J. Jpn. Bot. 87: 193–205 (2012)

Cytogenetic and Molecular Diversity of Cirsium arvense (Asteraceae) Populations in

a, a a Masoud Sheidai *, Elnaz Seif , Maryam Nouroozi b and Zahra Noormohammadi

aShahid Beheshti University, GC, Faculty of Biological Sciences, , IRAN; bBiology Department, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Poonak, Tehran, IRAN *Corresponding author: [email protected]

(Accepted on December 28, 2011)

Cirsium arvense populations showed 2n = 2x = 34 chromosome number, forming mostly bivalents, although some amount of univalents and quadrivalents were also formed. The Ghasemloo populations showed the highest value of total and terminal chiasmata, while the Khalkhal population showed the least values of the same parameters. The Meyandoab population had the highest value of intercalary chiasmata. Meiotic abnormalities including, laggard chromosomes, stickiness, micronuclei formation, unorganized chromosomes, multipolar cell formation and desynapsis were observed almost in all the populations studied. The occurrence of large pollen grains (possibly 2n pollen grains) was observed along with smaller (normal) pollen grains in some of the populations. B-chromosome (0–1) was observed only in the Taleghan population. RAPD and ISSR primers used produced many reproducible bands. Some of the populations showed the presence of specific bands, while some bands were present in all except one population. UPGMA and NJ trees of RAPD and ISSR data grouped the populations in two different clusters/groups, indicating their genetic difference which is discussed in details.

Key words: Cirsium arvense, genetic diversity, Iran.

The genus Cirsium Mill. (Asteraceae) Caucasus and the adjoining regions of Asia contains about 250 perennial, biennial or rarely Minor (Bureš et al. 2004). About 44 Cirsium annual and spiny species (Zomlefer 1994, species have been reported in Flora Iranica Bureš et al. 2004). Growing mainly in Northern (Rechinger 1979), which have been classified Hemisphere (Europe, North Africa, Siberia, in five sections. Hybrid plants persist primarily Central and East Asia, West, East and Central through vegetative growth, forming clusters of America). flowering shoots often connected by rhizomes. The genus is considered to be complex in Because hybrids are usually fertile, they can nature partly due to high degree of hybridization often produce introgressive hybrids with the occurring among different species and also parental species or triple hybrids with other taxa because of highly variable germplasm interacting (Bureš et al. 2004). with different environmental conditions. In Limited cytological studies have been fact several hybrids have been described from performed on the genus Cirsium (Frankton

