Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(1): 77-87, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1355 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Yinan Liu, Jiaqing Wang, Hongling Kang (2022) Random Amplified Polymorphic DNA profiling in detecting genetic variation in Malva L. species: edible and medicinal plants. Caryolo- gia 75(1): 77-87. doi: 10.36253/caryolo- gia-1355 Received: July 2, 2021 Accepted: August 17, 2021 Published: July 6, 2022 Copyright: © 2022 Yinan Liu, Jiaqing Wang, Hongling Kang. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, 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. Random Amplified Polymorphic DNA profiling in detecting genetic variation in Malva L. species: edible and medicinal plants Yinan Liu*, Jiaqing Wang, Hongling Kang 1 School of Life Engineering, Shenyang Institute of Technology, Fushun 113122, Liaoning, China *Corresponding author. E-mail: liuyinan@situ.edu.cn Abstract. Malva L. (mallow) is the genus within the Malvaceae Juss. family, which includes twenty- five-forty. species and several hybrids. This genus contains herbaceous annual, biennial, and perennial species that are native to regions of Africa, Asia, and Europe. Malva species contain a lot of mucilage, malvin, flavonoids, terpenoids, poly- saccharides, and vitamin. No detailed Random Amplified Polymorphic DNA (RAPD) studies were conducted to study Malva genetic diversity. Therefore, we collected and analyzed seven species from seven provinces of Iran regions. Overall, eighty-five plant specimens were collected. We showed significant differences in quantitative morpho- logical characters in plant species. Malva verticillata L. depicted unbiased expected heterozygosity (UHe) in the range of 0.053. Shannon information was high (0.67) in Malva parviflora L. Malva vericillata showed the lowest value, 0.083. The observed number of alleles (Na) ranged from 1.16 to 2.33 in Malva vericillata and Malva parvi- flora. The effective number of alleles (Ne) was in the range of 1.078-1.922 for Malva vericillata and Malva parviflora.Gene flow (Nm) was relatively low (0.63) in Malva. The Mantel test showed correlation (r = 0.76, p=0.0001) between genetic and geographical distances. We reported high genetic diversity, which clearly shows the Malva species can adapt to changing environments since high genetic diversity is linked to species adaptability. Present results highlighted the utility of RAPD markers and morphometry methods to investigate genetic diversity in Malva species.Our aims were 1) to assess genetic diversity among Malva species 2) is there a correlation between species genetic and geographical distance? 3) Genetic structure of populations and taxa. Keywords: population structure, gene flow, random amplified polymorphic DNA (RAPD), Malva species, network. INTRODUCTION The use of medicinal plants can be influenced by the economic condi- tion, the high cost of medicines and the difficult access to public consulta- tions. In addition to that, there is a difficulty of access by residents in rural areas to health care units located in urban areas. Moreover, the increase the trend for considering traditional knowledge that supports using natural 78 Yinan Liu, Jiaqing Wang, Hongling Kang resources as an alternative to synthetic drugs (Battisti et al., 2013). Malvaceae Juss. (‘the mallows’) is a botanical fam- ily with a rich diversity of species for textile, medicinal, and ornamental purposes. It consists of 4465 species and about 245 genera (Tate et al., 2005) and mallows pre- sent a cosmopolitan distribution, but with a high num- ber of species in the tropics. The principle economic use of Malvaceae plants is as a source of natural fibers, the family providing perhaps the worlds three most impor- tant fiber crops plants of the family are also used for food, beverages, timber, in traditional medicine and in horticulture (la Duke and Dobley, 1995; Erbano et al. 2015; Frankham 2005; Ellegren and Galtier 2016; Tur- chetto et al. 2016). Many researches have been published on the ecology, taxonomy, genetic, cytology, chemotax- onomy, physiology, seed germination