The cultivated wheat gene pool has a narrow genetic base for resistance to the contemporary virulent races, such as TTKSK (Jin and Singh, 2006; Singh et al., 2006; Newcomb et al., 2016), TRTTF (Olivera et al., 2012); and TKTTF (Olivera Firpo et al., 2017). In order to broaden the basis of stem rust resistance in wheat breeding programs, it is necessary to identify and introgress effective genes from all genepools of wheat. Wild relatives of wheat are a tractable source of stem rust resistance genes. Indeed, a number of resistance genes derived from wild relatives of wheat appeared to be more effective against the races in the Ug99 group than Sr genes of wheat origin (Singh et al., 2006; Jin et al., 2007). Aegilops is the most closely related genus to Triticum (Kimber and Feldman, 1987; Jiang et al., 1994) and comprises 23 species that include diploid, tetraploid, and hexaploid genomes (van Slageren, 1994). Aegilops species are known to be a rich source of stem rust resistance, and several stem rust resistance genes have been transferred into cultivated wheat (Friebe et al., 1996; Schneider et al., 2008; Liu et al., 2011a,b; Olson et al., 2013a,b).

Ease of hybridization and reduced linkage drag make introgression from species in the primary gene pool preferred by wheat breeders to incorporate new alleles in their breeding programs (Feuillet et al., 2008). However, species in the secondary and tertiary gene pools constitute an important reservoir of genetic variability (Qi et al., 2007). Aegilops species in the tertiary genepool have not been exploited to any great extent for wheat improvement, and for resistance to TTKSK and other virulent Pgt races in particular. The objective of this study was to evaluate a collection of nine Aegilops species in the tertiary gene pool of wheat for resistance to race TTKSK and other Pgt races.

# MATERIALS AND METHODS

#### Germplasm

A total of 1,422 accessions of nine Aegilops species (three diploid and six tetraploid) deposited at the USDA-ARS, National Small Grain Collection (NSGC), Aberdeen, ID, were evaluated in this study. Species, the number of accessions and country of origin of each Aegilops species are given in **Table 1**.

## Inoculation, Incubation, and Disease Assessment

With the objective of identifying multiple and diverse resistance genes in individual accessions, we evaluated this Aegilops collection against multiple races with different virulence spectrum and origin. All accessions were characterized for reaction to three virulent Pgt races: TTKSK (Kenya), TRTTF (Yemen), and TTTTF (United States). Accessions resistant to the three races were further evaluated for their reaction to four additional US races (TPMKC, RKRQC, QTHJC, and QFCSC). The race designations are based on the letter code nomenclature system (Roelfs and Martens, 1988; Roelfs et al., 1993; Jin et al., 2008). Avirulence/virulence profile of the Pgt isolates used in the disease assessments is summarized in **Table 2**. Disease evaluations were conducted in two independent experiments. In each experiment, five seedlings per accession were inoculated with each race on fully expanded primary leaves 8–9 days after planting. Details on inoculation procedures and disease assessment were described by Jin et al. (2007). Disease reactions were classified according to Stakman et al. (1962). Infection types (ITs) 0, 1, and 2 were considered as resistant reactions and ITs 3 and 4 were considered as susceptible. Wheat cultivar McNair 701 (Cltr 15288) was included as susceptible check. Analyses of association via contingency tables were conducted to assess potential relationships of resistance to different Pgt races.

#### Inheritance Study

Bi-parental crosses between selected resistant accessions and a susceptible accession in five Aegilops species were made and F2 progeny were produced by selfing F1 plants. Seventeen F2 populations (four from Ae. cylindrica, four from Ae. peregrina, six from Ae. triuncialis, two from Ae. umbellulata, and one from Ae. comosa) were evaluated for reaction to race TTKSK to determine the inheritance of resistance based on phenotypic ratios. Chi-square (χ 2 ) test was used to determine the goodness of fit to expected genetic ratios in the F2 generation.

# RESULTS

A wide array of infection types was observed across the Aegilops spp. and ranged from highly resistant (IT 0) to highly susceptible (ITs 3+ and 4). Low ITs (; or ;1−) were frequently observed in Ae. caudata, Ae. cylindrica, Ae. neglecta, Ae. peregrina and Ae. triuncialis, whereas ITs 2- and 2-; were predominant in Ae. biuncialis and Ae. geniculata. We observed a high percentage of resistance in this Aegilops collection as 977 (68.8%), 927 (65.2%), and 850 (59.8%) accessions produced low infection types to races TTKSK, TTTTF, and TRTTF, respectively (**Table 3**). Five hundred and fifty one (38.8%) accessions were resistant to the three races evaluated. The frequencies of accessions resistant to race TTKSK varied among the species: over 80% in six Aegilops species (Ae. caudata, Ae. cylindrica, Ae. geniculata, Ae. neglecta, Ae. peregrina, and Ae. triuncialis), and below 30% in three species (Ae. biuncialis, Ae. comosa, and Ae. umbellulata) (**Table 3**).

Pairwise association for resistance to races TTKSK, TRTTF, and TTTTF exhibited variation among species and pathogen races. Over 75% of the accessions of Ae. geniculata and Ae. neglecta were resistant to races TTKSK, TRTTF, and TTTTF (**Table 3**). Resistance to pairs of the three Pgt races in Ae. geniculata and Ae. neglecta were highly associated (**Table 4**), suggesting that accessions resistant to race TTKSK are likely to be resistant to races TRTTF and TTTTF. Association for the reaction to races TTSKS, TRTTF, and TTTTF was also observed

TABLE 1 | Number of accessions and country of origin of Aegilops species used in this study.

TABLE 2 | Isolate designation, origin, and virulence phenotype of Puccinia graminis f. sp. tritici races used to evaluate resistance in Aegilops spp.

1Race nomenclature was based on Roelfs and Martens (1988) and Jin et al. (2008).

TABLE 3 | Number and frequency of Aegilops accessions resistant to Puccinia graminis f. sp. tritici races TTKSK, TRTTF, and TTTTF at the seedling stage.

in Ae. triuncialis (**Table 4**). Resistance with race specificity was observed in accessions of the remaining species, most noticeably in Ae. cylindrica, where only one accession exhibited resistance to race TRTTF.

A group of 408 accessions resistant to races TTKSK, TRTTF, and TTTTF, were evaluated against US races TPMKC, RKRQC, QTHJC, and QFCSC. Three hundred ninety-six accessions remained resistant to all the races evaluated (**Supplementary Table 1**), indicating these accessions possess genes with broad spectrum resistance.

Sixty-five percent of the accessions evaluated in this study are native to Turkey or Greece. The frequencies of accessions resistant to all races from both countries were similar (30.6%) (**Table 5**). Higher frequencies of resistance were

TABLE 4 | Probability from contingency tables for association analysis of the reactions of accessions of Aegilops ssp. to races TTKSK, TRTTF, and TTTTF of Puccinia graminis f. sp. tritici.

aBased on P < 0.05.

TABLE 5 | Number and percentage of resistant Aegilops species accessions according to country of origin.

1Accessions resistant against all Pgt races (TTKSK, TRTTF, and TTTTF) evaluated.

obtained in accessions from Macedonia (35.8%), France (38.9%), Iraq (46.7%), and Montenegro (75.0%), but the numbers of accessions evaluated from these countries were significantly smaller.

Segregation ratios of the F2 progeny from biparental crosses between resistant and susceptible accessions indicated that resistance to race TTKSK in selected accessions is mostly conferred by single genes (**Table 6**). Eight resistant Aegilops accessions carry a single gene with dominant effect, whereas two resistant accessions carry a single gene with recessive effect. Two genes conferring resistance to race TTKSK were observed in three accessions of Ae. triuncialis. Inheritance with epistatic effect between two genes was also observed in four resistant parents. Segregation ratios of the F2 progeny of one Ae. triuncialis and one Ae. umbellulata resistant parent fit to a 9:7 ratio indicating the presence of a complementary gene action with duplicate recessive epistasis. Epistatic effect between two dominant genes was also observed in two Ae. peregrina resistant parents (**Table 6**), where the F2 progenies fit to a 11:5 ratio.

#### DISCUSSION

Races of P. graminis f. sp. tritici, such as the Ug99 race group, TKTTF and others detected from the contemporary Pgt populations worldwide, are a serious threat to bread and durum wheat production worldwide because of their broad virulence to many cultivars and rapid geographic spread. The limited number of stem rust resistance genes effective against these virulent races requires the identification of new sources of resistance. Different Aegilops species have contributed several stem rust resistance genes effective against race TTKSK including Sr32, 33, 45, 46, 47, 51, 53, SrTA10187 and SrTA10171 (Friebe et al., 1996; Schneider et al., 2008; Liu et al., 2011a,b; Olson et al., 2013a,b). However, only one gene, Sr53, is derived from Ae. geniculata in the tertiary genepool. Results from this study demonstrated that Aegilops species in the tertiary genepool of wheat are a rich source of resistance to race TTKSK and other Pgt races with broad virulence.

Although the overall frequency of resistant accessions in the entire Aegilops collection evaluated against races TTKSK, TRTTF, and TTTTF in this study was over 60%, we observed significant variation among species. Only two species (Ae. geniculata and Ae. neglecta) exhibited a high frequency of resistance (over 80%) against the three races. Interestingly in Ae. biuncialis, a species that also shares the same genome constitution as Ae. geniculata and Ae. neglecta (UUMM), the frequencies of resistance varied, exhibiting a high level of race specificity. Differences in the frequencies of resistance to stem, stripe, and leaf rust in species carrying the same genome have been also reported in the Section Sitopsis (SS genome) of Aegilops (Anikster et al., 2005; Scott et al., 2014). In species such as Ae. geniculata and Ae. neglecta where there is a high degree of association of the reactions to races

TABLE 6 | Segregation of F2 populations of bi-parental crosses of Aegilops spp. to race TTKSK of P. graminis f. sp. tritici.

aFemale parent/Male parent; (R) and (S) indicate the resistant and susceptible parent, respectively.

TTKSK, TRTTF, and TTTTF, it is highly likely that the genes that confer resistance to one race is also effective against the other races. The progeny populations via bi-parental crosses initiated through this study will be further developed and analyzed to understand the genetic relationships for resistance to different races in these selected accessions.