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Fig. 1. Map of Iran, showing localities of the Cirsium arvense populations studied. Population code. 1. Asara Karaj. 2. Porkan Karaj. 3. Kordan Tehran. 4. Taleghan Ghazvin. 5. Malard Karaj. 6. Shahrestanak Karaj. 7. Tafresh Markazi. 8. Markazi. 9. Kashan. 10. Ghasemloo West Azarbayejan. 11. Tabriz West Azarbayejan. 12. Khalkhal Ardebil. 13. Meyandoab West Azarbayejan. and Moore 1961, Tonian 1981a, 1981b, Tonian Different molecular markers including 1982, Dempsey 1994, Ghaffari 1999, Ozcan RAPD (Random Amplified Polymorphic et al. 2008) and only recently some cytological DNA) as well as ISSR (Intersequence Simple study concerned with chromosome pairing Repeats) have been used for studying genetic and B-chromosomes at the species level diversity and identification of hybrids different have been reported from Iran (Nouroozi et al. plant species (e.g., Chen et al. 2004, Sheidai et 2010, 2011). In these studies, some indication al. 2007, Wei et al. 2008, Tafvizei et al. 2010, of the occurrence of unreduced (2n) pollen Sheidaei et al. 2010), including Asteraceae taxa grains in other Cirsium species but not in C. (Ash et al. 2003, Dogan et al. 2007). arvense has been provided. Since C. arvense is of wide distribution in Iran and grows in Materials and Methods various environmental conditions, we planned Plant materials to study some of these populations for the Thirteen populations of Circium arvense potential of unreduced pollen grain formation (L.) Scop. were collected for cytological and and B-chromosome occurrence. Moreover the molecular studies (Fig. 1). present study reports molecular diversity of 13 populations of C. arvense growing in Iran for the Cytological studies first time. Meiotic studies were performed on young June 2012 Journal of Japanese Botany Vol. 87 No.3 195 flower buds collected using minimum 100 and primer extension for 2 min at 72ºC. A final metaphase/diakinesis pollen mother cells incubation for 10 min at 72ºC was performed (PMCs) and 500 anaphase and telophase cells to ensure that the primer extension reaction for data collection (Sheidai and Rashid 2007). proceeded to completion. Pollen stainability as a measure of fertility was The PCR amplified products were separated determined by staining minimum 1000 pollen by electrophoresis on a 2% agarose gels using grains with 2% acetocarmine: 50% glycerin 0.5 × TBE buffer (44.5 Mm Tris/Borate, 0.5 (1:1) for about 1/2 hr. Round complete pollens Mm EDTA, pH 8.0) or 6% polyacrylamide gels. which were stained were taken as fertile, while The gels were stained with ethidium bromide incomplete, shrunken pollens with no stain and visualized under UV light (Sambrook et were considered as infertile (Sheidai and Rashid al. 2001). A 100 bp DNA ladder (GeneRuler, 2007). Fermentas) was used as the molecular standard in order to confirm the appropriate RAPD RAPD analysis markers. These markers were named by primer Thirty decamer RAPD primers of Operon origin, followed with the primer number and the Technology (Alameda, Canada) belonging to size of amplified products in base pairs. OPA, OPH sets were used in this study. DNA extraction was done by using the CTAB method ISSR assay (Murry and Tompson 1980) with modification Total genomic DNA was extracted from described by De La Rosa et al. (2002). The PCR fresh leaves using the CTAB method by Murry reaction mixture consisted of 1 ng template and Tompson (1980) with the modification DNA, 1 × PCR buffer (10 mM Tris-HCL pH described by De la Rosa et al. (2002). Six 8.8, 250 mM KCL), 200 µM dNTPs, 0.80 ISSR primers used are (GA)9T, UBC810, µM 10-base random primers and 1 unit of Taq UBC811, UBC834, UBC849 and CA7GT polymerase, in a total volume of 25 µL. DNA commercialized by UBC (the University amplification was performed on a palm cycler of British Columbia). PCR reactions were GP-001 (Corbet, Australia). Template DNA was performed in a 25 µL volume containing 10 initially denatured at 92ºC for 3 min, followed mM Tris–HCl buffer at pH 8; 50 mM KCl; 1.5 by 35 cycles of PCR amplification under the mM MgCl2; 0.2 mM of each dNTP; 0.2 µM of following parameters: denaturation for 1 min a single primer; 20 ng genomic DNA and 3 unit at 920C, primer annealing for 1 min at 36ºC of Taq DNA polymerase (Bioron, Germany).

Table 1. Cytogenetic characters in the Cirsium arvense populations studied Locality 2n ROD RB I IV IX TX TOX B Taleghan 34 2.08 7.80 5.50 0.80 0.88 20.86 21.75 0–1 Tabriz 34 3.30 5.90 4.90 1.38 1.9 20.20 22.10 0 Ghasemloo 34 3.80 9.50 2.60 0.50 1.8 24.87 26.60 0 Tafresh 34 3.93 8.76 2.26 0.86 2.23 24.40 26.56 0 Mahallat 34 2.32 11.24 3.44 0.04 1.44 24.96 26.40 0 Khalkhal 34 4.23 6.73 5.80 0.13 1.60 18.23 19.90 0 Meyandoab 34 3.70 9.10 3.60 0.28 2.60 22.60 25.35 0 Abbreviations. ROD. Mean of rod bivalents. RB. Mean of ring bivalents. I. Mean of univalents. IV. Mean of quadrivalents. IX. Mean of intercalary chiasmata. TX. Mean of terminal chiasmata. TOX. Mean of total chiasmata. B. B-chromosomes. 196 植物研究雑誌 第 87 巻 第 3 号 2012 年 6 月

