Abstract: The genus Vicia L. (Fabaceae) comprises approximately 190 species in the world. It includes 47 annual or perennial species in Iran. The karyotype of two species and two subspecies of the genus from Northwest Iran were investigated. Aceto-iron-hematoxilin was used to stain chromosomes. Chromosome characteristics including number, long arm, short arm and lengths, total length of chromosome set, arm ratio index and relative chromosome length were measured using the Micromeasure software. Results showed that these species were different in chromosome number, karyotype formula and karyotype characteristics such as V. villosa (2n = 2x = 14), V. hyrcanica (2n = 2x = 12), V. sativa subsp. sativa (2n = 2x = 12) and V. sativa subsp. nigra (2n = 2x = 12).
INTRODUCTION
The genus Vicia L. (Fabaceae) comprises approximately 190 species in the world (ILDIS, 1999). The genus mostly located in Europe, Asia and North America, extending to the temperate regions of south America and tropical Africa (Maxted, 1993). Most of them are diploids with a basic number x = 5, 6 or 7 (Maxted et al., 1991; Darlington et al., 1945) while only six of them are polyploids (Cremonini et al., 1992). The chromosome numbers of half of the 166 species are known (Maxted, 1993; Maxted et al., 1991).
Maxted et al. (1991) have divided the genus into two subgenera, including Vicilla and Vicia. The subgenus Vicilla divided into 17 sections and the subgenus Vicia into 9 sections. Most of these species are annual, but a few of them belonging to the section Cracca are perennial (Yamamoto, 1973). Cytological investigations have mainly concerned the determination of chromosome number and the examination of chromosome morphology (Maxted et al., 1991; Sahin and Babag, 1990). Karyological studies have played an important role in solving taxonomic problems (Goday and Pimpinelli, 1986).
The objective of this study was construction of a detailed karyotype analysis of Vicia villosa, Vicia hyrcanica, Vicia sativa L. subsp. sativa, Vicia sativa L. subsp. nigra and Vicia lathyroides using aceto-iron-hemaoxilin staining method.
MATERIALS AND METHODS
Seed samples of vetches were collected from different parts of rangelands and farmlands of the Ardabil province (Northwest of Iran) in 2006. These vetches include Vicia villosa L., Vicia hyrcanica Fish and Mey., Vicia sativa L. subsp. sativa and Vicia sativa L. subsp. nigra. These vetches were identified according to Flora of Turkey (Davis, 1988). Seeds of vetches were germinated on moist blotting paper and the root tips were obtained. For karyotype analyses they were pretreated with 0.05% colchicines for 3 h at 25 °C, then fixed in 1:1 (v/v) solution of formalin 10% and chromic acid 1% for 36 h at 4 °C and stored in 70% ethanol at -20 °C. The root tips were hydrolyzed with 1 N NaOH at 60 °C for 8 min. They were stained with Aceto-iron-hematoxilin for 12 h at 25 °C. After each step root tips were washed briefly in distilled water. Meristematic region with 1 mm of length excised and macerated in cytase enzyme at room for 2 h. Squash preparations on slides were made in 45% acetic acid (Asghari-Zakaria et al., 2002).
Chromosomes were measured for long arm, short arm, chromosome lengths, total length of chromosome set, arm ratio index and relative chromosome length, on 10 enlarged well-spread metaphase cells, using Micromeasure software developed by the department of Colorado State University available on Internet At http://www.colostate.edu/Depts/Biology/Micromeasure.
Homologous chromosomes were identified based on the position of centromers. The nomenclature followed by Levan et al. (1964).
RESULTS AND DISCUSSION
Vicia villosa: Mitotic chromosomes are shown in Fig. 1. Karyotypic characters of the mitotic chromosomes are shown in Table 1. The analysis of karyotype showed that this species has 2n = 2x = 14 chromosomes (Fig. 1). Chromosome length in Vicia villosa ranged from 4.53 μm in chromosome 7 to 5.89 μm in chromosome 1. Furthermore, arm ratio index values ranged from 1.46 in chromosome 7 to 2.52 in chromosome 5 (Table 1). The ratio between the largest and the smallest chromosome was 1.30:1. The karyotype is made of seven pairs of chromosomes (one metacentric and six sub-metacentrics).
Chromosome 7 (SAT chromosome), a secondary constriction near centromeric region. With the viewpoint of Stebbins (1971) symmetry class, Vicia villosa fell in A2 category and showed the most symmetric karyotype.
Vicia sativa L. subsp. sativa: Mitotic chromosomes are shown in Fig. 2. Karyotypic characters of the mitotic chromosomes are shown in Table 2. The analysis of karyotype showed that this species has 2n = 2x = 12 chromosomes (Fig. 2). Chromosome length in Vicia sativa L. subsp. sativa ranged from 2.89 μm in chromosome 6 to 5.69 μm in chromosome 1. Furthermore, arm ratio index values ranged from 1.38 in chromosome 4 to 6.07 in chromosome 2 (Table 2). The ratio between the largest and the smallest chromosome was 1.97:1. The karyotype is made of six pairs of chromosomes (one metacentric and five sub-acrocentrics).
