Nova Biotechnol Chim (2022) 21(1): e1320 DOI: 10.36547/nbc.1320 1 Nova Biotechnologica et Chimica Genetic diversity and phylogenetic analyses of Vitex agnus-castus L. populations using ISSR-PCR and chloroplast DNA trnL intron and trnL-F sequences Emre Sevindik Faculty of Agriculture, Department of Agricultural Biotechnology, Aydın Adnan Menderes University, Cakmar, Turkey  Corresponding author: ph.d.-emre@hotmail.com Article info Article history: Received: 12th December 2021 Accepted: 2nd March 2022 Keywords: ISSR-PCR trnL intron trnL-F Phylogenetic Vitex agnus-castus Abstract In this study, genetic diversity and phylogenetic analysis of some Vitex agnus-castus L. populations were conducted based on ISSR-PCR and chloroplast DNA trnL intron, and trnL-F sequence analyses. Vitex agnus-castus populations were detected in Aydın province, Turkey. Fresh leaf samples from the populations were collected and brought to the laboratory for genomic DNA isolation. 15 ISSR primers were used to determine the genetic diversity ofe Vitex agnus-castus populations. A total of 138 bands were obtained in ISSR analysis, 85 of which were polymorphic and 53 were monomorphic. Polymorphism rate was determined as 61.59 %. trnC, trnD, trnE, and trnF primers were used for PCR amplification of the chloroplast trnL intron and trnL-F region. A total of 138 bands were obtained by ISSR analysis. For trnL intron analyses, nucleotide lengths of 13 populations were between 508 and 516. The average nucleotide composition consisted of 38.5 % T, 18.3 % C, 27.5 % A and 15.7 % G. In trnL-F assays, the nucleotide lengths of the 13 populations ranged from 330 to 353. The average nucleotide composition consisted of 29.4 % T, 18.1 % C, 32.9 % A and 19.6 % G. The results of the phylogenetic trees constructed using some trnL intron and trnL-F sequences of Vitex doniana, Vitex trifolia, Vitex triflora, Vitex turczaninowii, Vitex queenslandica, Vitex axillariflora, Vitex rotundifolia and Vitex negundo species obtained from NCBI were compared. As a result of the study, polymorphisms were obtained at a rate of 61.59% from the ISSR analysis. In addition, the phylogenetic relationship between chloroplast trnL intron and trnL-F sequences of Vitex agnus-castus populations along with the other species was revealed. Introduction Medical and aromatic plants have a long history of being used globally for medicinal purposes and have traditionally been an important component of health services throughout human history due to their health benefits (Eryigit et al. 2015; Chaachouay et al. 2019). The Vitex genus includes about 250 species and many of these have important medicinal effects (Yao et al. 2016). Vitex agnus-castus L., known as chasteberry, belongs to the Lamiaceae family (previously included in the Verbenaceae family), and it is a 1 – 3 m long highly branched small tree. Its trunk is covered with short, dense, soft and grey hairs. It is a deciduous shrub native to Europe and Central Asia (Stojković et al. 2011; Hürkul and Köroğlu 2018; Souto et al. 2020; Ilhan 2021). This plant is mailto:ph.d.-emre@hotmail.com Nova Biotechnol Chim (2022) 21(1): e1320 2 naturally found in many cities throughout Turkey, including Amasya, Antalya, Bursa, Muğla, Trabzon and Çanakkale (Yilar et al. 2016). Vitex agnus- castus, used in conventional medicine, has a long tradition in folk medicine, and its use dates back to 2,500 years ago (Sorensen and Katsiotis 1999; Lataoui et al. 2014; Uçak Koç et al. 2017). It is widely used in Anatolian folk medicine as diuretics, appetizers, antifungals, antispasmodics, for preterm labor and for pain relief (Ilhan 2021). Its fruits, flowers and leaves have been reported to contain flavonoids, tannins, iridoids and diterpenoids (Sağlam et al. 2007; Sarikurkcu et al. 2009). V. agnus-castus fruits have been used as a flavor and spice component