DISTRIBUTION AND CHARACTERIZATION OF AEGILOPS CYLINDRICA HOST FROM IRAN

Jointed goatgrass (Aegilops cylindrica Host; 2n = 4x = 28, CCDD) is a tetraploid remote relative of bread wheat (Triticum aestivum L; 2n=6x=42, AABBDD) with two genomes and 28 chromosomes. The diversity center of this species is in the Fertile Crescent and in central Asia and could also be found in many places in Iran. In this experiment, 359 accessions provided by National Plant Gene Bank of Iran (NPGBI) were used. Based on the geographical distribution, the highest distribution of A. cylindrica is found in North, West and North-West regions of Iran. The data on the distribution of A. cylindrica showed that its distribution centers in Iran are more than those reported in the previous studies. Chromosome counting showed that all A. cylindrica accessions are tetraploid (2n=4x=28). Results of factor analysis for nine morphological chromosome traits showed that karyotypic variation within accessions are related to the length of chromosomes and there is difference between the accessions for their total chromosome length, but the karyotype of different accessions are almost the same for the symmetry. Low coefficient of variation in morphological traits as well as symmetric karyotypes of A. cylindrica species observed in this study could lead us to predict that A. cylindrica could be a recently evolved species among the remote relatives of bread wheat. Introduction Jointed goatgrass, originated from two species, is native to the Mediterranean, Middle East, Asia, and was also introduced to the Great Plains and the Pacific northwest of the United States (Kimber and Feldman, 1987; van Slageren, 1994). It is a winter annual grass weed that infests over three million ha of winter wheat in the Pacific Northwest and Great Plains regions of the USA (Dewey, 1996). It reduces winter wheat yields by interference and lowers harvested grain quality. Average yield loss due to Aegilops cylindrica infestations has been estimated to be 25% (Anderson, 1993; Donald and Ogg Jr, 1991). It has also been estimated that the economic cost of A. cylindrica to winter wheat producers in the western United States is $145 million annually (Ogg, 1993). Jointed goat grass and winter wheat are closely related. Therefore, the development of selective herbicides to control this weed in winter wheat has been problematic. A. cylindrica is a bushy type plant with 20-40 cm long culms. It is characterized by narrow, 4-5 cm long, and glabrous to sparsely hairy leaves, a narrow, lanceolate and 6-9 cm long spike, almost ended with two incomplete spikelets. Each spike consists of 6-11 spikelets and breaks off Corresponding author: behnam.bakhshi@gmail.com, b.bakhshi@areeo.ac.ir Vegetable Research Center, Horticultural Sciences Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran. 3Seed and Plant Improvement Institute (SPII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran. Department of Agronomy and Plant Breeding, Faculty of Agricultural Science and Engineering, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran. Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Zanjan, Zanjan, Iran.


Introduction
Jointed goatgrass, originated from two species, is native to the Mediterranean, Middle East, Asia, and was also introduced to the Great Plains and the Pacific northwest of the United States (Kimber and Feldman, 1987;van Slageren, 1994). It is a winter annual grass weed that infests over three million ha of winter wheat in the Pacific Northwest and Great Plains regions of the USA (Dewey, 1996). It reduces winter wheat yields by interference and lowers harvested grain quality. Average yield loss due to Aegilops cylindrica infestations has been estimated to be 25% (Anderson, 1993;Donald and Ogg Jr, 1991). It has also been estimated that the economic cost of A. cylindrica to winter wheat producers in the western United States is $145 million annually (Ogg, 1993). Jointed goat grass and winter wheat are closely related. Therefore, the development of selective herbicides to control this weed in winter wheat has been problematic.
A. cylindrica is a bushy type plant with 20-40 cm long culms. It is characterized by narrow, 4-5 cm long, and glabrous to sparsely hairy leaves, a narrow, lanceolate and 6-9 cm long spike, almost ended with two incomplete spikelets. Each spike consists of 6-11 spikelets and breaks off entirely or disintegrates into segments at maturity. Each spikelet holds one to three seeds that are reddish-brown in color and reach maturity in mid-summer, Fig. 1). The genomic constitution of A. cylindrica was determined by the analyses of chromosome pairing, storage proteins, isozymes, and differences in restriction length patterns of repeated nucleotide sequences (Linc et al., 1999). Linc et al. (1999) identified the diploid species A. caudata L. (2n=2x=14, CC) and A. tauschii Coss. (2n=2x=14, DD) as the donor of the C and the D genome of A. cylindrica, respectively. Previously, the cytoplasm of A. cylindrica was shown to be contributed by A. tauschii (Maan, 1976;Tsunewaki, 1989) but, more recent analysis with chloroplast microsatellite markers has shown that both A. tauschii (D-type cytoplasm) and A. markgrafii (Greuter) K. Hammer. (C-type cytoplasm), now a synonym of A. caudata, have contributed their cytoplasms to A. cylindrica (Gandhi et al., 2005). High genome homology shared between A. cylindrica and its progenitor species and low intra-species polymorphism in A. cylindrica indicated it as a new species with little chromosome changes. The D genome chromosomes of A. cylindrica species are more similar to A. tauschii biotypes and D cr2 genome of hexaploid cytotype of A. crassa Boiss. than D genome of bread wheat (Badaeva et al., 2002;Caldwell et al., 2004). These results indicate that there are different versions of D genomes for both A. cylindrica and T. aestivum L. species.
