2023, Vol. 13, No. 3, 543 – 552 http://dx.doi.org/10.11594/jtls.13.03.13
How to cite:
Zakiyah AS, Monica R, Siswanto D, Arumingtyas EL (2023) Variation of Fruit Color in Cakra Hijau, G1/M8 and HV-149 Research Article
Variation of Fruit Color in Cakra Hijau, G1/M8 and HV-149 Chilli Pepper Cultivar: Physiology and Molecular Approach
‘Ainun Sayyidah Zakiyah, Rosina Monica, Dian Siswanto, Estri Laras Arumingtyas*
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya, Malang 65145, Indonesia
Article history:
Submission June 2021 Revised June 2021 Accepted November 2021
ABSTRACT
The fruit color of chili pepper is an important characteristic in identification and classification and is often used as the basis for determining consumer preferences.
Information on the relationship between chili fruit color and its molecular profile is very important in supporting selection activities in plant breeding. This study aims to identify genetic diversity associated with the fruit color of three genotypes of chili (Capsicum frutescens L.): Cakra Hijau, HV-149 and G1/M8, using Random Ampli- fied Polymorphic DNA (RAPD) and Simple Sequence Repeat (SSR). Morphological confirmation was carried out according to Capsicum descriptors. Nineteen RAPD markers and six SSR markers were used for genetic variability assessment. Genetic variation was analyzed using the unweighted pair group method with the arithmetic mean and the Jaccard similarity index. The three chili genotypes had different fruit colors at each maturation stage. The immature Cakra Hijau fruit is dark green and turns dark red as it ripens. The immature fruits of the G1/M8 line are light green and turn red when ripe. Finally, immature HV-149 fruits are dark green and yellow when ripe. The SSR markers used in this study were unable to show polymorphism. On the other hand, the RAPD marker successfully detected genetic variation in the three chili genotypes and resulted in a total of 49 alleles. The average value of polymor- phic information content of the RAPD primers used ranged from 0 to 0.296, with the highest index indicated by OPA-1. The dendrogram shows the separation of the three genotypes into two main clusters, with the first cluster consisting of the HV-149 variety and the second cluster consisting of Cakra Hijau and G1/M8 lines. This study revealed that there are genetic variations based on the morphological characteristics of fruit color at each ripening stage and RAPD band profile. The RAPD marker was more effective than the SSR marker for identifying the genetic diversity of fruit color in the three chilies studied.
Keywords: Capsicum frutescens L., Genetic variation, RAPD, SSR
*Corresponding author:
E-mail: [email protected]
Introduction
Chilli pepper (Capsicum frutescens L.) is a member of the Solanaceae family and originated in South and Central America, with Brazil as a di- versity centre for Capsicum [1]. Chilli peppers are widely used by the community as a source of spice, herbal medicine, and antibacterial agents [2]. The natural pigment of the chilli fruit is also beneficial for food coloring and flavour enhancers [3]. The benefits of chilli peppers make this crop an important horticultural crop in the world due to its wide diversity [4]. In Indonesia, two species of
Capsicum (C. annuum L. and C. frutescens L.) are highly cultivated and widely used. [5].
Other than the level of spiciness, the variations that stand out in chillies are their fruit shape and color. Chilli fruit color is an important character that is widely used in chilli classification [6, 7]. In the development of identification using image pro- cessing, the color and shape of chillies are im- portant characteristics [8]. Color is also a consid- eration for consumers in choosing the type of chilli according to their needs [9]. In addition to color
variations related to the type or variety of chillies, the maturity level of the fruit also determines the color of the chilies. The color of immature chilies ranges from white, yellow, light green and dark green, while the color of ripe fruit varies from red, orange and yellow [7]. Many pigments play a sig- nificant role in the color formation of the fruit of chilli peppers. The green color is mainly con- trolled by chlorophyll, and the mature red color may be controlled by carotenoids [10]. The chilli pepper fruit ripening process is mainly controlled by the y, c1 and c2 loci; the interaction between them causes different ripe fruit colors in each chilli pepper plant [7]. Anthocyanins are also reported to determine the purple color of chilli peppers [11].
