Cytological study of Hordeum bulbosum L. in Iran

 Introduction

The genus Hordeum consists of 32 species (45 taxa in total, including subspecies and cytotypes) including diploid (2n=2x=14), tetraploid (2n=4x=28) and hexaploid (2n=6x=42) cytotypes with a basic chromosome number of x=7 (Bothmer et al., 1995). The genus is classified into five genome groups, namely H, I, X, Y and XI (Taketa et al., 1999). In this study, genome designation followed that of Taketa et al. (2001), namely, H. vulgare and H. bulbosum both carry the H genome, so that H. marinum carries the X genome, while H. murinum has the Y genome, and the 25 remaining species share variants of the I genome (Taketa et al., 2005). H. bulbosum has been recognized as one of the two separate allogamous species ofthe genus, possessing a sporophytic incompatibility system (Bothmer et al., 1995). This species include two well-known cytotypes, diploid and tetraploid, with the latter being more widespread. The tetraploid cytotype is commonly considered as an autopolyploid (HHHH) (Xu and Snape, 1988; Chin, 1941; Papes and Bosiljevac, 1984).

The populations of bulbous barley grow widely in the mountainous and sub mountainous regions of Iran in the north, northeast, northwest, west, southwest and the south (except in the Central Plateau, northern Persian Gulf and southern Caspian Sea shores) (Bor, 1970) with different and under stressful environmental conditions.

Symeonidis et al., (1985) claimed that the chromosome set of bulbous barley originated from Greece which contains 16 metacentric including 4 satellited, 8 sub-metacentric and 4 telo-centric chromosomes. Nasirzadeh and Mirzaie Nadoushan (2005) reported that bulbous barley in north of Fars province is tetraploid with karyotype formulae (6m+1sm).

The aim of the present work was the evaluation of the cytotypes of H. bulbosum in Iran, characterization of the cytological and karyotypic details (numerical parameters) and their correlations with the geographic distribution of H. bulbosum.

Materials and Methods

Plant materials

A total of 32 specimens of H. bulbosum were randomly collected from various regions of Iran by the authors and were identified morphologically according to Bothmer et al., (1995) and analysed cytologically (Table 1).

Chromosome spread preparation

The seeds were germinated on paper tissue in pertidishes and the root tips selected for cytological experiments. Somatic chromosomes of meristematic root tip cells were treated from germinated seeds based on Agayev (1996) protocol with minor modifications. Briefly, pretreatment was carried out in saturated solution of Monobromonaphthalene, washed in distilled water for 30 min, fixed in Chromic Acid/Formaldehyde mixture (1/1) at about 4 ºC for 24 h, and finally washed under tap water for 3 h. Then the materials were transferred into 70% ethanol solution and kept refrigerated till staining process. For staining, the materials were transferred into distilled water for about 5-6 min and treated with 1N NaOH at 60 ºC for 10 min, washed in distilled water thoroughly for 30 min then stained in aceto-iron hematoxylin at 30 ºC for 24 h, washed in distilled water for at least 30 min, and macerated for 10-15 min in cellulase-pektinase enzyme solution at 37 ºC.

The roots were gently squashed in 45% acetic acid, on a slide glass and were observed and photographed under an Olympus AX-40 light microscope. At least, five cells were screened and the cells with good spread were used for analyzing and constructing karyograms. In order to characterize the karyotypic asymmetry, 5- 10 chromosome spreads from different individuals of each accession were examined. All chromosome sizes were measured with computer–aided program Image Tool 3.0. The parameters measured for each metaphase chromosome spread included Total Chromosome Length of the haploid complement (TCL), Mean Chromosome Length of the haploid complement (MCL), and Total Form percent (TF%: Ratio between the shortest arms of the chromosomes and their total length); the TF% value was considered to be close to 50% in most symmetric karyotypes and less than 50% based on the degree of asymmetry, (Huziwara, 1962), R (Ratio between the longest and the shortest arms of the chromosomes, Siljak-Yakovlev, 1986), S% (equals to length of the shortest chromosome dividede on length of the longest chromosome, Stebbins, 1971), Asymmetry index (AsI%=100 × ΣL/ΣTCL; where l is long arms in chromosome set and TCL is total chromosome length in chromosome set, Arano and Saito, 1980) and Karyotype formulae: according to their arm ratios (long/short) designated by the position of the centromere: 1 (metacentric; M), 1-1.7 (metacentric; m), 1.7-3 (submetacentric; sm), 3-7 (subtelocentric; st), and 7-39 (telocentric; t)] (Levan et al., 1964).

Table 1. Accessions of H. bulbosum (HB) collected from different places in Iran.

Results and Discussion

All of the studied populations were tetraploid (2n=4x=28) and the results of the analyzed parameters of karyotype of H. bulbosum supported the autopolyploidy origin of the species with nearly symmetric karyotype combining four homologous or near homologous genomes that were in accordance with previous reports (Chin, 1941; Morrison, 1959; Xu and Snape, 1988). Karyotype was nearly symmetrical with chromosomes varying in mean total chromosome lengths from 5.22 (in HB90SW from Dasht-e Felard at Chaharmahal va Bakhtiari province) to 15.04 μm (in B3W from Darrehshahr in Ilam province) (Table 2). The descriptions of karyotype formulae and their analyzed parameters results are shown in Tables 2 and 3, respectively.

Table 2. Karyotype analysis of the different populations of H. bulbosum species(n=chromosome number, TL=Total haploid chromatin length, MCL=Mean Chromosome Length, SE=Standard Error, TF%=Total Form percent, S%=Length of the shortest chromosome divided on length of the longest chromosome, R=ratio between the longest and the shortest arms of the chromosomes, AsI%=Asymetry index, *=Satellite.

