Nova Biotechnol Chim (2021) 20(2): e877 DOI: 10.36547/nbc.877 1 Nova Biotechnologica et Chimica Sequence analysis of chloroplast psbA-trnH region in Citrus L. (Rutaceae) species from the Aegean region of Turkey Emre Sevindik1, , Gaye Zeynep Canbolat1, İlayda İrem Moral1, Monika Sujka2 1Department of Agricultural Biotechnology, Faculty of Agriculture, Adnan Menderes University, South Campus, Cakmar, Aydin, Turkey 2Deptartment of Analysis and Food Quality Assessment, University of Life Sciences in Lublin, 8 Skromna St., Lublin 20-704, Poland  Corresponding author: ph.d-emre@hotmail.com Article info Article history: Received: 22nd February 2021 Accepted: 6th October 2021 Keywords: Citrus cpDNA psbA-trnH sequence analysis Abstract The aim of the study was to analyze the sequence of some citrus species found in the Aegean Sea region of Turkey based on the psbA-trnH cpDNA region. Genomic DNA was isolated from healthy and green leaves. Total genomic DNA was extracted using the GeneMark DNA isolation Plant Kit. The psbA-trnH region of chloroplast DNA was amplified using primers psbA and trnH. DNA sequences were edited using the Sequencher 5.4.6 and sequencing data were analyzed using the MEGA 6.0 software. The maximum likelihood (ML) tree was created to determine the relationships between Citrus taxa. Amplified cpDNA psbA-trnH sequences ranged between 426 and 470 nucleotides. The maximum likelihood phylogenetic tree was composed of two clades. The divergence values differed between 0.000 and 0.012. In addition, the sequences of some species belonging to the Rutaceae family were obtained from NCBI and a maximum likelihood tree was constructed. The phylogenetic relationship between the sequence data of some species belonging to the Rutaceae family taken from NCBI and Citrus species was revealed. © University of SS. Cyril and Methodius in Trnava Introduction Rutaceae Juss. is a large tree, bush and grass family comprising around 150 – 170 genera and nearly 2.040 species (Morton and Telmer 2014). The genus Citrus L. belongs to the Aurantioideae subfamily of the Rutaceae family. Citrus includes economically and commercially important fruit crops, major fruit plants of the world such as limons, mandarins, sour oranges, sweet oranges, squash, grapefruits, kumquats and others. (Groppo et al. 2008; Baig et al. 2009; Kumar et al. 2013). It is believed that especially the region extending from the north eastern India to eastward, from primary main origins of the genus Citrus are in South and Southeast Asia from Malaya Archipelago to China and Japan, and from south to Australia (Jena et al. 2009). The fruits and leaves of Citrus species contain a variety of essential oils containing biologically active compounds such as vitamin C, folic acid, potassium, flavonoid glycosides, coumarins, pectin, which are important for various nutrients and human diets (Othman et al. 2016; Bozkurt et al. 2017; Elkhatim et al. 2018; Liu et al. 2018). Molecular analysis to be conducted on genetic variation among the individuals of a population mailto:ph.d-emre@hotmail.com Nova Biotechnol Chim (2021) 20(2): e877 2 may offer a tool to monitor the genetic variability of a diminishing population and evaluate the genetic consequences of fragmentation on the remaining populations (Al-Qurainy et al. 2014). Chloroplast DNA (cpDNA) particularly includes non-coding regions which are maternally hereditary in angiosperms and have rich variability. These regions are significantly applied in various studies in molecular phylogenetic, population genetics and protection with a more comprehensive perspective (Chen et al. 2013; Gutiérrez-López et al. 2016). The psbA-trnH intergenic region is among the most variable regions of the chloroplast genome. This region is suitable for DNA barcoding studies, consists of two parts in evolutionary conservation (Štorchová and Olson 2007; Filiz and Koç 2012; Yılmaz 2020). The aim of the study was to determine the sequence analysis of selected Citrus L. taxa using the psbA-trnH cp (DNA) sequence in order to explain the phylogenetic relationships between them. Experimental Collection of plant materials and isolation of genomic DNA The fresh green leaves from 11 populations of five species of the genus Citrus: C. aurantium L., C. limon (L.) Burm. f., C. paradisi Macfad., C. reticulata