Aegilops tauschii Coss. (2n = 2x = 14) is a diploid wild species which is reported as a donor of the D-genome of cultivated bread wheat – Triticum aestivum L. (Kihara, 1944; McFadden and Sears, 1946). What is more, D-genome is also a part of several tetraploid (i.e., Ae. cylindrica Host and Ae. ventricosa Tausch.) and hexaploid [i.e., Ae. juvenalis (Thell.) Eig and Ae. vavilovii (Zhuk.)] Chennav. species of the genus Aegilops.

Among Aegilops species, Ae. tauschii has the widest geographic distribution, from Turkey on the West to Afghanistan and central Asia in the East, and thus adapted to diversified environmental conditions (Ogbonnaya et al., 2005; Kalia et al., 2016). Ae. tauschii encompasses two subspecies – Ae. tauschii Coss. subsp. strangulata (Eig) Tzvel. and Ae. tauschii subsp. Tauschii (Kalia et al., 2016). Subspecies strangulata is distributed from Transcaucasian region (Armenia, Azerbaijan) to the southeastern Caspian Sea region in Iran, whereas subspecies tauschii is native to the southwestern Caspian Iran and Afghanistan (Kihara et al., 1965; Ogbonnaya et al., 2005; Wang et al., 2013; Kalia et al., 2016). According to previous reports, it is assumed that Ae. tauschii ssp. strangulata is the ancestor of the wheat D-genome (Wang et al., 2013).

Recently, progressive genetic erosion and increasing number of races of pathogens destructive for cereals were observed. Hence, there is need to find new sources of variation for breeding of cereals. Ae. tauschii is an excellent source of novel genes to various biotic and abiotic stresses (Valkoun et al., 1985; Gill et al., 1986; Cox et al., 1992; Assefa and Fehrmann, 2004). Close evolutionary relationship, a variety of genes and relatively easy crossability make this species especially interesting for improvement of cultivated Triticeae species like wheat and triticale (Ogbonnaya et al., 2005). Considering breeding of cereals, one of the main goals is to transfer genes determining resistance to fungal diseases like leaf rust and powdery mildew from wild relatives into cultivated varieties (Gill et al., 1983, 1986; Dhaliwal et al., 1991).

Leaf rust, caused by Puccinia triticina Eriks., occurs on leaves and is one of the most destructive diseases of wheat in the world, because of the ability of the pathogen to adapt to diverse climatic conditions (Kolmer, 1996; Chen et al., 2013). It occurs regularly in the regions with humid and warm environmental conditions like continental Europe with a temperate climate and leads up to 30% of yield losses (Roelfs et al., 1992). It was reported that genetic engineering of host resistance is both profitable and safe for environment approach to control leaf rust (Assefa and Fehrmann, 2000; Kalia et al., 2016). According to the literature, Ae. tauschii is a source of four seedling resistance genes [Lr21 (1D), Lr32 (3D), Lr39 (2D), Lr42 (1D)] and one determining adult plant resistance [Lr22a (2D)].

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is second globally important leaf disease not only in wheat but also in triticale breeding, because populations of the pathogen are very dynamic and continually change their virulence structure. It is very common in Europe, parts of Asia, and the southeastern part of North America, with high rainfalls and maritime or semi-continental climate, where powdery mildew occurs annually and leads to significant yield losses (Wang et al., 2016). So far, the most feasible means of controlling the disease is by host resistance (Bennett, 1984; Wang et al., 2016). Over 70 powdery mildew resistance (Pm) alleles at 50 loci have been formally designated (Pm1–Pm54), whereas genes Pm2 (5D), Pm19 (7D), Pm34 (5D), and Pm35 (5D) were originated from Ae. tauschii (Cowger et al., 2012; Wang et al., 2016). What is more, Ae. tauschii might constitute also a valuable source of Pm43 gene (Majka et al., 2016), which was previously mapped on chromosome 2D of this species by Jia et al. (2013).

