# Molecular Characterization of Wild and Cultivated Strawberry (Fragaria × ananassa) through DNA Barcode Markers

Authors: Awais Qarni; Khushi Muhammad; Abdul Wahab; Amir Ali; Chandni Khizar; Izhar Ullah; Abeer Kazmi; Tahira Sultana; Asma Hameed; Muhammad Younas; Mehdi Rahimi
Journal: Genetics Research
 (2022)

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## Abstract

Background. DNA barcoding is a useful technique for the identification, conservation, and diversity estimation at the species level in plants. The current research work was carried out to characterize selected Fragaria species from northern Pakistan using DNA barcode markers. Methodology. Initially, the efficacy of eight DNA barcode markers was analyzed based on the amplification and sequencing of the genome of selected Fragaria species. The resultant sequences were analyzed using BLAST, MEGA 7.0, and Bio Edit software. The phylogenetic tree was constructed by using Fragaria current species sequences and reference sequences through the neighbor-joining method or maximum likelihood method. Results. Among eight DNA barcode markers, only two (ITS2 and rbclC) were amplified, and sequences were obtained. ITS2 sequence was BLAST in NCBI for related reference species which ranged from 89.79% to 90.05% along with Fragaria vesca (AF163517.1) which have 99.05% identity. Similarly, the rbclC sequence of Fragaria species was ranged from 96% to 99.58% along with Fragaria × ananassa (KY358226.1) which had 99.58% identity. Conclusion. It is recommended that DNA barcode markers are a useful tool to identify the genetic diversity of a species. Moreover, this study could be helpful for the identification of the Fragaria species cultivated in other regions of the world.


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## Body

## 1. Introduction

Fragaria is commonly known as wild-type strawberries, a genus of flowering plants in the Rosaceae family, including Potentilla and Duchesnea, which are closely related to Fragaria [1]. A total of seven chloroplast genomes of Fragaria species were completely sequenced [2]. Fragaria vesca whole genome has been sequenced and provides the first reference genome for Fragaria species and is established as the model system for Fragaria genome-related study [3].Wild strawberry is native to Bhutan, Afghanistan, China, Nepal, Sikkim, Myanmar, and Pakistan [4]. Cultivated strawberry is a well-liked fruit cultivated throughout the world and most economically important processed and fresh fruits, consumed for its pleasant flavor and nutrient content, and their global production reaches 9.1 M tones in 2016; annual growth has grown over 5% in the last decade [5].The medicinal claims of wild strawberries have been around for hundreds of years [6]. Fragaria nubicola plant juice is used in the treatment of profuse menstruation, tongue blemishes [7], stomach ulcers, as an antiseptic, healing of wounds, children's diarrhea, and urinary infections in different parts of Pakistan [8]. Tea is prepared from the roots and leaves of Fragaria nubicola and has antimicrobial and anti-inflammatory properties [4]. Traditionally, its rhizome is used to treat inflammation and contains ellagic acid, and various other glycosides, antioxidants, anti-inflammatory, anticancer agents, and anti-neurodegenerative properties are also present in Fragaria fruits [9].Fragaria species hold promising traits for selective breeding such as abiotic-stress tolerance and acclimation or disease and pest resistance [1]. Efforts to establish a phylogenetic relationship in Fragaria have been hampered by the low level of variability detected in the chloroplast DNA of Fragaria [10]. Evidence of wild and cultivated strawberries shows that flowering and running are genetically distinct processes but mutually exclusive because the predominance of one over the other is completely linked to genetic and environmental factors [11]. Numerous species with similar morphological traits are misused as official drugs, which leads to substitution and adulteration causing issues with product safety and quality control [12].DNA barcoding is an effective, rapid, inexpensive, and standard method for assessing and identifying various plant species [13]. In addition, this method can effectively identify unknown species or species having complex morphometric behavior [14]. This method is also used to study both interspecific and intraspecific variations. DNA barcoding was used to identify some of metazoan species by the sequences of cytochrome oxidase 1 (CO1) genes. DNA barcode regions, such as rbcLc, matK, ITS2, rpoB, rpoC, rbcLc, Ycf3, ITS2, and trnH-psbA, were used to identify flowering species [15]. According to CBOL (Consortium for the Barcode Life), the cpDNA regions (rbclC andITS) play a crucial role in the rapid identification of plant species.To the best of our knowledge to date, no study has been conducted to assess the genetic diversity among wild and cultivated strawberries. Similarly, no barcoding study is reported forFragaria nubicola Lindl. The objectives of the current study are to estimate the genetic diversity of wild and cultivated Fragaria through the molecular marker and to find out suitable DNA markers linking to wild and cultivated Fragaria species from Mansehra, Pakistan.