Race specificity was a common feature observed in this Aegilops collection, as five species exhibited a percentage of accessions resistant to all three races TTKSK, TRTTF, and TTTTF below 20% (**Table 3**), and have no association of the reaction of two out of three races. Previous studies also report race specificity in Aegilops species (Olivera et al., 2007; Scott et al., 2014). Since gene introgression from Aegilops species in the tertiary genepool is a long and laborious process, it is preferable to use accessions that carry stem rust resistance that is effective against multiple races. About 30% (396 accessions) were resistant against all the races evaluated, indicating the availability of potential sources of new and diverse stem rust resistance genes that could be very useful in wheat breeding programs. Most of these resistant accessions (84%) were from the tetraploid species Ae. geniculata, Ae. neglecta, and Ae. triuncialis. Additional studies are required to assess the diversity in these resistant accessions to allow the identification of donor accessions that are likely to contribute non-redundant stem rust resistance genes. Choosing resistant accessions from geographically diverse countries of origin and exhibiting different infection types for gene introgression is a first step to maximize the chances of capturing new and unique resistance genes (Anikster et al., 2005).

Sixty-five percent of the accessions evaluated in this study originated from Turkey or Greece, two countries having the largest numbers of Aegilops species. Turkey is known to be the center of diversity for Aegilops (Eig, 1929), and 17 out of the 23 Aegilops species have been identified in its territory (van Slageren, 1994). The nine species evaluated in this study are present in Turkey. The number and frequency of resistant accessions from Turkey and Greece (n = 285, 30.6%) from this study demonstrated that valuable sources of new genetic variation for stem rust resistance are present in these countries.

A prior knowledge on the inheritance of resistance in wild wheat relatives will facilitate alien gene introgression into wheat. We produced 17 biparental crosses to investigate the inheritance of TTKSK resistance. These populations will be further developed to map resistance genes and to develop closely linked markers within the wild species. Simple inheritance of stem rust resistance was found in most selected resistant accessions. Our result of a single dominant gene segregating in the Ae. umbellulata biparental F2 population from a cross between PI 542369 and PI 298905 was confirmed in an F3 population and mapped to chromosome 2U (Edae et al., 2016). A similar approach will be followed to characterize the resistance identified in this study. Two stem rust resistance genes were identified in three Ae. triuncialis resistant parents. Further studies are needed to characterize the effectiveness of each resistance gene. Multiple stem rust resistance genes with different resistance profile were reported in Ae. sharonensis (Olivera et al., 2008; Yu et al., 2017). A more complex inheritance of stem rust resistance with genes exhibiting epistatic effects was also observed in three Aegilops species. These results highlight the value of studying the genetics of stem rust resistance in the wild relative before attempting wide crosses for gene transferring.

Aegilops species in the tertiary genepool do not possess genome(s) homologous to the cultivated forms, and gene transfer through homologous recombination cannot be achieved with

these species (Harlan and de Wet, 1971). Cytogenetic techniques such as irradiation and chemical treatments, production of synthetic amphiploids, use of gametocidal chromosomes, or Ph1 gene mutants may be required for gene introgression into the cultivated forms (Friebe et al., 1996; Zaharieva and Monneveux, 2006). However, the introgression of alien chromatin to substitute for homoeologous chromosome segments has the potential of a simultaneous introduction of deleterious DNA that can affect agronomic and quality traits of wheat (Feuillet et al., 2008; Wulff and Moscou, 2014). New sequencing technologies, like Genotyping-By-Sequencing, have allowed the development of genetic linkage maps in wild relatives of wheat with non-previous available markers, and the identification of closely linked markers that can facilitate the gene transfer process by reducing the introgressed alien chromatin segment into elite materials (Edae et al., 2016, 2017). The sources of resistance identified from the tertiary genepool will also serve as targets for resistance gene cloning. Cloned genes and their delivery as transgenes in single or multiple resistance gene cassettes will completely resolve the linkage drag problem and ensure the effectiveness and durability of genes derived from more distant relatives of wheat (Wulff and Moscou, 2014). Today, new cloning techniques like mutational genomics (MutRenSeq) (Steuernagel et al., 2016) and association genetics with R gene enrichment sequencing (AgRenSeq) (Arora et al., 2018) allow a rapid and cheaper discovery and cloning of resistance genes. These technologies are opening new doors for fully exploiting the richness and diversity of wild relatives for wheat improvement.

#### REFERENCES

## AUTHOR CONTRIBUTIONS

PO and YJ were involved in the experimental design and manuscript preparation. PO performed the experiments and completed the data analysis. MR was involved in manuscript preparation and revision.

#### FUNDING

This research was funded by USDA-ARS and the Durable Rust Resistance in Wheat (OPP1144199) project administrated by Cornell University and funded by the Bill and Melinda Gates Foundation and the United Kingdom Department for International Development.

#### ACKNOWLEDGMENTS

We thank H. Bockleman, United States Department of Agriculture-Agricultural Research Service, National Small Grains Collection, Aberdeen, ID, for providing Aegilops spp. seeds.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01719/ full#supplementary-material

tritici from 2008 to 2014 reveals virulence to SrTmp in the Ug99 race group. Phytopathology 100, 986–996. doi: 10.1094/PHYTO-12-15-0337-R

**Conflict of Interest Statement:** 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.

Copyright © 2018 Olivera, Rouse and Jin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Evolution of the S-Genomes in *Triticum-Aegilops* Alliance: Evidences From Chromosome Analysis

#### *Alevtina S. Ruban1 and Ekaterina D. Badaeva2,3 \**

1 Laboratory of Chromosome Structure and Function, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany, 2 Laboratory of Genetic Basis of Plant Identification, Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia, 3 Laboratory of Molecular Karyology, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia

Five diploid Aegilops species of the Sitopsis section: Ae. speltoides, Ae. longissima, Ae. sharonensis, Ae. searsii, and Ae. bicornis, two tetraploid species Ae. peregrina (=Ae. variabilis) and Ae. kotschyi (Aegilops section) and hexaploid Ae. vavilovii (Vertebrata section) carry the S-genomes. The B- and G-genomes of polyploid wheat are also the derivatives of the S-genome. Evolution of the S-genome species was studied using Giemsa C-banding and fluorescence in situ hybridization (FISH) with DNA probes representing 5S (pTa794) and 18S-5.8S-26S (pTa71) rDNAs as well as nine tandem repeats: pSc119.2, pAesp\_SAT86, Spelt-1, Spelt-52, pAs1, pTa-535, and pTa-s53. To correlate the C-banding and FISH patterns we used the microsatellites (CTT)10 and (GTT)9, which are major components of the C-banding positive heterochromatin in wheat. According to the results obtained, diploid species split into two groups corresponding to Emarginata and Truncata sub-sections, which differ in the C-banding patterns, distribution of rDNA and other repeats. The B- and G-genomes of polyploid wheat are most closely related to the S-genome of Ae. speltoides. The genomes of allopolyploid wheat have been evolved as a result of different species-specific chromosome translocations, sequence amplification, elimination and re-patterning of repetitive DNA sequences. These events occurred independently in different wheat species and in Ae. speltoides. The 5S rDNA locus of chromosome 1S was probably lost in ancient Ae. speltoides prior to formation of Timopheevii wheat, but after the emergence of ancient emmer. Evolution of Emarginata species was associated with an increase of C-banding and (CTT)10-positive heterochromatin, amplification of Spelt-52, re-pattering of the pAesp\_SAT86, and a gradual decrease in the amount of the D-genome-specific repeats pAs1, pTa-535, and pTa-s53. The emergence of Ae. peregrina and Ae. kotschyi did not lead to significant changes of the S∗ -genomes. However, partial elimination of 45S rDNA repeats from 5S∗ and 6S∗ chromosomes and alterations of C-banding and FISH-patterns have been detected. Similarity of the Sv -genome of Ae. vavilovii with the S s genome of diploid Ae. searsii confirmed the origin of this hexaploid. A model of the S-genome evolution is suggested.

Keywords: wheat, *Aegilops,* S-genome of *Ae. speltoides*, S∗ -genome of other *Aegilops* species, chromosome, karyotype evolution, C-banding, FISH

#### *Edited by:*

István Molnár, Centre for Agricultural Research (MTA), Hungary

#### *Reviewed by:*

Elena Khlestkina, Institute of Cytology and Genetics, Russian Academy of Sciences, Russia Shuhei Nasuda, Kyoto University, Japan

> *\*Correspondence:* Ekaterina D. Badaeva<EMAIL_ADDRESS>
#### *Specialty section:*

This article was submitted to Plant Breeding, a section of the journal Frontiers in Plant Science

*Received:* 30 July 2018 *Accepted:* 12 November 2018 *Published:* 04 December 2018

#### *Citation:*

Ruban AS and Badaeva ED (2018) Evolution of the S-Genomes in Triticum-Aegilops Alliance: Evidences From Chromosome Analysis. Front. Plant Sci. 9:1756. doi: 10.3389/fpls.2018.01756

**47**

# INTRODUCTION

Evolutionary goat grasses, or Aegilops are closely related to wheat and contributed two of the three subgenomes of hexaploid bread wheat (Sears, 1969; Kihara, 1975; Feldman, 2001). The natural distribution area of the genus Aegilops L. covers the Mediterranean basin, southwestern and central Asia (Witcombe, 1983; Kimber and Feldman, 1987; Van Slageren, 1994; Kilian et al., 2011). Their center of origin is thought to be located in Transcaucasia (Hammer, 1980; Van Slageren, 1994), or in the Fertile Crescent (Kimber and Feldman, 1987). These regions contain the highest concentration of Aegilops species. Goat grasses inhabit a broad range of environments and are characterized by very wide adaptation.  Some Aegilops accessions are characterized by high grain quality and increased micronutrient content (Rawat et al., 2011; Farkas et al., 2014; Rakszegi et al., 2017) that can be used for wheat improvement. Although many agronomically useful genes have already been transferred from Aegilops to common wheat varieties or breeding lines (Knott and Dvorák, 1976; Schneider et al., 2008; Rawat et al., 2011; McIntosh et al., 2013; Zhang et al., 2015), their genetic potential in broadening genetic diversity of wheat is not fully exploited. Utilization of gene pool of Aegilops requires good knowledge of genetics and genomics of these species, including their karyotypes and chromosomal structures.