The morphological characteristics of chromosomes are shown in Figure 1. As presented in Table 2, the metacentric (M and m) chromosomes dominated the observed karyotypes with 79.46% and the second frequency belongs to the submetacentrics (20.09%). Only one population (HB81SW from Noorabad –e Mamasany in Fars province) had a sub-telocentric (st) chromosome with karyotype formulae (5m* + 1sm + 1st). No telocentric chromosome was observed (see Table 2).

Figure 1: Somatic chromosomes (karyotype) of 32 Iranian H. bulbosum (HB) populations (2n=4x=28).Mitosis squash photograph for accessions: HB24W, HB213W and HB221NW with showing Satellitedchromosoms are presented. Scale bar: 20 μm.

The populations HB106NE, HB201W and HB221W had 3 sub-metacentric, HB2W, HB22W (2M + 5m*) and HB23W (7m*) without sub-metacentric chromosome and other remaining populations (56.25%) had karyotype formulae of 6m+1sm including six metacentric and one sub-metacentric that were in accordance with Chin (1941), Linde-Laursen et al. (1990), Morrison (1959) and Vahidy and Jahan (1998) (Table 2). Nasirzadeh and Mirzaie Nadoushan (2005) have analyzed the karyotype parameters of H. bulbosum populations and have suggested that they originated from Fars province and showed that their karyotype formulae were 6m+1sm which was partly in agreement with the results of this study. Symeonidis and Lazaros (1985) reported that the karyotype of Greece populations of bulbous barely was 4m+2sm+1t. In this study, we have not found telocentric chromosome in tetraploids indicating that the karyotype of Iranian tetraploid bulbous barley is different from Greece populations. Our results showed that all populations have one metacentric or sub-metacentric satellited chromosome, except for HB90SW (from Dasht-e Felard in Chaharmahal va Bakhtiari province). Two populations (HB106NE from National Park of Golestan and HB202W from Ilam) had one submetacentric satellited chromosome with karyotype formulae (4M+3sm*). The presence of typical SM satellited chromosomes occurred more frequently among the studied populations of the Hordeum bulbosum (Rajhathy et al., 1964; Vosa, 1976; Coucoli and Symeonidis, 1980; Chin, 1941; Linde-Laursen et al., 1990; Morrison, 1959). As noted by Heneen (1977) and the different origin of the materials should be a logical explanation for the observed differences since SAT chromosomes in the Triticeae are well known to evident morphological variation the of shape and the indices among different populations or varieties of one species. The karyotype formulae polymorphism in homologous chromosomes of H. bulbosum could be correlated with their out-breeding nature. No B chromosome was observed among the materials studied.

The highest TL variation was found in HB215W population[SE (standard error) of MCL=2.1μm], and the lowest chromosome length variation was scored in HB209W population(SE of MCL=0.69 μm) (Table 2). The ratio between the longest and the shortest arms (R) ranged from 1.23 HB3W accessionto 1.69 in HB106NE accession(Table 2). Asymmetry Index (AsI%) ranged from 54.89 in HB2W populationto 62.19 in HB106NE population (Table 2). The degree of karyotype asymmetry as indicated by TF% values ranged from 37.1% (HB106NE and HB221NW accessions) to 45.1% (HB2W) (Table 2). As the TF% values were near to 50%, we can conclude that type of chromosomes were metacentric to submetacentric. Also the mean of S% (Stebbins 1971) indicating symmetry index was from 48.58% (HB215W) to 81.32 (HB217W) with mean of 70.1% indicating nearly symmetrical karyotype for H. bulbosum.

Based on the results of this study (the factors studied and the resulted asymmetry indices) HB221NW proved to have the most asymmetric karyotype (with the formulae of 1M* + 3m + 3sm) among the populations studied. Regarding the asymmetry indices observed in HB221NW it could be suggested that the karyotype asymmetry in this population was mainly affected by the place of the centromers rather than length of the chromosomes. HB2W with the least chromosomal arm ratio variability, showed the most symmetric karyotype (with the formulae of 2M + 5m*). Regarding all the analyzed factors, a high similarity were found between HB2W, HB14W, HB22W and HB23W (see Table 2).

The karyotype asymmetry can be a fine appearance of the general morphology of karyotype in plants (Romero Zarco, 1986). As Sharma (1990) has mentioned, symmetrical karyotypes are more primitive than asymmetrical ones and longer chromosomes than shorter ones; median centromers with chromosome arms of equal length are more primitive than chromosomes with arms of unequal length. From the chromosome length point of view, the longest chromosomes were found in HB3W that could be considered as most primitive population. We observed that the most asymmetric karyotypes within northeast populations (e.g. Golestan) and populations of the west of Iran had the most symmetric karyotypes. Therefore considering the above notions and the results of this study, it could be assumed that the oldest populations are in the slopes of Zagros Mountains (west of Iran) and the youngest ones occurred in the northeast of the country (Figure 2).

Figure 2: Distribution of collected accessions of Hordeum bulbosum (W=west, SW=southwest, N=north, NE=northeast, NW=northwest). Arrows indicate the distributions direction of H. bulbosum in Iran.

This suggestion is in accordance with the conclusion reported by Bothmer, et al. (1995) namely the H. bulbosum (4x) has originated from Greece and then distributed eastwards. Based on these results it can also be concluded that the Western populations (e.g. HB2W, HB14W, HB22W, HB23W and HB90SW) generally possessed the highest chromosomal length and the highest mean TCL (9.87 μm) and the most symmetric karyotypes are the oldest populations and the northeast populations with mean TCL of 8.5 μmare the youngest populations of H. bulbosum in Iran (Figure 2).