## 2. Method and Material

### 2.1. Study Area and Spatial Mapping

The genus (Fragaria) species were collected from different areas of district Mansehra, Pakistan, namely, Jabori, Garhi Habibullah, Baffa, Kund Bangla, Shogran, Battal, Balakot, Dodial, Chattar, and Qalandarabad (Figure 1). Sampling areas were recorded through the help of a GPS device, from where plants were collected; at the sampling time, plant location, attitude, and elevation were recorded from different areas (Table 1).Figure 1
Map of district Mansehra from where genus (Fragaria) species were collected and the areas of Mansehra included Jabori, Garhi Habibullah, Baffa, Kund Bangla, Shogran, Batal, Balakot, Dodial, Chattar, and Qalandarabad.Table 1
GPS location of different regions of Mansehra, Pakistan.
S. noLocationsLatitude (N)Longitude (E)Elevation (meter)1Jabori34.601073.764517352Garhi Habibullah34.401273.467817903Battal34.687073.745615184Baffa34.503573.100112075Shogran34.606373.850428706Balakot34.518973.634519857Qalandarabad34.302273.353411938Kund Bangla34.301873.215829759Dodial34.100473.1972112010Chattar34.423273.68732071


### 2.2. Plant Collection

Plant samples were collected with the help of taxonomists. The respective samples were collected in April, May, and June 2020 in triplicate from each collection area. Suitable zipper plastic bags were used for sample collection to save it for a long duration and the best result. The plant materials were transferred to the laboratory of the genetic department, Hazara University, Mansehra, before DNA extraction. Herbarium sheets of samples were kept at room temperature in the laboratory.


### 2.3. Storage of Plant Material

The plant samples were properly dried at room temperature when plants samples were completely dried and crushed with the help of mortar and pestle. The standard scientific protocol was used for the labeling of plant tissue and then pasted on the herbarium sheet and submitted for registration.


### 2.4. Extraction of Genomic DNA

The genomic DNA was extracted from several selected plants of every position by using the modified CTAB method [16]. CTAB buffer was heated before starting the extraction of genomic DNA. We added 800 μl, heated CTAB buffer (2% CTAB + tris buffer 100 ml + 1.6 g EDTA + 16.4 g NaCl+ 0.3%BME +2.4 g PVP+ 100 ml water) in each tube, and incubated at 56°C for day and night. Then, 500 μl PCI (phenol chloroform iso-amyl alcohol at a ratio of 24:1) was added and centrifuged for 20 minutes at 8,000 rpm. The upper layer (supernatant) was transferred to new fresh labeled tubes. Then, we added 500 μl cold iso-propanol (C3H8O) and mixed well until DNA precipitated. Tubes were spun for 15 minutes at 8,000 rpm, and the supernatant was discarded. The pellet was washed by adding 70% ethanol into each tube and centrifuged for 5 minutes at 10,000 rpm. The ethanol was discarded from tubes, and the tubes were kept at room temperature for drying. Then, we added 60 μl ddH2O in each tube to dilute genomic DNA.


### 2.5. Selection of DNA Barcode Markers

Different DNA barcode markers were used to amplify the desired fragment in the genome of selectedFragaria species DNA and checked the different barcode markers by using a specific sequence of DNA, namely, ITS2, matK, rbcL, ITS, rbclA, rbclC, Ycf3, and trnV (Table 2). The barcode markers were selected on the basis of previous literature related to Fragaria species [24–26].Table 2
Conditions of the selected markers and their sequences used in the current molecular study.
Sr.NoPrimersPrimer nameSequenceMelting temperature (°C)References1ITS2-FITS2-RITS-S2FITS-S3R5′ATGCGATACTTGGTGTGAAT 3′5′GACGCTTCTCCAGACTACAAT 3′52[17]2ITS-FITS-RITS-FITS-R5′GCCGTTAAGACCAGGGAT 3′5′TGATTCACGGGATTCTGC 3′50[18]3rbcl-FrbcL-R1f724r5′ATGTCACCACAAACAGAAAC3′5′TCGCATGTACCTGCAGTAGC 3′56[19]4rbclc-FrbCIC-RHRM_rbcLCFHRM_rbcLCR5′TAGACCTTTTTGAAGAAGGTTCTGT3′5′TGAGGCGGRCCTTGGAAAGTT 3′76[20]5rbclA-FrbclA-Ra-fa-r5′ATGTCACCACAAACAGAGACTAAAGC 3′5′CTTCTGCTACAAATAAGAATCGATCTC 3′63[21]6matK-FMatK-R2.1a forward3.2 reverse5′ATCCATCTGGAAATCTTAGTTC 3′5′ CTTCCTCTGTAAAGAATTC 3′51[22]7trnV-FtrnV-RtrnV_UAC-E1FtrnV_UAC-E1R5′ GTAGAGCACCTCGTTTACAC 3′5′GTGTAAACGAGGTGCTCTAC 3′56[23]8ycf3-Fycf3-Rycf3-E2Fycf3-E2R5′GGCCGTGATCTGTCATTAC3′5′TTCCGCGTAATTTCCTTC3′53.2[23]


