© 2008 The Japan Mendel Society Cytologia 73(3): 327–332, 2008
Comparative Study of Chromosome Morphology in Silybum marianum
Rasool Asghari-Zakaria1,*, Ali Reza Panahi2 and Majid Sadeghizadeh3
1 Department of Crop Production and Breeding, Faculty of Agriculture, Mohaghegh Ardabili University of Mohaghegh Ardabil, Iran 2 Department of Biology, Faculty of Science, Mohaghegh Ardabili University of Mohaghegh Ardabil, Iran 3 Department of Biology, Faculty of Science, Tarbiat Modarr,es University of Tarbiat Modarres Tehran, Iran
Received August 22, 2008; accepted November 19, 2008
Summary Karyotype and morphology of mitotic chromosomes in four populations of medicinal plant Silybum marianum collected from various geographical locations of Iran and two cultivars of this species were studied. Chromosome characteristics were measured from 10 complete metaphase cells using Micromeasure software. Results showed that S. marianum is a diploid species with 2n 2x 34 chromosomes. The karyotype consisted of six pairs of metacentric, ten pairs of submeta- centric, and one pairs of acrocentric chromosomes. One of the chromosomes (chromosome 1) had a secondary constriction on centromeric region of its short arm. Karyological characteristics of all ma- terials studied were similar to each other, however, among different genotypes some variations were observed on chromosome arm ratios and relative lengths. According to Stebbin’s asymmetry index, S. marianum is placed at 2B category.
Key words Asteracea, Cardiunea, Karyotype, Milk thistle.
Milk thistle (Silybum marianum) is a member of the Asteraceae, and is known by several other names, including Blessed milk thistle, spotted thistle, Mary’s thistle, Marian thistle, Holy thistle and variegated thistle. It is an annual or biannual herbaceous plant originally a native of Southern Eu- rope through to Asia, it is now found throughout the world (Blumenthal 1998). The seeds of this plant contain the 3-oxyflavone silymarin, an isomeric mixture of three flavonolignans (silychristin, silydianin and silybin). These compounds are of considerable pharmacological interest owing to their strong anti-hepatotoxic and hepatoprotective activity (Alikaridis et al. 2000, Wang et al. 2002). It has been shown that silymarin also acts as an anticholesterolaemic agent (Krecman et al.1998). The plant is valued for its medicinal value, but it is sometimes grown as an ornamental plant because of its unusual leaves. It is also cultivated as an oil crop for its oil bearing seeds (Goeden 1976). Milk thistle seed extracts have been used for over 2000 years in treating liver diseases, and modern research has confirmed their value. Seed extracts have been used for treating Amanita mushroom poisoning, for viral hepatitis, cirrhosis, and exposure to halogenated hydrocarbons (Bosisio et al. 1992, Gaedeke et al. 1996, Vogel et al. 1984, Wagner 1981). In conjunction with data from other approaches, chromosome information and numerical analysis based on karyotype data continues to be useful in assessing generic and tribal relationships in large and complicated families of plants (Carr et al. 1999). There are a few reports on the chro- mosome number of S. marianum (Ghaffari 1989, Kamel 2004, Mohamed 1997, Van Loon 1974). However, no published report could be found on detailed karyotype analysis of chromosomes in S. marianum. In the present study, an attempt was made to develop detailed karyotype of this species and compare it with a number of other species in the subtribe Carduinae of Asteracea family. * Corresponding author, e-mail: [email protected] 328 R. Asghari-Zakaria et al. Cytologia 73(3)
Table 1. Origin of S. marianum genotypes used in this study
Genotype Origin Latitude Altitude
G1 Population collected from Andimeshk region, Ahvaz, southwest of Iran 32°27 36 N 136 48°21 33 E G2 Population collected from Kaleibar region, Ahar, northwest of Iran 38°51 23 N 1420 47°02 10 E G3 Cn-seed cultivar obtained from gene bank of Iran — — G4 Population collected from Ghaemiyeh region, Shiraz, south of Iran 29°52 18 N 889 51°34 28 E G5 Budacalazy cultivar obtained from gene bank of Iran — — G6 Population collected from Moghan region, Ardabil, northwest of Iran 39°39 26 N 78 47°88 10 E
Materials and methods Seeds of four populations of S. marianum collected from various geographical locations of Iran and two cultivars of this species were provided by seed bank of Iran (Table 1) were used in this study. The root tips were pretreated in 0.05% solution of colchicine for 2.5 h at room temperature before they were fixed in Cromic acid-Formalin (1 : 1 v/v) at 4°C for 24 h. Staining with Hema- toxylin has been done as described earlier by Asghari-Zakaria et al. (2002). Chromosome measurements including long arm, short arm, and chromosome lengths, total length of chromosome set, arm ratio index and relative chromosome length were made from 10 en- larged well-spread metaphase cells for each population, using Micromeasure software developed by the Biology department of Colorado State University, available on Internet at http://www.colostate. edu.Depts.Biology. The nomenclature of chromosomes followed Levan et al. (1964), and chromo- somes were named as 1, 2, 3, … and 17 in descending order of length. Karyotype asymmetry was estimated using the ratio of the shortest/longest pair (R), the total form percent (TF), difference be- tween relative length of the longest and the shortest chromosomes (DRL), Relative length of the shortest chromosome (S), Stebbin’s (1971) asymmetry category, the intra-chromosomal (A1) and the inter-chromosomal asymmetry index (A2) indices (Romero Zarco 1986).
