Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(1): 3-10, 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-863 Citation: S. Sadeghian, A. Hatami, M. Riasat (2020) Karyotypic investigation concerning five Bromus Species from several populations in Iran. Caryolo- gia 73(1): 3-10. doi: 10.13128/caryolo- gia-863 Received: April, 2019 Accepted: February, 2020 Published: May 8, 2020 Copyright: © 2020 S. Sadeghian, A. Hatami, M. Riasat. This is an open access, peer-reviewed article pub- lished 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. Karyotypic investigation concerning five Bromus Species from several populations in Iran Sara Sadeghian, Ahmad Hatami, Mehrnaz Riasat Research Division of Natural Resources Department, Fars Agricultural and Natural Resources Research and Education Center, AREEO, Shiraz, Iran *Corresponding author: s.sadeghian@areeo.ac.ir Abstract. Karyotypes of five taxa (fourteen populations) of the genus Bromus from different geographic origins is presented: B. scoparius, B. japonicus, B. madritensis, B. rubens and B. tomentellus. The ploidy levels were different. B. scoparius and B. japoni- cus were found 2n=2x=14, B. madritensis and B. rubens were found 2n=4x=28 and B. tomentellus were found 2n=6x=42. Detailed karyotype analysis allows us to group the different populations and to postulate relationships among them. Keywords. Bromus, Chromosome, Karyology, Iran. INTRODUCTION The genus Bromus L. belongs tribe Bromeae and Poaceae family. The taxon includes about 160 annual and perennial species (Acedo and Liamas, 2001) distributed all over the world. Bromus species are distributed in tem- perate regions and are always exist with rangeland species (Verloove, 2012). It is an important rangeland plant species in Iran, which are placed in 6 sec- tions; Bromus, Genea, Nevskiella, Neobromus, Ceratochla and Pnigma (Bor, 1970) (Table 1). The Genea section is the widest section of the Bromus genus in terms of geographic distribution (sales, 1994). Bromus species are known as the species with various intra-specific ploidy levels and form different ecotypes with various characteristics. Hill (1965) recorded up to 112 chromosomes for B. erectus. Devesa et al. (1990) indicates the importance of cytological studies for understanding the evolu- tion of the genus Bromus. Naganowas ka (1993) used genetic distances esti- mated based on centromeric index and total chromosome length to investi- gate interrelationships of several species of Bromus. Yang and Dunn (1997) recorded various levels of polyploidy in B. inermis Leyss. Martinello and Schifino-Wittmann (2003) studied 14 accessions of Bromus auleticus. Their accessions were all hexaploid and the high symmetry and homogeneity of the karyotypes made it difficult to detect possible intraspecific differences. Massa et al. (2004) proposed a taxonomic treatment within Bromus sect. Ceratochloa of South America. Their plant materials included 28 hexaploid 4 Sara Sadeghian, Ahmad Hatami, Mehrnaz Riasat (2n=6x=42) populations and 2 octaploid (2n=8x=56) populations. Oja and Laarmann (2002) also recorded different ploidy levels within species of Bromus (2n=14, 28, 42 and 56). Sheidai and Fadaei (2005) studied ten populations of six Bromus species and the species pos- sess karyotypes varying from 2n = 2x = 14 (diploid) to 2n = 4x = 28 (tetraploid). Mirzaie-Nodoushan et al. (2006a) investigated kary- otypic of some Bromus species in Iran and indicated that populations of the species were differed in their karyo- typic characteristics and ploidy levels of the populations were varied from 2n=14 to 2n=84. Mirzaie-Nodoushan et al. (2006b) also reported evolutionary karyotypic variation in B. tomentellus populations in Iran and confirmed the existence of high levels of ploidy as well as existence of dodecaploid karyotypes in the species. Sadeghian and et al. (2010) studied nine populations of three Bromus species (B. danthoniae, B. sterilis and B. tectorum) and reported that all species were diploid with 2n=2x=14. Artico et al. (2017) also reported that the chromosomal number of B. Linnaeus was 2n = 6x = 42. Since the karyological information is the basic requirement of a breeding program, in this study, 14 populations of Bromus were surveyed for the karyologi- cal data as a part of an ongoing work on the populations. Cytogenetic studies play an important role in deter- mining the relationship between species especially wild and native plants and as a first step in the analysis of the phylogeny and evolution of species is relative. Con- sidering that the species studied in different climates of southwest Iran and Fars province are abundant they are considered as the main vegetation cover of these areas. Therefore, to investigate the relationship between spe- cies, these species have been used in this study. MATERIALS AND METHODS Fourteen populations of five Bromus species: B. tomentellus (three populations) belong to Pnigma sec- tion, B. madritensis (two populations) and B. rubens (three populations) belong to Genea section and B. sco- parius (three population) and B. japonicus (three popu- lation) belong to Bromus section were studied (Table 1). Voucher specimens were deposited in the Herbarium of Fars Research and Education Center for Agriculture and Natural Resources and in gene bank RIFR (Research Institute of Forest and Rangelands) of Iran. Root tip meristems from seedling obtained by the germination of ripe seeds collected from natural popu- lations (14 populations, representing 5 species) on wet filter paper in Petri dishes and left at 22°C temperature. When they reached 1-1.5 cm in length, rootlets were col- lected. The material was pretreated in %0.5 saturated α-Bromo naphthalene at 4°C for 4 h, fixed in %10 for- maldehyde and chromium trioxide (1:1 volume ratio) for 16 to 20 h at 4°C. Then, the roots tips were rinsed for 3 h in distilled water. Hydrolysis was carried out with NaOH (1 Normal) at 60ºC for 20-30 min (Sadeghian et al. 2010)) and used hematoxylin-iron for chromosome staining for 1-2 h. Squashed in a droplet of %45 acetic acid and lactic acid (10:1) (Wittmann 1965). At least, five well-spread metaphase plates from different individu- als were analyzed per population. The best metaphasical Table 1. The origin of materials used in chromosome studies of Bromus. Species (population) Section Origin Altitude Herbarium code B. japonicus (16462) Bromus Golestan, Maraveh tapeh, station 430 m 16462 B. japonicus (16525) Bromus Golestan, Gomayshan, seidabad -15 m 16525 B. japonicus (16587) Bromus Golestan, Tooskasetan 1216 m 16587 B. madritensis (3668) Genea Fars, Shiraz, Dasht-e Arjan 2000 m 3668 B. madritensis (Arjan) Genea Fars, Shiraz rosd of Dasht-e Arjan to Tang- e Abolhayat, about Kandee village 1300 m - B. rubens (15169) Genea Fars, Kazeroon, kotal dokhtar 1400 m 15169 B. rubens (15317) Genea Fars, Fasa, Mianjangal 1750 m 15317 B. rubens (2125) Genea Fars, Kazeroon 530 m 2125 B. scoparius (5983) Bromus Gilan, Talesh, subatan yelagh 1800 m 5983 B. scoparius (5984) Bromus Gilan, Talesh, khotbesara, lapehkara 1800 m 5984 B. scoparius (5985) Bromus Gilan, Masal 1900 m 5985 B. tomentellus (Bavanat) Pnigma Fars, Bavanat, Simakan, Lakposhti range 2300 m - B. tomentellus (Simakan) Pnigma Fars, Simakan, Lakposhti range 2350 m - B. tomentellus (Eghlid) Pnigma Fars, Eghlid, Dozkord, Pasahlaki 2200 m - 5Karyotypic investigation concerning five Bromus Species from several populations in Iran plates were selected and measured by Micromeasure 3.3 software (Reeves et al. 2000). In each mitotic metaphase (at least 5 plates) the arm’s length of each chromosome was measured. The following parameters were estimated in each metaphase plate to characterize the karyotypes numeri- cally: long arm (LA), short arm (SA), total length (TL), relative length percentage (RL %), arm ratio (AR), cen- tromeric index (CI) (Huziwara, 1962), value of relative chromatin (VRC). Karyotype asymmetry was estimated by three different methods namely, total form percentage (TF %) (Huziwara, 1962); difference of relative length (DRL), intra-chromosomal asymmetry index (A1) and inter-chromosomal asymmetry index (A2). Both indices (A1 and A2) (Romero Zarco, 1986) were independent to chromosome number and size. Also karyotypic evolu- tion has been determined using the symmetry classes of Stebbins (SC) (Stebbins, 1971). Karyotype formula was determined by chromosome morphology based on centromere position according to classification of Levan (Levan et al. 1964). For each population, karyo- grams were drawn based on length of chromosome size (arranged large to small). In order to determine the variation between popu- lations, one-way unbalanced ANOVA was performed on normal data and parameter means were compared by Duncan’s test. The principal components analysis (PCA) was performed to evaluate the contribution of each karyotypic parameter to the ordination of species. Clus- tering was performed using the unweighted pair group method with arithmetic (UPGMA) after calculation of Cophenetic correlation coefficient (r) to examine karyo- type similarity among populations. Numerical analysis was performed using SAS ver. 6.12 (1996), JMP ver. 3.1.2 (1995) and StatistiXL ver. 1.7 (2007) softwares. RESULTS There was no different among basis chromosome number of the species (x=7). The somatic chromosome numbers (2n), karyotype formula and parameters for the studied species are summarized in Table 2. Two species as B. scoparius and B. japonicus were diploid, two spe- cies as B. madritensis and B. rubens were tetraploid and one species as B. tomentellus was hexaploid. The studied species included metacentric (m) and sub-metacentric (sm) chromosomes regarding the chro- mosomal types (Table 2). Satellites were observed in one chromosomes pair in B. scoparius and B. japonicus and two chromosomes pairs in B. madritensis and B. rubens and for B. tomentellus species which has three chromo- somes pairs having satellites (Fig 1). According to the Stebbin’s bilateral table, populations of B. rubens (15317) included the highest value regarding the intra-chromo- somal asymmetry index (0.288) and was classified as group 1B and population of B. japonicus (16462) includ- ed the lowest value regarding the intra-chromosomal asymmetry index (0.180) and was classified as group 1A. The results of analysis of variance indicated that there was a significant difference (P≤1%) between the populations in terms of chromosomal traits (TL, LA, Table 2. Karyotypic characters of different Bromus taxa and population. Taxon (population) 2n A1 A2 %TF DRL VRC SC K.F. B. japonicus (16462( 2x=14 0.180 0.129 45.174 5.369 9.605 1A 12m+2sm B. japonicus (16525( 2x=14 0.197 0.145 44.700 6.131 8.704 1A 14m B. japonicus (16587) 2x=14 0.250 0.144 42.950 6.193 8.249 1A 14m B. madritensis (3668) 4x=28 0.206 0.213 43.022 4.353 5.358 1A 28m B. madritensis (Arjan) 4x=28 0.238 0.199 41.988 4.757 5.329 1A 28m B. rubens (15169) 4x=28 0.252 0.241 41.486 5.583 6.114 1B 28m B. rubens (15317) 4x=28 0.288 0.256 38.797 5.332 6.013 1B 28m B. rubens (2125) 4x=28 0.257 0.230 39.904 5.389 6.390 1B 24m+4sm B. scoparius (5983) 2x=14 0.233 0.116 42.493 4.722 8.166 1A 14m B. scoparius (5984) 2x=14 0.227 0.124 42.113 5.722 7.929 1A 12m+2sm B. scoparius (5985) 2x=14 0.214 0.126 44.062 5.479 6.866 1A 14m B. tomentellus (Bavanat) 6x=42 0.215 0.110 42.523 1.698 6.271 1A 38m+4sm B. tomentellus (Simakan) 6x=42 0.204 0.122 44.354 2.278 6.894 1A 42m B. tomentellus (Eghlid) 6x=42 0.225 0.140 41.278 2.146 6.878 1A 42m 2n: Diploid chromosome numbers A1: intrachromosome asymmetry index, A2: interchromosome asymmetry index, TF%: total form percent- age, DRL: difference of relative length, VRC: value of relative chromatin, symmetry classes (SC) of Stebbins and karyotype formula (K.F.). 