Phylogenetic comparison of the A genome using karyotype analysis in some Triticum species

Introduction

Plant nuclear genomes are enormously variable. Chromosome number, the degree of gene clustering, and chromosome size can all differ considerably, even between closely related species (Kellogg and Bennetzen, 2004).

The earliest work on wheat chromosomes relied on reconstructions made from microtomal serial sectioning of root tips. The development of squash technique reduced the possibilities of misinterpretations. Studies of the morphology of chromosomes in Triticum species have been made by many workers, including Schulz-Schaeffer and Haun (1961), Khan (1963), Kimber (1971), Johnson and Dhaliwal (1978), Kerby and Kuspira (1987), Miller (1987) and Khan (2005).

Because of different workers had to do without standardization of techniques in different laboratories, their reports showed inconsistencies in the standard karyotype analysis based on chromosome length and arm ratio still holds its merit (Jahan and Vahidy, 1989). The analysis of plant genomes has provided insight into how these evolutionary events have occurred and the rate at which evolution could have taken place. Wheat has played a major role in the development of the world civilization. The domestication of wheat was a major event in the world civilization because it allowed humans to change from nomadic hunter gathers to permanent residents of specific locations. Many useful genes from wild species are available to be used in breeding Programmers as source of genes for disease resistance applying different chromosomal engineering techniques involving backcrosses, "bridges" crosses and selection of cytological as well as desired agronomic characteristics (Stalker, 1980; Dhalival et al., 1986; Gale and Miller, 1987). By 1951, the work of Lilienfeld and colleagues had established many of the genomic relationships of the diploid and polyploid Triticum L. and Aegilops L. species on the basis of chromosome pairing in hybrids (Lilienfeld and Kihara, 1951). These studies have continued to the present and have accurately classified chromosome translocations (Naranjo, 1995; Naranjo et al., 1988; Maestra and Naranjo, 1999; Jauhar et al., 1991). Archaeological evidence has shown that Triticum turgidum L. (AABB) has been grown in both Mesopotamia (Tigris and Euphrates River Valley) and in the Nile River Valley 10,000 years ago. Because wild T. tauschii (Coss.) Schmalh. is found only in the mountain region of south Russia, west Iran and north Iraq, it is thought that the hybridization that produced T. aestivum L. have occurred in these regions. It has been suggested that this occurred as recently as 8,000 years ago which coincides with the development of collective settlements by man (Wazuddin and Driscollt, 1986).

The goal of the present study was to find relationships between Triticum species using karyotype analysis and molecular markers.

Materials and Methods

Plant materials

A total of 46 wheat accessions were collected for this study. The materials were taxonomically identified based on Rahiminejad and Kharrazyan (2005). The Karyotype analysis was performed on the accessions belonging to five species and two subspecies carrying A genome (Triticum monococcum L., T. boeoticum Boiss. subsp. thaodar (Reut.) Schiemann, T. boeoticum subsp. boeoticum, T. urartu Tumanian ex Gandilyan, T. durum Desf., T. turgidum and T. aestivum) (Table 1).

Chromosome spread preparation

Triticum seeds were germinated at 25ºC on moist filter paper in Petri dishes. Actively-growing root tips, 1 cm in length, were excised from the germinating seeds and pretreated with α-bromonaphthalene for 4-6 hours at 4ºC in refrigerator and fixed in chromic acid-formaldehyde mixture (1:1 of 1% chromic acid + 10% formaldehyde) at about 4ºC for 24 hours. The root tips were transferred into 70% ethanol solution and kept refrigerated till staining. Subsequently, the root tips were hydrolyzed in 1 N NaOH at 60ºC for 10 minutes, stained for 24 hours in hematoxylin stain at 30ºC, and squashed in 45% glacial acetic acid. Before squashing, the root tips were treated with cellulase-pectinase enzyme solutions at 37ºC for 10-15 minutes. The selected cells were photographed under an Olympus AX-40 light microscope. Karyotypes were obtained from well-spread metaphase plates.

All chromosomal sizes were measured with computer-aided program Micro Measure 3.3. Software (Reeves, 2001). Relative length in proportion to total genome length (RL), total chromosome length (TCL), mean chromosome length (MCL), arm ratio (short arm length/long arm length) with their respective standard errors and centromeric index (length of the short arm/total length of the chromosome X 100) (Arano and Sattio, 1980), total form percentage (TF%) (Huziwara, 1962), centromeric index mean of long and short arms (CI), asymmetry index (AsI%) (Arano and Sattio, 1980), S% (The shortest chromosome length/the longest chromosome length), DRL (difference of range relative length), intrachromosomal asymmetry index (A1) (Romero-Zarko, 1986), interchromosomal asymmetry index (A2) (Romero-Zarko, 1986), Stebbin’s classification (Stebbins, 1971) and karyotype formula (Levan et al., 1964) were calculated (Table 2). The data analyzed with Excel, SAS (version 14) and SPSS (version 16) statistical softwares.

Table1. List of scientific name, accessions codes^*, ploidy level, kind of genomes and collecting localityies used for comparative cytological study of Iranian Triticum species

^{*All samples are kept in the herbarium of Isfahan University}

Table 2. The means of karyotypic characters of forty-six mitotic chromosomes in diplo, tetra and hexaploids species of the genus Triticum used in this study (n = Chromosome number, TCL = Total haploid chromatid length, MCL = Mean of chromosomes length, TF% = Total form percent, Cent.Index = Centromeric Index (= S/(L+S)), Lon.Ch. = Longest chromosome length, Sho.Ch. = Shortest chromosome length, ML = Mean of large arms, MS = Mean of short arms, r = Arms ratio, AsI% = Asymmetry index, S% = Ratio between the shortest and longest of the chromosomes percent, DRL = Difference of range relative length, A1 = Intrachromosomal asymmetry, A2 = Interchromosomal asymmetry (C.V.) and RL = Relative length of chromosomes)

Results and Discussion

The results showed that about 33% of diploid accessions had sub-metacentric (sm) chromosomes in their Karyotype formula and other ones had only metacentric chromosomes. Tetraploid accessions had 2-4 sub-metacentrics (sm) and hexaploid accessions possessed 2-5 sub-metacentric (sm) chromosomes in their Karyotype formulae (Table 1).

