Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(1): 125-132, 2020 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-147 Citation: R. Partovi, A. Iaranbakhsh, M. Sheidai, M. Ebadi (2020) Popula- tion genetic studies in wild olive (Olea cuspidata) by molecular barcodes and SRAP molecular markers. Caryologia 73(1): 125-132. doi: 10.13128/caryolo- gia-147 Received: January 9, 2019 Accepted: February 23, 2020 Published: May 8, 2020 Copyright: © 2020 R. Partovi, A. Iar- anbakhsh, M. Sheidai, M. Ebadi. This is an open access, peer-reviewed arti- cle published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Population genetic studies in wild olive (Olea cuspidata) by molecular barcodes and SRAP molecular markers Rayan Partovi1, Alireza Iranbakhsh1,*, Masoud Sheidai2, Mostafa Ebadi3 1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran 2 Faculty of Life Sciences & Biotechnology, Shahid Beheshti University, Tehran, Iran 3 Department of Biology, Islamic Azad University, Damghan Branch, Damghan, Semnan Province, Iran *Correspondign author. E-mail: iranbakhsh@iau.ac.ir Abstract. Olive is an important horticultural plant having both cultivated and wild forms. The aim of the present study was investigating genetic diversity of 13 wild olive trees belonging four geographical populations in IRAN using SRAP neutral molecular markers as well as cp-DNA rpl intergenic sequences and ITS region. Genetic diversity parameters determined for 76 SRAP loci within the studied olive populations identi- fied the most variable loci. Population differentiation parameters determined for SRAP loci, identified 13 SRAP loci with Gst value of 1, that means they differentiate the stud- ied trees. PCoA analysis based on SRAP data separated olive trees from each other due to genetic difference. Distribution of the samples in PCoA plot indicated that the population 1 are more spread due to population genetic variability. However, the SRAP result reveals that these molecular markers can be used in population genetic investiga- tions and germ plasm analysis. AMOVA showed significant genetic difference among the studied olive populations. Cp-DNA analysis produced 366 bp long sequences, out of which 224 sites were segregating among the studied plants. The mean nucleotide diversity was 0.32. TCS network based on cp-DNA separated most of the studied pop- ulations. Therefore, it seems that cp-DNA rpl sequences is a suitable barcode molecular marker for population genetic studies. Phylogenetic tree of ITS data could partially dif- ferentiate wild olive population. In conclusion, a combined use of SRAPs and cp-DNA sequences are suggested for wild olive population genetic investigation. Keywords. SRAP, cp-DNA, ITS, Population genetic, Olive. INTRODUCTION Olive tree (O. europaea L.) of the genus Olea (O. europaea subsp. euro- paea var. europaea) is one of the most important horticultural crop plants. It is an ancient plant species with grate economic value (Zohary and Hopf 2000), and has both cultivated and wild forms. Oleaster (O. europaea sub- 126 Rayan Partovi et al. sp. europaea var. sylvestris Miller) is the Mediterranean wild olive and is possibly the progenitor of the culti- vated olive. The non-Mediterranean wild olives are geo- graphically isolated from the oleaster and show differ- ent morphological characters. Green (2002) grouped all morphological forms of wild olive in a single aggregate i.e. Olea europaea subsp. cuspidata, but the other inves- tigators consider these intra-specific forms as ecotypes both in Africa and Iran (Besnard et al. 2002; Sheidai et al. 2010). The occurrence