The Indonesian local chilli pepper, Cakra Hi- jau, is a commercial variety that has a deep green immature fruit and a red mature fruit [12]. The in- duction of the mutation process using Ethyl Me- thane Sulfonate (EMS) on the Cakra Hijau variety has produced the G1/M8 line with a distinct fruit maturation color compared with the Cakra Hijau variety [13]. The G1/M8 line has a light green fruit in the immature stage that turns red in the mature stage. Meanwhile, the immature fruit of HV-149 genotypes is deep green in color and turns yellow when it matures. Studies on the genetic properties of color variations and changes during the ripening process of chilli peppers have never been carried out. Information about the relationship between the morphological characteristics of color and ca- rotenoid content is not yet available. Molecular analysis that reveals variations in chilli has been frequently carried out. Genotypic characterization using RAPD and SSR molecular markers has suc- cessfully strengthened Capsicum diversity identi- fication [14, 15]. The RAPD markers effectively produce a high degree of polymorphism to identify genetic variability [16–19). Furthermore, SSR markers have been widely used because of their ability to detect multi-allelic forms of variation in inbred lines [19–20].
The objective of this research was to assess the genetic diversity among (C. frutescens L.) Cakra Hijau, the G1/M8 line and HV-149 using RAPD and SSR. Information about the relationship be- tween morphological and molecular characteris- tics greatly supports the breeding process, espe- cially in marker-assisted selection.
Material and Methods Plant material
The plant material consists of three cultivars:
Cakra Hijau, G1/M8 and HV-149, which have dis- tinct fruit color characteristics. The Cakra Hijau variety has deep green immature fruit and red ma- ture fruit. The G1/M8 line has light green imma- ture fruit that turns red when in the mature stage.
Meanwhile, HV-149 genotypes have a deep green immature fruit and yellow mature fruit. Ten chilli plants were grown from seeds using the hydro- ponic method in the same environmental condi- tions. When the chilli seedlings were 40–50 days old, they were transferred to soil planting media for further observation of their morphological characteristics and molecular analysis.
Morphological characteristic observation The morphological observation of chilli pep- pers was conducted based on the Capsicum de- scriptor. The morphological characters were ob- served in the plant's height, branches, canopy length, leaf, flowers, and fruits. Ten plants from each genotype were used for morphological eval- uation. The result was analyzed using statistical analysis with a significant value of α = 0.05. The data on the morphological characteristics was then categorized according to the chilli descriptor books, IPGRI, AVRDC and CATIE [21]. The data was presented in a dendrogram.
Carotenoid content analysis
Chilli pepper fruits were harvested based on maturity stages: mature stage ± 0–39 Days After Pollination (DAP) pre-mature stage (± 40–50 DAP) and mature stage (± 50–60 DAP). The ca- rotenoid extraction process was based on previous research [22]. A total of 0.5 gram of fruit pericarp was ground using a mortar and pestle, then ho- mogenized with 10 mL of 80% acetone. The ho- mogenate was filtered using filter paper, and the filtrate was measured using UV-spectrophotome- ter wavelengths of 480, 645 and 663 nm (Å). The measurement was carried out in three replicates for each fruit's maturation stages. The measure- ments were calculated to reveal the carotenoid content in each chilli pepper fruit.
Carotenoid (mg/g 𝐹𝑟𝑒𝑠ℎ 𝐹𝑟𝑢𝑖𝑡 𝑊𝑒𝑖𝑔ℎ𝑡) = Å480 + (0.114 x Å663) − (0.638 × Å645) ...(1) [22]
Genomic DNA extraction
Genomic DNA was isolated from 500 mg of young chilli leaves at the 3–4 leaf stage of the seedling from each genotype of Cakra Hijau, G1/M8 and HV-149. DNA extraction was per- formed using the Cetyl Trimethyl Ammonium Bromide (CTAB) method with some minor modi- fications [23]. DNA quantification was carried out using the ND-1000 Nanodrop Spectrophotometer machine.
RAPD analysis
A total of 19 RAPD primers (Operon Technol- ogies Inc., Alameda, California, USA) were used.
DNA amplification was carried out in a 20 µL re- action mixture containing 50 ng of DNA genome, 10 µL of GoTaq® Green 2× Master Mix (Promega No Cat: M7122), 1 µL RAPD primer, 1 µL of bo- vine serum albumin and 2 µL of nuclease free wa- ter. RAPD primers were amplified under polymer- ase chain reaction (PCR): pre-denaturation at 95ºC for 1 min, denaturation at 94ºC for 1 min, and an- nealing at 33–41ºC (on the basis of the Tm value of the primer, Table 1), extension at 72ºC for 1 min
and final extension at 72ºC for 5 min with 45 cy- cles. PCR products were separated by electropho- resis using 2.5% agarose gel stained with ethidium bromide.