So far, many efforts have been undertaken to characterize genetically diverse accessions of Ae. tauschii using SSR markers also in terms of disease resistance (Naghavi et al., 2009; Chhuneja et al., 2010). Despite a large number of analyzed accessions (13 and 85, respectively) and SSR markers (21 and 51, respectively) used in this study, none of the markers were assigned to resistance genes. Singh et al. (2012) evaluated 63 accessions of Ae. tauschii for marker-trait association using 35 SSR markers, and identified six SSR markers significantly associated with leaf rust resistance genes (Xcfd19, Xgdm35, Xgwm261, Xgwm515, Xcfd4, and Xgwm645). It was reported that using this markers the germplasm collections can be initially screened and characterized to identify candidate genetic stocks and genomic regions (Singh et al., 2012). What is more, some microsatellites markers associated with leaf rust resistance genes localized in D-genome were reported for bread wheat (Huang et al., 2003; Hiebert et al., 2007; Thomas et al., 2010).

Karyotype and C-banding patterns of Ae. tauschii chromosomes were reported by many authors (Chennaveeraiah, 1960; Gill and Kimber, 1974; Iordansky et al., 1978; Teoh and Hutchinson, 1983; Friebe et al., 1992). What is more, Ae. tauschii chromosomes were also examined using in situ hybridization with various probes, like pTa71 (18S-5.8S-26S rDNA), pTa794 (5S rDNA), pAs1 (Afa family) and pSc119.2 (Badaeva et al., 2002; Kwiatek et al., 2013; Molnar et al., 2014). However, in two of this studies only one accession of Ae. tauschii was taken under investigations. So far, only Friebe et al. (1992) and Badaeva et al. (2002) using C-banding technique revealed polymorphism between respectively 15 and 12 accessions of Ae. tauschii with a different origin. The number and distribution of repetitive sequences used in previous studies for fluorescent in situ hybridization were not sufficient to cover the whole karyotype of this species. What is more, the number and distribution of rDNA loci in the Triticeae species are reported to be a conservative feature (Badaeva et al., 1996). Because of the great agronomic importance of the D-genome in bread wheat, this genome was well-studied on the cytogenetic level using different probes especially repetitive sequences with oligonucleotides amongst them.

D-genome chromosomes of wheat cultivar Chinese Spring were studied in terms of distribution of synthetic oligonucleotides (2–3 bp repeats) (Cuadrado et al., 2000, 2008). Among 12 analyzed sequences only two allowed to obtain distinctive hybridization signals. (AAG)₅ revealed signals in chromosomes 1D, 2D and 7D, (AGG)₅ single and weak signals in chromosomes 2D, whereas two other sequences (AC)₈ and (GCC)₅ revealed dispersed signals scattered along the chromosomes (Cuadrado et al., 2008). Moreover, the presence of weak and single signals of GAA satellite sequence in chromosomes 1D, 2D, and 7D was also reported (Pedersen and Langridge, 1997; Komuro et al., 2013). On the other hand, fluorescence in situ hybridization (FISH) with repetitive sequences from BAC library of wheat (cv. Chinese Spring) revealed the presence of hybridization signals in D-genome and availability of sequences for Ae. tauschii karyotyping.

The main goal of this work was to examine the differences and similarities in chromosomes organization between the accessions of Ae. tauschii with a different origin in order to identify new sources of variation for breeding of Triticeae species especially resistance to fungal diseases. Therefore cytogenetic analyses were supported with molecular markers associated with genes determining resistance to leaf rust and powdery mildew.

Komuro et al. (2013) reported that combination of pTa-86 + pTa-535 + pTa-713 probes allows to discriminate all D-genome chromosomes of wheat without ambiguity including their orientation. What is more, by overlaying pTa-465, pTa-k566 and GAA FISH signals, wheat D-genome chromosomes were highly decorated. The main goal of this study was to examine the differences and similarities in chromosomes organization among accessions of Ae. tauschii with a different origin, which is believed as a potential source of genes especially determining resistance to fungal diseases (i.e., leaf rust) that could be used in wheat and triticale breeding.