of natural hybridization has been reported between different sub spices within the genus Olea. This holds true also for O. cuspidate and O. afri- cana (Besnard and Bervill 2000). Moreover, (Omrani- Sabbaghi et al. 2007) suggested hybridization of subsp. cuspidata and the cultivated olive in South Africa and Iran and (Sheidai et al. 2010) identified a population with intermediate morphological and molecular (RAP- Ds) characteristics. The wild relatives of crop plants (CWRs) constitute an important resource for improving agricultural pro- duction and for maintaining sustainable agro-ecosys- tems. Genetic material from CWRs has been utilized by humans for to improve the quality and yield of crops. For example, wild maize (Zea mexicana) is routinely grown alongside maize to promote natural crossing and improve yields. More recently, plant breeders have uti- lized CWR genes to improve a wide range of crops like rice (Oryza sativa), tomato (Solanum lycopersicum) and grain legumes (Hajjar and Hodgkin 2007). Therefore, A CWR can be defined as “a wild plant taxon that has an indirect use derived from its relatively close genetic relationship to a crop. The CWRs comprise a wonderful gene pool for future crop breeding programs. Since natural populations of CWRs are at risk and are threatened by habitat loss, deforestation, etc., popula- tion genetic study of these natural populations is impor- tant task as it provides insight about the genetic variabil- ity, population genetic structure, gene flow versus popu- lation fragmentation as well population genetic differen- tiation. The obtained information can be utilized in both breeding as well as conservation strategies of the CWRs. Recent population genetic studies use different molecular markers to investigate the genetic diversity as well as other population genetic features. This is also true for olive (Bracci et al. 2011), for example, Random Amplified Polymorphic DNA (RAPDs) (Sheidai et al. 2010) microsatellite (simple sequence repeat; SSRs) and inter simple sequence repeat; ISSR markers, Amplified Fragments Length Polymorphic markers (AFLPs) (Bal- doni et al. 2006), cp-DNA (Besnard et al. 2011). In the present study, genetic diversity, genetic diver- gence and genetic structure of four populations of O. euro- paea subsp. cuspidata from different localities are investi- gated using nrDNA ITS (Internal Transcribed Spacer) and SRAP (sequence-related amplified polymorphism) markers. Since, SRAP marker technique combines easiness, reliability, high variability, moderate throughput ratio and superficial sequencing of the selected bands, we used this technique to amplify coding regions of DNA to target open reading frames. MATERIALS AND METHODS Thirteen specimens belonging to four geographical populations of subspecies Olea europaea subsp. cuspi- data L. were collected from different localities that were placed between three provinces Bakhtiari, Boyer-Ahmad and Khuzestan. Details of geographical populations are given in Table 1. DNA extraction and PCR reactions DNA was extracted from dried leaf specimens (approximately 0.5 g material per sample) using CTAB (Cetyl trimethyl-ammonium bromide) activated char- coal protocol (Krizman et al. 2006 and Sheidai et al. 2013). Extracted DNA was run on 0.8% agarose gel. PCR reactions were carried in a 25μl volume containing 10 mM Tris-HCl buffer at pH 8; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM of each dNTP (Bioron, Germany); 0.2 μM of a single primer; 20 mg genomic DNA and 1 U of Taq DNA polymerase (Bioron, Germany). Table 1. List of 13 specimens of Olea europaea