Results and discussion Mitotic chromosomes and karyotypes of the six S. marianum genotypes are shown in Fig. 1 and karyotypic characters of the 17 mitotic chromosomes are shown in Table 2. The data on kary- otype formulae, difference between relative length of the longest and the shortest chromosomes, total form percentage, relative length of the shortest chromosome, ratio of the longest to the short- est chromosome, intra-chromosomal and inter-chromosomal asymmetry indices for each genotype are also presented in Table 3. Results showed that S. marianum is a diploid species with 2n 2x 34 chromosomes. This is in agreement with finding of Kamel (2004), Mohamed (1997), Ghaffari (1989) and Van Loon (1974) who showed that the chromosome number of this species was 2n 34. The karyotype con- sisted of six metacentric, ten submetacentric, and one acrocentric chromosomes. One of the chro- mosomes (chromosome 1) had more or less large secondary constriction on centromeric region of its short arm. The SAT chromosome had minor variation in size among cells and genotypes, proba- bly due to differences in chromosome contraction. Arm ratio index of chromosomes ranged from 1.23 in chromosome 4 to 3.94 in chromosomes 9. Karyological characteristics of all materials stud- ied were similar to each other, however, some variations were observed on chromosome arm ratios and relative lengths among different genotypes. Chromosomes 2, 4, 6, 7 and 8 were metacentric, 2008 Chromosomes of S. marianum 329
Fig. 1. Somatic metaphase chromosomes and karyotypes of Silybum marianum stained with aceto-iron- hematoxilin. The chromosome 1 shows secondary constriction on its centromeric region, Scale bars 3 mm. 330 R. Asghari-Zakaria et al. Cytologia 73(3)
Table 2. The karyotype characteristics of 17 mitotic chromosomes of Silybum marianum based on the means of the genotypes.
Chromosome Relative length (%) Long arm (mm) Short arm (mm) Arm ratio index
1 8.41 0.57 1.83 0.51 1.22 0.38 1.54 0.31 2 8.04 0.27 1.59 0.37 1.31 0.35 1.24 0.13 3 7.51 0.43 1.77 0.48 0.95 0.30 1.88 0.21 4 7.25 0.59 1.44 0.39 1.19 0.38 1.23 0.08 5 7.13 0.46 1.73 0.48 0.83 0.17 2.05 0.31 6 6.68 0.44 1.40 0.33 1.04 0.35 1.40 0.19 7 6.18 0.28 1.34 0.37 0.89 0.23 1.49 0.17 8 5.99 0.46 1.29 0.38 0.86 0.21 1.50 0.18 9 5.82 0.51 1.65 0.46 0.46 0.20 3.89 1.14 10 5.34 0.44 1.19 0.23 0.70 0.14 1.72 0.14 11 5.23 0.32 1.17 0.22 0.69 0.17 1.76 0.43 12 5.07 0.48 1.14 0.25 0.66 0.15 1.79 0.71 13 4.71 0.26 1.16 0.30 0.52 0.13 2.24 0.53 14 4.53 0.14 1.14 0.29 0.47 0.11 2.43 0.48 15 4.16 0.54 0.98 0.17 0.49 0.13 2.12 0.62 16 4.14 0.40 1.01 0.21 0.45 0.07 2.24 0.36 17 3.81 0.33 0.91 0.21 0.44 0.08 2.06 0.38
: mean standard error.
