Table Of ContentJ. Jpn. Bot. 87: 193–205 (2012)
Cytogenetic and Molecular Diversity of Cirsium arvense
(Asteraceae) Populations in Iran
Masoud sheidai a, *, Elnaz seiF a, Maryam nouroozi a
and Zahra noormohammadib
aShahid Beheshti University, GC, Faculty of Biological Sciences, Tehran, 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
—193—
194 植物研究雑誌 第87巻 第3号 2012年6月
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. Mahallat 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) T, UBC810,
9
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 MgCl ; 0.2 mM of each dNTP; 0.2 µM of
2
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) T producing the highest
9
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) T and bands 2000 and 5000 bp of the the primer UBC810, bands 760, 1100 and 200
9
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) T. The Kordan population was the UPGMA and NJ dendrograms of ISSR data
9
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) T, which was present in the other Ghasemloo and Mahallat in the second cluster,
9
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.