### 2.6. Gel Electrophoresis

The quality of isolated genomic DNA ofFragaria species was examined and checked through 1% agarose gel electrophoresis.100 ml of distilled water was taken in the flask, and we dissolved 1 gram of agarose powder and added 2000 μl 50XTAE buffer and shook it well. We put the flask in the microwave for 1 minute and then kept the flask at room temperature to solidify. We added 25 μl of ethidium bromide before solidifying the solution of agarose gel. Then, we transferred the prepared solution to gel tray and put combs for production of wells. The combs were removed after solidification of gel, and then gel was transferred to a gel tank. Gel tank had 600 ml of 1XTAE buffer. 5 μl of genomic DNA and 2 μl of dye were loaded in each well of gel. Run the gel for around 30 minutes at a voltage of 80 volts. Then, gel was run on UV lights to check the quality of genomic DNA and for documentation of the gel image.


### 2.7. PCR Reaction Reagents

The amplification of targeted DNA barcodes in the genome of selectedFragaria species was amplified using (Thermo Fisher Scientific, #F-548S) and followed the manufacture protocol. To obtain the target DNA fragment, the prepared PCR reaction mixture was placed in the ABI thermocycler, and the standard optimized program was adjusted in the mixture. The amplified PCR products were further confirmed by 1.5% agarose gel; gel photographs were taken in the UV apparatus (Table 3).Table 3
Reagents of PCR and their volume.
Sr. NoReagentsQuantity (μl)110 × Taq buffer2.52MgCl22.53DNTPs3.04Primer (F & R)2.05Taq DNA Polymarase0.56Templet DNA2.57ddH2O12Total Volume25


### 2.8. Sequencing and Phylogenetic Analysis

The obtained PCR results were correctly labeled for commercial sequencing and sent to BGI (Beijing Genomic Institute) for nucleotide sequencing. Later, the successful sequences were further analyzed by using BLAST (Basic Local Alignment Search Tool) at NCBI (database), Bio Edit, and MEGA 7.0 software for the construction of the phylogenetic tree. The phylogenetic trees were constructed of the investigated sequences through different methods (viz. maximum likelihood (ML), neighbor-joining (NJ), and maximum parsimony (MP) methods).


## 2.1. Study Area and Spatial Mapping

The genus (Fragaria) species were collected from different areas of district Mansehra, Pakistan, namely, Jabori, Garhi Habibullah, Baffa, Kund Bangla, Shogran, Battal, Balakot, Dodial, Chattar, and Qalandarabad (Figure 1). Sampling areas were recorded through the help of a GPS device, from where plants were collected; at the sampling time, plant location, attitude, and elevation were recorded from different areas (Table 1).Figure 1
Map of district Mansehra from where genus (Fragaria) species were collected and the areas of Mansehra included Jabori, Garhi Habibullah, Baffa, Kund Bangla, Shogran, Batal, Balakot, Dodial, Chattar, and Qalandarabad.Table 1
GPS location of different regions of Mansehra, Pakistan.
S. noLocationsLatitude (N)Longitude (E)Elevation (meter)1Jabori34.601073.764517352Garhi Habibullah34.401273.467817903Battal34.687073.745615184Baffa34.503573.100112075Shogran34.606373.850428706Balakot34.518973.634519857Qalandarabad34.302273.353411938Kund Bangla34.301873.215829759Dodial34.100473.1972112010Chattar34.423273.68732071


## 2.2. Plant Collection

Plant samples were collected with the help of taxonomists. The respective samples were collected in April, May, and June 2020 in triplicate from each collection area. Suitable zipper plastic bags were used for sample collection to save it for a long duration and the best result. The plant materials were transferred to the laboratory of the genetic department, Hazara University, Mansehra, before DNA extraction. Herbarium sheets of samples were kept at room temperature in the laboratory.


## 2.3. Storage of Plant Material

The plant samples were properly dried at room temperature when plants samples were completely dried and crushed with the help of mortar and pestle. The standard scientific protocol was used for the labeling of plant tissue and then pasted on the herbarium sheet and submitted for registration.


## 2.4. Extraction of Genomic DNA

The genomic DNA was extracted from several selected plants of every position by using the modified CTAB method [16]. CTAB buffer was heated before starting the extraction of genomic DNA. We added 800 μl, heated CTAB buffer (2% CTAB + tris buffer 100 ml + 1.6 g EDTA + 16.4 g NaCl+ 0.3%BME +2.4 g PVP+ 100 ml water) in each tube, and incubated at 56°C for day and night. Then, 500 μl PCI (phenol chloroform iso-amyl alcohol at a ratio of 24:1) was added and centrifuged for 20 minutes at 8,000 rpm. The upper layer (supernatant) was transferred to new fresh labeled tubes. Then, we added 500 μl cold iso-propanol (C3H8O) and mixed well until DNA precipitated. Tubes were spun for 15 minutes at 8,000 rpm, and the supernatant was discarded. The pellet was washed by adding 70% ethanol into each tube and centrifuged for 5 minutes at 10,000 rpm. The ethanol was discarded from tubes, and the tubes were kept at room temperature for drying. Then, we added 60 μl ddH2O in each tube to dilute genomic DNA.