Navratilova et al. (2003) showed that the karyotype of Vicia
sativa L. subsp. sativa composed of one
| Fig. 1: | Somatic metaphase chromosomes of Vicia villosa stained with aceto-iron-hematoxilin |
| Fig. 2: | Somatic metaphase chromosomes of Vicia sativa L. subsp. sativa stained with aceto-iron-hematoxilin |
| Table 1: | The mean karyotypic characters of seven mitotic chromosomes in Vicia villosa |
| *SAT-chromosome | |
| Table 2: | The mean karyotypic characters of six mitotic chromosomes in Vicia sativa subsp. sativa |
| *SAT-chromosome | |
| Table 3: | The mean karyotypic characters of six mitotic chromosomes in Vicia sativa subsp. nigra |
| *SAT-chromosome | |
| Fig. 3: | Somatic metaphase chromosomes of Vicia sativa L. subsp. nigra stained with aceto-iron-hematoxilin |
metacentric, four subacrocentrics and one acrocentric chromosome. It seems that they used another criterion for naming chromosomes.
Chromosome 2 and Chromosome 3 (SAT chromosomes), are distinguishable from other chromosomes through a satellite located on the telomeric region of their long arm. In the viewpoint of Stebbins (1971) symmetry class, Vicia sativa L. subsp. sativa fell in A3 category.
Vicia sativa L. subsp. nigra: Mitotic chromosomes are shown in Fig. 3. Karyotypic characters of the mitotic chromosomes are shown in Table 3. The analysis of karyotype showed that this species has 2n = 2x = 12 chromosomes (Fig. 3). Chromosome length in Vicia sativa subsp. nigra ranged from 4 μm in chromosome 6 to 8.28 μm in chromosome 1. Furthermore, arm ratio index values ranged from 2.65 in chromosome 6 to 4.81 in chromosome 3 (Table 3). The ratio between the largest and the smallest chromosome was 2.07:1. The karyotype is made of six pairs of chromosomes (two submetacentrics and four subacrocentrics).
Weber and Schifino-Wittmann (1999) have shown that the karyotype of Vicia
sativa subsp. nigra (synonym: Vicia angustifolia L.)
composed of six acrocentric chromosomes and Chromosome 5 has a secondary
| Fig. 4: | Somatic metaphase chromosomes of Vicia hyrcanica stained with aceto-iron-hematoxilin |
constriction in the long arm. The results of this study revealed that the genome of this species has six pairs of chromosomes (two submetacentrics and four subacrocentrics) and Chromosome 5 had a secondary constriction in the long arm. It seems that they used another criterion for naming chromosomes.
Schifino-Wittmann et al. (1994) described V. angustifolia with 2n = 14 but no such chromosome number was found in accessions included in this study.
Chromosome 5 (SAT chromosomes), are distinguishable from other chromosomes through a satellite located in the telomeric region of the long arm. In the viewpoint of Stebbins (1971) symmetry class, Vicia sativa subsp. nigra fell in B4 category. Vicia sativa subsp. nigra showed the most asymmetric karyotype.
Vicia hyrcanica: Mitotic chromosomes are shown in Fig.
4. Karyotypic characters of the mitotic chromosomes are shown in Table
4. The analysis of karyotype showed that this species has 2n = 2x
= 12 chromosomes (Fig. 4). Chromosome length in, Vicia
hyrcanica ranged from 8.37 μm in chromosome 6 to 11.60 μm
in chromosome 1. On the other hand, arm ratio index values ranged from
2.54 in chromosome 1 to 3.40 in chromosome 5 (Table 4).
The ratio between the largest and the smallest chromosome was 1.39:1,
Vicia hyrcanica has the largest genome
| Table 4: | The mean karyotypic characters of six mitotic chromosomes in Vicia hyrcanica |
| *SAT-chromosome | |
| Table 5: | Karyotypic characters of four species and subspecies in Vicia |
among the species investigated in this study. It possesses six pairs of chromosomes (four submetacentrics and two subacrocentrics).
Chromosome 2 (SAT chromosome), had a secondary constriction near centromeric region. Yamamoto (1973) showed that in V. hyrcanica (2n = 12) the total chromosome length was long. One long subterminal chromosome and one middle sized submedian chromosome which had a long satellite connected to the short arm were observed. The rest of the chromosomes were all subterminal. In the viewpoint of Stebbins (1971) symmetry class, Vicia hyrcanica fell in A4 category (Table 5).
CONCLUSIONS
The results of this study revealed that these species were different in chromosome number, karyotype formula and karyotype characteristics.