subsp. cuspidata L. from four populations accompanied by their distribution, altitude, longi- tude and herbarium number. No. Localities Altitude Latitude Longitude Voucher no. 1 Chaharmahal and Bakhtiari Province, Dehedz – Lordgan, Iran 1713 31°31’18” 50°28’26” HSBU2018700 2 Kohgiluyeh and Boyer-Ahmad Province, Khersaan Road, Iran 1380 31°26’59” 50°28’57” HSBU2018705 3 Chaharmahal and Bakhtiari Province, Lordgan, Monj, Gachahan, Iran 1151 31°26’48” 50°32’19” HSBU2018711 4 Chaharmahal and Bakhtiari Province, Lordgan, Monj, Gachahan, Iran 1592 35°55’41” 57°41’53” HSBU2018712 127Population genetic studies in wild olive (Olea cuspidata) by molecular barcodes and SRAP molecular markers SRAP study Five sequences related amplified polymorphism (SRAP) primer pairs including forward primers: Me1, Me2, Me3, Me4, Me5 and reverse primers: Em1, Em2, Em3, Em4, Em5 were used (Feng et al. 2014). PCR reac- tions were carried in a 25μl volume containing 10 mM Tris-HCl buffer at pH 8; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM of each dNTP (Bioron, Germany); 0.2 μM of a single primer; 20 ng genomic DNA and 1 U of Taq DNA polymerase (Bioron, Germany). Following programs used for amplification of SRAP region in a PCR reac- tion: 94°C, 1 min, 35°C, 1 min, and 72°C, 1 min for first five cycles then 5 min initial denaturation step 94°C, fol- lowed by 40 cycles of 1 min at 94°C; 1 min at 55°C and 2 min at 72°C and a final extension at 72°C for 7-10 mi ITS study The complete ITS region was amplified using forward ITS5 (5’- GGA AGT AAA AGTCGT AAC AAG G- 3’) and reverse primers ITS4 (5’- TCC GCT TAT TGA TAT GC- 3’) (White et al. 1990). Following program used for ampli- fication of nuclear region in a PCR reaction: 5 min initial denaturation step 94°C, followed by 40 cycles of 1 min at 94°C; 1 min at 53.5°C and 2 min at 72°C. The reaction was completed by final extension step of 7 min at 72°C. Cp- DNA study The intergenic spacer of chloroplast genome rpl16 was amplified and sequenced with universal primers fol- lowing the methodology of (Shaw et al. 2005; Timme et al. 2007). Each 20 µl of PCR tube contained 10 µl of 2x PCR buffer, 0.5 mM of each primer, 200 mM of each dNTP, 1 Unit of Taq DNA polymerase (Bioron, Germa- ny), and 1 µl of template genomic DNA at 20 ng µl-1 . The amplification reaction was performed in Techne thermocycler (Germany) with the following program: 2 min 94°C, 1 min at 94°C; 1 min at 54°C and 1min at 72°C. The reaction was completed by final extension step of 6 min at 72°C. Data Analyses SRAP bands were coded as binary characters (pres- ence = 1, absence = 0) and used for genetic diversity anal- ysis. Data obtained were analyzed for the genetic diversity parameters like, Nei’s gene diversity (H), Shannon infor- mation index (I), number of effective alleles, and percent- age of polymorphism (Weising et al. 2005; Freeland et al. 2011). Principal coordinate analyses (PCoA) were per- formed using PAST ver. 2.17 (Hammer et al. 2012). Nei’s genetic distance was used among populations. AMOVA (Analysis of molecular variance) test (with 1000 permutations) as implemented in GenAlex 6.4 (Peakall and Smouse 2006). The phylogenetic methods used to investigate the species relationships were Maximum parsimony (MP), Maximum likelihood (ML), Networking and Bayesian approaches. The ITS sequences were firstly aligned and used to test the proper nucleotide substitution model as applied in MEGA 7. Program (Tamura et al. 2012). Net- working was performed using Splits Tree 4 program (Huson and Bryant 2006) and Bayesian analysis was done using BEAST software v1.6.1 (Drummond et al. 