6 Sara Sadeghian, Ahmad Hatami, Mehrnaz Riasat a d g j m b c e f h i n k l Figure 1. Representative mitotic plates of Bromus – (a) B. scoparius (5983), 2n=2x=14, (b) B. scoparius (5984), 2n=2x=14, (c) B. scopari- us (5985) 2n=2x=14, (d) B. japonicus (16462), 2n=2x=14, (e) B. japonicus (16525), 2n=2x=14, (f ) B. japonicus (16587), 2n=2x=14, (g) B. rubens (15169), 2n-4x=28, (h) b. rubens (15317), 2n=4x=28, (i) B. rubens (2125), 2n=4x=28, (j) B. tomentellus (Bavanat), 2n=6x=42, (k) B. tomentellus (Simakan), 2n=6x=42, (l) B. tomentellus (Eghlid), 2n=6x=42, (m) B. madritensis (3668). 2n=4x=28, (n) B. madritensis (Arjan), 2n=4x=28. 7Karyotypic investigation concerning five Bromus Species from several populations in Iran SA) which revealed large variations among the germ- plasms in regard to studied traits. Symmetry type of Stebbins (1971) and asymmetry indices of Romero-Zarco (1986) are given in Table 2. Difference in the relative length percentage (DRL) of the highest and the smallest chromosomes varied from 6.19 in B. japonicus (16587) to 1.69 in B. tomentellus (Bavanat). According to Table 2, B. rubens (15317) was placed in 1B and had the highest values of intra- chro- mosomal asymmetry index. Similarly, high DRL value leads to more changes in the construction of chromo- somes. It had the lowest TF%. The TF% and A1 values had inverse ratio (Table 2). The mean value of chromosome’s long arm was varied from 5.27 in B. japonicus (16462) to 2.92 in B. madritensis (3668). Averages of chromosome’s short arm were different from 2.24 in B. madritensis (Arjan) to 4.34 in B. japonicus (16462). The total length of the chromosome was varied from 9.61 in B. japonicus (16462) to 5.17 in B. madritensis (Arjan) and the mean value of chromosome’s arm ratio was in range from 1.41 in B. rubens (15317) to 1.22 in B. japonicus (16462) (Table 3). The results showed that the highest VRC amongst all populations was obtained for B. japonicus (16462) and the lowest was obtained for B. madritensis (Arjan). Based on intra-chromosomal asymmetry, some popula- tions had the most asymmetrical and evolutionary kar- yotype. According to inter-chromosomal asymmetry, B. rubens (15317) had the most asymmetrical karyotype in all of the populations. The ratio of long arm/short arm chromosomes (AR) showed a high significant difference among some species belong to different sections, while other species are not clearly distinct (Table 3). Diploid species of B. japonicus (16462) for instance, had the lowest AR value (1.22), the highest TF% value (45.17) and the lowest A1 value (0.18), exhibiting the most symmetrically karyotypes, while B. rubens (15317) with the highest AR value (1.41), the low- est TF% value (38.80) and the highest A1 value (0.29) were introduced as the most asymmetrical karyotypes (Table 3). The pattern of variation of A1 and A2 values has been compared with the pattern of Stebbins’ system. The statistical comparison based on completely ran- domized design showed that there were significant dif- ferences among the populations for TL, LA and SA traits (P ≤ %1) (Table 4). The principal component analysis (PCA) of the kar- yotypic parameter shows the first two principal compo- nents account for 0.81% of total variance. Component one (0.59%) put emphasized on the A1, A2 and DRL. While component two (0.23%) accentuates, chromosome total length, long arm length, short arm length and TF% values which had the highest coefficients of Eigen vec- tors (Table 5). The diagram of the population’s dispersion, based on two first components showed that the populations separated in four groups, which completely fits with the results obtained through the average grouping analysis method (Fig. 2). The dendrogram obtained from the cytogenetic studies of 14 populations of Bromus indicated the for- Table 3. Mean of chromosomes analysis of Bromus population. Populations TL LA SA AR CI