in meals, as a hormone-like remedy for menstrual problems and a mild sedative and digestive tool in Iranian traditional folk medicine (Ghannadi et al. 2012). The aromatic leaves are used as a spice (Stojković et al. 2011; Mari et al. 2015). Genetic diversity is a prerequisite for the short and long-term survival of plant species in their natural environment (Verma et al. 2017). Morphological, biochemical, and DNA markers are used to determine the genetic diversity of plants (Sevindik et al. 2020). DNA-based molecular markers offer many advantages in determining genetic diversity. Among the polymerase chain reaction (PCR) based marker techniques, the ISSR technique is one of the simplest and most widely used markers. It is also a marker system utilized in the determination of genetic relationships and linkage mapping (Alansi et al. 2016; Atasagun et. al. 2018; Güngör et al. 2020). Chloroplast (cp) is an important organelle for green plants as it is the place where photosynthesis, carbon fixation and pigment synthesis take place. The Cp is an independent genome (cp genome) with a preserved circular structure and a low molecular weight (Zhou et al. 2019; Munyao et al. 2020; Zhu et al. 2021). Chloroplast DNA (cpDNA), which is inherited by the female in most angiosperm plants, is generally accepted as a single non-recombinant inheritance unit and is widely used to reveal plant phylogenies in interspecies studies (Taberlet et al. 1991; Gielly and Taberlet 1994; Dizkirici et al. 2019; Sun et.al. 2019). Non-coding regions of the chloroplast genome are used in molecular systematic and plant population genetic studies (Kalmer and Tekpınar 2017). Within this study the chloroplast regions with both variable and highly conserved primer sequences were used. The first of these regions is the trnLUAA intron; and the second is the trnL(UAA)- F(GAA) intergenic spacer (IGS). Noncoding cpDNA trnL-F is located between trnL (UAA) 3' exon and trnF (GAA) gene. In addition, it is more variable towards the coding regions, and some studies have revealed that it shows higher variations and more frequent mutations than the coding regions (Taberlet et al. 1991; Hartana 2010). The trnL intron is the first group I intron identified in chloroplast DNA and is also the first intron identified to cut a tRNA gene (Bakker et al. 2000). In this study, we performed a genetic diversity analysis using ISSR and cpDNA trnL intron and trnL-F region markers for some Vitex agnus-castus populations grown in the Aydın region of Turkey. Experimental Plant materials, genomic DNA isolation and PCR Leaf samples belonging to the Vitex agnus-castus populations to be used in this study were collected from the center, Koçarlı, Çine, Germencik, İncirliova, and Köşk districts of Aydın province (Fig. 1). Fig. 1. Location of collecting places in the Aydın/Turkey (https://www.google.com/maps). Leaf samples of the collected Vitex agnus-castus plant were brought to the laboratory and prepared for genomic DNA isolation. A commercial kit (GeneMark) was used for the genomic DNA isolation from the plant specimens. The obtained gDNA samples were stored at -20 °C. The primers, PCR mix and protocol used for the amplification of https://www.google.com/maps Nova Biotechnol Chim (2022) 21(1): e1320 3 ISSR-PCR, cpDNA trnL intron and trnL-F regions are given in Table 1 and Table 2. Table 1. Primers used in the ISSR-PCR reactions, PCR protocols and their Tm degrees. ISSR Primers DNA Sequences (5’-3’) Tm [oC] PCR Amplification (35 cycle) Amplification UBC-831 5’-CTCTCTCTCTCTCTCTT-3’ 50 °C 94 °C/1min 94 °C/1min 48-55 °C/1min 72 °C/1 min 72 °C/10min + UBC-830 5’-TGTGTGTGTGTGTGTGG-3’ 52 °C + UBC-807 5'-AGAGAGAGAGAGAGAGT-3' 50 °C + UBC-808 5’-AGAGAGAGAGAGAGAGC-3’ 52 °C + UBC-836 5'-AGAGAGAGAGAGAGAGYA-3’ 52 °C + UBC-856 5'–ACACACACACACACACYA-3’ 52 °C + UBC-853 5'- TCTCTCTCTCTCTCTCRT-3' 