2012a, b). RESULTS In total, 76 SRAP bands were obtained in olive trees studied. Some of these bands were common while, few bands were private in these trees. For example, SRAP bands 51, 52 and 76 occurred only in trees of population 4, while SRAP band 7 happened only in one of the trees in population 1. Similarly, SRAP band 4 was observed in the tree of population 3. Genetic diversity parameters determined for all SRAP loci within the studied olive populations identi- fied the most variable loci. The loci with highest value of gene diversity (H) and Shanon information index (I) are the most diverse SRAP loci (Table 2). The mean value obtained for H = 0.34, while I = 0.52. Population differentiation parameters determined for SRAP loci in the studied olive trees (Table 3), iden- tified the loci with highest migration/ exchange value (Nm) and also SRAP loci with the highest differentia- tion value (Gst). In total, 13 SRAP loci had Gst value = 1, that means they differentiate the studied trees. Similarly, SRAP loci with Nm>1 are considered highly migrated among the populations. PCoA analysis of the studied olive trees after 99 times permutation, based on SRAP data is presented in Figure 1. PCoA plot clearly separates olive trees of the studied populations from each other due to genetic dif- ference. Distribution of the samples in PCoA plot indi- cates that olive trees of population 1 are more spread due to within population genetic variability. However, in general, the SRAP result reveals that these molecular markers can be used in population genetic investigations and germ plasm analysis of olive. Nei, genetic distance determined among olive trees based on SRAP data (Table 4), revealed that the genetic distance among trees of the population 1 varies from 128 Rayan Partovi et al. 0.30 to 0.54, while it varies from 0.46 to 0.76 in olive trees of population 2. AMOVA showed significant genetic difference (Phipt = 0.43, P =0.01) among the studied olive populations. It also revealed that 43% of total genetic variation was due to among population genetic difference, whereas, 57% occurred due to within population genetic variability. Cp-DNA analysis We obtained 366 bp long sequences, out of which 224 sites were segregating among the studied plants. The mean nucleotide diversity (p) was 0.32. TCS network of the studied olive trees Figure. 2) separated most of the studied populations. For instance, trees of the popula- tion 2 and the population 4 were grouped together, while trees of population 1 were scattered in between these two populations. Therefore, it seems that cp-DNA (rpl16) sequences are a suitable barcode molecular marker for population genetic studies of olive. There has been no report of rpl16 sequences for the cultivates olive. Therefore, we could not compare these two forms together. Table 2. Genetic variability parameters for SRAP loci studied in olive populations. Locus Sample Size Ne H I 5 13 1.9187 0.4788 0.6718 8 13 1.9299 0.4818 0.6749 30 13 1.9683 0.4920 0.6851 36 13 1.9882 0.4970 0.6902 39 13 1.8989 0.4734 0.6663 53 13 1.9882 0.4970 0.6902 54 13 1.8943 0.4721 0.6650 55 13 1.9928 0.4982 0.6913 57 13 1.9562 0.4888 0.6819 58 13 1.9216 0.4796 0.6726 59 13 1.8943 0.4721 0.6650 64 13 1.8943 0.4721 0.6650 65 13 1.9865 0.4966 0.6898 67 13 1.9562 0.4888 0.6819 72 13 1.8943 0.4721 0.6650 Mean 13 1.5918 0.3492 0.5242 St. Dev 0.2911 0.1268 0.1506 Ne = Effective number of alleles. H = Nei’s (1973) gene diversity. I = Shannon’s Information index [Lewontin (1972)]. Table 3. Genetic differentiation parameters in the olive trees stud- ied based on SRAP loci. Locus