## 2.5. Selection of DNA Barcode Markers

Different DNA barcode markers were used to amplify the desired fragment in the genome of selectedFragaria species DNA and checked the different barcode markers by using a specific sequence of DNA, namely, ITS2, matK, rbcL, ITS, rbclA, rbclC, Ycf3, and trnV (Table 2). The barcode markers were selected on the basis of previous literature related to Fragaria species [24–26].Table 2
Conditions of the selected markers and their sequences used in the current molecular study.
Sr.NoPrimersPrimer nameSequenceMelting temperature (°C)References1ITS2-FITS2-RITS-S2FITS-S3R5′ATGCGATACTTGGTGTGAAT 3′5′GACGCTTCTCCAGACTACAAT 3′52[17]2ITS-FITS-RITS-FITS-R5′GCCGTTAAGACCAGGGAT 3′5′TGATTCACGGGATTCTGC 3′50[18]3rbcl-FrbcL-R1f724r5′ATGTCACCACAAACAGAAAC3′5′TCGCATGTACCTGCAGTAGC 3′56[19]4rbclc-FrbCIC-RHRM_rbcLCFHRM_rbcLCR5′TAGACCTTTTTGAAGAAGGTTCTGT3′5′TGAGGCGGRCCTTGGAAAGTT 3′76[20]5rbclA-FrbclA-Ra-fa-r5′ATGTCACCACAAACAGAGACTAAAGC 3′5′CTTCTGCTACAAATAAGAATCGATCTC 3′63[21]6matK-FMatK-R2.1a forward3.2 reverse5′ATCCATCTGGAAATCTTAGTTC 3′5′ CTTCCTCTGTAAAGAATTC 3′51[22]7trnV-FtrnV-RtrnV_UAC-E1FtrnV_UAC-E1R5′ GTAGAGCACCTCGTTTACAC 3′5′GTGTAAACGAGGTGCTCTAC 3′56[23]8ycf3-Fycf3-Rycf3-E2Fycf3-E2R5′GGCCGTGATCTGTCATTAC3′5′TTCCGCGTAATTTCCTTC3′53.2[23]


## 2.6. Gel Electrophoresis

The quality of isolated genomic DNA ofFragaria species was examined and checked through 1% agarose gel electrophoresis.100 ml of distilled water was taken in the flask, and we dissolved 1 gram of agarose powder and added 2000 μl 50XTAE buffer and shook it well. We put the flask in the microwave for 1 minute and then kept the flask at room temperature to solidify. We added 25 μl of ethidium bromide before solidifying the solution of agarose gel. Then, we transferred the prepared solution to gel tray and put combs for production of wells. The combs were removed after solidification of gel, and then gel was transferred to a gel tank. Gel tank had 600 ml of 1XTAE buffer. 5 μl of genomic DNA and 2 μl of dye were loaded in each well of gel. Run the gel for around 30 minutes at a voltage of 80 volts. Then, gel was run on UV lights to check the quality of genomic DNA and for documentation of the gel image.


## 2.7. PCR Reaction Reagents

The amplification of targeted DNA barcodes in the genome of selectedFragaria species was amplified using (Thermo Fisher Scientific, #F-548S) and followed the manufacture protocol. To obtain the target DNA fragment, the prepared PCR reaction mixture was placed in the ABI thermocycler, and the standard optimized program was adjusted in the mixture. The amplified PCR products were further confirmed by 1.5% agarose gel; gel photographs were taken in the UV apparatus (Table 3).Table 3
Reagents of PCR and their volume.
Sr. NoReagentsQuantity (μl)110 × Taq buffer2.52MgCl22.53DNTPs3.04Primer (F & R)2.05Taq DNA Polymarase0.56Templet DNA2.57ddH2O12Total Volume25


## 2.8. Sequencing and Phylogenetic Analysis

The obtained PCR results were correctly labeled for commercial sequencing and sent to BGI (Beijing Genomic Institute) for nucleotide sequencing. Later, the successful sequences were further analyzed by using BLAST (Basic Local Alignment Search Tool) at NCBI (database), Bio Edit, and MEGA 7.0 software for the construction of the phylogenetic tree. The phylogenetic trees were constructed of the investigated sequences through different methods (viz. maximum likelihood (ML), neighbor-joining (NJ), and maximum parsimony (MP) methods).