Blanco, C. sinensis (L.) Osbeck, were collected from Aegean region (Aydın, Muğla, İzmir, Manisa-Salihli) in Turkey (Fig.1) and brought to the plant biotechnology laboratory at Aydın Adnan Menderes University. Genomic DNA was isolated using the DNAeasy Plant Kit (GeneMark) based on the manufactures protocol. Then the samples were stored at -20 °C. Fig. 1. Location of collecting places in the Aegean region, Turkey (https://www.google.com/maps). PCR amplification and sequencing The entire psbA-trnH region was amplified using the Biometra Personal Thermal Cycler. The PCR reactions were performed using psbA and trnH primers designed by Sang et al. (1997) and Tate and Simpson (2003) (Table 1) for amplification of the psbA-trnH region of cpDNA. The https://www.google.com/maps Nova Biotechnol Chim (2021) 20(2): e877 7 amplification process was carried out in 25 µL of PCR reaction volume, which contained 5.0 µL master mix (0.75 U of Taq DNA polymerase, reaction buffer, 2 mM MgCl2, 250 µM dNTP), 1 µL for psbA and 1 µL for trnH primers, approximately 2.0 µL of total genomic DNA, and 17 µL of ddH2O. Gel electrophoresis in 0.8 % agarose gel run in 1.0X Tris-Borate-EDTA buffer was used to separate amplicons that were stained with ethidium bromide and visualized u s i n g a UV transilluminator (Fig. 2). Purified PCR products were sequenced at Labbiotek (İzmir, Turkey) using an ABI 3130XL genetic analyser (Applied Biosystems, Foster City, CA, USA) with a BigDye cycle sequencing kit (Applied Biosystems). DNA sequences were edited both manually and by the Sequencher 5.4.6 programs. Sequences for individual samples were obtained through at least 3 independent sequencing runs whereas sequences for each taxon were based on at least 3 independent specimens. When all the sequences of a taxon were identical, only one sequence (based on one specimen) was included in the phylogenetic analysis. Table 1. cpDNA psbA-trnH primers and PCR amplification. Primers Sequences (5’-3’) PCR amplification (35 cycles) psbA 5’-GTTATGCATGAACGTAATGCTC-3’ (Sang et al. 1997) 94 oC/5 min 94 oC/45 s 50 oC/45 s 72 oC/1 min 72 oC/10 min trnH 5’-CGCGCATGGTGGATTCACAATCC-3’ (Tate and Simpson 2003) Fig. 2. Amplified cpDNA psbA-trnH region. Aligment and phylogenetic analysis cpDNA psbA-trnH sequences were aligned using the MEGA 6.0 alignment software (Tamura et al. 2013). Sequences distance values and maximum likelihood (ML) trees were created using MEGA 6.0 (Tamura et al. 2013). To evaluate the degree of support for given clades, a bootstrap analysis (1000 replicates) was applied (Felsenstein 1985). Additionally, Tetradium austrosinense (KR533457.1), Tetradium ruticarpum (MK419238.1), Melicope degeneri (EU493198.1), Melicope polybotrya (EU493202.1), Clausena anisate (MK260868.1), Clausena lenis (KR533453.1), Murraya exotica (GQ435449.1), Murraya paniculata (GU135341.2), Glycosmis lucida (KR533450.1), Glycosmis pentaphylla (GQ435452.1), Zanthoxylum molle (MF070225.1), and Zanthoxylum bungeanum (MF070227.1) psbA- trnH sequences were obtained from NCBI and were used to reveal phylogenetic relationships among Citrus species. Results and Discussion Phylogenetics aims to reveal the evolutionary relationship between all groups of organisms in the form of ancestor-lineage relationships and has become a powerful tool and starting point in many areas of biology such as taxonomy, biogeography, and developmental genetics (Wei et al. 2014; Sarıçam and Müştak 2015; Sevindik and Okan 2020). Recently, phylogenetic has get into a rapidly expanding area thanks to major improvements in nucleic acid and protein sequencing techniques and analyses (Patwardhan et al. 2014). The sequence analyses are very useful for phylogenetic analysis in cases when the morphological characters are insufficient for information. Sequence analysis methods are used in a variety of 3 Nova Biotechnol Chim (2021) 20(2): e877 2 fields ranging from detecting the geographic origins of the living organisms to finding molecular evidence of the phylogenies (Inal et al. 2017). There are many studies in Citrus using various molecular markers, e.g. RAPD (Coletta Filho et al. 1998; Nicolosi et al. 2000), ISSR (Fang and Roose 1997), AFLP (Al-Nadabi et al. 2018), SSR (Jannati et al. 2009), as well as chloroplast DNA (Nicolosi et al. 2000; Penjor et al. 2010; Wali et al. 