Sample Size Ht Hs Gst Nm 2 13 0.2633 0.2381 0.0958 4.7202 3 13 0.3921 0.3145 0.1981 2.0240 4 13 0.3750 0.0000 1.0000 0.0000 7 13 0.0514 0.0472 0.0813 5.6481 10 13 0.2633 0.2381 0.0958 4.7202 13 13 0.3750 0.0000 1.0000 0.0000 14 13 0.1669 0.1162 0.3036 1.1472 17 13 0.2000 0.1746 0.1270 3.4365 22 13 0.0514 0.0472 0.0813 5.6481 23 13 0.3750 0.0000 1.0000 0.0000 24 13 0.1064 0.0873 0.1791 2.2910 28 13 0.1794 0.1508 0.1595 2.6340 31 13 0.2086 0.1634 0.2164 1.8105 32 13 0.3750 0.0000 1.0000 0.0000 34 13 0.1000 0.0944 0.0557 8.4721 36 13 0.5000 0.0000 1.0000 0.0000 39 13 0.3750 0.0000 1.0000 0.0000 41 13 0.1064 0.0873 0.1791 2.2910 43 13 0.2086 0.1634 0.2164 1.8105 44 13 0.5000 0.0000 1.0000 0.0000 45 13 0.3750 0.0000 1.0000 0.0000 47 13 0.1794 0.1508 0.1595 2.6340 48 13 0.1669 0.1162 0.3036 1.1472 50 13 0.3750 0.0000 1.0000 0.0000 51 13 0.3750 0.0000 1.0000 0.0000 52 13 0.3750 0.0000 1.0000 0.0000 53 13 0.5000 0.0000 1.0000 0.0000 58 13 0.4226 0.3434 0.1875 2.1669 62 13 0.3625 0.2744 0.2431 1.5564 68 13 0.2086 0.1634 0.2164 1.8105 69 13 0.1000 0.0944 0.0557 8.4721 75 13 0.5000 0.0000 1.0000 0.0000 76 13 0.3750 0.0000 1.0000 0.0000 Mean 13 0.3680 0.1308 0.6445 0.2758 St. Dev 0.0172 0.0085 Figure 1. PCoA plot of olive trees based on SRAP data revealing genetic separation of populations. 129Population genetic studies in wild olive (Olea cuspidata) by molecular barcodes and SRAP molecular markers ITS sequence analysis We obtained 183 bp long sequences in ITS region with 150 variable sites. The analysis revealed the pres- ence of 8 haplotypes in ITS with haplotype diversity, Hd: 0.80. An Olive tree 7, 9, 11 and 13 had similar sequences and forms a single haplotype group. The nucleotide distance (p distance) of the stud- ied trees (Table 5), revealed that p distance among olive trees of population 1 varied from 0.36 to 0.54, while the same value in population varied from 0.01 to 0.49. Maximum likelihood phylogenetic tree (ML) (Fig- ure 3) of the studied olive trees based on ITS sequences revealed that, trees of population 1, differ in their ITS sequences and were grouped in a separate clade. Howev- er, trees of populations 2, 3, and 4 were placed together in a single unresolved clade. This result indicates that ITS sequences can be used along with cp-DNA barcodes in olive population genetic studies. Comparing phylogenetic trees of SRAP markers, Cp-DNA and ITS sequences produced quart let distance = 0.56 and test performed based on the most agreeable sub-trees (MAST) (Figure. 4) revealed that, these mark- ers do differentiate some of the olive trees and place them in distinct clades. Joint phylogenetic ITS analysis of wild popula- tions and randomly selected cultivated olives (Figure. 5) revealed the genetic separation of these olives from each other. ITS sequences could differentiate different olive trees of wild populations but not the cultivars from each other. DISCUSSION Genetic structure analysis of both cultivated and wild olive is important for breeding and conservation purposes (Baldoni et al. 2006). Olive cultivars can be considered as varieties of unknown origin, currently propagated vegetative by cutting or grafting. Analysis of nuclear and cytoplasmic DNA polymorphisms in Medi- terranean oleaster populations has shown that eastern oleaster populations differ greatly from those of the west Mediterranean (Besnard et al. 2001), while the genet- Table 4. Nei genetic distance among the studied olives. Pop 1 2 3 4 5 6 7 8 9 10 11 12 2 0.30 3 0.39 0.42 4 0.44 0.46 0.33 5 0.54 0.40 0.52 0.42 6 0.65 0.54 0.57 0.62 0.40 7 0.56 0.62 0.66 0.64 0.65 0.54 8 0.76 0.70 0.81 0.77 0.71 0.44 0.69 9 0.65 0.64 0.68 0.73 0.67 0.49 0.57 0.57 10 0.60 0.53 0.60 0.68 0.50 0.41 0.48 0.55 0.43 11 0.60 0.55 0.73 0.60 0.57 0.65 0.80 0.82 0.69 0.53 12 0.41 0.43 0.57 0.62 0.63 0.60 0.72 0.65 0.56 0.65 0.67 13 0.47 0.46 0.57 0.52 0.63 0.63 0.68 0.69 0.56 0.68 0.71 0.17 Figure 2. TCS network of olive trees based on cp-DNA sequences revealing almost separation of the studied populations. 