## 3. Results and Discussion

The Rosaceae family is a diverse and widely spread family of flowering plants, consisting of 91 genera and around 3000 species. Members of the Rosaceae family are perennial, herbs, and shrubs and native to northern temperate and tropical countries [27]. The biologically important plant species must be identified and evaluated, which plays an important role in understanding the evolutionary history of plant species.This study shows that DNA barcoding is an efficient tool for species identification for many taxonomic groups of vascular plants. DNA barcoding is a better technique for distinguishing species. The DNA barcoding technique offers the way to study the phylogenetic relationship and genetic diversity between species populations based on short conserved nucleotide sequences of the genome [28, 29]. An expert taxonomist can solve the problem by using the DNA barcoding technique when the known species do not closely match the unknown species. In this study, the DNA barcoding markers such as ITS2, matK, rbcL, ITS, rbclA, rbclC, Ycf3, and trnV were applied against the high quality of Fragaria species DNA. Out of these markers, only two markers (ITS2 and rbclC) were successfully amplified, and sequences were obtained from BGI (Beijing Genomic Institute) China (Figures 2 and 3). The ITS2 marker showed 64% suitability, and rbclC showed 28.57% suitability against investigated Fragaria species.Figure 2
Alignment ofFragaria species of the ITS2 marker.Figure 3
Alignment ofFragaria species of the rbclC marker.The large scale phylogenetic exploration ofFragaria (strawberry) species was analyzed through 454 sequencing platforms and the Fluidigm access array method and then found the discrepancy among two wild octoploid species, i.e., F. chiloensis and F. virginiana [30]. In the current study, the genomic DNA of selected samples was extracted, and amplification of the targeted barcode region in the genome of selected Fragaria species was carried out through various PCR conditions. The amplified PCR products of the selected Fragaria species of the ITS2 marker can be seen in Figure 4. In a previous study, Potter et al. reported that on the ITS marker, Fragaria nubicola (AF163517) showed similarity with Fragaria vesca (AF163485), and Fragaria × ananassa (AF163494) showed similarity with Fragaria virginiana (AF163479). On the trnL marker, Fragaria nubicola (AF163562) showed similarity with Fragaria vesca (AF163542) and Fragaria × ananassa (AF163538) [31]. While in the current study, ITS and trnL markers were found unsuitable to determine diversity. Similarly, in a previous report, Fragaria × ananassa (JX117907) showed similarity with Fragaria chiloensis (JX402801) on the ycf3 marker through the maximum likelihood method [32], but no results were obtained by using the ycf3 marker in the current study. Potter et al. reported that Fragaria vesca (AF288102) showed similarity with potentilla anserine (AF2881113) through maximum parsimony on the matK marker [33], while no results were obtained on the matK marker as well in the current study.Figure 4
Amplified PCR products of selectedFragaria species of the ITS2 marker and 1 kb DNA ladder.