and economic uses of family Malvaceae such as (El-Rjoob and Omari 2009) in ecology; in taxonomy (Tate et al., 2005), in chemotax- onomy (Blunden et al., 2001; Gomez et al. 2005; Cires et al. 2013, Esfandani-Bozchaloyi et al. 2018a, 2018b, 2018c, 2018d) and in genetic researches (Baum et al., 2004) studied the pollen. The Malva genus has 25-40 species and it can be considered as an annual and/or biannual herb. Flow- ers with an epicalyx and 8-15 reticulated mericarps are the typical one (Fryxell, 1988; DellaGreca, et al., 2009). In medicine, mallow species are used in the treatment of respiratory, urinary, and digestive problems as they have high bactericidal, antiulcerogenic, anti-inflamma- tory, hepatoprotective, and antidiabetic activities (Pan- dey et al, 2012). The Malva genus is morphologically very diverse, but some species are hardly distinguish- able based on morphological features (Escobar et al., 2009). Several studies have been conducted to clarify the taxonomic affiliation of Malva species using differ- ent features, such as molecular data (nuclear ribosomal DNA (rDNA), internal transcribed spacer (ITS) region, intron–exon splice junction (ISJ), and inter simple sequence repeat polymerase chain reaction (ISSR) mark- ers) (Celka, et al., 2010), differentiation of seed and seed coat structure (El Naggar, 2001), morphology of pollen grains (El Naggar, 2004), epidermal structures and stem hairs (Akçin, and Özbucak, 2006), and plant morpholog- ical traits (Michael et al., 2009). The variability in mallow species is due, at least in part, to hybridization. Natural crossings between Malva pusilla Sm. and Malva neglecta Wallr., Malva alcea L., and Malva moschata L. as well as Malva sylvestris L. and Malva neglecta were found in Europe. Ray (1995) stated that hybridization or polyploidy is probably a factor in the evolution of these species, but this aspect has not been investigated so far. The taxonomy and systematics of the Malva genus are still unclear and very compli- cated. Taxonomic doubts have appeared because of the high level of homoplasty in morphological traits that are usually used as diagnostic features (Escobar García, et al.,2009). Based on the flower structure, Dalby (1968) divided the Malva genus into two sections: Bismalva (with Malva alcea, Malva excisa Rchb., and Malva mos- chata) and Malva (Malva neglecta, Malva pusilla, Malva sylvestris, and Malva verticillata) A different classifica- tion based on ITS molecular markers as well as fruit morphology and seed structure was reported by Ray (1995), and two groups were distinguished: malvoid and lavateroid. A similar division was proposed by Escobar Garcia et al. (2009) based on five ITS molecular mark- ers (matK plus trnK, ndhF, trnL-trnF, and psbA-trnH). These genetic relationships and the classification of Mal- va species were also confirmed by Celka et al. (2010) and Lo Bianco et al. (2017) based on ITS and ISSR molecular markers along with seed image analysis. Genetic diver- sity studies are usually tapped due to molecular markers. Molecular markers are an excellent method to disentan- gle phylogenetic association between species and popu- lation. Among molecular methods or markers, RAPD (Random Amplified Polymorphic DNA) are sensitive to detect variability among individuals of species. RAPD method is cost-effective and can work with limited sam- ple quantities. In addition to this, RAPD can amplify and target genomic regions with potential and several markers (Esfandani-Bozchaloyi et al. 2017). Taxonomical systematics studies were conducted in the past to identify the Malva species. According to the best of our knowledge, there is no existing RAPD data on genetic diversity investigations in Iran. We stud- ied seventy samples. Our aims were 1) to assess genetic diversity among Malva species 2) is there a correlation between species and geographical distance? 