Results obtained for Ae. tauschii chromosomes revealed many similarities between analyzed accessions, however, some hybridization patterns were specific for accessions which become from cognate regions of the World. The most noticeable differences were observed for accessions from China which were characterized by presence of distinct signals of pTa-535 in the interstitial region of chromosome 3D, less intensity of pTa-86 signals in chromosome 2D, as well as lack of additional signals of pTa-86 in chromosomes 1D, 5D, or 6D (Figure 1). These results were in compliance with results of Wang et al. (2013) who reported that landraces of Ae. tauschii of Chinese region and other wheats of the Far Eastern origin appeared homogeneous and separate from landraces that originated in western Asia.

Chromosomes 1D and 2D of Ae. tauschii are of great importance because on this chromosomes many resistance genes have been mapped (Gill et al., 2008; Majka et al., 2016). In wheat, those chromosomes were found to be highly polymorphic, whereas in all wheat D-genome chromosomes most genetic diversity was localized in the terminal parts of chromosomes and was correlated with high recombination rates (Akhunov et al., 2010; Wang et al., 2013). It was assumed that wheat polymorphisms must have been introgressed into wheat from Ae. tauschii (Wang et al., 2013). The high content of genes is directly proportional to the low content of repetitive sequences along the entirety of chromosomes 1D and 2D what was in accordance with the scarcity of hybridization sites of pTa probes which are repetitive sequences. Especially terminal region of the short arm of chromosome 2D is characterized by high recombination rate. This region was reported to be exposed to chromosome rearrangements, though such places in the genome may constitute a hot spots (Luo et al., 2009, 2013; Majka et al., 2016). Tau-16 and Tau-17 are the only accessions that lacks signals of the pTa-86 signals in chromosome 2D (Figure 1). What is more, results obtained for the rest of Ae. tauschii chromosomes confirm the patterns of genetic diversity among D-genome chromosomes and arms of wheat where diversity is high in terminal region of the long arm of chromosome 4D, and both terminal regions of chromosome 6D. In the contrary, very low levels of diversity were reported for the whole chromosomes 3D and 5D, three-quarters of chromosome 4D, and terminal regions of 6D (Akhunov et al., 2010). Friebe et al. (1992) reported a large amount of polymorphic variation in C-bands size, as well as in the intra-chromosomal distribution pattern of these bands between different accessions of Ae. tauschii. It is known, that C-banded regions are composed of highly repetitive DNA sequences and the location of hybridization sites in this work are similar to the C-banding distribution in chromosomes of Ae. tauschii reported by Friebe et al. (1992). Moreover, it was shown, that Ae. tauschii TA 2462 (origin – Mazandaran province, Iran) carry a reciprocal translocation T1DS.7DL and T7DS.1DL basing on C-banding and hybridization patterns of pAs1 probe (Friebe et al., 1992). What is more, Hohmann and Lagudah (1993) using C-banding technique reported the same reciprocal interchange between chromosomes, with the breakpoints located in the centromeric region of Ae. tauschii accession AUS 18902 with Iranian origin. The same translocation was present in Tau-13 (this work; origin – Mazandaran province, Iran), what indicates a common origin of all accessions (Figure 3).

In this study Ae. tauschii chromosomes showed similar hybridization patterns to wheat D-genome chromosomes, however, some differences were also observed among both species. It is worth to mention, that combination of pTa-86 + pTa-535 + pTa-713 probes allows to discriminate all Ae. tauschii chromosomes and their orientation according to Komuro et al. (2013). Terminal parts of chromosomes 1DL, 5DS, and 6DS carried less distinct pTa-535 signals in comparison to wheat cv. Chinese Spring. The crucial differences concern the distribution and intensity of pTa-86 probe. The typical pattern for this repetitive sequence revealed signals in terminal regions of chromosomes (short arms) 2D, 3D, and 4D (Figure 1). What is more, in about 43% of analyzed accessions strong hybridization signals in short arm of chromosome 5D, were identified (Figure 1). In addition, no differences were observed for pTa-713 probe. The hybridization patterns of all probes established on Tau-13 chromosomes were similar to those observed in wheat Chinese Spring. This accession was collected in Mazandaran province in Iran, what is in relevance with the results obtained by Wang et al. (2013) who report that southwestern and southern Caspian accessions of Ae. tauschii appears to be the main source of the wheat D-genome.