Fig. 2. Representative meiotic cells in the Cirsium arvense populations. A–C. Metaphase cells in the Tabriz, Tafresh and Meyandoab populations. D, E. Metaphase cells showing chromosomes stickiness in the Khalkhal and Tabriz populations. F, G. Meiocytes showing complete desynapsis in the Khalkhal and Tafresh populations. H–J. Multipolar cells in the Tafresh, Khalkhal and Mahallat populations. K, L. Potential unreduced pollen grains (larger size pollen grains) in the Khalkhal and Meyandoab populations. Scale bar = 10 µm.

Amplifications reactions were performed in Data analysis Techne thermocycler (Germany) with following In order to determine significant difference program: 5 min initial denaturation step 94°C, in meiotic characteristics, paired sample χ2 30 s at 94°C; 1 min at 50°C, 1 min at 72°C. test was performed. Principal components The reaction was completed by final extension analysis (PCA) was performed to show meiotic step of 7 min at 72°C. Amplification products differences among the populations studied. were visualized by running on 2% agarose gel, RAPD and ISSR bands obtained were treated following ethidium bromide staining. Fragment as binary characters and coded accordingly size was estimated by using a 100 base pairs (bp) (presence =1, absence = 0). Jaccard similarity molecular size ladder (Fermentas, Germany). as well as Nei’s genetic distance (Nei 1973) was determined among populations studied June 2012 Journal of Japanese Botany Vol. 87 No.3 197 and used for grouping of the genotypes by metaphase I stickiness. UPGMA (Unweighted Paired Group with Similarly the Mahallat populations showed Arithmetic Average) and NJ (Neighbor-Joining) the highest percentage of metaphase II cells clustering methods and ordination based on showing chromosome stickiness (20%), principal coordinate analysis (PCO) and PCA followed by the Taleghan population (15%). The (Podani 2000, Weising et al. 2005). NTSYS Tabriz population was the only one not showing Ver. 2.02 (1998) and DARwin ver. 5 (2008) metaphase II stickiness. Complete chromosome was used for clustering and PCO analyses. In clumping was mostly observed in the Taleghan, order to determine molecular difference between Ghasemloo and Tafresh populations. populations, AMOVA test was performed. For Different degrees of univalent formation test of agreement between RAPD and ISSR trees to complete desynapsis (all univalents) were obtained, Mantel test was performed (Podani observed in the populations studied (Fig. 2, F, 2000) by GENALEX 6 (Peakall and Smouse G). Chromosomes stickiness during anaphase 2006). I and II leading to the formation of anaphase bridges in some of the cases were observed Results in. The number and thickness of these bridges Cytology varied among different meiocytes. Laggard Cirsium arvense populations showed 2n = chromosomes of different numbers occurred 2x = 34 chromosome number (Fig. 2, A–C), in Taleghan and Mahallat populations. These supporting Nouroozi et al. (2010, 2011). All laggards may be the reason for micronuclei populations formed mostly bivalents, although formation in telophase I and II. some amount of univalents and quadrivalents Anaphase abnormalities leading to the were also formed (Table 1). formation of multipolar cells were observed The Ghasemloo populations showed the in all populations (Fig. 1, H–J). The highest highest value of total and terminal chiasmata percentage of triads occurred in the Ghasemloo (26.60 and 24.87, respectively), while the population followed by the Taleghan population. Khalkhal population showed the least values This latter population also showed the highest of the same parameters (19.90 and 18.23, percentage of pentads and hexads. χ2 test showed respectively). The Meyandoab population had significant difference for meiotic abnormalities the highest value of intercalary chiasmata (2.60). among the populations studied. Various meiotic abnormalities including, The occurrence of large pollen grains laggard chromosomes, stickiness, micronuclei (possibly 2n pollen grains) was observed along formation, unorganized chromosomes, with smaller (normal) pollen grains in five multipolar cell formation and desynapsis were populations of Taleghan, Tabriz, Ghasemloo, observed almost in all populations studied (Table Tafresh and Meyandoab (Fig. 1, K, L), all 1, Fig. 2, D–L). showing triad formation (Fig. 1, H, J). The large Chromosomes stickiness occurred in both pollen grains comprised about 1% of pollen metaphase of meiosis I and II. This occurred grains in these populations. B-chromosome (0– between two bivalents or more, forming 1) was observed only in the Taleghan population. complete clump in some of the cases (Fig. 1, Chi square test showed that the Khalkhal D, E). The highest percentage of metaphase population has significantly lower chiasma I cells showing chromosome stickiness was frequency compared to that of the Tafresh, observed in Tabriz population (42%), followed Mahallat and Ghasemloo populations (χ2 = by Meyandoab population (25%). Taleghan 14.70, p<0.01). Significant difference was population was the only one not showing observed for meiotic abnormalities among 198 植物研究雑誌 第 87 巻 第 3 号 2012 年 6 月