52 °C + UBC-892 5’- TAGATCTGATATCTGAAT-3’ 52 °C - UBC-810 5' -GAGAGAGAGAGAGAGAT-3' 50 °C + UBC-826 5’-ACACACACACACACACC-3’ 52 °C + UBC-811 -GAGAGAGAGAGAGAGAC-3’ 53 °C + UBC-834 5’-AGAGAGAGAGAGAGAYT-3’ 52 °C + UBC-873 5’-GACAGACAGACAGACA-3’ 48 °C + UBC-855 5’-ACACACACACACACAC 52 °C + UBC-880 5’-GGAGAGGAGAGGAGA-3’ 55 °C + Table 2. trnL intron and trnL-F primers used in this study with their designers and PCR protocols. Primer name 5’ to 3’ Primer sequence PCR Amplification (35 cycle) Based on (the source publication) Forward trnc 5’-CGAAATCGGTAGACGCTACG-3’ 94 °C/4min. 94 °C/1min 50 °C/1min. 72 °C/1 min. 72 °C/10min. Taberlet et al. 1991 Reverse trnd 5’-GGGGATAGAGGGACTTGAAC-3’ Taberlet et al. 1991 Forward trnLe 5’-GGTTCAAGTCCCTCTATCCC-3’ Taberlet et al. 1991 Reverse trnFf 5’-ATTTGAACTGGTGACACGAG-3’ Taberlet et al. 1991 1 % agarose gel 1X TBE buffer was used for electrophoresis of PCR products. 5 µl of the reaction mixture in PCR tubes was added to 1 µL loading buffer (Loading Dye Solution) and mixed, and 6 µL of this mixture was placed in the wells of the gel. After loading 3 kb DNA marker into the first well, the device was subjected to electrophoresis at 100 V for 90 min. Then, the DNA bands were imaged under UV light and their photos were taken (Fig. 2 – 4). Fig. 2. Gel image of ISSR-PCR bands amplified with UBC- 811 primer. Fig. 3. Gel image of PCR bands amplified with cpDNA trnL intron. Fig. 4. Gel image of PCR bands amplified with cpDNA trnL- F region. ISSR-PCR analyses Nova Biotechnol Chim (2022) 21(1): e1320 2 After the PCR analyses, DNA bands were scored as follows: “1” was given if there was DNA and “0” was given if there was no DNA in the bands. A “?” was given for missing data. Using bands, the genetic relationships of Vitex agnus-castus populations used in the study were analysed using the PAUP 4.0b10 (Swofford 2001) program. cpDNA trnL intron and trnL-F sequences Service procurement from biotechnology company Triogen (Istanbul, Turkey) was obtained for PCR reactions and purification. To ensure healthy analyses, it was necessary to visually check the accuracy of DNA sequences one by one. To this end, professional computer programs BioEdit (Hall 1999) and Finch TV, which are frequently used in molecular systematic studies worldwide, were utilized. A maximum likelihood phylogenetic tree was constructed using the MEGA 6.0 (Tamura et al. 2013) to extract phylogenetic relationships among the sequenced Vitex agnus-castus populations. To evaluate the degree of support for given clades, a bootstrap analysis (1,000 replicates) was applied (Felsenstein 1985). In addition, the genetic distance matrix between populations was also performed using the same software. Results and Discussion In recent years, some PCR-based molecular markers have been developed and tested in genetic studies of various organisms (Poyraz et al. 2016). In previous studies, genetic diversity, phylogenetic and molecular analyses of Vitex species have been conducted using RAPD (Sevindik et al. 2019), ISSR (Gıachıno and Avcı 2017; Saedyani et al. 2020), rps14 gene (Ayaz et al. 2020; Malik et al. 2021), nrITS DNA, chloroplast ndhF, trnH-psbA, trnG-trnS sequences (Bramley et al. 2009; Sun et al. 2019), chloroplast rbcL and ndhF sequences (Wagstaff et al. 1998), and chloroplast matK and psbA-trnH intergenic spacer (Phoolcharoen and Sukrong 2013). ISSR Analysis In recent years, PCR-based DNA marker systems such as AFLP, RFLP, RAPD, SSR and ISSR have been common for investigating the genetic makeup of populations, genetic characterization, genetic diversity, divergence and phylogenetic studies (Li and Ge 2001; Gajera et al. 2010; Chen et al. 2017; Hocaoglu-Ozyigit et al. 2020). ISSR markers have been proposed as a new source of genetic markers that are inherited in Mendelian fashion and are scored as dominant markers (Paul