All the accessions without T.duru-1, T.aest-73 and T.aest-47 (2A category), were placed in 1A category of Stebbins (1971) asymmetry categories (not showed in these Tables). Total chromosome length depended on the ploidy levels and chromosome numbers and consequently this parameter was too variable (Table 2).

TCL values varied between genotypes (Table 2). The TCL value ranged from 48.316 µm (T. urartu) with MCL of 7.606 ± 0.63 µm to (T. aestivum) with MCL of 11.475 ± 0.88 µm.

The most variable chromosomes in length SE of MCL were found in T. turgidum (SE of MCL = 1.94 µm) whereas the most similar chromosomes were scored in T. boeoticum subsp. thaodar (SE of MCL = 0.13 µm) (Table 2). The TF% value ranged from 43.29 (T. monococcum) to 40.66 (T. turgidum) (Table 2). The CI ranged between 0.431 (T. monococcum) and 0.404 (T. turgidum). The longest and the shortest chromosome length were found in T. aestivum. The highest mean length arm was in T. aestivum. and the lowest mean was in T. urartu. The arm ratio ranged from 1.27 in T. monococcum to 1.47 in T. turgidum. The ASI% varied from 56.69 (T. monococcum) to 59.35 (T. turgidum). The SI% ranged from 75.41 (T. boeoticum subsp. thaodar) to 50.418 (T. aestivum). DRL in T. monococcum was the highest (4.201) and in T. aestivum was the lowest (3.099). In general, A1 and A2 values showed the high degree of karyotype symmetry in the majority of the genotype studied (Razik Kamel, 2006). A1 ranged from 0.305 (T. aestivum) to 0.232 (T. monococcum) and A2 ranged from 0.102 (T. boeoticum subsp. boeoticum) to 0.166 (T. aestivum) (Table 2). Among the diploid species, the TCL value varied from 74.758 µm (T. boeoticum subsp. boeoticum) to 48.316 µm (T. urartu). Between tetraploid accessions the TCL value ranged between 156.210 µm (T. durum) and 130.133 (T. turgidum). Based on the result of this study, it could be concluded that generally, the chromosomal length in the T. urartu was shorter than other species of the genus Triticum. In this species, minimum value of TF%, CI and A1 belonged to T. boeoticum subsp. thaodar and maximum showed in T. monococcum. Also among this group (diploids) arm ratio (r) and AsI% in T. monococcum were the lowest and in T. boeoticum subsp. thaodar was the highest. Results showed that coefficient of variability for intra-specific chromosome length variation of short and long arms (Table 3) was higher for short arms and that it can be concluded that short arms were more effective in TCL than long arms. Karyotype studies were principally based on the idea that symmetrical karyotypes were more primitive than asymmetrical ones; and this holds true for longer chromosomes compared to shorter ones; median centromeres with arms of equal length are more primitive than chromosomes with arms of unequal length; low basic numbers give rise to higher ones. These features are based on the comparison between karyotypes of known relative antiquity, as determined through classical taxonomy (Sharma, 1990).

Table 3. Coefficient of variability for intra-specific chromosome length variation of short and long arms for Triticum species used in this study

The scatter diagram based on A1 and A2 (Romero-Zarco, 1986) constructed three groups of karyotype asymmetry in the accessions studied: 1- T. aestivum (a) with the highest asymmetrical karyotype, 2- T. monococcum, T. boeoticum subsp. thaodar and T. boeoticum subsp. boeoticum with the lowest asymmetrical karyotype and 3- T. urartu, T. turgidum and T. durum with an intermediate between two previous groups (Figure 2) (Jalilian and Rahiminejad, 2011).

Figure 2. Scatter diagram show the relationships between the Triticum species based on the intrachromosomal (A1) and interchromosomal (A2) asymmetry indices. Values of A1 and A2 are summarized in Table 2. (m. T. monococcum; u. T. uratu; b.b. T. boeoticum subsp. boeoticum; b.t. T. boeoticum subsp. thaodar; d. T. durum; t. T. turgidum; a. T. aestivum)

In the UPGMA dendrogram (Figure 3), the five Triticum species studied were divided into three groups: (1) A: (T. monococcum and T. urartu), (2) B: (T. boeoticum subsp. thaodar and T. boeoticum subsp. boeoticum), and (3) C: (T. durum, T. turgidum and T. aestivum) (Figure 3) (Akhavan and Saeidi, 2010; Jalilian and Rahiminejad, 2011).

Figure 3. The UPGMA dendrogram (used Euclidian Distance) shows relationships between Triticum species based on chromosomal characters (see Table 2). T.mono. T. monococcum; T.urar. T. urartu; T.b.t. T. boeoticum subsp. thaodar; T.b.b. T. boeoticum subsp.boeoticum; T.duru T. durum; T.turgi. T. turgidum; T.aest. T. aestivum.

According to the results, it might be suggested that T. durum was more primitive than T. turgidum and T. monococcum could be considered as a donor of A genome to T. durum and T. aestivum. This hypothesis verified SSR analysis (Ehtemam et al., 2010; Keshavarzi et al., 2012).

Acknowledgments

The authors wish to thank the Office of Graduate Studies and the Department of Biology, of the University of Isfahan for their support.