### 3.1. Molecular Phylogenetic Analysis ofFragaria by the ITS2 Marker

DNA barcoding, which utilizes the ribosomal DNA ITS2 region as a tag to identify species, has gained a lot of attention recently [34]. ITS2 has several benefits including strong universality, low intraspecific variance but significant interspecific divergence, and a short fragment length (200 bp) over other suggested DNA barcodes such as psbA-trnH, matK, rbcL, and ITS [35–37]. DNA barcoding technology is used to identify the known as well as unknown species of berry fruit products. It provides much accurate information regarding species which is to be recognized [38]. In a recent study, approximately 37 species of F. nilgerrensis belonging to five diverse genetic groups were identified through molecular markers which further helped in identification, classification, and evaluation of its whole genetic pool within germplasm [39]. To the best of our knowledge, this is the first time that the ITS2 regions have been utilized to identify Fragaria species.The molecular phylogenetic analysis ofFragaria of the ITS2 marker was conducted through different methods. The phylogenetic tree was constructed using the bootstrap mode among wild Fragaria and cultivated Fragaria species through three different methods. The tree was distributed into 3 clades. ITS2 sequence was BLAST in NCBI (database) for related reference species which ranges from 89.79% to 99.05% along with Fragaria vesca (AF163517.1) which have 99.05% identity and 1e.517 E value and 69% query coverage. In all phylogenetic trees, the wild-type Fragaria nubicola Lindl from Jabori, KundBangla, Battal and Fragaria vesca from Balakot were together in clad 1. Cultivated Fragaria × ananassa Duch from Dodial, Qalandarabab, and Baffa was placed in clad 2. In the 3rd clad, Potentilla indica from Shogran and Garhi Habibullah was positioned together with 0.048 branch length (Figures 5–7). The phylogenetic evolution of ten wild species of Fragaria was identified through complete chloroplast genome sequencing by clustering Fragaria species into two clades and explored their common ancestor and divergent species among them [40].Figure 5
Molecular phylogenetic tree of wild and cultivated species ofFragaria with the ITS2 marker through the maximum likelihood method.Figure 6
Phylogenetic tree among wild and cultivatedFragaria species of the ITS2 marker through the neighbor-joining method.Figure 7
ITS2-based phylogenetic tree among wild and cultivated Fragaria species through the maximum parsimony method.Phylogenetic analysis ofFragaria species through the neighbor-joining method revealed that one clad was formed in each linkage tree. In clad 1, Fragaria × ananassa from Battal, Dodial, and Baffa was together while Potentilla indica from Shogran showed diversity but due to low bootstrap value and branch length (0.022), and it is also placed in the similar clad (Figures 8 and 9). On the parsimony method, there are no parsimony informative sites. The sequences were got in the FASTA format and used for alignment through MEGA 7 (Cluster W) software. The selected Fragaria sequences and reference sequences were aligned and trimmed by removing the irregular sequences and then used for molecular phylogenetic analysis (Figure 10).Figure 8
Phylogenetic tree of cultivatedFragaria species sequences of (rbclC) through the maximum likelihood method.Figure 9
Phylogenetic tree of currentFragaria species sequences of rbclC through the neighbor-joining method.Figure 10
Alignment ofFragaria species sequences by the ITS2 marker with related reference species.Similarly, for phylogenetic analysis ofFragaria species with related reference species, the phylogenetic tree was constructed through the maximum likelihood method by using the bootstrap mode and the highest log-likelihood which are -630.3100. In clad 1, Fragaria × ananassa Duch from Dodial, Qalandarabad, and Baffa and the reference species (KT695219.1, AF163498.1, JN999221.1, MG235200.1, and MN601853.1) were together. Fragaria nubicola Lindl from Jabori, Battal, KundBangla, Fragaria vesca from Balakot, Fragaria nubicola (AF163517.1), Fragaria vesca (KX166972.1, MG235113.1), Fragaria moschata (AF163520.1), Fragaria orientalis (MH711106.1), Fragaria pentaphylla (AF163500.1), and Fragaria nipponica (KF873772.1) were together in clad 2. The Potentilla indica from Shogran and Garhi Habibullah were together in clad 3, while Drymocallis arguta (MG236177.1) was placed out of the group (Figures 11 and 12).Figure 11
Phylogenetic tree ofFragaria species of (ITS2) with related reference Fragaria species through the maximum likelihood method.Figure 12
Phylogenetic tree of selectedFragaria species (ITS2) with related reference Fragaria species through the neighbor-joining method.Phylogenetic tree analysis was also conducted through the maximum parsimony method by using a bootstrap mode; consistency index recorded was 0.921958 for all sites. In the phylogenetic tree,Potentilla indica from Shogran and Garhi Habibullah, Fragaria nubicola from Jabori, KundBangla, and Battal, Fragaria vesca from Balakot, reference species MH711106.1, KF873772.1, AF163500.1, AF163517.1, and MG236177.1 were placed together in clad 1. Fragaria × ananassa from Qalanderabad and Baffa and other reference species such as KX166972.1, MG235113.1, AF163520.1, MG235200.1, JN999221.1, AF163494.1, AF163498.1, MN601853.1, and KT695219.1 were together in clad 2, while Fragaria × ananassa Duch from Dodial was placed out of the group as it showed diversity (Figure 13).Figure 13
Phylogenetic tree of selectedFragaria sequences of the ITS2 marker with reference sequences by using the maximum parsimony method.


### 3.2. Molecular Phylogenetic Analysis ofFragaria by Using the rbclC Marker

TherbclC sequence of Fragaria species was BLAST in NCBI (database) with related reference species and prepared in the FASTA format. According to the results, alignments ranged from 96% to 99.58% along with Fragaria × ananassa Duch (KY358226.1) which has 99.58% identity and 0.0 E-values with 95% query coverage. The sequences of selected Fragaria species and related reference sequences were aligned and trimmed by the removal of irregular nucleotide sequence than used for phylogenetic analysis (Figure 14).Figure 14
Basic local alignment of the selectedFragaria species (rbclC) marker with related reference sequences.The phylogenetic tree constructed through the maximum likelihood method by using the bootstrap mode had a branch length of -1363.6855. TheFragaria × ananassa from Dodial, Baffa, and Batal and reference species JX118090.1, JX118093.1, JX18097.1, and JX118099.1 were found together in clad 1. Fragaria chilonosis (JX118100.1) and Fragaria daltoniana (JX118103.1) were placed in cladding 2, while Potentilla reptans (HM850287.1) and Potentilla erecta (KF602205.1) from Shogran were placed in clad 3 (Figure 15).Figure 15
Phylogenetic tree analysis ofFragaria species (rbclC) with reference species through using the maximum likelihood method.The phylogenetic tree constructed with the neighbor-joining method had 0.07867073 branch lengths.Fragaria × ananassa Duch from Dodial, Baffa, and Battal and reference species JX228090.1, JX118093.1, JX118094.1, and JX118097.1 were placed together in clad 1.Fragaria nipponica (JX11099.1), Fragaria chilonesis (JX118100.1), and Fragaria daltoniana (JX118103.1) were placed in clad 2, whereas Potentilla indicia, Potentilla reptans (HM850287.1), and Potentilla erecta (KF602205.1) collected from Shogran were placed in clad 3 (Figure 16).Figure 16
Phylogenetic tree of selectedFragaria (rbclC) species with related reference sequences through the neighbor-joining method.The phylogenetic tree constructed by applying the maximum parsimony method contained a consistency index of 0.752598 for all sites. In the phylogenetic tree,Potentilla indica from Shogran, Potentilla erecta (KF602205.1), Potentilla reptans (HM850287.1), Fragaria chilonesis (JX118100.1), and Fragaria daltoniana (JX118103.1) are the part of clad 1. The Fragaria × ananassa Duch from Battal and reference species JX118099.1, JX118093.1, JX118097.1, and JX118090.1 were placed in clad 2. The Fragaria × ananassa from Baffa and Fragaria virginiana (JX118094.1) were placed in clad 3, while Fragaria × ananassa collected from Dodial showed diversity and therefore placed out of the group (Figure 17).Figure 17
Phylogenetic tree analysis of theFragaria species (rbclC) marker with related reference species through the maximum parsimony method.