130 Rayan Partovi et al. ic diversity of cultivated populations shows a complex patchy pattern (Owen et al. 2005). The present investi- gation also revealed genetic difference between Iranian wild populations and the cultivated olive forms. Based on the frequency and distribution of poly- morphisms, several authors suggested that many olive cultivars have been produced from naturally cross-bred genotypes (Besnard et al. 2001), while, others, due to the great genetic distance between populations of wild olives and cultivars, suggested that many local cultivars may have an allochthonous origin (Angiolillo et al. 1999; Bronzini de Caraffa et al. 2002). Genetic diversity of both cultivated and wild olives has been investigated by using different molecular mark- ers (Bracci et al. 2011), revealing the genetic structure of these olive forms. In the present study, SRAP and cp-DNA rpl sequences could be used in wild olive dif- Table 5. P nucleotide distance among olive plants based on ITS sequences. 1 2 3 4 5 6 7 8 9 10 11 12 13 1 - 2 0.53 - 3 0.52 0.43 - 4 0.56 0.50 0.36 - 5 0.54 0.51 0.46 0.45 - 6 0.49 0.36 0.25 0.36 0.33 - 7 0.48 0.35 0.24 0.35 0.32 0.01 - 8 0.48 0.35 0.24 0.35 0.33 0.01 0.00 - 9 0.48 0.35 0.24 0.35 0.33 0.01 0.00 0.00 - 10 0.48 0.35 0.24 0.35 0.33 0.01 0.01 0.01 0.01 - 11 0.48 0.35 0.24 0.35 0.33 0.01 0.00 0.00 0.00 0.01 - 12 0.48 0.35 0.24 0.35 0.32 0.01 0.00 0.00 0.00 0.01 0.00 - 13 0.48 0.35 0.24 0.35 0.32 0.01 0.00 0.00 0.00 0.01 0.00 0.00 - Figure 3. ML phylogenetic tree of the studied olive trees based on ITS sequences. Figure 4. Most agreeable sub-trees (MAST) plot showing the com- mon clades differentiated by ITS, Cp-DNA and ISSR trees. 131Population genetic studies in wild olive (Olea cuspidata) by molecular barcodes and SRAP molecular markers ferentiation. Cp-DNA polymorphisms is used for phy- logeographic, population genetic and forensic analyses in plants, but detecting cp-DNA variation is some- times challenging, limiting the applications of such an approach (Besnard et al. 2011). According to our knowl- edge rpl16 sequences were only used in genetic variabil- ity assessment of tissue culture regenerated olive plants (Kangarloo et al. 2016) and not in olive population genetic investigation. Therefore, our study is the first time report on application of the cp-DNA sequences for wild olive population differentiation. Besnard et al. (2001) used eight complete sequences of cp-DNA genomes of Olea in their study. The reported low nucleotide divergence between olive cp-DNA line- ages, not exceeding 0.07%. Based on these sequences, markers were developed for studying two single nucle- otide substitutions and length polymorphism of 62 regions (with variable microsatellite motifs or other indels). They used these markers to study the cp-DNA variation in cultivated and wild Mediterranean olive trees. The discriminating power of cp-DNA variation was particularly low for the cultivated olive tree with one predominating haplotype, but more diversity was detected in wild populations. This is almost in agree- ment with present study findings. Besnard et al. (2001) and Pérez-Jiménez et al. (2013) suggested that cp-DNA markers will have applications for a comparative study of the dynamic of wild olive tree populations in different environments, such as archipelagos and Saharan moun- tains. Such information may be relevant for defining appropriate strategies of prospection and in situ conser- vation of the wild olive tree. In conclusion, the present study revealed that a com- bination of neutral molecular markers, like SRAPs and cp-DNA sequences are powerful markers to differentiate wild olive populations. 