A. cylindrica has wide distribution from Western Europe to East Asia and even North America. The diversity center of this species is in the Fertile Crescent and in central Asia and could also be found in many places in Iran (van Slageren, 1994). A. cylindrica has spreaded westward to Greece, Bulgaria, Romania, Kosovo, Montenegro, Serbia, and Hungary. Northwards, it is distributed in the Caucasus region and along the Black Sea coast. Though rare, this species is also present in the western arc of the Fertile Crescent involving Lebanon, Jordan, Syria, northern Iraq, and northwestern Iran (van Slageren, 1994).
The geographic distribution of A. cylindrica encompasses and extends beyond areas, where it's diploid progenitors, A. tauschii and A. markgrafii can be found (Fig. 2). (C). The geographic coordinates were obtained from the system-wide information network for genetic resources (SINGER; http://singer.cgiar.org/). Throughout its range of distribution, A. cylindrica is considered as a weedy species, particularly in common wheat fields, where it chronically infests fields in the Mediterranean, the Middle East, Europe, and the United States of America (Dewey, 1996;Ogg and Seefeldt, 1999;van Slageren, 1994). Jointed goatgrass has also been suggested as a source of genetic variation for wheat improvement (Bouhssini et al., 1998;Farooq et al., 1992;Iriki et al., 2001) because it is a close relative of common wheat and both species carry the D genome donated by A. tauschii (Kimber and Zhao, 1983;Riley and Law, 1965). In addition, natural hybridization between wheat and jointed goatgrass suggests a potential for gene flow between these species under field conditions (Gandhi et al., 2006;Zemetra et al., 1998). Thus, there is considerable interest in understanding various aspects of the evolution of A. cylindrica for its better management and use. The D genome of hexaploid wheat has been shown to be more closely related to the D genome of A. tauschii subsp. strangulata (Eig) Tzvelev than to A. tauschii subsp. tauschii Coss. (Dvorak et al., 1998;Lubbers et al., 1991;Pestsova et al., 2001), whereas the D-type plastome and the D genome of A. cylindrica are more closely related to A. tauschii subsp tauschii than to A. tauschii subsp. strangulata (Gandhi et al., 2005).
Although molecular genetic diversity and ploidy level of A. tauschii has been reported before (Bakhshi et al., 2010;Levan et al., 1964), no extensive study has been done to identify cytogenetic and morphologic characteristics of this species. We have collected many accessions from different regions of Iran that it could be remarkable to identify potential genetic diversity among these accessions. In this study, we analyzed chromosomes features of A. cylindrica as well as distribution and morphological characteristics of this species which is widely distributed in Iran. Since this species is a relative of bread wheat it is important to identify available genetic diversity in this species to be used in necessary condition in bread wheat breeding programs.

Materials and Methods
Plant material: 359 accessions were used in this experiment, which were provided from National Plant Gene Bank of Iran (NPGBI). These accessions were collected from sixteen provinces of Iran viz., West Azarbaijan, East Azarbaijan, Ardebil, Zanjan, Qazvin, Kurdistan, Hamedan, Kermanshah, Ilam, Lorestan, Chaharmohal Bakhtiari, Mazandaran, Tehran, Esfahan, Semnan and Khorasan. A total of 23 traits were evaluated, 16 of which were qualitative and seven were quantitative. Evaluation of all traits was conducted using three replications of each accession. The mean and mode was calculated for quantitative and qualitative traits, respectively. Estimated statistical parameters traits were calculated for quantitative traits. Shannon and Weaver diversity index were calculated for measuring qualitative traits. Non-standard values of diversity index (Hc) and standard diversity index (SDI) was calculated as follows (Hennink and Zeven, 1990): In this formula, for certain traits, such as c, n, including the number of phenotypic classes and Pi is equal to the frequency of bushes.