Fig. 3. PCA plot of meiotic data in the Cirsium arvense populations. populations studied too. The primer OPC-08 produced 6 specific PCA plot showed cytogenetic differences bands, primer OPC-10 produced 5 specific of the populations studied (Fig. 3). Two bands, while some other primers produced 2 and populations of Taleghan and Mahallat are placed 3 specific bands and the primers OPR-08, OPI-1 in different positions in the PCA plot, far from and OPC-05 produced 1 specific band. the other populations. Similarly Tabriz and Some of the populations showed the presence Khalkhal are placed far from the others. of specific bands, for example only Kordan population had band 1400 bp of RAPD primer RAPD analysis OPI-12, band 1100 bp of the primer OPC-05 and Out of 20 RAPD primers used, 13 primers band 1300 bp of the primer OPC-10. produced reproducible bands (Fig. 4). In total The Tabriz population showed specific bands 178 RAPD bands (loci) were obtained out of of 400 bp of the primer OPR-08, while Asara which only 8 bands ranging in size from 500– population was the only population having band 2000 bp were common in all 13 populations 700 bp of the primer OPM-10, band 900 bp studied and 180 bands were polymorph. of the primer OPC-08 and 300 bp of OPA-02. Among the primers used OPC-08 produced Kashan population had the band of 500 bp of the the highest number of bands (23), while primers primer OPA-02. OPI-12 produced the lowest number of bands Some bands were present in all except one (10). The Primer OPC-08 also produced the population, for example bands 690 bp of the highest number of polymorphic bands (23). primer OPM-10 and 550 bp of OPC-05 were June 2012 Journal of Japanese Botany Vol. 87 No.3 199

Fig. 4. OPA04 RAPD primer profile of the Cirsium arvense populations. Abbreviations. L. Ladder. 1. Asara. 2. Porkan. 3. Kordan. 4. Taleghan. 5. Malard. 6. Shahrestanak. 7. Tafresh. 8. Ghasemloo. 9. Mahallat. 10. Tabriz. 11. Kashan. 12. Khalkhal. 13. Meyandoab. N. No DNA.

Fig. 5. NJ tree of RAPD data in the Cirsium arvense populations. absent only in Taleghan population, the bands therefore it is discussed bellow. 550 bp of the primer OPC-05, bands 650 bp In general two major clusters were obtained. of the primers OPC-08 and OP1-12 were only The populations of Asara, Shahrestanak, Porkan, absent in Kashan population. Malard, Kordan and Taleghan formed the UPGMA and NJ dendrograms of RAPD first major cluster, out of which, Kordan and data produced similar results supported by PCO Taleghan show more RAPD similarity and the ordination plot (Figs. 5, 6). The Cophenetic Asara population differ the most from the other correlation of NJ tree was higher (r = 0.98) and populations. 200 植物研究雑誌 第 87 巻 第 3 号 2012 年 6 月

Fig. 6. PCA plot of RAPD data in the Cirsium arvense populations.