In addition to the great potential for wheat breeding, goat grasses can also be an attractive model for studying mechanisms of reticulate evolution. Depending on taxonomical system, the genus Aegilops is classified into 20 (Kihara, 1954), 22 (Zhukovsky, 1928; Eig, 1929; Van Slageren, 1994), 24 (Kimber and Feldman, 1987), 25 (Chennaveeraiah, 1960), or 26 species (Witcombe, 1983). These species are split into sections based on morphological criteria or genome composition. At present, the system suggested by Van Slageren (1994) is commonly accepted; therefore, we will follow this nomenclature. According to it, 10 Aegilops species are diploid and 12 – polyploid, that were formed as a result of hybridization of different diploid progenitors.

Based on a series of pioneering works of the famous Japanese geneticist (Kihara, 1937, 1949, 1954, 1957, 1963; Lilienfeld, 1951; Kihara et al., 1959), diploid Aegilops were divided into three major genomic groups, C, D, and S. The C-genome group included two species; the D-genome group included four species; and the S-genome group consisted of three species of the Sitopsis (Jaub. & Spach) Zhuk. section: Ae. longissima Schweinf. & Muschl. (including Ae. sharonensis Eig), Ae. bicornis (Forssk.) Jaub. & Spach, and Ae. speltoides Tausch (Kihara, 1937, 1949; Lilienfeld, 1951). A new diploid species of the Sitopsis section— Ae. searsii Feldman and Kislev ex Hammer, has been discovered later by Feldman and Kislev (1977). Analysis of the karyotype, meiotic chromosome pairing, pollen fertility and seed set in Ae. longissima x Ae. searsii hybrids showed that Ae. searsii possesses the S∗ -genome (Feldman et al., 1979; Yen and Kimber, 1990a).

Thus, current taxonomy recognizes five diploid species carrying the S-genome: Ae. speltoides including ssp. ligustica (Savign.) Fiori (SS) and ssp. speltoides Boiss., Ae. bicornis (Sb S b ), Ae. searsii (Ss S s ), Ae. sharonensis (SshS sh), and Ae. longissima (Sl S l ) (Van Slageren, 1994; Kilian et al., 2011; Feldman and Levy, 2015). These species are morphologically similar, but can be easily distinguished by their habitat, climatic adaptation, and distribution areas. Based on differences in spike morphology, Eig (1929) divided the Sitopsis group into two sub-sections, Truncata and Emarginata. Subsection Truncata includes only one species–Ae. speltoides (SS), which grows in central, eastern, and northern part of the Sitopsis area. This species consists of two forms, ligustica and auscheri, which differ in their fruiting spike and the mode of seed dispersal (Eig, 1929; Zohary and Imber, 1963), but are similar in karyotype structure (Chennaveeraiah, 1960). Their hybrids are fully fertile and show complete meiotic chromosome pairing (Zohary and Imber, 1963). Ae. speltoides has the lowest nuclear DNA content (1C = 5.81 ± 0.123 pg) within the Sitosis group (Eilam et al., 2007) and differs significantly from Emarginata species in its chromosome morphology (Chennaveeraiah, 1960), Giemsa C-banding (Teoh and Hutchinson, 1983; Friebe and Gill, 1996; Friebe et al., 2000) and FISH patterns (Yamamoto, 1992a,b; Jiang and Gill, 1994b; Badaeva et al., 1996a,b; Salina et al., 2006b; Raskina et al., 2011; Belyayev and Raskina, 2013).

The subsection Emarginata includes four species: Ae. bicornis, Ae. searsii, Ae. sharonensis, and Ae. longissima, which grow in the central and southern part of the Sitopsis section habitat (Feldman and Kislev, 1977). Study of the chromosome pairing of intraspecific hybrids (Kihara, 1954, 1963; Feldman et al., 1979; Yen and Kimber, 1989, 1990a,b,c), similarity of karyotype structure (Riley et al., 1958; Chennaveeraiah, 1960), the number and distribution of 5S and 45S rDNA loci (Yamamoto, 1992a,b; Badaeva et al., 1996b), and the distribution of pSc119.2 sequence (Badaeva et al., 1996a) suggest a close relationship of Emarginata species, although they differ from each other in genome size (Eilam et al., 2007) and C-banding patterns (Friebe and Gill, 1996).

Morphologically, Ae. bicornis is the most primitive species in this group (Eig, 1929). It is more difficult to produce hybrids with Ae. bicornis than with other Aegilops of the S-genome group (Kimber and Feldman, 1987). Genome size of Ae. bicornis (1C = 6.84 ± 0.097 pg) is only little larger than that of Ae. searsii (1C = 6.65 ± 0.091 pg), and is lower than of Ae. longissima (1C = 7.48 ± 0.082 pg) or Ae. sharonensis (1C = 7.52 ± 1.000 pg) (Eilam et al., 2007). Morphologically Ae. searsii resembles Ae. longissima, but differs from it in a number of morphological traits which are considered as evolutionary advanced (Feldman and Kislev, 1977). Ae. longissima x Ae. searsii hybrids exhibit meiotic irregularities and are highly sterile (Feldman et al., 1979). By contrast, the F1 hybrids Ae. longissima x Ae. sharonensis are fertile and show complete chromosome pairing in meiosis. Isolation of these species is caused by different ecological requirements (Feldman and Levy, 2015). According to other hypothesis (Waines and Johnson, 1972), Ae. sharonensis could be a hybrid between Ae. longissima and Ae. bicornis. Ae. longissima carries a speciesspecific 4S∗ /7S∗ translocation (Tanaka, 1955; Yen and Kimber, 1990b; Friebe et al., 1993; Naranjo, 1995), while no structural rearrangements have been identified in other species of this group (Yen and Kimber, 1989, 1990a,b,c; Maestra and Naranjo, 1997, 1998; Luo et al., 2005; Dobrovolskaya et al., 2011).

The similarity of Emarginata species and separate position of Ae. speltoides within the Sitopsis section was confirmed by molecular analyses of nuclear and cytoplasmic DNA. Based on the variation of repeated nucleotide sequences (RNS) Dvorák and Zhang (1992) showed that the Sitopsis species are phylogenetically similar, but Ae. speltoides is clearly separated from species of the Emarginata group. RAPD- and AFLP analyses revealed that Ae. speltoides forms a cluster with polyploid wheats, which is separated from other Sitopsis species (Kilian et al., 2007, 2011; Goryunova et al., 2008). Study of organellar DNAs by PCR-single-strand conformational polymorphism (PCR-SSCP) revealed high similarity of Ae. bicornis - Ae. sharonensis - Ae. longissima plasmons and their distinctness from plasmon of Ae. speltoides (Wang et al., 1997).

Comparative sequence analysis provided further insights into the evolution of Triticum and Aegilops and allowed the estimation of divergence time of different genomic groups. Comparison of chloroplast (Yamane and Kawahara, 2005; Golovnina et al., 2007; Gornicki et al., 2014; Middleton et al., 2014; Bernhardt et al., 2017) and nuclear DNA sequences (Petersen et al., 2006; Salse et al., 2008; Marcussen et al., 2014) strongly suggest that Ae. speltoides occupies a basal position on the phylogenetic tree of Aegilops/Triticum (Petersen et al., 2006; Kawahara, 2009). Probably Ae. speltoides diverged from the progenitor of the Triticeae much earlier than diploid wheat and Aegilops species (Yamane and Kawahara, 2005; Salse et al., 2008; Gornicki et al., 2014; Middleton et al., 2014; Bernhardt et al., 2017). Estimates obtained from the analyses of nuclear DNA sequences placed the possible divergence time within the period from ∼7 MYA (Marcussen et al., 2014) to 3.5–2.7 MYA (Dvorák and Akhunov, 2005; Salse et al., 2008). Estimates obtained from chloroplast DNA favored a more recent origin of Ae. speltoides – 4.1–3.6 MYA (Bernhardt et al., 2017) to 2.67 ± 1.1. MYA (Middleton et al., 2014). Marcussen et al. (2014) supposed that the Dgenome lineage (which indeed included D, M, and S∗ genome species, Sandve et al., 2015) emerged through ancient homoploid hybridization between A and S genomes. The members of Emarginata group are thought to radiate from common ancestor approximately 1.0–2.0 MYA (Ae. searsii) – 1.4 MYA (Ae. bicornis) – to 0.4 MYA (Ae. sharonensis) (Marcussen et al., 2014; Feldman and Levy, 2015).

Hypothesis that the B and G genomes of polyploid wheats originated from a diploid S-genome Aegilops species was put forward in the middle XXth (Sears, 1956; Riley et al., 1958). Different taxa were suggested as potential progenitors of polyploid wheat (Haider, 2013). All species of the Sitopsis section have been considered as the B-genome donors: Ae. speltoides (Sarkar and Stebbins, 1956; Tanaka et al., 1979; Bahrman et al., 1988; Kerby et al., 1990; Daud and Gustafson, 1996; Maestra and Naranjo, 1998; Yan et al., 1998; Blake et al., 1999; Rodríguez et al., 2000a; Haider, 2013), Ae. bicornis (Sears, 1956), Ae. longissima (Tanaka, 1956; Konarev et al., 1976; Konarev, 1980; Peacock et al., 1981), Ae. searsii (Feldman and Kislev, 1977; Nath et al., 1983, 1984; Kerby et al., 1990; Liu et al., 2003), Ae. sharonensis (Kushnir and Halloran, 1981) or yet unknown species of the Emarginata group (Kerby et al., 1990). Molecular analyses of common wheat genome and genomes of related species confirmed the ancestry of wheat B- genome from Ae. speltoides or the species close to it (Talbert et al., 1991; Petersen et al., 2006; Goryunova et al., 2008; Salse et al., 2008; Marcussen et al., 2014). Based on the analysis of nuclear or plastid DNA, ancient tetraploid emmer could emerge 0.4–0.8 MYA (Huang et al., 2002; Dvorák and Akhunov, 2005; Yamane and Kawahara, 2005; Golovnina et al., 2007; Gornicki et al., 2014; Marcussen et al., 2014; Middleton et al., 2014; Bernhardt et al., 2017).