## 3.1. Molecular Phylogenetic Analysis ofFragaria by the ITS2 Marker

DNA barcoding, which utilizes the ribosomal DNA ITS2 region as a tag to identify species, has gained a lot of attention recently [34]. ITS2 has several benefits including strong universality, low intraspecific variance but significant interspecific divergence, and a short fragment length (200 bp) over other suggested DNA barcodes such as psbA-trnH, matK, rbcL, and ITS [35–37]. DNA barcoding technology is used to identify the known as well as unknown species of berry fruit products. It provides much accurate information regarding species which is to be recognized [38]. In a recent study, approximately 37 species of F. nilgerrensis belonging to five diverse genetic groups were identified through molecular markers which further helped in identification, classification, and evaluation of its whole genetic pool within germplasm [39]. To the best of our knowledge, this is the first time that the ITS2 regions have been utilized to identify Fragaria species.The molecular phylogenetic analysis ofFragaria of the ITS2 marker was conducted through different methods. The phylogenetic tree was constructed using the bootstrap mode among wild Fragaria and cultivated Fragaria species through three different methods. The tree was distributed into 3 clades. ITS2 sequence was BLAST in NCBI (database) for related reference species which ranges from 89.79% to 99.05% along with Fragaria vesca (AF163517.1) which have 99.05% identity and 1e.517 E value and 69% query coverage. In all phylogenetic trees, the wild-type Fragaria nubicola Lindl from Jabori, KundBangla, Battal and Fragaria vesca from Balakot were together in clad 1. Cultivated Fragaria × ananassa Duch from Dodial, Qalandarabab, and Baffa was placed in clad 2. In the 3rd clad, Potentilla indica from Shogran and Garhi Habibullah was positioned together with 0.048 branch length (Figures 5–7). The phylogenetic evolution of ten wild species of Fragaria was identified through complete chloroplast genome sequencing by clustering Fragaria species into two clades and explored their common ancestor and divergent species among them [40].Figure 5
Molecular phylogenetic tree of wild and cultivated species ofFragaria with the ITS2 marker through the maximum likelihood method.Figure 6
Phylogenetic tree among wild and cultivatedFragaria species of the ITS2 marker through the neighbor-joining method.Figure 7
ITS2-based phylogenetic tree among wild and cultivated Fragaria species through the maximum parsimony method.Phylogenetic analysis ofFragaria species through the neighbor-joining method revealed that one clad was formed in each linkage tree. In clad 1, Fragaria × ananassa from Battal, Dodial, and Baffa was together while Potentilla indica from Shogran showed diversity but due to low bootstrap value and branch length (0.022), and it is also placed in the similar clad (Figures 8 and 9). On the parsimony method, there are no parsimony informative sites. The sequences were got in the FASTA format and used for alignment through MEGA 7 (Cluster W) software. The selected Fragaria sequences and reference sequences were aligned and trimmed by removing the irregular sequences and then used for molecular phylogenetic analysis (Figure 10).Figure 8
Phylogenetic tree of cultivatedFragaria species sequences of (rbclC) through the maximum likelihood method.Figure 9
Phylogenetic tree of currentFragaria species sequences of rbclC through the neighbor-joining method.Figure 10
Alignment ofFragaria species sequences by the ITS2 marker with related reference species.Similarly, for phylogenetic analysis ofFragaria species with related reference species, the phylogenetic tree was constructed through the maximum likelihood method by using the bootstrap mode and the highest log-likelihood which are -630.3100. In clad 1, Fragaria × ananassa Duch from Dodial, Qalandarabad, and Baffa and the reference species (KT695219.1, AF163498.1, JN999221.1, MG235200.1, and MN601853.1) were together. Fragaria nubicola Lindl from Jabori, Battal, KundBangla, Fragaria vesca from Balakot, Fragaria nubicola (AF163517.1), Fragaria vesca (KX166972.1, MG235113.1), Fragaria moschata (AF163520.1), Fragaria orientalis (MH711106.1), Fragaria pentaphylla (AF163500.1), and Fragaria nipponica (KF873772.1) were together in clad 2. The Potentilla indica from Shogran and Garhi Habibullah were together in clad 3, while Drymocallis arguta (MG236177.1) was placed out of the group (Figures 11 and 12).Figure 11
Phylogenetic tree ofFragaria species of (ITS2) with related reference Fragaria species through the maximum likelihood method.Figure 12
Phylogenetic tree of selectedFragaria species (ITS2) with related reference Fragaria species through the neighbor-joining method.Phylogenetic tree analysis was also conducted through the maximum parsimony method by using a bootstrap mode; consistency index recorded was 0.921958 for all sites. In the phylogenetic tree,Potentilla indica from Shogran and Garhi Habibullah, Fragaria nubicola from Jabori, KundBangla, and Battal, Fragaria vesca from Balakot, reference species MH711106.1, KF873772.1, AF163500.1, AF163517.1, and MG236177.1 were placed together in clad 1. Fragaria × ananassa from Qalanderabad and Baffa and other reference species such as KX166972.1, MG235113.1, AF163520.1, MG235200.1, JN999221.1, AF163494.1, AF163498.1, MN601853.1, and KT695219.1 were together in clad 2, while Fragaria × ananassa Duch from Dodial was placed out of the group as it showed diversity (Figure 13).Figure 13
Phylogenetic tree of selectedFragaria sequences of the ITS2 marker with reference sequences by using the maximum parsimony method.


