05 was applied to determine significant differences in gene expression between the three varieties, and standard deviations were calculated for the means of the biological replicates.
Compliance with ethics requirements. This article does not contain any studies with human or animal subjects.

Results
Chemical analysis of stevia sweeteners. HPLC was used to determine the levels of stevioside and rebaudioside A. The results shown in Table (2) and Fig. (1A-C) indicate that the highest stevioside content was observed in the Sponti variety (21.46%), followed by China1 (0.18%) and finally Egy1 (12.27%). The range from lowest to highest value was 12.27 to 21.46% (with a 9.19% increase observed for the Sponti variety, Table 2). The highest levels of rebaudioside A were observed in China1 and Egy1 (15.54% and 14.48%, respectively), while a value of 13.02% was observed for the Sponti variety (Table 2).
PCR amplification of UGT76G1. The UGT76G1 gene was amplified from the stevia varieties by PCR using the designed primers (UGT76G1 F and R). The PCR assay showed an amplification product of the expected size (1.51 kb), as shown in Fig. (2). These data are consistent with 33 , in which a product with same molecular weight was detected.
UGT76G1 gene sequencing and phylogenetic analysis. The polymerase chain reaction amplification products of the UGT76G1 gene were sequenced and analyzed to determine nucleotide similarity among the three www.nature.com/scientificreports www.nature.com/scientificreports/ stevia varieties, as shown in Fig. (3). Data were submitted to GenBank for identification of the accession numbers for each sequence (Table 3, Figs 4 and 5). The partial sequence of the UGT76G1 gene was aligned and compared to the sequences in GenBank. A dendrogram was generated using MEGA5 software to examine the phylogenetic relationship of UGT76G1 among the three stevia varieties, namely, Egy1, China1 and Sponti. The observed similarity might be the result of the existence of a common ancestor for Egy1 and Sponti, and this ancestor might differ from the ancestor of China1 (Fig. 6). The generated genetic similarity dendrogram for the three stevia varieties classified the populations into two major groups: Group I (Egy1 and Sponti) and Group II (China1). The results showed that Egy1 and Sponti belong to the same cluster (Figs 6 and 7). Table (4) and Fig. (8) indicate that the gene expression of UGT76G1 varied significantly between the three stevia varieties under investigation. Based on the obtained results, China1 showed the highest gene expression level, exhibiting a ΔΔCq value of 0.178, followed by Egy1, which exhibited a relatively low expression level (ΔΔCq value of 0.119). The lowest gene expression level was reported for the Sponti variety, which showed a ΔΔCq value of 0.074, and these values were significantly different for the three varieties (p = 0.05). The relative gene expression of UGT76G1 was significantly  www.nature.com/scientificreports www.nature.com/scientificreports/ high between China1 and Egy1 (value of 0.002), followed by China1 and Sponti and Egy1 and Sponti (0.0001 and 0.0007, respectively). These results support the finding obtained using HPLC analysis that showed a significant increase in rebaudioside A concentrations in China1 compared to Sponti.

Discussion
The stevioside content observed in this study was higher than that reported by Parris et al. 39 , in which the concentration ranged from 2.8-5.49% 25 and 6.98-12.16%. For rebaudioside A content, China1 exhibited the highest value (15.54%), while Sponti exhibited the lowest value (13.02%). A low value for rebaudioside content was also reported by 40 . The variation in stevioside content and rebaudioside A content was further speculated to be associated with increasing altitude, resulting in decreasing temperature and in turn in accumulation of stevioside.    Table 3. Accession numbers of the UGT76G1 gene in GenBank.
www.nature.com/scientificreports www.nature.com/scientificreports/ Recently, stevia has received much attention. Rebaudioside A represents 30-40% of total glycosides 41 , but in the current study, the rebaudioside A levels of the stevia varieties China1, Egy1 and Sponti were 15.54%, 14.48% and 13.02% of the total glycosides, respectively. Therefore, these varieties are considered good materials for the study of the synthesis of steviol glycosides. The results of the present study are consistent with those of previous studies that determined stevioside, rebaudioside A and steviol levels via different methods 42 . HPLC and NIR spectroscopy models have been used to directly measure the steviol glycoside content in S. rebaudiana Bertoni to decrease the cost and complexity of operation 36 . The UGT76G1 gene was amplified from the stevia varieties by PCR using the designed primers (UGT76G1 F and R). The PCR assay showed an amplification product of the expected size (1.51 kb), which is in accordance with Li et al. 24 . The phylogenetic analysis indicated that the three sequences were grouped with UGT76G1 in the same cluster, indicating a close relatedness between UGTSr and UGT76G1. The phylogenetic analysis showed a close relationship between UGTSr and UGT76G1 43 Stevia rebaudiana UGT76G1). In a previous study, a mutation in the UGT76G1 gene was found to cause a reduction in rebaudioside A accumulation to 0.2% compared to normal plants, which usually exhibited 30-40% accumulation of total glycosides 33 . It was concluded that these three UGTs were not expressed at higher levels than any of the other UGTs. The expression of these genes was in the following order: UGT85C2 > UGT76G1 > UGT74G1. In the present study, we used qRT-PCR, a highly sensitive and www.nature.com/scientificreports www.nature.com/scientificreports/ specific method, to accurately measure the transcript levels of the three varieties 44 . Increased transcript levels of the UGT76G1 enzymes increased the level of the final product of the biosynthetic pathway, rebaudioside A 45,46 . Rebaudioside A accumulation is one of the most important traits contributing to the economic value of stevia crops 47 . www.nature.com/scientificreports www.nature.com/scientificreports/

Conclusion
The present study investigated the relationship among three Stevia varieties using HPLC and molecular techniques. Sequence analysis was used to determine the nucleotide similarity of the stevia varieties China1, Egy1, and Sponti. The three varieties were clustered into two major groups and showed close similarity to UGT76G1. Phylogenetic relationships among different sequences of S. rebaudiana UGT76G1 were studied. A dendrogram of genetic similarities among the three varieties was constructed. The results indicated that the three varieties were clustered into two major groups: Group I (Egy1 and Sponti) and Group II (China1). This similarity might be the   Table 4. Relative gene expression of the stevia UGT76G1 gene in three varieties using RT-quantitative PCR. www.nature.com/scientificreports www.nature.com/scientificreports/ result of the existence of a common ancestor for Egy1 and Sponti, and this ancestor might be different from the ancestor of China1. The results obtained for the gene expression of UGT76G1 indicated that China1 showed the highest gene expression levels compared to the other two varieties, and the gene expression level of UGT76G1 was significantly higher in China1 than in the two varieties.