The origin of the G-genome of Triticum timopheevii Zhuk. from the S-genome of Ae. speltoides was first hypothesized by Giorgi and Bozzini (1969) based on comparison of chromosome morphologies and was later confirmed by numerous studies including chromosome pairing analysis of intraspecific hybrids (Shands and Kimber, 1973; Tanaka et al., 1979; Maestra and Naranjo, 1999; Rodríguez et al., 2000a), comparison of C-banding (Badaeva et al., 1996a) and ISH patterns (Jiang and Gill, 1994a,b; Salina et al., 2006b), isozyme profiles (Konarev et al., 1976; Nakai, 1978; Jaaska, 1980), AFLP- (Kilian et al., 2007, 2011) and RFLP-analyses (Dvorák and Zhang, 1990; Talbert et al., 1991; Dvorák, 1998), sequencing of nuclear (Huang et al., 2002) and cytoplasmic DNA (Sasanuma et al., 1996; Yamane and Kawahara, 2005; Golovnina et al., 2007; Gornicki et al., 2014). These studies revealed that Ae. speltoides is more closely related to the G genome of T. timopheevii than to the B-genome of common wheat and suggested that ancient T. timopheevii could emerge approximately 0.4 MYA (Huang et al., 2002; Gornicki et al., 2014).

The S∗ -genome is identified in two tetraploid Aegilops species belonging to the section Aegilops L.: Ae. peregrina (Hach. in Fraser) Maire & Weiller (=Ae. variabilis Eig, UpUp S p S p ) and Ae. kotschyi Boiss. (UkUk S k S k ). Based on the "analyzer" method H. Kihara (1954) proposed that Ae. peregrina is a hybrid between Ae. umbellulata Zhuk. and a diploid species of the Sitopsis group (Lilienfeld, 1951), although conventional chromosome staining did not reveal the S∗ -genome in these species (Chennaveeraiah, 1960). Cytoplasmic genomes of Ae. peregrina and Ae. kotschyi are most closely related to the cytoplasmic genome of Ae. searsii (Ogihara and Tsunewaki, 1988; Siregar et al., 1988). However, meiotic analysis of the F1 hybrids between Ae. kotschyi and induced autotetraploid of three Sitopsis species showed that Ae. kotschyi shared the S∗ genome with Ae. longissima (Yen and Kimber, 1990d). Yu and Jahier (1992) come to the same conclusion based on chromosome pairing analysis in hybrids of Ae. variabilis (=Ae. peregrina) with different Sitopsis species. RFLP profiles of RNS suggested that the S∗ genome of Ae. peregrina and Ae. kotschyi could have originated from Ae. longissima or Ae. sharonensis or the species immediately preceding the divergence of these diploids (Zhang et al., 1992). C-banding and FISH analyses confirmed highest similarity of the S∗ -genome of these tetraploids with Ae. longissima or Ae. sharonensis (Jewell, 1979; Jewell and Driscoll, 1983; Friebe et al., 1996; Badaeva et al., 2004; Zhao et al., 2016).

Ae. vavilovii (Zhuk.) Chennav. (D1D1X crX crS v S v ) is a hexaploid taxa belonging to section Vertebrata Zhuk. emend Kihara, complex Crassa. Ae. vavilovii originated from hybridization of tetraploid Ae. crassa Boiss. with a species of Emarginata group, possibly Ae. longissima (Kihara, 1963; Kihara and Tanaka, 1970). Originally Ae. vavilovii was treated as a subspecies of hexaploid Ae. crassa, and its taxonomic rank was raised to independent biological species by Chennaveeraiah (1960). Although this author was unable to determine genome constitution of Ae. vavilovii, he noticed a pairwise similarity of the satellite chromosomes in karyotype of this species.

Yen and Kimber (1992) failed to identify the exact donor of the Sv -genome of Ae. vavilovii based on analysis of chromosome pairing in the F1 hybrids of Ae. vavilovii with induced autotetraploids of the Sitopsis species and proposed that the Sv genome is substantially modified. By using molecular markers (Talbert et al., 1991) showed that the Sv -genome of Ae. vavilovii is related to the S∗ -genome of Emarginata group. Data collected by molecular methods (Zhang and Dvorák, 1992), C-banding and FISH analyses (Badaeva et al., 2002; Zhang et al., 2002) confirmed, that Ae. vavilovii contains the Sv -genome that could probably derive from Ae. searsii (Badaeva et al., 2002).

Because of the genetic relatedness of the S-genome Aegilops species and polyploid wheats as well as of their potential for wheat improvement, they have been attracting the attention of researchers over the past century. Numerous intraspecific hybrids have been created to transfer desired genes from Aegilops to wheat (Schneider et al., 2008). Sets of addition, substitution or translocation wheat-Aegilops lines, including Ae. speltoides (Friebe et al., 2000; Liu et al., 2016), Ae. searsii (Pietro et al., 1988; Friebe et al., 1995), Ae. sharonensis (Olivera et al., 2013), Ae. longissima (Friebe et al., 1993), and polyploid Ae. peregrina, (Jewell and Driscoll, 1983; Friebe et al., 1996; Yang et al., 1996) and Ae. kotschyi (Rawat et al., 2011) were obtained and characterized using a combination of C-banding and analyses with the group-specific molecular or isozyme markers. As a result of these studies, the genetic classifications were developed for Cbanded chromosomes of several S-genome species (Friebe and Gill, 1996).

 Probe pSc119.2 was used most frequently (Badaeva et al., 1996a, 2002, 2004; Molnár et al., 2016; Zhao et al., 2016), however, the pSc119.2 signals are located predominantly in subtelomeric chromosome regions, thus hindering unequivocal chromosome identification. Probe pAs1, which proves to be highly informative for many Aegilops species, is not very useful for the S-genome analysis owing to a small number of detected sites (Badaeva et al., 1996a). In most papers FISH-labeled Aegilops chromosomes were classified based on their morphology, which is not sufficient to determine their correspondence to the genetic nomenclature of C-banded chromosomes. Owing to this, it was necessary to find FISH markers for the precise identification of all S-genome chromosomes and coordination of classification systems.

Recently, Komuro et al. (2013) isolated and characterized a number of repetitive DNAs from the wheat genome, which can potentially be used for molecular-cytogenetic analysis of wheat and Aegilops species. Several new sequences have been described in other papers (Salina et al., 1998, 2009; Adonina et al., 2015; Badaeva et al., 2015; Zhao et al., 2016). In this study we characterized the S genomes of diploid and polyploid Triticum and Aegilops species using C-banding and FISH with a set of "classical" [pSc119.2, pAs1, pTa71, pTa794, Spelt-1, Spelt-52] and novel [pAesp\_SAT86, (CTT)n, (GTT)n, pTa-535, pTa-s53] probes in order to assess evolutionary changes in the Triticum-Aegilops alliance.

## MATERIALS AND METHODS

#### Plant Material

Five diploid (Aegilops speltoides, Ae. longissima, Ae. sharonensis, Ae. searsii, Ae. bicornis), two tetraploid (Ae. peregrina and Ae. kotschyi) and one hexaploid (Ae. vavilovii) Aegilops species carrying the S-genome have been examined in comparison with two tetraploid wheats, T. timopheevii and T. dicoccoides. The list of accessions, their ploidy level, genome constitution and the origin are given in **Table S1**.

#### DNA Probe*s*

Following probes were used for FISH:

Plasmid clones pTa71 - a 9 kb long sequence of common wheat encoding 18S, 5.8S and 26S rRNA genes including spacers (Gerlach and Bedbrook, 1979), pTa794 – a 420 bp long sequence of wheat containing the 5S rRNA gene and intergenic spacer (Gerlach and Dyer, 1980), pAs1 - a 1 kb fragment derived from Ae. tauschii and belonging to Afa family (Rayburn and Gill, 1986), pSc119.2 – a 120 bp long sequence isolated from rye (Bedbrook et al., 1980), pTa-s53 – a 587 bp DNA fragment isolated from common wheat (Komuro et al., 2013), Spelt-1 – a 150 bp fragment isolated from Ae. speltoides (Salina et al., 1997), Spelt-52 (homolog of pAesKB52) – a 276 bp long DNA fragment isolated from Ae. speltoides (Salina et al., 2004a), and pAesp\_SAT86 - a new satellite family with a monomer length of 86 bp isolated from Ae. speltoides genomic DNA (Badaeva et al., 2015) and showing 91-94% similarity to wheat repeat pTa-713 described in Komuro et al. (2013) were labeled with dUTP-ATTO-488, dUTP-ATTO-550, dUTP-ATTO-647N by nick-translation using an Atto NT Labeling Kit (Jena Bioscience, Germany) or with FITC (fluorescein-12-dUTP, Roche, Germany) or biotin (biotin-16-dUTP, Roche, Germany) by nick-translation using the Nick Translation Mix (Roche, Germany) according to manufacturers' instruction.

Probe pTa535-1 was used as 5′ 6-carboxyfluorescein (6- FAM) or 6-carboxytetramethylrhodamine (TAMRA) end-labeled (MWG, Germany) oligo probe (5′ -AAA AAC TTG ACG CAC GTC ACG TAC AAA TTG GAC AAA CTC TTT CGG AGT ATC AGG GTT TC-3′ ) (Komuro et al., 2013; Tang et al., 2014).

The oligo-(CTT)10 or complementary oligo-(GAA)10 probes [thereafter (CTT)n] were labeled with 5/6-Sulforhodamine 101- PEG3-Azide or 6-Carboxyfluorescein Azide by click chemistry (Baseclick, Germany).

The oligo-(GTT)9 probe labeled at the 3′ -end with fluorescein-12-dUTP was synthesized in the Laboratory of Biological Microchips at the Engelhardt Institute of Molecular Biology, Moscow, Russia.

## Giemsa C-Banding Method

The Giemsa C-banding method described in Badaeva et al. (1994) was used for analysis. Seeds were soaked in water for 24 h at room temperature and then kept at 4◦C overnight on wet filter paper in Petri dishes. For the next 24 h Petri dishes were placed at 24◦C. Roots were cut and treated with 0.05% colchicine for 3 h. Further, roots were fixed in 45% acetic acid for 4 h, washed with distilled water and treated with 0.2 N HCl for 15 min at 4◦C and for 5 min at 60◦C. After overnight treatment with a 4 mg/ml Cellulysine (Fluka, Switzerland) solution at 24◦C root meristems were squashed in drop of 45% acetic acid. Slides were frozen in liquid nitrogen and coverslips were removed. After that slides were placed into 96% ethanol at room temperature. Chromosomes of wheat were classified according to nomenclature suggested in Gill et al. (1991), Badaeva et al. (2016); chromosomes of Aegilops species were classified according to the nomenclature of Friebe et al. (1993, 1995, 1996, 2000), Friebe and Gill (1996). Karyotype of one typical accession per each species was taken as standard for alignment of C-banding and FISH patterns.