2013; Oueslati et al. 2016; Uchoi et al. 2016; Sevindik and Yalçın 2018) and nuclear ribosomal DNA (nrDNA) markers (Amar et al. 2014; Sun et al. 2015). In this study, we obtained psbA-trnH sequences ranging from 426 to 470 nucleotides for 11 specimens. The highest number of nucleotides was determined for Citrus sinensis (Aydın) (470 bp) w h i l e the lowest for Citrus sinensis (Bergama) (426 bp). Average nucleotide composition of psbA-trnH was 42.3 % T, 11.7 % C, 28.8 % A, and 17.2 % G. (Table 2). Table 2. Length and A, T, G and C contents of cpDNA psbA-trnH sequences of Citrus taxa. Taxa psbA-trnH A [%] T [%] G [%] C [%] C. paradisi (Aydın) 455.0 28.8 42.0 16.9 12.3 C. limon (Aydın) 452.0 29.0 42.7 17.5 10.8 C. sinensis (Salihli) 450.0 28.7 42.4 17.1 11.8 C. sinensis (Muğla) 464.0 28.4 41.8 17.9 11.9 C. sinensis (Aydın) 470.0 28.5 41.5 17.9 12.1 C. reticulata (Bergama) 466.0 29.2 41.0 17.6 12.2 C. reticulata (Aydın) 448.0 29.5 41.5 17.6 11.4 C. aurantium (Aydın) 453.0 28.9 41.5 17.2 12.4 C. paradisi (İzmir) 434.0 28.3 43.1 16.4 12.2 C. limon (Muğla) 430.0 28.6 44.2 16.7 10.5 C. sinensis (Bergama) 426.0 28.6 43.7 16.7 11.0 Avg. 449.8 28.8 42.3 17.2 11.7 The total length of the aligned psbA-trnH sequence matrix was 483 nucleotides. The genetic distance method based on psbA-trnH set was performed with MEGA 6.0 software. The lowest sequence divergence among the ingroup taxa was 0.000 while the highest was 0.012 (Table 3). Table 3. Pairwise sequence distances among some Citrus taxa for cpDNA psbA-trnH sequences using MEGA 6.0 software distance matrix. Species 1 2 3 4 5 6 7 8 9 10 11 C. paradisi (Aydın) - C. limon (Aydın) 0.007 C. sinensis (Salihli) 0.005 0.002 C. sinensis (Muğla) 0.005 0.002 0.000 C. sinensis (Aydın) 0.005 0.002 0.000 0.000 C. reticulata (Bergama) 0.012 0.010 0.007 0.007 0.007 C. reticulata (Aydın) 0.012 0.010 0.007 0.007 0.007 0.000 C. aurantium (Aydın) 0.012 0.010 0.007 0.007 0.007 0.000 0.000 C. paradisi (İzmir) 0.002 0.005 0.002 0.002 0.002 0.010 0.010 0.010 C. limon (Muğla) 0.010 0.002 0.005 0.005 0.005 0.012 0.012 0.012 0.007 C. sinensis (Bergama) 0.005 0.002 0.000 0.000 0.000 0.007 0.007 0.007 0.002 0.005 - Using the MEGA 6 program, Tajma's Neutrality Test (Tajima 1989) was calculated based on psbA- trnH sequences of Citrus species. Numbers of sequences (m) gave one segregation site (S) revealing very low nucleotide diversity (π) of 0.005690 (Table 4). Maximum likelihood (ML) tree was generated using psbA-trnH sequences of certain Citrus species distributed in Turkey. It consists of two large clades. Clade 1 is divided into two subclades, A and B. Subclade A consists of C. paradisi (Aydın), C. paradisi (İzmir), C. limon (Muğla), C. limon (Aydın) species, whereas all C. sinensis populations are placed in subclade B. Clade 2 consists of C. reticulata (Aydın), C. aurantium (Aydın) and C. reticulata (Bergama) species (Fig. 3). 4 Nova Biotechnol Chim (2021) 20(2): e877 7 Table 4. Tajima’s Neutrality Test Values based on cpDNA psbA-trnH of date Citrus species. No. of sequences “m” No. of segregating sites “S” Ps=S/n Θ = ps/a1 nucleotide diversity ‘’ π ‘’ Tajima test statistic ‘’D’’ 11 7 0.017115 0.005843 0.005690 -0.106371 So far, many studies have been done on Citrus using a wide range of gene regions and markers. Wali et al. (2013) carried out the phylogenetic analysis of Citrus species using cpDNA rps14 genes. They situated C. reticulata and C. aurantium C. sinensis var. malta, and C. sinensis var. fruiter species in one branch, and C. limon and C. sinensis var. mousami species in another branch. In our study, C. reticulata and C. aurantium were detected in Clade 2, whereas populations of C. sinensis and C. limon in Clade 1. Wali et al. (2013) concluded that rps14 gene sequence was highly protected in Citrus species, and it did not provide much information to form the phylogeny of Citrus species. Sevindik and Yalçın (2018) performed phylogenetic analysis of some Citrus species widespread in the Aegean Region based on cp(DNA) trnL intron and trnL-F sequences and they located C. aurantium (Aydın), C. reticulata (Bergama) and C. reticulata (Aydın) species in the same branch of the phylogenetic tree (Fig. 3). These three species were also situated in the same branch of our phylogenetic tree formed on the basis of psbA-trnH sequences. Analysis of trnL intron