Table 3. Karyotype formulae (KF), Difference between relative length of the longest and the shortest chro- mosomes (DRL), Total form percentage (TF), Relative length of the shortest chromosome (S%), Ratio of the longest to the shortest chromosome (R), the intra-chromosomal asymmetry index (A1) and the inter-chromosomal asymmetry index (A2) and Stebbin’s asymmetry category (ST) in six genotypes of Silybum marianum
Genotype KF TF DRL S% R A1 A2 Stebbins
G1 7m 9sm 1st 38.36 5.52 3.67 2.49 0.39 0.29 2B G2 7m 9sm 1st 36.49 5.08 3.45 2.48 0.43 0.27 2B G3 7m 9sm 1st 35.66 4.59 3.68 2.25 0.45 0.26 2B G4 10m 6sm 1st 38.04 4.54 3.64 2.35 0.38 0.23 2B G5 8m 8sm 1st 37.07 4.91 3.84 2.28 0.42 0.27 2B G6 5m 10sm 2st 33.87 3.51 4.34 1.81 0.49 0.18 2A
m metacentric, sm submetacentric, st acrocentric. chromosomes 3, 5, 14 and 16 were submetacentric, and chromosome 9 was acrocentric in all of genotypes. Other chromosomes showed variation in morphology over genotypes. For example, chromosome 10 was submetacentric in all of genotypes except for G3 and G5 and chromosome 12 was metacentric in all of genotypes except for G3 and G6 and so on (Table 2). The G6 genotype had two acrocentric chromosomes (chromosomes 9 and 15), but other genotypes had only one chro- mosome of this type (Table 2). According to relative length of chromosomes, some variations were also observed among genotypes. Chromosome 1 was the longest chromosome in all of the genotypes except for G6 and chromosome 17 was the smallest one in all of the genotypes except for G5 (Table 2). The rank of other chromosome differed among the genotypes probably due to differences in chromosome con- traction or structural modifications. According to asymmetry indices, the category of all genotypes is same except for G6, in which the values of difference between relative length of the longest and the shortest chromosomes, total form percentage, ratio of the longest to the shortest chromosome, and inter-chromosomal asymmetry index were smaller than other genotypes (Table 3). In general, karyotype of S. mari- 2008 Chromosomes of S. marianum 331 anum placed on 2B class of Stebbin’s (1971) asymmetry categories. Silybum belongs to the Carduinae subtribe of Cardueae tribe in Asteraceae family. Some 1600 species in about 36 genera as Arctium, Carduus, Cirsium, Notobasis, Onopordum and Silybum are placed in this subtribe (Bremer 1994, Bures et al. 2004). Within the subtribe, some of the thistles are found, e.g. in the diverse genera Carduus (approx. 90 species), Cirsium (200–300 species) or Onopordum (approx. 60 species), which have a large natural distribution area and have been distrib- uted further by human influence. Other large genera within the subtribe predominantly occur in mountain areas and often show high degrees of endemism, like Cousinia (approx. 700 species), Saussurea (approx. 400 species) or Jurinea (approx. 200 species) (Knapp 1987, Bremer 1994). The most frequent chromosome number within the genus Cirsium is 2n 34 (reported for approx. 69% of the species); 2n 68 is also relatively common (approx. 10% of species), and less frequent counts include 2n 30 and 2n 32 (both approx. 5%). Other related genera are Carduus (prevalent 2n 16, 22), Cousinia (2n 26, 24, 36), Saussurea (2n 26, 36, 52) and Jurinea (2n 30, 32, 34, 36). In ad- dition, the chromosome number in Arctium, Notobasis and Onopordum mostly is as 2n 36, 2n 34, 2n 34, respectively (Ghaffari et al. 2006, Goldblatt and Johnson 1990, 1991, 1994, 1996, 1998, Krasnikov et al. 2003, Lovkvist and Hultgard 1999, Sheidai et al. 2006, Susanna et al. 2003). According to the chromosome number, S. marianum is mostly closer to Onopordum and Noto- basis genera in Carduinae subtribe. However, it is essential to study phylogenetic relationships of this species with other genera in the subtribe by means of molecular approaches as done with other species in Cardueae tribe by Häffner and Hellwig (1999) using DNA sequence data of the Internal Transcribed Spacer (ITS) regions of the nuclear ribosomal DNA.
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