#### Fluorescence *in situ* Hybridization

Detailed protocols of the pretreatment of the materials, fixation and chromosomal preparation are given in Badaeva et al. (2017). Briefly, seeds were germinated in Petri dishes on wet filter paper at 24◦C in dark. Roots were excised when 2 cm long, treated with ice-cold water for 24 h, and fixed with ethanol:acetic acid (3:1) fixative for at least 4 days at room temperature. Before slide preparation roots were stained in 2% acetocarmine for 15 min. Meristems were cut off and squashed in a drop of 45% acetic acid. Slides were frozen in liquid nitrogen and coverslips were removed with a razor blade. The slides were kept in 96% ethanol in a freezer.

Hybridization mixture contained 1 g dextran sulfate dissolved in 1 ml of distilled water, 5 ml deionized formamide, 1 ml of 20x SSC, 1 ml Herring sperm DNA (10 mg/ml, Promega, USA). Per slide 40–60 ng of each labeled probe were added to 18 µl hybridization mixture. Post hybridization washes were carried out as follows: for probes labeled with biotin or fluorescein the slides were washed in 0.1x SSC 2 × 10 min, then in 2x SSC 2 × 10 min at 42◦C. Slides hybridized with directly labeled probes were washed at 58◦C in 2x SSC for 20 min. The probes labeled with fluorescein were detected using antifluorescein/Oregon green R , rabbit IgG fraction, Alexa Fluor R 488 conjugated antibody (Molecular Probes, USA). Biotin was detected with sptreptavidin-Cy3 (Amersham Pharmacia Biotech, USA). The slides were counter-stained with DAPI (4′ ,6-diamidino-2-phenylindole) in Vectashield mounting media (Vector laboratories, Peterborough, UK) and examined with a Zeiss Imager D-1 microscope. Selected metaphase cells were captured with an AxioCam HRm digital camera using software AxioVision, version 4.6. Images were processed in Adobe PhotoshopR , version CS5 (Adobe Systems, Edinburgh, UK). For classification, chromosomes were aligned with the C-banding patterns based on the hybridization patterns of labeled CTT- and GTT-satellite sequences.

## RESULTS

#### Analysis of Diploid Species

According to the C-banding and FISH patterns of nine probes, five diploid species of the Sitopsis section split into two groups corresponding to taxonomically recognized sub-sections Truncata (Ae. speltoides) and Emarginata (Ae. longissima, Ae. sharonensis, Ae. searsii, Ae. bicornis).

#### Sub-section *Truncata: Ae. speltoides*

The karyotype of Ae. speltoides consists of metacentric or submetacentric chromosomes; the chromosome pairs 1S and 6S carry large satellites in their short arms (**Figure S1**). All chromosomes contain large Giemsa-positive pericentromic heterochromatin, prominent subtelomeric C-bands, and some small or medium sized interstitial bands. Giemsa-patterns allowed the identification of all Ae. speltoides chromosomes. We observed significant variations of Giemsa bands between plants within and between accessions. Heteromorphism of homologous chromosome has been recorded in all studied genotypes (**Figure S1**).

The (CTT)10 clusters (**Figure 1**, CTT) are located in proximal and interstitial chromosome regions, overlapping with Giemsa N-bands (Jiang and Gill, 1994b). No (CTT)10 signals were found in the sub-telomeric parts of the chromosomes possessing Cbands. The (GTT)9 probe forms prominent proximal clusters (**Figure 1**, GTT, **Figures 2**, **3f**), often exceeding the size of (CTT)n-signals. The abundance of the GTT-microsatellite is an important diagnostic feature of Ae. speltoides chromosomes.

The pSc119.2 labeling patterns are represented by subtelomeric and interstitial signals allowing the discrimination of all Ae. speltoides chromosomes. Some hybridization sites are found in all genotypes, whereas other vary in the presence and signal size (**Figure 1**). Based on dual-color FISH with (CTT)10 and pSc119.2 probes we corrected previously published classification of pSc119.2-labeled chromosomes (Badaeva et al., 1996a) according to the genetic nomenclature (Friebe et al., 2000). In particular, the chromosomes 2S and 3S have been renamed.

Major NORs are detected on chromosomes 1S and 6S, and one pair of 5S rDNA loci are mapped on the chromosome 5S (**Figures 1**, **2**). In addition, accession TA1873 shows one minor site on the long arm of one 5S chromosome.

Repeat pAesp\_SAT86 exhibits significant variation of labeling patterns between Ae. speltoides genotypes (**Figure 1**). Two sites located in the short arm of 4S and pericentromeric region of 7SL are permanent (**Figure 1**, yellow arrows). In genotypeTS89 this repeat is transferred to the long arm of 4S, probably due to a

C2-1–C2-2–genotypes from Nahal Mearot, Israel; G2.46-1–G2.46-3–genotypes from Ramat haNadiv, Israel. Permanent pAesp\_SAT86 loci are indicated with yellow

pericentric inversion. Several facultative pAesp\_SAT86 sites were found in more than one genotype (**Figure 1**, red arrows), while some signals were detected in single genotypes on either one or

arrows; polymorphic sites are shown with red arrows.

both homologous chromosomes. The labeling patterns of Spelt-1 and Spelt-52 probes are highly polymorphic (**Figure 1**). The Spelt-1 sequence is located in subtelomeric regions of either one or two chromosome arms. The number of loci per diploid genome varied from six (TS89 Katzir and No2 from Turkey) to 27 (PI 542274 from Turkey). Genotypes differ from each other in the size and chromosome location of the Spelt-1 clusters. The Spelt-52 signals of variable size are located in distal chromosome regions, proximally to Spelt-1. The number of Spelt-52 clusters per diploid genome varied from eight to 22 (**Figure 1**), the size and chromosomal distribution are highly polymorphic. Genotypes differ from each other in a ratio of Spelt-1/Spelt-52 repeats. Thus, the Spelt-1 could significantly prevail over Spelt-52, or the Spelt-52 could be more abundant (**Figure 1**).

Only few inconsistent, dot-like pTa-535 signals have been detected in Ae. speltoides (**Figure 1**). No hybridization was found with pAs1 and pTa-s53 probes.

#### Sub-section Emarginata

Four species of the Emarginata sub-section have a similar karyotype, which is distinct from that of Ae. speltoides (**Figures S1**, **S2**). Chromosome pairs 5S∗ and 6S∗ carry unequal satellites: large on 6S∗ and small on 5S∗ chromosomes (**Figure S2**). Most Ae. sharonensis genotypes collected in Keshon (Israel) are heterozygotes (**Figures S2c1,c2**) indicating that open pollination is common in this population.

The karyotypes of Emarginata species differ in heterochromatin content detected by Giemsa staining. Ae. bicornis and Ae. searsii showed small-to-medium C-bands located in interstitial chromosome regions (**Figures S2a1–b4**). Ae. sharonensis and Ae. longissima exhibit prominent pericentromeric and subtelomeric and many interstitial Cbands (**Figures S2c1–d10**). C-banding patterns allowed the chromosome identification in all Emarginata species. A speciesspecific translocation between 4S∗ and 7S∗ is found in all Ae. longissima accessions.

The (CTT)10-hybridization pattern (**Figures 3d,e,i,j**, **4**, **5**) corresponds to the C-banding pattern. As expected, Ae. bicornis and Ae. searsii carry predominantly small CTTsignals (**Figures 3e,j**), while Ae. sharonensis and Ae. longissima possess prominent pericentromeric and distinct interstitial CTT-clusters. In contrast to Ae. speltoides, the (GTT)9 probe hybridizes poorly on the chromosomes of Emarginata species. Probably, accumulation of heterochromatin in this evolutionary lineage was mainly due to amplification of CTT-repeat, contributing to an increase of nuclear DNA content in Ae. sharonensis/Ae. longissima genomes as compared to Ae. bicornis/Ae. searsii (Eilam et al., 2007).

Emarginata species display similar pSc119.2 hybridization patterns consisting of subtelomeric signals of variable size in one or both chromosome arms. Interstitial loci were rarely found

(**Figures 4**, **5**). Permanent interstitial sites are found on 2S∗ S (Ae. sharonensis and Ae. bicornis), 4SlL (Ae. longissima), and 7Ss (Ae. searsii) only. The pSc119.2 cluster in the middle of 5S∗ S is present in all Ae. searsii accessions and some Ae. longissima and Ae. sharonensis lines (**Figure 4**). One or two polymorphic pSc119.2 sites were rarely observed on 1SbL and 4SbL of Ae. bicornis.

The number and location of 5S and 45S rDNA loci in Emarginata species is similar and differ from that in Ae. speltoides (**Figures 1**, **4**). Major NORs are located on 5S∗ S and 6S∗ S and permanent minor NORs are found on 1S∗ S (**Figure 4**). Additional minor sites were detected in the terminal region of 6S∗L of all Ae. searsii accessions and some lines of Ae. bicornis and Ae. longissima. All species possess two 5S rDNA loci located in the short arms of chromosome 1S∗ and 5S∗ , distally (1S∗ ) or proximally (5S∗ ) to the 45S rDNA loci.

The distribution of pAesp\_SAT86 signals is species- and chromosome-specific. An intraspecific polymorphism was

FIGURE 3 | Distribution of (CTT)10 and (GTT)9 probes (red and green colors respectively) on chromosomes of wheat and Aegilops species: (a), Triticum dicoccoides (IG 46396); (b), T. araraticum (IG 116164); (c), Ae. kotschyi (TA2206); (d), Ae. longissmia (AE 904); (e), Ae. searsii (AE 1071); (f), Ae. speltoides (C1, Technion park, Israel); (g), Ae. vavilovii (K-3637); (h), Ae. peregrina (C11, Nahal Mearot, Israel); (i), Ae. sharonensis (C6, Keshon, Israel); (j), Ae. bicornis (K-666). Scale bar, 10 µm.

detected in Ae. bicornis and Ae. longissima, labeling patterns are virtually invariable in Ae. searsii (**Figures 4**, **5**). Distribution of pAesp\_SAT86 clusters on Ae. sharonensis and Ae. longissima chromosomes is similar and differs from Ae. bicornis and Ae. searsii, which, in turn, are clearly distinct from each other. No similarity between homoeologous chromosomes of different species has been observed, though the chromosome 3Ss (Ae. searsii) shows almost the same distribution of pAesp\_SAT86 sequence as the chromosomes 2S∗ of Ae. sharonensis and Ae. longissima.