3) Genetic structure of populations and taxa 4) Are the Malva spe- cies able to exchange genes? MATERIALS AND METHODS Plant materials Seven Malva species were collected from differ- ent regions of Iran (Table 1). These species were studied via morphological and molecular methods. Eighty-five plant samples (nine-fifteen per plant species) were exam- ined for morphometry purposes (Figure 1). The random amplified polymorphic DNA analysis method was lim- ited to eighty-five samples. We focused on the following species Malva neglecta Wallr., Malva pusilla Sm., Malva 79Random Amplified Polymorphic DNA profiling in detecting genetic variation in Malva L. species Table 1. List of the investigated taxa including origin of voucher specimens. Taxa Locality Latitude Longitude Malva neglecta Wallr. West Azerbaijan, Kaleybar 38°5’46.4604” 46°16’23” Malva parviflora L. Hormozgan, Bandar Abbas 27°33’12” 56°44’16” Malva pusilla Sm. Khuzestan, Behbahan 30°17’01” 50°54’10” Malva sylvestris L. Esfahan, Ardestan on road to Taleghan 32°15’44” 51°16’33” Malva verticillata L. Kerman, Hamun-e Jaz Murian 27°10’13” 58°33’19” Malva nicaeensis All. Mazandaran, 40 km Tonekabon to janat abad 35°10’16” 51°55’18” Malva aegyptia L. Golestan, Gorgan 35°13’19” 52°10’31” Figure 1. Presence of species in different regions of Iran. 80 Yinan Liu, Jiaqing Wang, Hongling Kang sylvestris L., Malva verticillata L., Malva nicaeensis All., Malva aegyptia L. and Malva parviflora L. According to previous references, all the species were identified (Esco- bar García, et al., 2009; Ray, 1995 ). Morphometry In total thirty-eight morphological (ten qualitative, twenty-eight quantitative) characters were studied .̀ Five to ten plant specimens were randomly studied or mor- phological analyses. Data were transformed (Mean= 0, variance = 1) prior to ordination . Euclidean distance was implemented to cluster and ordinate plant species (Podani 2000). Random Amplified Polymorphic DNA We extracted DNA from fresh leaves. Leaves were dried. DNA extraction was carried out according to the previous protocol (Esfandani-Bozchaloyi et al. 2019). DNA quality was checked on an agarose gel to con- firm the purity. We amplified the DNA with the aid of RAPD primers (Operon technology, Alameda, Canada). These primers belonged to OPA, OPB, OPC, OPD sets. We selected those primers (10) which could show clear bands and polymorphism (Table 2). Overall, the poly- merase chain reaction contained 25μl volume. This 25 volume had ten mM Tris-HCl buffer, 500 mM KCl; 1.5 mM MgCl2; 0.2 mM of each dNTP; 0.2 μM of a single primer; 20 ng genomic DNA and 3 U of Taq DNA poly- merase (Bioron, Germany). We observed the following cycles and conditions for the amplification. Five minutes initial denaturation step was carried out at 94°C after this forty cycles of 1 minute at 94°C were observed. Then 1-minute cycle was at 52-57°C followed by two minutes at 72°C. In the end, the final extension step was per- formed for seven to ten minutes at 72°C. We confirmed the amplification steps while observing amplified prod- ucts on a gel. Each band size was confirmed according to 100 base pair molecular ladder/standard (Fermentas, Germany). Data analyses We used an Unweighted pair group method with arithmetic mean (UPGMA) and Ward methods. Ordi- nation methods such as multidimensional scaling and principa l coordinate ana lysis were a lso performed (Podani 2000). The morphological difference among species and population was assessed through analy- sis of variance (ANOVA). PCA analysis (Podani 2000) was done to find the variation in plant population morphological traits. Multivariate and all the neces- sary calculations were done in the PAST software, 2.17 (Hammer et al. 2001). To assess genetic diversity, we encoded RAPD bands as present and absent. Numbers 1 and 0 were used to show the presence and absence of bands. It is essential to know the polymorphism information content and marker index (MI) of prim- ers because these parameters ser ve to obser ve poly- morphic loci in genotypes (Ismail et al. 2019). Marker index was calculated according to the previous proto- col (Heikrujam et al. 2015). Other parameters such as the number of polymorphic bands (NPB) and effec- tive multiplex ratio (EMR) were assessed. Gene diver- Table 2. RAPD primers and other parameters. Note: TNB - the number of total bands, NPB: the number of polymorphic bands, PPB (%): the percentage of polymorphic bands, PI: polymorphism index, EMR, effective multiplex ratio; MI, marker index; PIC, polymorphism information content for each of CBDP primers. Primer name Primer sequence (5’-3’) TNB NPB PPB PIC PI EMR MI OPA-05 5’-AGGGGTCTTG-3’ 13 12 92.31% 0.54 8.21 10.23 4.55 OPA-06 5’-GGTCCCTGAC-3’ 17 17 100.00% 0.47 7.32 11.55 4.18 OPB-01 5’-GTTTCGCTCC-3’ 11 9 96.89% 0.43 6.56 9.34 7.17 OPB-02 5’-TGATCCCTGG-3’ 13 12 95.81% 0.34 4.21 6.60 5.59 OPC-04 5’-CCGCATCTAC-3’ 12 12 100.00% 0.47 3.37 9.55 3.25 OPD-02 5’-GGACCCAACC-3’ 11 11 100.00% 0.56 4.86 11.88 3.45 OPD-03 5’-GTCGCCGTCA-3’ 9 7 84.99% 0.43 3.51 8.43 3.85 OPD-05 5’-TGAGCGGACA-3’ 15 13 93.84% 0.66 4.66 11.33 4.67 OPD-08 5’-GTGTGCCCCA-3’ 12 11 94.91% 0.48 5.21 12.50 5.65 OPD-11 5’-AGCGCCATTG-3’ 14 13 95.74% 0.67 5.66 9.57 5.37 Mean 12.7 11.7 95.88% 0.55 5.5 9.4 4.8 Total 127 117 81Random Amplified Polymorphic DNA profiling in detecting genetic variation in Malva L. species sity associated characteristics of plant samples were calculated. These characteristics include Nei ’s gene diversity (H), Shannon information index (I), number of effective alleles (Ne), and percentage of polymor- phism (P% = number of polymorphic loci/number of total loci) (Shen et al. 2017). Unbiased expected het- erozygosity (UHe), and heterozygosity were assessed in GenAlEx 6.4 software (Peakall and Smouse 2006). Neighbor-joining (NJ) and networking were studied to fathom genetic distance plant populations (Huson and Br yant 2006; Freeland et al. 2011). The Mantel test was carried out to find the correlation between genetic and geographical distances (Podani 2000). As we were interested in knowing the genetic structure and diversity, we also investigated the genetic differ- ence between populations through AMOVA (Analysis of molecular variance) in GenAlEx 6.4 (Peakall and Smouse 2006). Furthermore, gene flow (Nm) was esti- mated through Genetic statistics (GST) in PopGene ver. 1.32 (Yeh et al. 1999). We also did STRUCTURE anal- ysis to detect an optimum number of groups. For this purpose, the Evanno test was conducted (Evanno et al. 2005). First data were scored as dominant markers (ISSR) so we used from STRUCTURE analysis for esti- mate the parameters that related to gene flow among studied population. Burn-in = 10000, and 10 runs were performed for relationship between Genetic structure and distance of geographical. Ma ximum likelihood method and Bayesian Information Criterion (BIC) was studied by structure analysis (Falush et al. 2007; Evan- no et al. 2005; Meirmans 2012). RESULTS Morphometry Significant ANOVA results (P <0.01) showed differ- ences in quantitative morphological characters in plant species. Principal component results explained 67% variation. First component of PCA demonstrated 49% of the total variation. Leaf morphology and traits such as calyx length, calyx width positively correlated with corolla length, corolla color (>0.7). The second and third components explained floral characters such as corolla apex, seed length and number of segment stem leaves. Unweighted pair group method with arithmetic mean (UPGMA) and principal coordinate analysis (PCoA) plots showed symmetrical results (Figure 2, Figure 3). Generally, plant specimens belonging to different spe- cies were separated from each other due to differences in morphology. Morphological characters divided Malva species into two groups, as evident in the UPGMA tree (Figure 2). Populations belonging to Malva aegyptia were in the first group. On the other hand, the second group consisted of two sub-groups. Malva pusilla and Malva verticillata formed the first sub-group. Malva neglecta, Malva sylvestris, Malva parvif lora, Malva nicaeensis formed the second sub-group. These groups and sub- groups were formed due to morphological differences among the individuals of Malva. Our PCoA results also confirmed the application of morphological charac- ters in separating and clustering the species in separate groups (Figure 3). Identical results were also reported in the UPGMA tree (Figure 2). Species identification and genetic diversity The primers, i.e., OPC-04, OPB-01, OPA-05 and OPD-11 could amplify plant (Malva species) DNA (Fig- ure 4). 