SSR analysis
The six pairs of SSR primers (Table 2) were used to differentiate all samples tested. The ampli- fication process used 5 µL of GoTaq® Green 2x Master Mix (Promega No Cat. M7122), 0.5 µL forward and 0.5 µL reverse SSR primer, 1 µL of 50 ng DNA genome and 3 µL nuclease free water.
The PCR running programme consists of 32 cycles with a pre-denaturation of 94ºC for 1 min, dena- turation at 94ºC for 1 min, annealing (refer to Ta- ble 2 for the temperature), extension at 72ºC for 1 min and final extension at 72ºC for 5 min. The am- plicon product is visualized by electrophoresis us- ing 2.5% agarose gel. The size of the amplification product was estimated using a 100 bp DNA ladder (Jena Bioscience, Cat. No.: M-214S).
Construction of phylogenetic trees
For the construction of the phylogenetic tree, the morphological characteristic data were classi- Table 1. RAPD Primer Sequence [17]
Primer
Name Primer Sequence (5′–3′) Ta (ºC)
OPA-17 GACCGCTTGT 35
OPU-10 ACCTCGGCAC 40
OPD-19 CTGGGGACTT 33
OPU-19 GTCAGTGCGG 38
OPW-03 GTCCGGAGTG 36
OPA-11 CAATCGCCGT 36
OPA-01 CAGGCCCTTC 37
OPA-02 TGCCGAGCTG 40
OPB-03 CATCCCCCTG 35
OPB-06 TGCTCTGCCC 40
OPF-09 CCAAGCTTCC 33
OPF-11 TTGGTACCCC 33
OPF-14 TGCTGCAGGT 38
OPF-18 TTCCCGGGTT 37
OPW-04 CAGAAGCGGA 35
OPD-13 GGGGTGACGA 38
OPB-11 GTAGACCCGT 33
OPL-05 ACGCAGGCAC 41
OPB-03 CATCCCCCTG 35
OPB-04 GGACTGGAGT 33
Table 2. SSR Primer Sequence [14]
Primer
Name Primer Sequence (5′–3′) Ta (ºC) CA-19 F: CCG CAA TGG CAG
TAT GAT CT
R: CGG CTC TAT CTA CAA CGG TG
55.1 CA-26 F: CGC ATA TAG GCA
GAT CAA AT
R: TGA CTC AAA TGC TCT CTG AA
50.6 CA-52 F: TAG CAG AGG ACC
AGT TAG CA
R: ATG TTC TGA GTC CAC GAT GC
55.1 CA-56 F: CTT CGC ATA TGG
CAG ATC A
R: TCT CTG TGG CTG ACT CAA AT
52.7 CA-62 F: CGC ATA TAG GCA
GAT CAA AT
R: GGT CAG ACT ACG ACT CTC TCA
51 CA-96 F: CGC ATA TAG GCA
GAT CAA AT
R: AAT CTC TGT GGC TGA CTC AA
51.8
fied into scores of 1, 2, 3 and 4 based on the chilli descriptor books, IPGRI, AVRDC and CATIE. If a sample shows a characteristic belonging to cate- gory 1, then it will be scored as "1", and "0" for categories 2, 3 and 4; if the sample is included in category 2, then it will be scored "1" for that cate- gory, and "0" for categories 1, 3 and 4 etc.
The RAPD and SSR electrophoresis gel am- plification results were analyzed to determine the reproducibility of each marker. Each marker's pol- ymorphic information content (PIC) value was de- termined by converting binary data through a for- mula expressed in the equation below. The pres- ence of a band is scored as "1", and the absence of a band is scored as "0". Only clear and unambigu- ous RAPD and SSR bands were scored. The data matrix of the morphological characteristics and the RAPD and SSR markers was calculated using Jaccard's similarity index. The dendrogram was conducted using the unweighted pair group method with the arithmetic mean (UPGMA) clus- ter algorithm. The analysis was conducted using PAST 4.09 software.