The Spelt-1 repeat was not found in any Emarginata species, and Spelt-52 is detected in Ae. sharonensis and Ae. longissima only (**Figures 4B–G**). Signals of variable size are located in terminal regions of either one or both arms of all chromosomes except 6S∗ . Only two interstitial loci are found in the long arms of 2S∗ and 4S∗ . Distribution of Spelt-52 is highly diverse and polymorphisms are often observed even between homologous chromosomes. Depending on genotype, the number of signals ranges from 0 to 14. Most Ae. longissima accessions carry a Spelt-52 site in the long arm of 5S∗ , while it is absent in six out of 8 Ae. sharonensis accessions (**Table S2**). No other differences in labeling patterns were found between these species.

Distinct signals of the D-genome specific probes pAs1, pTa-535 or pTa-s53 are revealed in Ae. bicornis and Ae. searsii only (**Figures 4A**, **5**). Two small pTa-535 sites are found in the distal parts of 2Sb S and 7SbL chromosomes of Ae. bicornis; the first one overlaps with pTa-s53, and the second—with pAs1 sites. The pTa-535 probe hybridizes to subterminal regions of five pairs of Ae. searsii chromosomes, 1SsL and 6SsL exhibiting the largest signals. A relatively intense pAs1 signal is detected in a terminus of 4Ss S and few very weak interstitial signals are observed on 1SsL, 3SsL, and 7SsL. Faint, dispersed, non-specific pAs1 signals are distributed in distal halves of Ae. sharonensis and Ae. longissima chromosomes, while pTa-s53 and pTa-535 did not hybridize to the chromosomes of these species.

# Analysis of Polyploid Species: Wheats

Differences between emmer and Timopheevii wheat are mainly due to species-specific translocations identified in both evolutionary lineages (Naranjo et al., 1987; Liu et al., 1992; Jiang and Gill, 1994a; Maestra and Naranjo, 1999; Salina et al., 2006a). The (CTT)10 signals on T. araraticum and T. dicoccoides chromosomes (**Figures 3a,b**, **6a,c,h**) mainly correspond to the C-bands, whereas (GTT)9 forms large clusters in proximal regions of all B- and G-genome chromosomes (**Figure 2**, G,B; **Figures 3a,b**); their positions mainly overlapped with the location of (CTT)10 clusters. A similar pattern is also observed in Ae. speltoides (**Figure 3f**).

Although the pSc119.2 hybridization patterns in these two wheat species are distinct and species-specific, they share some

FIGURE 6 | Hybridization patterns of (CTT)10 (a,c,h, green), pSc119.2 (b,i,j, green), pTa-535 (c,i, red), pTa-s53 (b,h, red), pAesp\_SAT86 (f,h, red), NORs (d,e, green), and 5S rDNA (d,e, red), 5S rDNA (f, green); Spelt-1 (j,g, red) and Spelt-52 (g, green) on metaphase chromosomes of T. dicoccoides, IG 46396 (b–d,f,j) and T. araraticum, K-59940 (a,e,h,g,i). Position of pTa-s53 hybridization sites on T. dicoccoides chromosomes (c), huge cluster of pTa-535 sequence on the chromosome 4GS (i) and Spelt-1 site on T. dicoccoides chromosome 2AL (j) are indicated with arrows. Scale bar, 10 µm.

FIGURE 7 | Distribution of repetitive DNA families on chromosomes of Ae. peregrina left side and Ae. kotschyi (right side of the figure): (a), C11 (Nahal Mearot, Israel); (b), C8 (Haifa, Carmel, Israel); (c), K-61; (d), C9 (Keshon, Israel); (e), TA2206; (f), K-91; (g), hybrid Ae. umbellulata TU04 × Ae. sharonensis TH02; (h), K-2905. Probe combinations are given on the top; signal color corresponds to probe name. The S-genome chromosomes are shown on the top, the U-genome–on the bottom part of the figure.

similar features. As in Ae. speltoides, pSc119.2 signals are located in interstitial and subtelomeric regions of orthologous chromosome allowing a complete chromosome identification.

Two chromosome pairs of T. araraticum and T. dicoccoides carry major NORs (**Figures 6d,e**). These are 1B and 6B in T. dicoccoides and 6G and 6At in T. araraticum (transfer of NORs from 1G to 6At is due to species-specific translocation 1G-4G-6At in Timopheevii lineage, Jiang and Gill, 1993). Group 1 and 5 chromosomes of T. dicoccoides display eight 5S rDNA signals (**Figure 6d**), but only six - in T. araraticum (chromosomes 1At , 5At , and 5G, see **Figure 6e**). Chromosome 5S of Ae. speltoides shows one 5S rDNA locus, therefore 1S likely lost the 5S rDNA locus in the progenitor of Ae. speltoides after the formation of ancient emmer, but prior to the divergence of Timopheevii wheat.

The pAesp\_SAT86 clusters are found on both A and B/G genome chromosomes (**Figures 6a,f**), T. dicoccoides and T. araraticum show different labeling patterns and both exhibit broad intraspecific polymorphisms (Badaeva, unpublished). A large pAesp\_SAT86 signal is found on 1BS of all emmer (**Figure 6f**) and common wheat (Komuro et al., 2013), but it is absent on 1G of T. timopheevii (**Figure 6a**). By contrast, huge 3GL- and 7GL-located pericentromeric pAesp\_SAT86 clusters are missing on the homoeologous chromosomes of emmer wheat. At the same time, similar labeling patterns were observed on 4B/4G, 5B/5G, and 6B/6G of these species.

Very weak Spelt-52 signals were seen on 1GS and large on 6GL of T. araraticum. The same sequence was not detectable in wild emmer. Two faint Spelt-1 signals were revealed on the chromosome pair 2A of T. dicoccoides (**Figure 6j**), whereas ten clear signals were observed on chromosomes 6At S, 1GL, 4GL, 5GL, and 6GS of T. araraticum (**Figure 6g**).

Probe pTa-535 hybridized predominantly on the A-genome chromosomes of both wheat species (**Figures 6b,i**, red color). A large pTa-535 cluster was found on the short arm of 4G of T. araraticum (**Figure 6i**, indicated with arrows). Overlapping, small pAs1/ pTa-535 signals are detected in distal halves of 3GL and 3BL. In addition, faint pAs1 signals were found in the satellite of 1B, in the middle of 6BS and 7BL of wild emmer. T. araraticum carries small pAs1 loci on 5GL and 7GL and in the satellite of 6At (data not shown). Only weak pTa-s53 signals were observed on chromosomes 3AS and 5AL of T. dicoccoides (**Figure 6c**), and no hybridization was found on T. araraticum (**Figure 6h**).

## Polyploid *Aegilops*: *Ae. peregrina and Ae. kotschyi*

Ae. peregrina and Ae. kotschyi are both tetraploids with the same genome constitutions UUS∗ S ∗ . Their C-banding patterns are generally similar, however, some differences in morphology and heterochromatin distribution on chromosomes 2S∗ , 4S∗ , and 7S∗ are observed (**Figure S3**). According to C-banding patterns, Ae. peregrina carries 4S-7S∗ translocation and therefore the Sp genome is originated from Ae. longissima. The Sk genome of Ae. kotschyi is more diverged from the S∗ -genomes of Emarginata species, but shares similar structure and C-banding pattern of chromosome 4S∗ with Ae. sharonensis (**Figures S2**, **S3**).

FISH with (CTT)10 and (GTT)9 probes reveals large CTTclusters on all chromosomes, but only few weak GTT-signals on some U and S∗ -genome chromosomes of both species (**Figures 3c,h**). Distribution of (CTT)10 probe corresponds to the C-banding patterns (**Figure 7**, **Figure S3**), and dualcolor FISH allows aligning of the CTT/C-banding and pSc119.2-FISH patterns (**Figure 5**). Positions of pSc119.2 clusters on chromosomes of the two species are similar except for 4S∗ , which carries two prominent subtelomeric signals in Ae. kotschyi, but one huge cluster in the short and two smaller sites in the long arm in Ae. peregrina (**Figure 7**). Labeling patterns varies between the accessions. Owing to subterminal location of pSc119.2 sites and polymorphism of labeling patterns, applicability of the pSc119.2 probe for chromosome identification of Ae. peregrina and Ae. kotschyi is limited.

Distribution of 45S and 5S rDNA loci on Ae. peregrina and Ae. kotschyi chromosomes is similar **Figure 7**, (NOR+5S) and corresponds to that in the parental species. Signal size of pTa71 probe (45S rDNA) on 5Sk and especially 6Sk chromosomes of Ae. kotschyi is significantly smaller than on the orthologous chromosomes of Ae. peregrina, which can be an indicative of more extensive gene loss at the respective loci.

FISH reveals similar hybridization patterns of pAesp\_SAT86 probe on chromosomes of Ae. peregrina and Ae. kotschyi. According to dual-color FISH, the largest pAesp\_SAT86 signals are located on chromosomes 1S∗L (polymorphic), 2S∗L, 1US, 6UL, and 7UL. Chromosomes 3S∗ and 4S∗ carry medium and 5S∗ S, 6S∗L, and 4US – faint signals (**Figure 7**). Labeling patterns of chromosomes 3S∗ , 4S∗ , 6S∗ , and 7S∗ are polymorphic. In contrast to Ae. peregrina and diploid Emarginata species, the chromosome 6Sk of Ae. kotschyi carries large pAesp\_SAT86 cluster in the short arm.

The Spelt-1 repeat is not found in these tetraploid species, while Spelt-52 is revealed only in few accessions of Ae. kotschyi and Ae. peregrina. Small Spelt-52 clusters are observed on four out of seven S∗ -genome chromosome: 3S∗ S, 4S∗L, 6S∗L, and 7S∗L. Number of signals varies from two to six (**Figure 7**), nearly half of genotypes we examined do not exhibit any hybridization. This is strictly different from what is observed in a newly synthesized hybrid Ae. umbellulata x Ae. sharonensis, in which 12 distinct Spelt-52 signals are observed in either one or both arms of chromosome pairs 1S∗ , 2S∗ , 3S∗ , and 7S∗ (**Figure 7g** ).

Very few weak pAs1 and pTa-535 signals are located predominantly on the U-genome chromosomes of Ae. kotschyi and Ae. peregrina (**Figure 7**), while the pTa-s53 sequence is totally absent.