Seven other populations form the second ISSR analysis major cluster, including Tafresh, Ghasemloo, The six ISSR primers used in this experiment Tabriz, Meyandoab, Mahallat, Kashan and produced 83 reproducible bands (Fig. 7), out Khalkhal. Out of which, Kashan and Khalkhal of which 80 bands were polymorph and 3 and also Ghasemloo and Tabriz show more bands were monomorph. The highest number similarity in RAPD while the Tafresh population of polymorphic bands was obtained for the differ the most from the other populations. ISSR primer CA7G (20), while ISSR primers PCA plot reveals RAPD molecular diversity UBC811 produced only 9 polymorphic bands. of the populations in more details (Fig. 6). The In total 16 specific bands were obtained, Porkan population stands far from the other with ISSR primer (GA)9T producing the highest populations, to which the Asara population joins No. (5) and primer UBC811 producing only 1 with some distance. Meyandoab stands far in the specific band. Some of the populations showed other side of the plot, to which the Khalkhal and the presence of specific ISSR band/locus, for Tabriz populations are joined. example, Shahrestanak population was the only June 2012 Journal of Japanese Botany Vol. 87 No.3 201

Fig. 7. CA7GT ISSR profile of the Cirsium arvense populations. Abbreviations: N. No DNA. 1. Meyandoab. 2. Khalkhal. 3. Kashan. 4. Tabriz. 5. Mahallat. 6. Ghasemloo. 7. Tafresh. 8. Shahrestanak. 9. Malard. 10. Taleghan. 11. Kordan. 12. Porkan. 13. Asara. L. Ladder.

Fig. 8. NJ tree of ISSR data in the Cirsium arvense populations. population having ISSR band 2100 bp of the primer UBC810. The Porkan population was primer UBC849, band 1500 bp of the primer the only one showing ISSR band 2300 bp of (GA)9T and bands 2000 and 5000 bp of the the primer UBC810, bands 760, 1100 and 200 202 植物研究雑誌 第 87 巻 第 3 号 2012 年 6 月

Fig. 9. PCA plot of ISSR data in the Cirsium arvense populations. bp of the primer CA7GT, band 110 bp of the the other populations. primer (GA)9T. The Kordan population was the UPGMA and NJ dendrograms of ISSR data only population showing bands 2300 and 5000 produced similar results supported by PCO bp of the primer UBC810 and only Taleghan ordination plot. The Cophenetic correlation population had the band 600 bp of the same of NJ tree was higher (r = 0.96), therefore it is primer. discussed bellow (Figs. 8, 9). Some ISSR bands were present in all In general two major clusters were obtained, populations except one, for example, ISSR band separating Porkan, Asara, Kordan, Shahrestanak, 300 bp of the primer UBC810 was present in all Taleghan and Malard in one cluster and the populations except in the Malard and Kashan other populations in the second major cluster. population did not have the band 600 bp of the Kordan and Shahrestanak in the first cluster and primer (GA)9T, which was present in the other Ghasemloo and Mahallat in the second cluster, populations. Meyandoab population did not have show more similarity in ISSR characteristics. the bands 700 and 850 bp of the primer CA7GT, Mantel test performed between RAPD and while the Malard population did not show the ISSR trees showed t = 0.03, p = 0.37, showing band 300 bp of the primer UBC810 occurring in agreement between the two trees obtained. June 2012 Journal of Japanese Botany Vol. 87 No.3 203