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Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 73, Issue 1 - 2020 Firenze University Press Karyotypic investigation concerning five Bromus Species from several populations in Iran Sara Sadeghian, Ahmad Hatami, Mehrnaz Riasat High genetic diversity and presence of genetic structure characterise the endemics Ruta corsica and Ruta lamarmorae (Rutaceae) Marilena Meloni1, Caterina Angela Dettori2, Andrea Reid3, Gianluigi Bacchetta2,4,*, Laetitia Hugot5, Elena Conti1 Cytogenetic effects of C6H4 (CH3)2 (xylene) on meristematic cells of root tips of Vicia faba L. and mathematical analysis Cihangir Alaca1, Ali Özdemir1, Bahattın Bozdağ2, Canan Özdemir2,* Clethodim induced pollen sterility and meiotic abnormalities in vegetable crop Pisum sativum L. Sazada Siddiqui*, Sulaiman Al-Rumman Temporal Analysis of Al-Induced Programmed Cell Death in Barley (Hordeum vulgare L.) Roots Büşra Huri Gölge, Filiz Vardar* Genetic diversity, population structure and chromosome numbers in medicinal plant species Stellaria media L. VILL. Shahram Mehri*, Hassan Shirafkanajirlou, Iman Kolbadi A new diploid cytotype of Agrimonia pilosa (Rosaceae) Elizaveta Mitrenina1, Mikhail Skaptsov2, Maksim Kutsev2, Alexander Kuznetsov1, Hiroshi Ikeda3, Andrey Erst1,4,* Study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (Ocimum basilicum L.) Irina Petrescu1, Ioan Sarac1, Elena Bonciu2, Emilian Madosa1, Catalin Aurelian Rosculete2,*, Monica Butnariu1 Chemical composition, antioxidant and cytogenotoxic effects of Ligularia sibirica (L.) Cass. roots and rhizomes extracts Nicoleta Anca Şuţan1,*, Andreea Natalia Matei1, Eliza Oprea2, Victorița Tecuceanu3, Lavinia Diana Tătaru1, Sorin Georgian Moga1, Denisa Ştefania Manolescu1, Carmen Mihaela Topală1 Phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm Bombyx mori induced by microsporidian infection Hungund P. Shambhavi1, Pooja Makwana2, Basavaraju Surendranath3, Kangayam M Ponnuvel1, Rakesh K Mishra1, Appukuttan Nair R Pradeep1,* Electrophoretic study of seed storage proteins in the genus Hypericum L. in North of Iran Parisa Mahditabar Bahnamiri1, Arman Mahmoudi Otaghvari1,*, Najme Ahmadian chashmi1, Pirouz Azizi2 Melissa officinalis: A potent herb against EMS induced mutagenicity in mice Hilal Ahmad Ganaie1,2,*, Md. Niamat Ali1, Bashir A Ganai2 Population genetic studies in wild olive (Olea cuspidata) by molecular barcodes and SRAP molecular markers Rayan Partovi1, Alireza Iaranbakhsh1,*, Masoud Sheidai2, Mostafa Ebadi3 In Vitro Polyploidy Induction in Persian Poppy (Papaver bracteatum Lindl.) Saeed Tarkesh Esfahani1, Ghasem Karimzadeh1,*, Mohammad Reza Naghavi2 Long-term Effect Different Concentrations of Zn (NO3)2 on the Development of Male and Female Gametophytes of Capsicum annuum L. var California Wonder Helal Nemat Farahzadi, Sedigheh Arbabian*, Ahamd Majd, Golnaz Tajadod A karyological study of some endemic Trigonella species (Fabaceae) in Iran Hamidreza Sharghi1,2, Majid Azizi1,*, Hamid Moazzeni2 Karyological studies in thirteen species of Zingiberacaeae from Tripura, North East India Kishan Saha*, Rabindra Kumar Sinha, Sangram Sinha