𝑃𝑜𝑙𝑦𝑚𝑜𝑟𝑝ℎ𝑖𝑐 𝐼𝑛𝑓𝑜𝑟𝑚𝑎𝑡𝑖𝑜𝑛 𝐶𝑜𝑛𝑡𝑒𝑛𝑡 (PIC) = 2 × 𝑓𝑖 × (1 − 𝑓𝑖)……… (2) [24]
Where fi = (sum of alleles or band) / no samples.
Results and Discussions
Clustering Result for Morphological Character The morphological characteristics were ob- served for three chilli peppers in this study. There were variations in the morphological characteris- tics observed, specifically the stem, leaf, flower and fruit. HV-149 is the highest chilli plant among them; for the leaf character, it was found that the G1/M8 line has the largest leaf diameter (Figure 1).
The fruit of each chilli pepper genotype showed variations in color at each maturation stage. It was found that the Cakra Hijau variety was characterised by a deep green color in its im- mature stage and a deep red color in its mature stage. The G1/M8 line is a chilli plant mutant that arose from EMS of the Cakra Hijau variety and has a light green color when it is immature and turns red when it matures [5, 13]. For the HV-149 variety, the fruits have a deep green color in the immature stage and yellow in the mature stage (Figure 2). The color variation in each maturation stage is closely related to the amount of carotenoid content [25].
Figure 1. Chili pepper plant and leaves (C. frutescens L.). (A) Cakra Hijau variety; (B) G1/M8 line; (C) HV- 149 (Taiwan Kuning) variety.
The total carotenoid content of chilli pepper fruits greatly varies. The carotenoid content in im- mature fruits is approximately 0.81–1.9 mg/g fresh fruit weight (FW), approximately 1.24–2.57 mg/g FW in the pre-mature stage and in the range of 2.45–2.81 mg/g FW in the mature fruit stage (Figure 3). The carotenoid contents in mature chilli pepper fruits in the HV-149 variety are not significantly different from that in Cakra Hijau and G1/M8, despite differences in the color of the mature fruit. This phenomenon is not in accord- ance with a previous finding that carotenoid con- tent causes alteration of color from the immature to the mature fruit stage and carotenoid content will gradually increase during the fruit develop- ment process [26]. The different amounts of carot- enoid compounds in the fruit chromoplasts deter- mined the color of ripe chillies, which have yel- low, orange, red or dark red colors [27]. The fact that there is no difference in carotenoid content
while there is a variation in fruit color indicates that differential gene function and expression might be evidenced in carotenoid biosynthesis as stated by other researchers [28].
The carotenoid compound will gradually in- crease from the immature to the mature stage in each fruit according to the natural biosynthesis process of the fruit pigment [29]. The highest total carotenoid content in mature fruits was found in the Cakra Hijau genotype (2.81 mg/g FW) and the lowest was found in the HV-149 genotype which has a yellow mature fruit color. Compared with other genotypes in the same fruit maturation stage, HV-149 had the highest total carotenoid content in its immature (1.9 mg/g FW) and pre-mature fruits (2.5 mg/g FW) (Figure 3).
The differences in the carotenoid accumula- tion pattern in each genotype are due to the ability of fruits to synthesis carotenoids during the ripen- ing process regardless of the species [30]. The Figure 2. Chilli pepper in each maturation stage (C. frutescens L.) immature, pre-mature and mature. (A)
Cakra Hijau variety; (B) G1/M8 line; (C) HV-149 (Taiwan Kuning) variety; DAP: Day After Polli- nation.
Figure 3. Carotenoid content in chili pepper plants
chilli pepper genotypes, fruit maturation stage and other environmental conditions during the grow- ing process affect the varied amounts of carote- noid accumulation by suppressing the formation of carotenoid precursors in the chilli fruit [29]. The UPGMA cluster analysis, based on Jaccard's Co- efficient with 1000 bootstraps, produced a dendro- gram with two major clusters with a similarity co- efficient between 0.885–0.99 (Figure 4). The clus- ter was separated into two cluster groups with a cut-off similarity coefficient between 0.885 and 0.9. The first cluster consisted of the HV-149 va- riety, and the second consisted of the G1/M8 line and the Cakra Hijau variety. The genotypes close to the similarity coefficient of 0.885 were more dissimilar than the genotypes close to the similar- ity coefficient of 0.99. This result shows that G1/M8 is closely related to Cakra Hijau based on
morphological data, with a cut-off similarity coef- ficient of 0.92.