and trnL-F sequences revealed that C. limon (Aydın) and C. paradisi (Aydın) belong to a Fig. 4. The maximum likelihood tree generated using cpDNA psbA-trnH sequences and other species sequences retrieved from NCBI. 5 Fig. 3. The maximum likelihood tree generated using cpDNA psbA-trnH sequences. Nova Biotechnol Chim (2021) 20(2): e877 2 subsidiary group, whereas C. limon (Muğla) is situated in a separate branch. Generally, according to our results, populations of the same species are found to be together. The study by Sevindik and Yalçın (2018) showed that, based on trnL intron analysis, C. paradisi (Aydın) and C. paradisi (İzmir) populations are located in different branches, however the results of trnL-F analysis indicated that these populations are in the same branch. Our analysis of psbA-trnH sequence showed that both populations are in Clade 1. In case of populations of C. sinensis species, the above mentioned authors reported that they belong to different branches, both in the trnL intron tree and in the trnL-F tree. In psbA-trnH tree, all populations of all C. sinensis species were collected in one group. According to psbA-trnH results, populations belonging to the same species form a group, while this did not happen with trnL intron and trnL-F sequences (Sevindik and Yalçın, 2018). Uchoi et al. (2016) analysed phylogenetically Citrus species distributed in India using rbcL and matK sequences. They applied the maximum parsimony (MP) and neighbor-joining (NJ) methods. In the NJ bootstrap consensus tree formed based on rbcL sequence, C. reticulata and C. sinensis were grouped together, while C. limon and C. aurantium species were in different groups. In turn, in the NJ phylogenetic tree based on the matK sequences, C. aurantium and C. reticulate were in one group and the species C. sinensis and C. limon were in different groups. Our research has shown that C. reticulata and C. aurantium species are located in Clade 2, which is partially consistent with the NJ tree formed with the matK sequence. Sun et al. (2015) investigated the taxonomy and phylogeny of 26 Citrus materials of 22 Citrus species using sequence analysis of the ITS region of nrDNA. In their research, C. aurantium and C. sinensis were found in one group, while C. limon belonged to a separate group. According to our results, C. aurantium populations belong to Clade 2 and C. sinensis and C. limon populations to Clade 1. However, the ITS sequences results obtained by Sun et al. (2015) are not consistent with ours. Penjor et al. (2010) determined the phylogenetic relations of Citrus and its relatives using maximum parsimony and neighbour-joining methods and cpDNA rbcL gene sequence. They placed C. aurantium, C. limon, C. sinensis and C. paradisi in one group. We found that C. limon, C. sinensis and C. paradise populations belong to Clade 1, while C. aurantium to Clade 2. The phylogenetic relationship of some species of Rutacee family (taken from NCBI) with five Citrus species investigated in this study is given in Fig. 4. As it is seen from the tree, Citrus species are separated from Tetradium ruticarpum, Melicope degeneri, Melicope polybotrya, Clausena anisate, Clausena lenis, Murraya exotica, Murraya paniculata, Glycosmis lucida, Glycosmis pentaphylla, Zanthoxylum molle and Zanthoxylum bungeanum species. Penjor et al. (2010) grouped Citrus species with Fortunella, Poncirus, Microcitrus, Eremocitrus, and Clymenia. Groppo et al. (2008) determined cpDNA in rps16 intron analysis and situated Citrus, Poncirus and Microcitrus in one group, whereas trnL-F analysis revealed that Balsamocitrus, Afraegle, Citrus and Clausena form one group. Conclusion In this study, phylogenetic analysis of Turkish Citrus L. taxa using cpDNA psbA-trnH sequences was performed to elucidate phylogenetic relationships. According to the results of the study, the separation of Citrus species in the phylogenetic tree obtained with psbA-trnH sequence data was realized. However, it has been found that cpDNA psbA-trnH sequence populations of species belong together. The data obtained from this study will guide phylogenetic studies on both Citrus and different plant species. Acknowledgements This research was supported by the TUBITAK 2018/2209- A. (Project number: 1919B011800889). 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