et al. 2020). These markers have the role of analyzing genetic diversity through the classification of varieties (Kiani and Siahchehreh 2018). In ISSR analysis 15 primers were used of which 14 yielded positive results while 1 primer did not (Table 1). A total of 138 bands were obtained in ISSR analysis, 85 of which were polymorphic and 53 were monomorphic. Polymorphism rate was determined as 61.59 %. The UPGMA tree generated based on the ISSR dataset consisted of two clades (Fig. 5). Fig. 5. The UPGMA tree generated using ISSR data. Clade 1 was divided into 2 subclades. Subclade A consisted of only the Çine purple flowered population. Çine purple flowered population was found together with Koçarlı white flowered, Çakmar white flowered, Germencik purple flowered and Erbeyli white flowered populations in RAPD analysis (Sevindik et al. 2019). In the subclade B, Çine white flower, Çine pink flower, Çakmar pink flowered, Aydın purple flowered and Aydın white flowered appeared in the same group. According to Sevindik et al. (2019), these populations were found together according to the RAPD results. In subclade B, Çakmar purple flowered, Germencik purple flowered, Erbeyli white flowered, Çakmar white flowered and İncirliova purple flowered populations emerged together. According to Sevindik et al. 4 Nova Biotechnol Chim (2022) 21(1): e1320 3 (2019), Germencik purple flowered, Çakmar white flowered and Erbeyli white flowered were found together, while İncirlova purple flowered and Çakmar purple flowered populations detected in separate groups. Clade 2 consisted of Köşk purple flower and Koçarlı white flower populations. According to Sevindik et al. (2019), both populations emerged in different groups. According to PAUP analysis, the closest genetic distances were between Germencik purple flowered and Erbeyli white flowered populations with a value of 0.10145; and the greatest genetic distance was between Çine purple flowered and Koçarlı white flowered populations with a value of 0.36957 (Table 3). Giachino and Avci (2017) used 6 of 21 ISSR primers for Vitex agnus-castus populations collected from various parts of the Yunt Mountain of Manisa. The discrimination power of ISSR primers was evaluated by calculating various marker parameters such as percent polymorphism, polymorphism information content (PIC), resolving power (RP), and marker index (MI). In the study, 31 (65.95 %) of a total of 47 useable bands were found to be polymorphic. The polymorphic band ratios of the primers varied from 25 % to 85.7 %. Polymorphism information content (PIC) values of 6 primers ranged from 0.22 to 0.35, and the mean PIC value was calculated as 0.30. The resolution power (RP) values ranged between 0.5 and 3.25, and the marker index (MI) ranged between 0.11 and 1.72. Saedyani et al. (2019) determined genetic diversity for 19 Vitex agnus-castus genotypes using 13 ISSR primers. In their study, 74 bands were obtained and the polymorphism rate was 95% on average. They determined the genetic distance of the genotypes between 0.195 and 0.593 using the Dice coefficient. cpDNA trnL intron and trnL-F sequence analysis Phylogenetic analyses of molecular sequences are part of many molecular and phylogenetic biology studies (Anisimova et al. 2013). The cpDNA trnL- F region is located in the large single-copy region of the chloroplast genome and is widely used for phylogenetic analyses (Hocaoglu-Ozyigit et al. 2020). For the cpDNA trnL intron, the base lengths of Vitex agnus-castus populations ranged from 508 to 516. The genetic distance between populations was between 0.000 and 0.002 (Table 4). The mean nucleotide ratio for the trnL intron was 38.5 % T, 18.3 % C, 27.5 % A, and 15.7 % G. Using the MEGA 6.0 program, Tajma's Neutrality Test (Tajima 1989) was calculated based on cpDNA trnL intron sequences of Vitex agnus-castus