DRL %TF A1 A2 B. japonicus (16462) 9.61 5.27 4.34 1.22 0.46 5.37 45.17 0.18 0.13 B. japonicus (16525) 8.71 4.82 3.90 1.24 0.45 6.13 44.70 0.20 0.15 B. japonicus (16587) 8.24 4.71 3.55 1.32 0.43 6.19 42.95 0.25 0.14 B. madritensis (3668) 5.23 2.92 2.31 1.27 0.44 4.35 43.03 0.21 0.21 B. madritensis (Arjan) 5.17 2.94 2.24 1.32 0.43 4.76 41.99 0.24 0.20 B. rubens (15169) 5.91 3.38 2.54 1.33 0.42 5.58 41.49 0.25 0.24 B. rubens (15317) 5.62 3.29 2.34 1.41 0.41 5.33 38.80 0.29 0.26 B. rubens (2125) 5.97 3.42 2.55 1.35 0.42 5.39 39.90 0.26 0.23 B. scoparius (5983) 8.02 4.55 3.48 1.31 0.43 4.72 42.49 0.23 0.12 B. scoparius (5984) 7.62 4.29 3.34 1.29 0.43 5.72 42.11 0.23 0.12 B. scoparius (5985) 6.87 3.84 3.03 1.27 0.44 5.48 44.06 0.22 0.13 B. tomentellus (Bavanat) 6.08 3.42 2.67 1.28 0.43 1.69 42.52 0.22 0.11 B. tomentellus (Simakan) 6.90 3.84 3.06 1.26 0.44 2.28 44.35 0.20 0.12 B. tomentellus (Eghlid) 6.52 3.68 2.84 1.30 0.43 2.15 41.28 0.22 0.14 TL: total length of chromosome, LA: long arm, SA: short arm, AR: arm ratio, CI: centromeric index, DRL: difference of relative length, TF%: total form percentage, A1: intra-chromosome asymmetry index, A2: inter-chromosome asymmetry index. 8 Sara Sadeghian, Ahmad Hatami, Mehrnaz Riasat mation of five clusters in the Euclidean distance of 0.05. The first cluster consists of the B. tomentellus popula- tions (Simakan, Eghlid and Bavanat). The populations of Bavanat and Eghlid showed the most kinship, and the population of Simakan is located in a relatively short distance to these two populations. The second cluster consists of B. rubens (15317) and B. rubens (2125) popu- lations which showed a close kinship. The third cluster consists of the populations B. madritensis (3668, Arjan) and B. rubens (15169), the two populations of 3668 and Arjan are closely related to each other, with a relatively short distance from the population of 15169. The popu- lations of B. scoparius (5983, 5984, 5985) and B. japoni- cus (16587) formed the fourth cluster. The populations of 5983 and 5984 are located close to each other with a rel- atively short distance from the population of 5985. The population of 5985 is located in a longer distance than the three mentioned populations. The two populations of B. japonicus (16462 and 16525) with a short distance from each other, formed the fifth cluster (Fig. 3). DISCUSSION This study reveals a detailed picture of the chro- mosome features in five Bromus species of Iran. The knowledge of chromosome numbers, karyotype evolu- tion, ploidy level and genome size can provide additional information that not only gives further insight into the functioning of the genome, but also have considerable predictive powers. In this genus, the basic chromosome number is x=7, as were found for fourteen populations of five species of Bromus (2n=2x=14, 2n=4x=28 and 2n=6x=42). This study confirmed that the Bromus species show great var- iations in the number of chromosomes. At the interspe- cific level, quantitative and qualitative data allowed us the differentiation of several of the taxa studied. Among species, the most variable characters were the number of “m”, and “sm” chromosomes, as well as the number and position of satellites (Table 2; Fig. 1). As a result, the spe- cies also could be differentiated by the number, type and position of satellites. This study revealed that three populations of B. rubens and B. madritensis were tetraploid (2n=4x=28) species (Table 2 and Fig. 1). This is in agreement with the results of an investigation recorded by Sheidai and Fadaei (2005). Three populations of B. tomentellus was the only hexaploid (2n=6x=42), but Mirzaie-Nodoushan et al. Table 4. The results of variance analysis for karyotypic data based on CRD design. S.O.V D.F TL Mean of squares LA SA AR CI DRL TF A1 A2 Populations 13 11.50** 2.677** 2.032** 0.014** 0.001** 10.383** 9.7ns 0.004ns 440.55** Error 56 0.50 0.131 0.115 0.009 4.64E-04 