119 polymorphic bands were generated and amplified. Amplified products ranged from 100 to 3000 bp. We recorded the highest polymorphic bands for OPA-06. OPD-03 had the lowest polymorphic bands. The average polymorphic bands ranged to 11.9 for each primer. The polymorphic information content (PIC) had Figure 2. UPGMA clusters of morphological characters revealing species delimitation in Malva species. 82 Yinan Liu, Jiaqing Wang, Hongling Kang values in the range of 0.34 (OPB-02) to 0.67 (OPD- 011). Primers had 0.55 average polymorphic information con- tent values. Marker index (MI) values were 3.25 (OPC-04) to 7.17 (OPB-01), with an average of 4.8 per primer. Effec- tive multiplex ratio (EMR) values are useful to distin- guish genotypes. In our study, we reported 6.60 (OPB- 02) to 12.50 (OPD-08) EMR values. EMR values aver- aged 9.4 per primer (Table 2). All the necessary genetic features calculated of seven Malva species are shown (Table 3). Malva verticillata depicted unbiased expected heterozygosity (UHe) in the range of 0.053. Shannon information was high (0.67) in Malva parviflora. Malva verticillata showed the lowest value, 0.083. The observed Figure 3. PCoA plot morphological characters revealing species delimitation in Malva species. Figure 4. Gel Electrophoresis image of DNA fragments of Malva species. L = Ladder 100 bp. Arrows show polymorphic bands.1,8,15,22: Malva neglecta 2,9,16,23: Malva parviflora 3,10,17,24: Malva pusilla. 4,11,18,25: Malva sylvestris 5,12,19,26: Malva verticillata 6,13,20,27: Malva nicaeensis7,14,21,28: Malva aegyptia. 83Random Amplified Polymorphic DNA profiling in detecting genetic variation in Malva L. species number of alleles (Na) ranged from 1.16 to 2.33 in Malva verticillata and Malva parviflora. The effective number of alleles (Ne) was in the range of 1.078-1.922 for Malva veritcillata and Malva parviflora. Gene flow (Nm) was relatively low (0.63) in Malva. Analysis of Molecular Variance (AMOVA) test high- lighted genetic differences among Malva species (P = 0.001). AMOVA showed that 50% of genetic variation was among the species. Relative less variation (12%) was reported within the species (Table 4). Genetic similar- ity and dissimilarity assessed through Genetic statis- tics (GST) showed significant differences i.e., (0.567, P = 0.001) and D_est values (0.876, p = 0.001). The neighbor-joining tree and MDS plot of Mal- va populations based on RAPD data produced similar results therefore only neighbor-joining tree is presented and discussed (Fig. 5). NJ net tree revealed that the sev- en species are well differentiated on the genetic grounds. In both UPGMA and NJ trees, samples of the Malva aegyptia were placed far from each other. Malva pusilla was placed close to Malva verticillata , and far from Mal- va aegyptia . In both analyses, Malva nicaeensis showed closer affinity with Malva sylvestris, Malva parviflora. Genetic distance of the two subsp. was estimated to be 1.66 by Kimura 2p distance. Gene flow (Nm) was relatively low (0.63) in Malva species. Genetic identity and phylogenetic distance in the Rindera members are mentioned (Table 5). Malva verticillata and Malva nicaeensis were genetically closely related (0.856) to each other. Malva nicaeensis and Mal- va aegyptia were dissimilar due to low (0.694) genetic similarity. The mantel test showed correlation (r = 0.76, p=0.0001) between genetic and geographical distances. The Evanno test showed ΔK =6 (Figure 6). Figure 6, showed the genetic details of the Malva species. Accord- ing to STRUCTURE analysis Malva pusilla and Malva aegyptia were closely related to common alleles (Figure 6). The rest of the Malva species are genetically differen- tiated due to different allelic structures (Figure 6). The neighbor-joining plot also showed the same result. Lim- ited gene flow results were supported by K-Means and STRUCTURE analyses too. We could not identify sub- stantial gene flow among the Malva species. This result is in agreement with grouping we obtained with Neigh- bor- joining (Figure 5), as these populations were placed