populations. Numbers of sequences (m) yielded one segregation site (S) revealing very low nucleotide diversity (π) of 0.001422 (Table 5). The maximum likelihood phylogenetic tree constructed using 13 populations consisted of two large groups (Fig. 6). The first clade is divided into two subgroups. Subclade A consisted of İncirliova purple flowered, Koçarlı white flowered, Germencik purple flowered, Çine purple flowered, Çakmar white flowered and Çakmar purple flowered, and this group was supported with a bootstrap value of 55 % (Fig. 6). In the study of Sevindik et al. (2019), Çakmar purple flowered and İncirliova purple flowered populations were found in a separate group, while Koçarlı white flowered, Germencik purple flowered, Çine purple flowered and Çakmar white flowered populations were found in the same group. Subclade B consisted of Çakmar pink flowered, Çine pink flowered, Erbeyli white flowered and Köşk purple flowered, and this group was supported with a 60 % bootstrap value (Fig. 6). Sevindik et al. (2019) found Erbeyli white flowered and Köşk purple flowered populations in separate groups, while Çakmar pink flowered and Çine pink flowered populations in the same group. Clade 2 consisted of Aydın white flowered, Aydın purple flowered and Çine white flowered populations. Within this group, Aydın white flowered and Aydın purple flowered populations were monophyletic with a bootstrap value of 63 % (Fig. 6). 5 Nova Biotechnol Chim (2022) 21(1): e1320 3 Table 3. Pairwise genetic distance matrix obtained from PCR with ISSR primers. Populations 1 2 3 4 5 6 7 8 9 10 11 12 13 Çine purple flower - 0.19565 0.23915 0.22464 0.20455 0.24638 0.27536 0.30435 0.26087 0.26812 0.26812 0.31159 0.36957 Çine White flower 27 - 0.17391 0.15952 0.13636 0.23913 0.22464 0.21014 0.18116 0.18841 0.20290 0.26087 0.33333 Çine pink flower 33 24 - 0.13043 0.15152 0.15217 0.16667 0.19565 0.19565 0.18841 0.20290 0.27536 0.31884 Çakmar pink flower 31 22 18 - 0.12879 0.13768 0.18116 0.23913 0.21014 0.20290 0.20290 0.27536 0.36232 Aydın purple flower 27 18 20 17 - 0.15152 0.13636 0.20455 0.19697 0.19637 0.18939 0.28788 0.29545 Aydın white flower 34 33 21 19 20 - 0.17391 0.27536 0.24638 0.22464 0.23913 0.32609 0.35507 Çakmar purple flower 38 31 23 25 18 24 - 0.17391 0.17391 0.12319 0.13768 0.25362 0.25362 Çakmar White flower 42 29 27 33 27 38 24 - 0.11594 0.13768 0.13768 0.19565 0.29710 İncirliova purple flower 36 25 27 29 26 34 24 16 - 0.13768 0.13768 0.19565 0.32609 Germencik purple flower 37 26 26 28 26 31 17 19 19 - 0.10145 0.21739 0.26087 Erbeyli white flower 37 28 28 28 25 33 19 19 19 14 - 0.15942 0.23188 Köşk purple flower 43 36 38 38 38 45 35 27 27 30 22 - 0.20290 Koçarlı white flower 51 46 44 50 39 49 35 41 45 36 32 28 - 6 Nova Biotechnol Chim (2022) 21(1): e1320 2 Table 4. Pairwise genetic distance matrix obtained from cpDNA trnL intron sequences. Populations 1 2 3 4 5 6 7 8 9 10 11 12 13 Aydın purple flower - Aydın white flower 0.000 Cakmar pink flower 0.002 0.002 Cakmar purple flower 0.002 0.002 0.002 Cakmar white flower 0.002 0.002 0.002 0.000 Cine pink flower 0.002 0.002 0.000 0.002 0.002 Cine purple flower 0.002 0.002 0.002 0.000 0.000 0.002 Cine white flower 0.002 0.002 0.002 0.002 0.002 0.002 0.002 Erbeyli white flower 0.002 0.002 0.000 0.002 0.002 0.000 0.002 0.002 Germencik purple flower 0.002 0.002 0.002 0.000 0.000 0.002 0.000 0.002 0.002 Incirliova purple flower 0.002 0.002 0.002 0.000 0.000 0.002 0.000 0.002 0.002 0.000 Kocarlı white flower 0.002 0.002 0.002 0.000 0.000 0.002 0.000 0.002 0.002 0.000 0.000 Kösk purple flower 0.002 0.002 0.000 0.002 0.002 0.000 0.002 0.002 0.000 0.002 0.002 0.002 - Table 5. Tajima’s Neutrality Test Values based on cpDNA trnL intron of date Vitex agnus-castus populations. No. of sequences “m” No. of segregating sites “S” Ps=S/n Θ = ps/a1 nucleotide diversity ‘’ π ‘’ Tajima test