Chromosome counting: Following the technique, developed at International Maize and Wheat Improvement Center (CIMMYT) institute (Mujeeb-Kazi and Miranda, 1985), root tips were collected between 9 AM to 10:30 AM, and then placed in a Petri dish, on a filter paper moistened with α-bromonaphthalene pre-treatment solution. The samples were pre-treated about 2.5 to 3.5 hours, but generally 3 hrs as pre-treatment time was used, that resulted satisfying chromosome contraction and high mitotic index. After pre-treatment, the root tips were transferred to vials, containing 0.2% aceto-orcein and refrigerated (4°C) until they were being used. Afterwards, the root tips were transferred to 2% aceto-orcein, in order to intensify the staining for 2 days before squashing. After staining the aceto-orcein was removed from the vial and 45% acetic acid was added to fill about a quarter of the vial. Vial was heated over a flame to bring the contents to a slow boiling. After boiling, the vial contents (45% acetic acid + root tip) were transferred into an evaporating dish.
A root tip was taken from it and placed over on filter paper to remove extra acetic acid. Apical root tip measuring 2-2.5mm was cut and placed on dry microscope slide. The root tip was squashed by an arrow-head needle, and a small drop of 45% acetic acid was quickly added to the squashed tissue. The slide was then slightly warmed and a cover glass was placed gently over the macerated cellular area. The cover glass slides were gently dabbed with coarse filter paper, the slide was heated slightly, placed between folded filter paper on a flat surface and thumb pressure applied directly to the cover glass. After squashing, the slide was suitable for observing chromosomes by microscope.
Karyotype preparation: In order to prepare karyotypes of A. cylindrica, 12 accessions of A. cylindrica were used (Table 1). Study of karyotypes: Chromosomes were named according to the location of centromere in the chromosome (Levan et al., 1964). In this study, comparison between karyotypes in different accessions of a species was performed by comparing their symmetry. The Stebbins method (Stebbins, 1971) has been used for determining the degree of symmetry. Additionally total form percentage index (TF) (Forni-Martins, 1994), the relative percentage of the longest chromosome to the shortest chromosomes (S) (Bennardello, 1994), coefficient chromosomal length variations (CV) (Sheidai et al., 1996, the average ratio of long to short arm (R) (Bennardello, 1994) and range of chromosome length variation (V) (Datta and Agarwal, 1992) were calculated. Evaluation of karyotype evolution was calculated using Dispersion Index (DI) to show a few differences that were not visible in Stebbins indicators (Lavania and Srivastava, 1999). Factor analysis for morphological features of chromosomes based on principal component analysis and Varimax rotation was also conducted. For measuring different parts of the chromosomes and analyzing morphological data of chromosomes, Micromeasure software (Reeves, 2001) and the SPSS software were used, respectively.

Results and Discussion
Geographical distribution of A. cylindrica accessions in Iran: Investigation on collecting location and geographical distribution of A. cylindrica accessions reveals that this species predominantly grows in the range of 800 to 2000 meters altitude. Thus this species is adapted to mountainous ecosystems and not to low altitude ecosystems of Caspian shores, southern shore, Khuzestan and Ilam. The results of the geographical distribution using ILWIS software also showed that the highest distribution of A. cylindrica was in north, west and north-west regions, including East Azarbaijan, West Azarbaijan and Kermanshah provinces, contrasting to that of the southern and southeast regions which showed the lowest distribution (Fig. 3). However, it may be found anywhere on the mountainous areas of Alborz and Zagros. Some populations could even be found on briny margins of Urmialake and Semnan, Northern Khorasan and around Qom. Whereas, A. cylindrica doesn't grow on salty deserts of central and southern Iran. While, it is abundant in central Iranian land, A. cylindricais usually found in the more elevated northern strip of the central Iranian desserts and not in the more arid region of southern and central areas. The present investigation on the geographical distribution of A. cylindrica showed that the distribution centers are more than that reported in a previous study conducted in Iran (Gandhi et al., 2005). In the present study, in addition to north, west and northwest, northeast and southwest of Iran have also been identified as distribution centers for this species. The results also showed that this species mostly present in mountain ecosystems and it is not found at low altitude ecosystems such as the margins and the southern coast ecosystems of Caspian Sea. Previous studies indicated that the evolution of bread wheat occurred in high altitudes of Caspian Sea ecosystems (Jaaska, 1981;Nakai, 1978). On the other hand, the results of this study showed that the A. cylindrica species has a wide distribution in this region. With this explanation, the possibility of challenging unproven hypothesis that A. cylindrica had a potential to be as a donor of hexaploid wheat D genome (Asghar et al., 2001) might be more acceptable.