AMOVA test performed between populations genotypes. placed in the two major clusters in both RAPD As stated before, there were bands which and ISSR markers showed significant difference occurred in some of the populations but were (p<0.002) for ISSR with 14% of variance absent in the others. Since, even single base between two groups and 86% within populations change at the primer annealing site is manifested in each group, and (p<0.001) for RAPD with as appearance or disappearance of RAPD and 21% of variance between the two groups and ISSR bands, these bands may indicate the 79% within populations. occurrence of genetic changes in the genome of the populations through the loss or rearrangement Discussion of some of their nucleotides. Chromosomal Cytology crossing over during meiosis may result in loss All the populations studied showed 2n = 2x of primer attachment pair sites in the offspring = 34 chromosome indicating that no polyploid leading to novel RAPD pattern in the offspring population we could encounter. The populations (Smith et al. 1996). We also observed significant studied differed in meiotic behavior and chiasma change in chiasma frequency and distribution as frequency. These results indicate a change in well as heterozygote translocations in C. arvense meiotic control over chromosome pairing and populations which may be responsible for chiasma formation during species diversification. RAPD/ISSR diversity. Variation in chiasma frequency and localization Both RAPD and ISSR trees obtained are in is genetically controlled (Quicke 1993) and has general agreement separating the populations been reported in several plant species as well studied in two groups, indicating their genetic as in crop plant varieties (Rees and Dale 1974, difference, which is also partly supported by Rees and Jones 1977). cytogenetic analysis. It is interesting to say that All the populations studied although populations of Asara, Porkan, Kordan, Taleghan, are diploid, shows that the occurrence of Malard and Shahrestanak are placed in three quadrivalents as a result of heterozygote neighbor provinces of Tehran, Ghazvin and translocation. Such heterozygote translocations Alborze, which form the first group/cluster in lead to the formation of new linkage groups. RAPD and ISSR analyses, while, populations Therefore both mechanisms of a change in of Tafresh and Mahallat (), chiasma frequency and the occurrence of Ghasemloo and Meyandoab (West Azarbayejan), heterozygote translocations in the C. arvense Tabriz (East Azarbayejan, Khalkhal (Ardebil) populations with the same chromosome number and Kashan (Isfahan) which comprise the may be a mean for generating new forms of second group/cluster are located in much farther recombination influencing the variability within distance from the populations of first cluster/ natural populations in an adaptive way (Rees group. Therefore we can suggest that different and Dale 1974). populations have gained different genetic variations which may be used in populations Molecular analyses adaptations as also discussed in cytogenetic The presence of RAPD and ISSR section before. polymorphic bands in the populations studied Therefore, the present study shows indicates the presence of genetic polymorphism cytogenetic and molecular diversity available in these genotypes. Moreover the occurrence in the C. arvense populations which is possibly of specific bands/loci only in some of the related to their geographical distribution and populations illustrates the occurrence of unique adaptive nature of these changes, however, insertion/deletion in DNA material of these whether these genetic diversity bring about 204 植物研究雑誌 第 87 巻 第 3 号 2012 年 6 月 morphological changes in the populations Cardueae) taxa from Turkey. Caryologia 61: 375–382. studied and if we can recognize any intraspecific Peakall R. and Smouse P. E. 2006. 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a a a b M. Sheidai , E. Seif , M. Nouroozi , Z. Noormohammadi： イランにおけるセイヨウトゲアザミ（キク科）の細胞学 的及び分子的多様性 ての集団に見られた．大花粉粒（非減数と推定される） イラン産セイヨウトゲアザミ Cirsium arvense (L.) はより小型の（尋常な）花粉粒と共にいくつかの集団で Scop.（キク科）の 13 集団を調べた結果，染色体数が 観察された．セイヨウトゲアザミにおける大花粉粒の存 2n = 2x = 34 で，一価染色体や四価染色体も一部ある 在は初めての報告となる．B 染色体は Taleghan 集団に ものの，大部分が二価染色体をもつことが分かった． のみ見られた．抽出した DNA の RAPD と ISSR バン Ghasemloo 集団は高頻度の全体そして端部キアズマを ディングパターン解析の結果，調べた 13 集団に遺伝的 示した．一方，Khalkhal 集団はキアズマ形成では最低 に異なる二つのクラスターが認められた． の頻度を示した．Meyandoab 集団は介在キアズマの頻 （a イラン・Shahid Beheshti 大学生物科学部， 度が最も高かった．減数分裂における異常は，調べた全 b イラン・Islamic Azad 大学基礎科学部生物部門）