statistic ‘’D’’ 13 1 0.001980 0.000638 0.001422 2.700233 7 Nova Biotechnol Chim (2022) 21(1): e1320 3 Fig. 6. The maximum likelihood tree generated using cpDNA trnL intron sequences. According to Sevindik et al. (2019), these three populations were in the same group. There are both similarities and differences between the UPGMA dendrogram generated by RAPD analysis and the phylogenetic trees constructed with trnL intron sequences. Additionally, Vitex doniana (MK187249.1), Vitex trifolia (AJ505539.1), Vitex triflora (MK797715.1), Vitex turczaninowii (MG836415.1), Vitex queenslandica (MG836414.1), Vitex axillariflora (MG836413.1), Vitex rotundifolia (AB817427.1; AB817638.1; AB817574.1) and Vitex negundo (DQ304786.1; DQ304787.1) species were added to the analyses after obtaining their trnL intron sequences from NCBI. A maximum likelihood phylogenetic tree was generated and the relationship of Vitex agnus- castus populations with other species was revealed. The phylogenetic tree consists of 2 clades (Fig. 7). The first clade consists of Vitex agnus-castus populations, Vitex axillariflora, Vitex trifolia, Vitex rotundifolia and Vitex negundo species formed and is supported by a bootstrap value of 95 %. Clade 2 is divided into two subclades. Subclade A, consists of Vitex triflora and Vitex doniana species and is supported by a bootstrap value of 97 %, and subclade B, consists of Vitex turczaninowii Vitex queenslandica species and is supported by a 75 % bootstrap value (Fig. 7). Fig. 7. The maximum likelihood tree generated using cpDNA trnL intron sequences and other species sequences retrieved from NCBI (Bootstrap values greater than 50 % are given above branches). For the cpDNA trnL-F region, the base lengths of Vitex agnus-castus populations ranged from 330 to 353. The genetic distance matrix between populations turned out to be 0.000 (Table 6). 8 Nova Biotechnol Chim (2022) 21(1): e1320 3 Table 6. Pairwise genetic distance matrix obtained from cpDNA trnL-F sequences. Populations 1 2 3 4 5 6 7 8 9 10 11 12 13 Aydın purple flower Aydın white flower 0.000 Cakmar pink flower 0.000 0.000 Cakmar purple flower 0.000 0.000 0.000 Cakmar white flower 0.000 0.000 0.000 0.000 Cine pink flower 0.000 0.000 0.000 0.000 0.000 Cine purple flower 0.000 0.000 0.000 0.000 0.000 0.000 Cine white flower 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Erbeyli white flower 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Germencik purple flower 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Incirliova purple flower 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Kocarlı white flower 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Kösk purple flower 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 - 9 Nova Biotechnol Chim (2022) 21(1): e1320 3 The mean nucleotide ratio for the trnL-F region was 29.4 % T, 18.1 % C, 32.9 % A, and 19.6 % G. Using the MEGA 6.0 program, Tajma's Neutrality Test (Tajima 1989) was calculated based on cpDNA trnL-F sequences of Vitex agnus-castus populations. Numbers of sequences (m) gave one segregation site (S) revealing very low nucleotide diversity (π) of 0.000000 (Table 7). Table 7. Tajima’s Neutrality Test Values based on cpDNA trnL –F of date Vitex agnus-castus populations. No. of sequences “m” No. of segregating sites “S” Ps=S/n Θ = ps/a1 nucleotide diversity ‘’ π ‘’ Tajima test statistic ‘’D’’ 13 0 0.000000 0.00000E+000 0.000000 n/c The maximum likelihood tree constructed using trnL-F sequences consists of two clades (Fig. 8). Clade 1 is divided into 3 subclades in itself. Subclade A consists Aydın purple flowered, Aydın white flowered, Çakmar pink flowered, Çakmar purple flowered, Çine pink flowered and Çine purple flowered populations. Fig. 8. The maximum likelihood tree generated using cpDNA trnL-F sequences. Sevindik et al. (2019) detected the Çine purple flowered