# Polyploid *Aegilops*: *Ae. vavilovii*

The hexaploid species Ae. vavilovii with the genome constitution D1D1X crX crS v S v is characterized by a medium amount of Giemsa bands. Small and medium sized bands are distributed predominantly in interstitial chromosome regions (**Figure 8**). Two chromosome pairs are submetacentrics with small satellites, which morphologically correspond to 5S∗ . Two other pairs are metacentrics with large satellites, which is typical for chromosome 6S∗ . The C-banding pattern of Ae. vavilovii is similar to the parental species: Ae. crassa (Badaeva et al., 1998, 2002) and Ae. searsii (Friebe et al., 1995; Friebe and Gill, 1996). Intraspecific variations due to chromosomal rearrangements were identified in two of the three accessions of Ae. vavilovii.

The distribution of (CTT)10 signals is generally similar to the observed C-banding patterns (**Figure 8**). The (GTT)9 probe results in distinct signals on five pairs of the Sv genome

chromosomes and small to medium clusters on seven pairs of the D1 and Xcr genome chromosome. (GTT)9 signals only partially overlap with (CTT)10 loci (**Figure 3g**).

Probe pSc119.2 hybridized with all Sv and some Xcr genome chromosomes. Signals are located in subterminal chromosome regions; interstitial sites were found in the middle of 5Sv S and in the distal region of 4SvL and 7SvL (**Figure 8**).

Probe pTa71 revealed eight major and eight minor NOR sites in Ae. vavilovii. The major NORs are located on group 5 and 6 chromosomes belonging to Sv and Xcr genomes. Minor NORs mapped on all three pairs of group 1 chromosomes and, surprisingly on 6D1 S. Six of 5S rDNA sites are located on group 1 and 5 chromosomes. An additional, minor 5S rDNA locus is detected in the proximal region of an unknown small metacentric chromosome (**Figure 8**, shown with arrow).

The pAesp\_SAT86 signals of different sizes were detected on many Ae. vavilovii chromosomes; the number of loci varies from one to three per chromosome (**Figure 8**). Distribution of pAesp\_SAT86 sites on 2Sv , 3Sv , and 7Sv is different from Ae. searsii, while the remaining chromosomes of these genomes show similar labeling patters.

The A/D-genome-specific probes pAs1, pTa-535, and pTas53 hybridize mainly to the D1 -genome and partially to Xcr genome chromosomes of Ae. vavilovii (**Figure 8**). The Sv genome possesses the lowest amount of these sequences. Small pAs1 signals were observed only in terminal regions of 4Sv S and 6Sv S. Neither Spelt-1, nor Spelt-52 hybridization sites were detected in Ae. vavilovii.

# DISCUSSION

Karyotype analysis as a tool for studying evolutionary processes must be based on an unified chromosome nomenclature. The first classification of chromosomes according to their homoeologous relationships and genome affinities was developed for common wheat by Sears (1954), and since then it is used as standard in genetic and cytogenetic studies of the Triticeae. Although the nomenclature of Giemsa C-banded chromosomes is now available for many Aegilops species, including Ae. speltoides, Ae. searsii, Ae. longissima, and Ae. peregrina (Friebe and Gill, 1996), their correspondence to the distribution of FISH probes is not known.

In order to link C-banding and FISH patterns (Jiang and Gill, 1993) developed a method of sequential C-banding and in situ hybridization analysis. An alternative approach was suggested by Pedersen and Langridge (1997), who used the barley probe pHvG38 containing a GAA-satellite sequence for the identification of wheat chromosomes. Later this sequence was successfully used for the FISH analyses of wheat, barley, rye and some other cereal chromosomes (Pedersen et al., 1996; Cuadrado et al., 2000; Vrána et al., 2000; Cuadrado and Jouve, 2002; Kubaláková et al., 2005; Kato, 2011; Komuro et al., 2013; Adonina et al., 2015; Badaeva et al., 2016), however it was rarely applied for Aegilops species (Molnár et al., 2005, 2016; Mirzaghaderi et al., 2014).

The CTT–labeling patterns of Aegilops chromosomes obtained in our study basically correspond to their C-banding patterns. Therefore, we used the CTT-signals as landmark to identify chromosomes according to the genetic nomenclature. This allowed us to compare karyotypes based on chromosome homoeology and to trace chromosomal changes that could have occurred over the course of species evolution.

#### *Ae. speltoides* and Polyploid Wheats Are Cytogenetically Distinct From the S ∗ -Genome of Other Diploid and Polyploid *Aegilops* Species

Based on C-banding and FISH patterns it is possible to divide the S-genome chromosomes of diploid and polyploid wheat and Aegilops species into two distinct groups. The first one includes Ae. speltoides and polyploid wheat. The second contains four diploid species of the Emarginata sub-section and three polyploid Aegilops, in agreement with molecular phylogenetic analyses (Yamane and Kawahara, 2005; Golovnina et al., 2007; Goryunova et al., 2008; Salse et al., 2008; Gornicki et al., 2014; Marcussen et al., 2014; Middleton et al., 2014; Feldman and Levy, 2015). The main diagnostic features of these groups can be described as follows.

1. The satellite chromosomes of the S-genome of Ae. speltoides and B/G-genomes of polyploid wheats belong to homoeologous groups 1 and 6 (Dvorák et al., 1984; Friebe et al., 2000). The satellites are large and nearly equal in size (Chennaveeraiah, 1960). The satellite of T. timopheevii chromosome 1G is transferred to 6At as a result of a speciesspecific translocation (Jiang and Gill, 1994a; Rodríguez et al., 2000b; Dobrovolskaya et al., 2011). Major 45S rDNA sites are located on the short arms of group 1 and 6 chromosomes (**Figure 2**) (Yamamoto, 1992a,b; Jiang and Gill, 1994b; Badaeva et al., 1996b; Raskina et al., 2011; Belyayev and Raskina, 2013; Molnár et al., 2016). In addition to major NORs, Jiang and Gill (1994b) revealed minor 45S rDNA loci in the long arm of chromosome 1B of common and durum wheat, 1G of T. timopheevii and 1S of Ae. speltoides, which were never observed in other S∗ -genome Aegilops species. Diploid Emarginata species possess two pairs of satellite chromosomes assigned to genetic groups 5 and 6 (Friebe et al., 1993, 1995; Friebe and Gill, 1996); satellites significantly differ in size (Chennaveeraiah, 1960). The secondary constrictions of 5S∗ and 6S∗ are suppressed in polyploid Ae. peregrina and Ae. kotschyi, but are extended in hexaploid Ae. vavilovii. FISH with the probe pTa71 revealed major 45S rDNA sites on 5S∗ and 6S∗ chromosomes of diploid and polyploid Aegilops species, but signal sizes were significantly reduced in tetraploid Ae. peregrina and Ae. kotschyi (**Figures 4**, **7**). Permanent minor 45S rDNA loci were present on chromosome 1S∗ , and additional minor site was detected in the terminus of 6S∗L of all Ae. searsii and some Ae. bicornis and Ae. longissima accessions (**Figure 5**). Earlier we also found minor 45S rDNA locus in a terminus of the short arm of an unknown chromosome, probably 3Sl , of Ae. longissima, accession TA1912 (Badaeva et al., 1996b). These observations are in agreement with previously published results (Yamamoto, 1992a,b; Friebe et al., 1993; Badaeva et al., 1996b, 2002, 2004).

# Different Families of Repetitive DNA Show Different Evolutionary Rates

Our data and previous findings imply that the evolutionary rate varies between different families of repetitive DNAs. Despite distinct differences between Ae. speltoides/polyploid wheats and other S∗ -genome Aegilops species in the distribution of rDNA probes, the patterns of 45S and 5S rDNA loci was highly conserved within each group. Only minor intra-and inter-specific variations were observed,

(1) Regarding the appearance of minor NORs, which occur at similar positions on the orthologous chromosomes (Yamamoto, 1992a,b; Badaeva et al., 1996b), and

(2) The decrease of signal size on the S∗ -genome chromosomes of tetraploid Aegilops species (Yamamoto, 1992a,b; Badaeva et al., 2004). Such signal reduction could be explained by uniparental elimination of genes (Shcherban et al., 2008).

The distribution of the rye-derived pSc119.2 repeat is also found to be relatively conserved within each of the two Sgenome groups. This sequence with a 120 bp-long repeat unit is broadly distributed in the Triticaea and some Aveneae species and constitutes large and evolutionary old component of their genomes (Contento et al., 2005). The repeat units isolated from wheat, rye, barley and Aegilops species showed 70-100% similarity to each other. Nucleotide sequences of pSc119.2 repeat units are not species-specific, and one site may contain diverse members of the family (Contento et al., 2005). The authors proposed that these individual pSc119.2 sites are transferred as blocks and can be translocated within the genome resulting in position variation and site numbers. Similar was observed in our material.  Therefore, a terminal location of pSc119.2 satellite family is probably a more primitive character compared to interstitial locations. Intercalary pSc119.2 sites are typical for Ae. speltoides (Badaeva et al., 1996a; Molnár et al., 2016), B- and G-genomes of polyploid wheats (Jiang and Gill, 1994a; Schneider et al., 2003), and rye (Cuadrado and Jouve, 2002); the rye genome being highly rearranged relative to wheat (Liu et al., 1992). Strong differences in the distribution of pSc119.2 sites in the R and S genome chromosomes suggest that transposition of this repeat proceeded in genomes of rye and Ae. speltoides independently, likely, after their radiation from the ancestral form.

Comparison of C-banding patterns with the distribution CTT+ GTT-microsatellite sequences shows that heterochromatin blocks detected by Giemsa staining in different Triticum and Aegilops species could have different sequence composition. Thus, Ae. speltoides chromosomes carry prominent proximal and telomeric C-bands and only few intercalary bands, which is considered as primitive karyotype structure (Stebbins, 1971). Only proximal bands overlap with both (CTT)10 and (GTT)9 clusters. The GTT-repeat is more abundant in these chromosomal regions. Intercalary C-bands correspond to CTT-signals, and probably they are composed by this microsatellite mainly. Neither (CTT)10, nor (GTT)9 signals were detected in telomeric heterochromatin, which is enriched in Spelt-1 and Spelt-2 repeats.