close to each other. As evidenced by STRUCTURE plot based on admixture model, these shared alleles comprise Table 3. Genetic diversity variables of Malva (N = number of sam- ples, Na= number of different alleles, Ne = number of effective alleles, I= Shannon’s information index, He = gene diversity, UHe = unbiased gene diversity, P%= percentage of polymorphism in popu- lations). Taxon N Na Ne I He UHe %P Malva neglecta 10.000 1.500 1.311 0.279 0.267 0.187 50.00% Malva parviflora 9.000 2.333 1.922 0.670 0.333 0.417 83.33% Malva pusilla 12.000 1.500 1.441 0.330 0.233 0.233 50.00% Malva sylvestris 13.000 1.333 1.232 0.196 0.200 0.133 33.33% Malva verticillata 10.000 1.167 1.078 0.083 0.150 0.053 16.67% Malva nicaeensis 15.000 1.200 1.462 0.337 0.290 0.240 50.00% Malva aegyptia 15.000 1.433 1.196 0.150 0.183 0.090 19.67% Table 4. Analysis of molecular variance (AMOVA) of the studied species. Source Df SS MS Est. Var % Among Regions 5 42.297 12.648 0.337 20% Among Pops 15 96.827 8.802 0.774 50% Among Indiv 59 64.383 2.130 0.363 18% Within Indiv 65 14.500 0.204 0.204 12% Total 133 215.007 1.678 100% df: degree of freedom; SS: sum of squared observations; MS: mean of squared observations; EV: estimated variance. Figure 5. Integer NJ net tree produced while using RAPD data. Table 5. The Nei genetic similarity (Gs) estimates using RAPD markers. pop1 pop2 pop3 pop4 pop5 pop6 pop7 1.000 pop1 0.766 1.000 pop2 0.760 0.764 1.000 pop3 0.750 0.730 0.827 1.000 pop4 0.774 0.797 0.762 0.794 1.000 pop5 0.733 0.770 0.727 0.707 0.856 1.000 pop6 0.679 0.722 0.750 0.704 0.719 0.698 1.000 pop7 84 Yinan Liu, Jiaqing Wang, Hongling Kang very limited part of the genomes in these populations and all these results are in agreement in showing high degree of genetic stratification within Malva popula- tions. DISCUSSION The Malva is a relatively complex taxonomic group, and several morphological characters make it difficult to identify and classify Malva species (Ray 1995; Escobar García, et al., 2009). Given the complexity, it is necessary to explore other methods that could complement the traditional taxonomical approach (Erbano et al. 2015). Advent and developments in molecular techniques have enabled plant taxonomists to utilize molecular proto- cols to study plant groups (Erbano et al. 2015; Abeshu & Zewdu 2020.; Amar et al 2021; Beltran et al. 2021). We examined genetic diversity in Malva by morphologi- cal and molecular methods (Das et al 2021; Gutierrez- Pacheco et al 2021; Hindersah et al 2021; Jordaan & Rooyen et al 2021). We mainly used RAPD markers to investigate genetic diversity and genetic affinity in Mal- va. Our clustering and ordination techniques showed similar patterns. Morphometry results clearly showed the utilization or significance of morphological charac- ters in Malva species. PCoA plot results also confirmed the application of morphological characters to sepa- rate Malva species. The present study also highlighted that morphological characters such as corolla color, leaf shape, leaf length, stamens position, leaf margin and corolla lenght could delimit the Malva group. The Malva species highlighted morphological differences. We argue that such a dissimilarity was due to differences in quan- titative and qualitative traits. In our study, morphology and genetic diversity in seven taxa of Malva species are given in detail for the first time. The aim of the present study was to find diag- nostic features to separate species of Malva in Iran. Mor- phological characters are considered as an useful tool for the identification of the species, as indicated previously Ray (1995). Malvaceous germplasm has been variously investi- gated by different molecular marker techniques but the earlier studies either focused on the comparison of the Malvaceae with other families in the order Malvales or to explore the genetic relationships and diversity within and among population and limited number of species in the same genus. Very little attention has been given to the analysis at interspecific and intergeneric levels. La Duke and Dobley (1995) has the only worth mentioning work in this regard. Their results showed