population in a different group, while all other populations were determined in the same group. In subclade B, Erbeyli white flowered and Köşk purple flowered populations were sister groups, while İncirliova purple flowered populations were close to these two populations. Sevindik et al. (2019) identified three populations in different groups. Subclade C consists of only the Germencik purple flowered population. Clade 2 consisted of Çine white flowered and Koçarlı white flowered populations, which was supported by a bootstrap value of 99 %. According to Sevindik et al. (2019), these two populations were included in separate groups. In this study, a maximum likelihood phylogenetic tree was generated using trnL-F sequences of Vitex trifolia (AJ505539.1), Vitex turczaninowii (MG836415.1), Vitex queenslandica (MG836414.1) and Vitex axillariflora (MG836413.1) from NCBI. The relationship of Vitex agnus castus populations with other species was revealed. The phylogenetic tree consists of 2 clades (Fig. 9). The first clade consists of Vitex agnus castus populations and Vitex trifolia species, and this group has a bootstrap value of 94 %. Clade 2 consists of Vitex turczaninowii, Vitex queenslandica and Vitex axillariflora species. In trnL intron analysis, Vitex agnus-castus populations coexisted with Vitex axillariflora and Vitex trifolia species. In addition, in trnL-F analysis, Vitex turczaninowii, Vitex queenslandica and Vitex axillariflora were found in one group. Vitex axillariflora and Vitex trifolia species were found together with Vitex agnus-castus populations, and Vitex turczaninowii and Vitex queenslandica species were found in a separate group in trnL intron analysis. Malik et al. (2021) determined the phylogenetic relationship of some species belonging to the Lamiaceae family with the chloroplast rps14 gene. Vitex agnus-castus and Vitex trifolia species were found in the same group in the study. In our trnL intron and trnL-F sequence studies, Vitex agnus- castus and Vitex trifolia were found in the same group (Fig. 7 and Fig. 9). Ayaz et al. (2020) performed phylogenetic analysis of some species belonging to Lamiaceae in Pakistan using the rps14 gene. In their study, Vitex agnus-castus var pseudo- negundo and Vitex negundo species were found in the same group. In the ML tree constructed using trnL intron sequences and sequences taken from 10 Nova Biotechnol Chim (2022) 21(1): e1320 2 NCBI, the Vitex agnus-castus populations were in the same clade with Vitex axillariflora and Vitex negundo. Wagstaff et al. (1998) used chloroplast rbcL and ndhF sequences to reveal the phylogenetic relationships of some Lamiaceae taxa. Vitex agnus-castus species formed a sister group with Petitia domingensis species in the strict consensus tree of rbcL and ndhF sequences. Fig. 9. The maximum likelihood tree generated using cpDNA trnL-F sequences and other species sequences retrieved from NCBI (Bootstrap values greater than 50 % are given above branches). Conclusion As a result of the study, approximately 61.59 % polymorphism were detected among Vitex agnus- castus populations based on ISSR analyzes. In general, the results of this research aiming to analyze the genetic diversity and phylogenetic analysis of Vitex agnus-castus populations through ISSR, cpDNA trnL intron and trnL-F sequence comparisons will be used to determine the phylogenetic relationship between Vitex agnus- castus species and other species. Acknowledgments This research was supported by the Aydın Adnan Menderes University (Project no:ZRF-20001). Conflict of Interest The authors declare that they have no conflict of interest. Reference Alansi S, Tarroum M, Al-Qurainy F, Khan S, Nadeem M (2016) Use of ISSR markers to assess the genetic diversity in wild medicinal Ziziphus spina-christi (L.) 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