3.987 6.869 0.002 86.161 %C.V. 2.57 0.61 0.48 0.01 5.51E-04 5.19 7.40 2.57E-03 152.93 **: Significant at 1%. Table 5. Specific values of variance percentage and coefficients of specific vectors in analysing main components. Name of traits First component Second component SA 0.93 0.32 LA 0.87 0.44 TL 0.90 0.38 AR 0.05- 0.72 CI 0.89 -0.27 A1 0.82- 0.52 A2 -0.77 0.22 DRL 0.09 0.79 TF 0.89 -0.23 Specific values 5.28 2.07 Percentage of Variance 0.59 0.23 Cum Percentage of Variance 0.59 0.81 Princ1 P ri n c2 5.02.50.0-2.5-5.0 4 3 2 1 0 -1 -2 B.t. Eghlid B.t. Simak anB.t. Bav anat B.s. 5985 B.s. 5984 B.s. 5983 B.r. 2125 B.r. 15317 B.r. 15169 B.m. A rjan B.m. 3668 B.j. 16587 B.j. 16525 B.j. 16462 Score Plot Figure 2. Scatter plot of 14 populations for the first two principals. 9Karyotypic investigation concerning five Bromus Species from several populations in Iran (2006b) recorded different ploidy levels within this spe- cies (2n=42, 70 & 84). Three populations of B. japonicus and B. scoparius species studied were diploid (2n=2x=14), supporting the earlier report of Safari et al. (2017). The present study confirmed that the Bromus species show great variations in the number of chromosomes both at inter and intra-specific levels. This kind of genetic and cytogenetic variability can confer an adaptive advantage against variable climate and other ecological elements in the region (Mirzaie-Nodoushan et al. 2006a). The Duncan’s test applied to the chromosome mor- phometric traits (LA, SA, TL, AR, DRL, TF%, A1 and A2) showed a highly significant difference among all examined populations of different sections (Table 3). The study revealed cytogenetic differences (P≥1%) in ANO- VA for karyological data as well as the ratio of long arms to short arms among populations. So these results indi- cate a significant quantitative change in amount of chro- matin in Bromus species diversification (Tables 2 and 4). Considering the changes of intrachromosome asym- metry index (A1) among diploid and tetraploid spe- cies, the lowest value exists in the diploid (B. japonicus, 16462) and the highest value exists in the tetraploid spe- cies (B. rubens, 15317) (Table 2). The results of analysis of variance showed that except for A1 and TF%, there was a significant difference (P≤1%) between genotypes in terms of chromosomal traits (LA, SA, TL, AR, DRL and A2) which indicated large variations among the germplasms in regard to studied traits. Cluster analysis based on chromosomal charac- teristics separated, the fourteen investigated popula- tions of Bromus species into two major groups consist- ent of statistical analysis of chromosome morphometric traits (Fig. 3). The first group has eight populations of Bromus species (B. madritensis, B.rubens and B. tomen- tellus) which are tetraploid and hexaploid (2n=4x=28 & 2n=6x=42) and belong to Genea and Pnigma sec- tions. The second group has six populations of Bromus species (B. scoparius and B. japonicus) that are diploid (2n=2x=14) and belong to Bromus section. Cluster anal- ysis based on cytological data showed that the popula- tions with the lowest metric distance may lead to use populations in crosses for inducing the highest genetic variations (Fig.3). However, grouping of the Bromus pop- ulations based on karyotypic data, agrees with either the taxonomic treatment of the genus Bromus of the same species based on morphological characters. Figure 3. Dendogram of 14 populations of Bromus by analyzing nine karyotipic parameters using Ward ‘s cluster analysis method. 10 Sara Sadeghian, Ahmad Hatami, Mehrnaz Riasat These genomic differences could be used for breed- ing purposes. 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Mitotic instabilities in tetra- ploid, hexaploid and octaploid Bromus inermis. Can. J. Genet. Cytol., 19: 550-553. 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. 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