that, the genetic relation- ships and diversity within and between 12 malvaceous species belonging to five genera are investigated by using the Amplified fragment length polymorphism (AFLP). Shaheen et al., (2009) with used AFLP (Amplified fragment length polymorphism) marker to explore phe- netic relationships and diversity within and between 13 Malvaceae species belonging to 5 different genera. Their primary objective of the study was to evaluate the taxo- nomic potential, usefulness and applicability of AFLP marker system to reconstruct genetic relationships at interspecific and intergeneric level in Malvaceae. Two primer pairs produced a total of 73 bands, of which 70 were polymorphic. According to Celka et al (2010) two categories of DNA markers were used to determine genetic relation- ships among eight Malva taxa. A maximum parsimony analysis validated the division of the genus Malva into the sections Bismalva and Malva. The species classified into those sections formed separate clusters. Malva mos- chata was a distinctive species in the section Bismalva, as confirmed by previous genetic research based on ITS and cpDNA sequence analyses. The applied markers Figure 6. STRUCTURE plot of RAPD data in Malva populations studied. 1. Malva neglecta; 2. Malva parviflora; 3. Malva pusilla; 4. Malva sylvestris; 5. Malva verticillata; 6. Malva nicaeensis; 7. Malva aegyptia. 85Random Amplified Polymorphic DNA profiling in detecting genetic variation in Malva L. species revealed a very high level of genetic identity between Malva alcea and Malva excisa and enabled molecular identification of M. alcea var. fastigiata. Jedrzejczyk and Rewers (2020) applied flow cytom- etry and inter simple sequence repeat polymerase chain reaction (ISSR-PCR) for fast and accurate species identi- fication. Genome size estimation by flow cytometry was proposed as the first-choice method for quick accession screening. Out of the 12 tested accessions, it was possi- ble to identify six genotypes based on genome size esti- mation, whereas all species and varieties were identified using ISSR markers. Flow cytometric analyses revealed that Malva species possessed very small (1.45–2.77 pg/2C), small (2.81–3.80 pg/2C), and intermediate (11.06 pg/2C) genomes, but the majority of accessions pos- sessed very small genomes. The relationships between the investigated accessions showed the presence of two clus- ters representing malvoid and lavateroid group of spe- cies. Their results showed that Flow cytometry and ISSR molecular markers can be effectively used in the identifi- cation and genetic characterization of Malva species. Until now, molecular studies using ISSR markers conducted in the Malva genus have only included a few species (Celka, et al., 2012). All primers used in ISSR- PCRs for the Malva genus revealed 100% polymorphism between all accessions. Therefore, it was possible to iden- tify all tested species. Moreover, for Malva verticillata taxon, it was possible to distinguish all studied varieties. The usefulness of most of the used ISSR primers was also confirmed in Ocimum L., Origanum L. and Men- tha L. identification (Lo Bianco, et al., 2017). The sys- tematics of the Malva genus and closely related genera is complicated. Moreover, the relationships obtained from molecular studies do not confirm traditional classifica- tion (Escobar García, et al., 2009). So far, only molecular analysis relying on rDNA ITS sequences and ISSR mark- ers have shed light on taxonomical relationships between Malva species (Escobar García, et al., 2009). Phylogenetic analyses of rDNA ITS sequences indicated the presence of two well-supported clusters within the mallow species (malvoid and lavateroid clades), which is consistent with the presented data. Molecular markers (R APD) and morphometr y analysis were useful to study genetic diversity and pop- ulation structure in Malva species identification. All the species had distinct genetic differentiation. Present results highlighted isolation and limited gene flow are the main deterministic factors that shape the Malva population. 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