Statistical parameters for the traits of A. cylindrica species: Rachis width, spikelet seed number, plant height, kernel width and leaf number of rachis traits showed the highest phenotypic coefficient of variation, with 13.14, 11.33, 10.85, 10.43 and 10.11 percent, respectively. Most of these traits have been also observed among traits with high diversity in other collected accessions in Iran including A. tauschi and A. crassa (Aghaei et al., 2008;Ranjbar et al., 2007). Furthermore, maturity date, spikelet length and length of spikelet glume indicated the lowest phenotypic coefficient of variation with 4.80, 5.02, and 5.61 percent. Most standard deviations were also related to plant height, flowering date and maturity date and the lowest standard deviation were related to rachis width, spikelet width and width of spikelet glume (Table 2). White and flour form kernel, brown and glabrous glumes, moderate fragility spikes, brown stamens and standing bushes were predominantly observed in the research field. Furthermore, growth habit showed the most variation using non-standard and standard diversity index of Shannon-Weaver (Peet, 1974). Thus, according to Shannon-Weaver Diversity index, growth habit could be introduced as the most effective qualitative trait to distinguish A. cylindrica populations (Table  3). Relatively low level of phenotypic variation coefficients for different traits were obtained in this study showing that this species is relatively new. However, high genetic diversity has been observed for A. tauschi in northern area of Iran (Aghaei et al., 2008).  Karyotype analysis of accessions: Chromosome counting showed that most of A. cylindrica accessions are tetraploid (2n = 4x = 28). Furthermore, cytogenetic studies showed no aneuploid and B chromosome, but difference in chromosome length. Collecting place and accession numbers are presented in Table 1 and chromosomes count, satellites count in karyotype and karyotypic formulae are shown in Table 5. The metaphase cell and ideogram of various accessions are presented in Figs. 4-15. All investigated accessions had a satellite in the short arm of chromosomes no. 8. The presence of satellite in the same pair of chromosomes is also reported (Karataglis, 1989). Considerable variation was observed in total length of chromosomes (TLC) and the average length of chromosomes (C). High variation in length of chromosomes may be a sign of genome adaptation of this species to those places from where they have been collected. The largest chromosome (11.33 micrometer) was found in Zanjan accession and the smallest chromosome (4.87 micrometer) in Urmia accession and both of them were sub-metacentric. Maximum and minimum of long arm to short arm ratio was observed in Islam Abad Gharb (2.129) and in Shirvan (1.78) accessions, respectively. For total form percentage index (TF), the maximum (35.32) and minimum (31.18) TF was observed in Ardabil and Zanjan accessions, respectively. This data shows that karyotypes of Ardebil and Zanjan accessions have the highest and the lowest symmetry. The highest coefficient of variation (CV) was found in accessions from Naghadeh and the lowest in the Javanrud accessions. All of the accessions that were collected from the northwest and the west of Iran, belonged to A2 position of the Stebbins Table, indicating a relatively symmetrical karyotype for recently evolved species with short evolutionary history. Also, distribution index of chromosome (DI) showed that the Hashtrud accession had the highest DI (7.43) and Zanjan accession had the lowest DI (18.5). This observation indicated that the Hashtrud accession had the highest symmetry in contrast to Zanjan accession. DI could be more reliable than other indicators because three important karyotypic criteria, including variation in chromosomes length, centromere position and relative size of chromosomes, are involved in DI calculation (Table 6). Range of chromosome length variation, total length of chromosome, average of chromosome length, total length of chromosomes (TLC), centromere index, and standard deviation of chromosome length traits had the largest factor coefficients in the first factor. Because most of these traits depend on the chromosomes length, this factor is named length of chromosomes. This factor presents 65.83 percent of the total variance that shows there is great diversity for traits related to chromosome length among accessions. In the second factor, CV of chromosome length, minimum of short arm to long arm ratio percentage and distribution index The results of factor analysis of 9 chromosomal morphological traits showed that the first two factors had Eigen values greater than one with 65.83 to 88.39 percent variance and these two factors were responsible for diversity in the accessions (Tables 7-8). traits had the largest  factor coefficients. Thus, the second factor was named as karyotype symmetry duo to all of these traits indicated karyotype symmetry of the accessions. The second factor accounted for 22.55 percent of the total variance, indicating that there was no great difference between accessions in terms of symmetry and confirming the placement of accessions at the 2A position of the Stebbins table. Therefore, the results of factor analysis show that karyotypic variation within accessions is related to the length of chromosomes and there is difference between accessions for their total chromosome length; but the karyotype of different accessions are same for their symmetry and they are relatively symmetrical. As morphological studies were conducted, low coefficient of variation coupled with symmetric karyotype indicates A. cylindrica as a recently evolved species. A. cylindrica diversity centers are mostly located in the Northwest regions where the highest numbers of collection sites are distributed. We also observed that A. cylindrica accessions of Iran should be treated as a recently evolved species due to low diversity in morphological traits and symmetric karyotypes.