The immature fruits of the Cakra Hijau and HV-149 varieties have a deep green color, while those of the G1/M8 genotype have a light green color. When HV-149 genotype fruits reach ma- turity, they turn yellow, whereas those of the Cakra Hijau and G1/M8 genotypes are red, as shown in the dendrogram. In identifying and quan- tifying carotenoid compounds in chilli pepper fruits, the carotenoid content between several Capsicum cultivars is varied [26]. On the other hand, significant differences were found in mature fruits, especially in the HV-149 variety, which has yellow mature fruit. A clear difference in fruit color in the mature stage affected the relationship between those genotypes.
Identification of genetic variability based on RAPD Markers
Among the 19 RAPD primers used in this study, only 13 primers showed clear and unambig- uous polymorphic bands (Figure 5). A total of 49 alleles were formed from the RAPD amplification process. The band pattern from RAPD amplifica- tion could show informative genetic characters among the samples identified [31]. In this re- search, the amplification results revealed 63.2%
monomorphic bands and 36.7% polymorphic bands. Although this data did not show high ge- netic diversity, it clustered the G1/M8 mutant with the initial line, Cakra Hijau, and separated from the introduction line HV-149.
The RAPD primer that produced the highest number of polymorphic bands was the OPA-02 marker. OPA-02 marker has 60% polymorphism
(Table 3), with polymorphic bands formed at 1500, 1000 and 400 bp (Figure 5). The number of DNA amplification band results depended on the primer attached to the DNA template, noted by the presence or absence of an amplification product from a single locus [31]. The RAPD amplification results from the OPB-4 and OPA-02 primers in the HV-149 variety have the highest number of poly- morphic bands compared with other genotypes.
The differences in the polymorphism results may be caused by the genetic variation that exists among different genotypes. A high number of pol- ymorphism bands also represented great diversity in the plant DNA sample [32].
The dendrogram on the RAPD amplification results showed the same pattern as the dendrogram on the morphological data analysis (Figure 6).
Figure 4. Dendrogram based on morphological characteristics
Two major clusters formed: the first cluster con- sisted of the HV-149 variety, the second cluster consisted of the Cakra Hijau variety and the G1/M8 line with Jaccard's similarity coefficient value of 0.74. Genetic similarity indicates the
close relationship between the Cakra Hijau variety and the G1/M8 line genotypes. High genetic simi- larity may increase the possibility of a relationship between the genotypes. Meanwhile, small genetic similarities may decrease the possibility of a rela-
Figure 5. RAPD marker banding pattern of different Capsicum varieties on 2.5% agarose gel. M: DNA Ladder 100 bp; NTC: Non-Template Control; CH: Cakra Hijau Variety; GM: G1/M8 line; HV: HV-149 (Tai- wan Kuning) variety; OP: name of RAPD primers.
Table 3. PIC Values for RAPD Primers
Primer Name MB PB Total Number of Band %P PIC
OPD-13 3 2 5 40 0.17
OPB-11 2 1 3 33 0.14
OPL-05 3 1 4 25 0.11
OPB-03 2 1 3 33 0.14
OPB-04 3 1 4 25 0.11
OPA-17 3 1 4 25 0.11
OPU-10 3 3 6 50 0.22
OPD-19 1 1 2 50 0.22
OPU-19 2 1 3 33 0.148
OPW-03 2 1 3 33 0.148
OPA-11 2 2 4 50 0.22
OPA-01 3 0 3 0 0
OPA-02 2 3 5 60 0.267
Total 31 18 49
Mean 2.28 1.43 3.71 38
Note: MB: monomorphic Band; PB: Polymorphic Band; %P: Percent of Polymorphic bands; PIC: Polymorphic Information Content
tionship with the genotype [31]. Overall, the den- drogram result shows the tested genotypes grouped based on their fruit maturation color and where they were collected from. The HV-149 va- riety was collected from Taiwan, and the Cakra Hijau variety (C. frutescens L.) was domesticated in Indonesia [5]. The G1/M8 line is a mutant gen- otype from Cakra Hijau.