## 3.2. Molecular Phylogenetic Analysis ofFragaria by Using the rbclC Marker

TherbclC sequence of Fragaria species was BLAST in NCBI (database) with related reference species and prepared in the FASTA format. According to the results, alignments ranged from 96% to 99.58% along with Fragaria × ananassa Duch (KY358226.1) which has 99.58% identity and 0.0 E-values with 95% query coverage. The sequences of selected Fragaria species and related reference sequences were aligned and trimmed by the removal of irregular nucleotide sequence than used for phylogenetic analysis (Figure 14).Figure 14
Basic local alignment of the selectedFragaria species (rbclC) marker with related reference sequences.The phylogenetic tree constructed through the maximum likelihood method by using the bootstrap mode had a branch length of -1363.6855. TheFragaria × ananassa from Dodial, Baffa, and Batal and reference species JX118090.1, JX118093.1, JX18097.1, and JX118099.1 were found together in clad 1. Fragaria chilonosis (JX118100.1) and Fragaria daltoniana (JX118103.1) were placed in cladding 2, while Potentilla reptans (HM850287.1) and Potentilla erecta (KF602205.1) from Shogran were placed in clad 3 (Figure 15).Figure 15
Phylogenetic tree analysis ofFragaria species (rbclC) with reference species through using the maximum likelihood method.The phylogenetic tree constructed with the neighbor-joining method had 0.07867073 branch lengths.Fragaria × ananassa Duch from Dodial, Baffa, and Battal and reference species JX228090.1, JX118093.1, JX118094.1, and JX118097.1 were placed together in clad 1.Fragaria nipponica (JX11099.1), Fragaria chilonesis (JX118100.1), and Fragaria daltoniana (JX118103.1) were placed in clad 2, whereas Potentilla indicia, Potentilla reptans (HM850287.1), and Potentilla erecta (KF602205.1) collected from Shogran were placed in clad 3 (Figure 16).Figure 16
Phylogenetic tree of selectedFragaria (rbclC) species with related reference sequences through the neighbor-joining method.The phylogenetic tree constructed by applying the maximum parsimony method contained a consistency index of 0.752598 for all sites. In the phylogenetic tree,Potentilla indica from Shogran, Potentilla erecta (KF602205.1), Potentilla reptans (HM850287.1), Fragaria chilonesis (JX118100.1), and Fragaria daltoniana (JX118103.1) are the part of clad 1. The Fragaria × ananassa Duch from Battal and reference species JX118099.1, JX118093.1, JX118097.1, and JX118090.1 were placed in clad 2. The Fragaria × ananassa from Baffa and Fragaria virginiana (JX118094.1) were placed in clad 3, while Fragaria × ananassa collected from Dodial showed diversity and therefore placed out of the group (Figure 17).Figure 17
Phylogenetic tree analysis of theFragaria species (rbclC) marker with related reference species through the maximum parsimony method.


## 4. Conclusion and Recommendation

The result of this study showed that DNA barcoding is a useful technique for the identification and investigation of unknown species. Initially, we studied eight barcode markers among these two DNA barcode markers (ITS2, rbclC) which were successfully amplified and showed the significant result. The genetic diversity of selected Fragaria species showed similarities with related reference species. Furthermore, Potentilla indica from Shogran showed diversity which is the wild relative of Fragaria strawberry.To fully understand genetic diversity, the following recommendations can be considered:(i)
More DNA markers should be used to understand the genetic diversity of Fragaria species(ii)
DNA barcoding markers would be used against different populations for the identification of specific locus and genetic diversity within and between populations



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*Source: 9249561-2022-10-11.xml*