pTa-86 clone is a homolog of pSc119.2 sequence which was derived from S. cereale (Bedbrook et al., 1980; Komuro et al., 2013). The typical hybridization pattern obtained for Ae. tauschii chromosomes revealed six strong hybridization signals in terminal parts of chromosomes 2D, 3D, and 4D (short arms) (Figure 1). That results were in coincidence with hybridization pattern of pSc119.2 obtained for Ae. tauschii accession MvGB605 by Molnar et al. (2014) and for accession Tau-20 by Kwiatek et al. (2013), where additional signals in terminal region of chromosome 4DL were also observed. However, both papers did not reveal any distinct signals of pSc119.2 in chromosomes 1D, 5D and 6D, what confirms that this hybridization pattern is specific only for some accessions with a different origin. Majka et al. (2017), hybridized gDNA of rye to chromosomes of Ae. tauschii (Tau-20) what reveals chromosome labeling and six terminal signals in chromosomes without 5S and 35SrDNA loci (1D and 5D). In this paper we also observed six distinct hybridization signals obtained for pTa-86 probe, what is consistent with GISH results and confirms homology of pTa-86 with pSc119.2 sequence derived from S. cereale (Figure 1).

High polymorphism between analyzed accessions and extensive allelic variation were revealed using molecular markers associated with resistance genes. In case of most of the markers localized in chromosomes 1D and 2D, the diversity of banding patterns was high. Additionally, results obtained for markers located in the distal part of the short arm of chromosome 3D also revealed the high diversity of obtained banding patterns. What is more, the differences in number and size of the bands were observed even between accessions with close origin. It should be mention that most of the analyzed molecular markers, thus resistance genes, were mapped to the distal parts of D-genome chromosomes (Somers et al., 2004; Liu et al., 2014). Only two of them Xgwm296 and Xgwm539 were mapped to the interstitial parts of chromosomes, but the polymorphism of obtained banding patterns was low. That confirms the high polymorphism of chromosomes 1D and 2D, as well as high recombination rates at the ends of D-genome chromosomes (Akhunov et al., 2010; Luo et al., 2013). What is more, Naghavi et al. (2009) using SSR markers reported that genetic variability in the genome of Ae. tauschii were higher than in D-genome of bread wheat. Therefore, this wild species might constitute a source of agronomically important genes that could be transferred into wheat germplasm (Naghavi et al., 2009). Singh et al. (2012) reported that only one of the known, major leaf rust resistance genes Lr39 was declared to be associated with marker Xgdm35, through the conservative test. They declared that 180 bp product obtained for Ae. tauschii is directly related to the resistance. What is more, Wiśniewska et al. (2007) obtained 250 bp product for wheat cultivar Thatcher, whereas Sun et al. (2009) show that Xgdm35 amplified polymorphic products (229–265 bp in range) in diverse wheat germplasm (239 bp in wheat cultivar Chinese Spring). This reports were in compliance with results obtained in this study and indicates that Tau-4, Tau-5, Tau-6, Tau-10, Tau-11, Tau-13, Tau-19, Tau-20, and Tau-21 can consist a source of leaf rust resistance, whereas Tau-18 has similar organization of Xgdm35 locus in comparison to wheat cultivar Thatcher-Lr39 (Figure 4).

Obtained results imply, that there is a moderate or high level of polymorphism in the genome of Ae. tauschii. We showed the similarities and differences of repetitive sequences organization are determined by geographical origin. Therefore, selected accessions of Ae. tauschii might constitute an accessible source of variation for improvement of Triticeae species like wheat and triticale.

MM initiated and designed the study. MK and HW initiated and designed the project. MM and JM made the mitotic chromosome preparations followed by FISH analysis. MM performed the molecular marker analysis and analyzed the data. MM wrote the paper.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.