Every molecular marker has different effec- tiveness based on its ability to show polymor- phism. The effectiveness of molecular markers can be identified from the PIC value. Molecular markers with PIC values between 0 and 0.5 indi- cate good marker quality that can be used for ge- netic variation analysis between samples.
The RAPD primer used in this study has a PIC value between 0 and 0.5, indicating that the pri- mers are good for determining genetic variations in the tested genotype (Table 3). If the PIC value is high, allelic variation will be high. The highest PIC value of a primer allows the same primer to amplify different loci even in the same species [24].
Genetic diversity can be identified by the gen- otyping-clustering technique [32]. Clustering analysis based on morphological characteristics may not exactly represent the genetic relationship since the morphological characteristics might be altered by environmental conditions [33, 34]. Mo- lecular analysis is needed to validate the clustering analysis results [35].
The dendrogram results based on morphologi- cal characteristics and RAPD have different Jac- card's similarity coefficients. The dendrogram based on morphological characteristics has a sim- ilarity coefficient between 0.885 and 0.99, while RAPD has a similarity coefficient range between
0.7 and 0.95, which is lower. This indicates that the differences among genotypes based on molec- ular genetics are more diverse than their morpho- logical characteristics. The difference in Jaccard's similarity index is shown by a difference in the amount of co-occurring data (indicated as 1 in the data matrix and the null data in the matrix). This similarity coefficient describes the relatedness among the genotypes in the range of 0–1. The gen- otypes close to the 0 similarity coefficient were more dissimilar than the genotypes close to the one similarity coefficient [36]. Thus, this study proves that genetic similarity identification based on RAPD molecular marker amplification is more effective in chilli peppers than in the morphologi- cal characteristics identification approach. RAPD has been confirmed as a superior morphological diversity identification tool to observe the genetic variability in C. annuum L. varieties around the world [37]. However, an extensive future study is still needed to distinguish the Cakra Hijau variety and the G1/M8 genotype based on carotenoid gene expression to support the clustering result based on the morphological characteristics and RAPD molecular markers.
Analysis for genetic information based on the SSRs
This study used six pairs of SSR primers to de- termine the genetic diversity of the studied chilli peppers. SSR primer amplification results using the PCR method produced clear and specific bands, but the band pattern for each type of plant used in this study is the same (Figure 7). Bands formed from the amplification process ranged from 100 bp to 300 bp. Six SSR markers employed in this study were not effective in distinguishing Figure 7. SSR banding pattern of different Capsicum varieties. M: DNA Ladder 100 bp; NTC: Non Template
Control; CH: Cakra Hijau Variety; GM: G1/M8 line; HV: HV-149 (Taiwan Kuning) variety; CA:
name of SSR primer.
the tested genotypes.
The amplification results of the same molecu- lar markers may give different results when ap- plied to different plant samples. Other researchers have used similar SSR markers (Ca-19, Ca-26, Ca- 52 and Ca-96) for their study and were able to dif- ferentiate between a group of EMS-induced chilli mutant plants and their original types (Cakra Hijau variety) [14]. The SSR primer (Table 2) was de- signed based on the flanking region of the repeti- tive region of C. annuum L. and has been proven to be applicable for C. frutescens L. and C.
chinense Jaq, as well as suitable for the identifica- tion of genetic variations in chilli plants [38].
However, the data showing the existence of ge- nomic uniformity between the three genotypes based on SSR markers contradicted the RAPD re- sults that showed polymorphism between the three genotypes. This phenomenon may be due to the weakness of SSR markers, which sometimes mis- classify heterozygous alleles as homozygous al- leles. This error can cause SSR markers not to identify genetic variations between genotypes ac- curately. Compared with SSR, RAPD has a high ability to detect polymorphisms because it ampli- fies genomic DNA with short arbitrary primers, al- lowing it to produce many polymorphic bands [38].
Conclusion
The color of the mature chilli fruit was related to the RAPD profile of that variety. Varieties that have red fruits are grouped in the same cluster.
However, fruit color during the immature and ma- ture stages has no relationship with the anthocya- nin content. The anthocyanin content generally in- creases when the fruit ripens. In contrast to RAPD, the SSR marker in this study was unable to show any variations.
Acknowledgement
This research was funded by the Doctor and Professor Research Grant of Universitas Brawi- jaya, Indonesia.
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