The C-banding patterns of the S∗ -genome Emarginata species and their polyploid derivatives are very similar to their CTThybridization patterns indicating that this sequence is a major component of Giemsa-positive heterochromatin. The GTTmicrosatellite is present in much lower quantities, and only few C-bands contain this sequence solely. Species of this genomic group exhibit drastic differences in the content of C-positive heterochromatin. Diploid Ae. bicornis, Ae. searsii, and hexaploid Ae. vavilovii are low heterochromatic; the (CTT)10-signals are small and located mainly in the intercalary chromosome regions. Karyotypically Ae. searsii is distinct from other diploid species and its divergence was accompanied mainly by heterochromatin re-pattering visualized by Giemsa-staining and FISH with the CTT-microsatellite probe. The genomes of Ae. sharonensis, Ae. longissima, Ae. kotschyi, and Ae. peregrina are highly heterochromatic; prominent C-bands and CTTsignals are distributed in proximal and intercalary chromosome regions (**Figure 5**). Thus, massive amplification of the CTTrepeat occurred at the stage of radiation of Ae. sharonensis and Ae. longissima, resulting in an increase of nuclear DNA (Eilam et al., 2007) and the amount of heterochomatin.

Three tandemly repeated DNA families, pAesp\_SAT86, Spelt-1, and Spelt-52 show the highest rate of evolution in the Triticum-Aegilops group. pAesp\_SAT86 sequence is detected in all S-genome species (**Figures 1**, **3**, **7**, **8**) and the B/G genomes of polyploid wheat (**Figure 6**). The labeling patterns are extremely variable in Ae. speltoides (**Figure 1**) and differ from polyploid wheat species which, in turn, are distinct from each other (Komuro et al., 2013; Badaeva et al., 2016). Diploid Emarginata species and their polyploid derivatives display species-specific patterns of pAesp\_SAT86 probe (**Figures 4**–**8**). Ae. bicornis shows the highest degree of intraspecific pAesp\_SAT86-polymorphism, while little variation has been observed in Ae. searsii, Ae. sharonensis (**Figure 4**), Ae. kotschyi (**Figure 7**), and Ae. vavilovii (data not shown). Ae. bicornis and Ae. searsii differ from each other and from other species of this group (**Figure 5**). Ae. sharonensis is more similar with Ae. longissima and Ae. peregrina in the distribution of pAesp\_SAT86 clusters and only slightly different from Ae. kotschyi. The pTa-713 (homolog of pAesp\_SAT86) hybridization patterns of Ae. peregrina reported by Zhao et al. (2016) is consistent with our results, though there are some discrepancies in chromosome designations.

The Spelt-1 repeat is found in Ae. speltoides and the B/G genomes of polyploid wheats. In Ae. speltoides it comprises nearly 2% of the nuclear genome (105–106 copies). The copy number of constituent sequence related to Spelt-1 is ∼40–60 reduced in genomes of tetraploid wheats, and ∼1200–2400 times reduced in genomes of other Triticeae (Pestsova et al., 1998; Salina et al., 1998; Salina, 2006). Minor amounts of Spelt-1 exist in genomes of rye, cultivated barley, most diploid and polyploid wheat as well as Aegilops species indicates that this sequence was already present in minor quantities in the common ancestor of the Triticeae (Salina et al., 1998). High homology (97–100%) of individual repetitive units implies that massive amplification of Spelt-1 repeat occurred in ancient Ae. speltoides after radiation from the common ancestor of the Triticeae (Salina et al., 1998; Salina, 2006). Spelt52 is homologous to the pAesKB52 repeat isolated earlier from Ae. speltoides by Anamthawat-Jonsson and Heslop-Harrison (1993). This repeat consists of monomers of two types, Spelt52.1 and Spelt52.2, which share a homologous stretch of 280 bp and have two regions without sequence similarity of 96 and 110 bp, respectively. Ae. speltoides displays intraspecific variation in the occurrence of Spelt52 monomer types, whereas Ae. longissima, Ae. sharonensis, and Ae. bicornis showed no interspecific variation (Salina et al., 2004a). The Spelt-52 is abundant in Ae. speltoides accounting for approximately 1% of nuclear genome (Anamthawat-Jonsson and Heslop-Harrison, 1993; Salina, 2006) and is also highly represented (1.0 × 104 – 2.5 × 105 copies) in Ae. longissima and Ae. sharonensis, but it present in minor quantities in Ae. bicornis and Ae. searsii (Salina, 2006).

Coincidently with previous findings (Salina et al., 2006b; Raskina et al., 2011; Belyayev and Raskina, 2013), we observed significant intraspecific variation of Spelt-1 and Spelt-52 labeling patterns (**Figure 2**). Strict differences in a ratio of Spelt-1/ Spelt-52 repeats detected between genotypes can be due to geographical origin of the material. Earlier, Raskina et al. (2011) found that the amount of Spelt-1 and, in lower extent, the Spelt-52 repeat decreases in marginal populations of Ae. speltoides.

Although pAs1 and pTa-535 repeats are abundant in some cereal genomes (Rayburn and Gill, 1986; Badaeva et al., 1996a; Taketa et al., 2000; Komuro et al., 2013), they are poorly represented in the S genomes of Triticum and Aegilops. Thus, we failed to detect any pAs1signals in Ae. speltoides, but Molnár et al. (2016) revealed small pAs1 signals on the chromosome 3S. Wheat chromosomes 3BL - 3GL and 7BL - 7GL possess pAs1 and pTa-535 clusters in similar positions (Schneider et al., 2003; Badaeva et al., 2016), although they are not detected in Ae. speltoides. Probably, these loci were present in the genome of ancient Ae. speltoides, but they were eliminated after radiation of polyploid wheats. The pAs1 and pTa-535 repeats are also poorly represented in genomes of Emarginata species. Two distinct interstitial pAs1 sites overlapping with either pTa-535, or with pTa-s53 loci are found in Ae. bicornis. pAs1 and pTa-535 are less abundant in Ae. searsii, Ae. sharonensis and Ae. longissima. Only chromosome 1Sk of tetraploid Ae. kotschyi contains a distinct pAs1/pTa-535 cluster, and these sequences are absent from the S ∗ genomes of Ae. peregrina and Ae. vavilovii.

#### Evolution of the S-Genome

Summarizing our data and the results of other authors (Kihara, 1954; Chennaveeraiah, 1960; Kihara and Tanaka, 1970; Yen and Kimber, 1990b; Zhang and Dvorák, 1992; Zhang et al., 1992; Dvorák, 1998; Feldman and Levy, 2015), the following scenario of the S-genome evolution can be suggested (**Figure 9**).

According to molecular phylogeny, Ae. speltoides is the most distinct diploid Aegilops, which diverged from the common ancestor very early, prior to the split of diploid wheat and Aegilops species (Salse et al., 2008; Gornicki et al., 2014; Marcussen et al., 2014; Feldman and Levy, 2015). Divergence of Ae. speltoides from an ancestral form was not associated with major translocations, because neither meiotic analysis (Rodríguez et al., 2000a), nor microsatellite mapping (Dobrovolskaya et al., 2011) detected structural chromosmal rearrangements in the Sgenome. However some genomic changes not causing linkage group perturbations did probably occur at the early stages of Ae. speltoides speciation. As was shown earlier, major NORs in Triticum and Aegilops species are located on group 1, 5, and 6 chromosomes (Appels et al., 1980; Appels and Honeucutt, 1986), while 5S rDNA loci are located separately from NORs in the short arms of group 1 and 5 chromosomes (Appels et al., 1980; Dvorák et al., 1989). The chromosome 5S of Ae. speltoides and B/G genome of polyploid wheats does not contain 45S rDNA loci, therefore the loss of respective NOR probably occurred prior to formation of ancient emmer. Other early genomic changes of ancient Ae. speltoides included the transposition of the pSc119.2 repeat from subtelomeric to interstitial chromosome regions and also the amplification of Spelt-1 repeat.

As mentioned above, Ae. speltoides and the B/G genomes of polyploid wheats are characterized by the abundance of GTTmicrosatellite (Cuadrado et al., 2000; Badaeva et al., 2016), which is poorly represented in diploid wheats (Badaeva et al., 2015) and most Aegilops species (**Figure 3**). This difference can be caused by massive amplification of GTT-repeat in the ancient Ae. speltoides. Alternatively, this repeat could be eliminated from the progenitor of wheat and Aegilops species. Taking into consideration the abundance of GTT-repeat in rye and Hordeum (Cuadrado and Jouve, 2002, 2007; Dou et al., 2016), the second scenario seems to be more likely. The progenitor of Ae. speltoides probably possessed minor amounts of Spelt-52 and the D-genome specific repeats pTa-535, or pAs1, as they are still present in Ae. speltoides and the B/G genomes of polyploid wheats (Schneider et al., 2003; Badaeva et al., 2016; Molnár et al., 2016). However, these sequences could be of the A-genome origin, which spread to the S-genome following allopolyploidization.

The emergence of tetraploid emmer was accompanied by the species-specific translocation involving the chromosomes 4A-5A-7B (Naranjo et al., 1987; Liu et al., 1992; Maestra and Naranjo, 1999). In addition to structural chromosome rearrangements, other genetic and epigenetic changes occurred in a newly formed polyploid, including inactivation of the 45S rDNA loci on the Agenome chromosomes, re-distribution of Giemsa C-bands and repetitive DNA families on both A and B-genome chromosomes. Evolution of polyploid wheat resulted in polymorphisms of various DNA sequences and heterochromatin patterns that were described in many publications (Friebe and Gill, 1996; Schneider et al., 2003; Badaeva et al., 2016).

Subsequent evolution of Ae. speltoides occurred independently of polyploid emmer and was accompanied by several transposon insertions (Salse et al., 2008) and the loss of the 5S rDNA locus on the chromosome 1S, which is present in emmer and common wheat (Mukai et al., 1990), but absent in T. timopheevii (Badaeva et al., 2016) and modern Ae. speltoides. Although T. timopheevii derived from the same parental species as emmer, different parental genotypes were involved in the origin of these two lineages (Golovnina et al., 2007). Timopheevii wheat emerged much later, than ancient emmer - nearly 0.4 MYA (Gornicki et al., 2014) and its formation was accompanied by different speciesspecific translocation involving the chromosomes 1G-4G-6At + 3At -4At (Jiang and Gill, 1994a; Maestra and Naranjo, 1999; Rodríguez et al., 2000b; Dobrovolskaya et al., 2011). As a result, a major NOR was translocated from chromosome 1G to 6At , and a massive cluster of the A/ D-genome specific repeat pTa-535 appeared on the short arm of chromosome 4G (**Figure 6g**). Existence of Spelt-52 sites and a spread of Spelt-1 to most T. timopheevii chromosomes (Salina et al., 2006b; Zoshchuk et al., 2007; Badaeva et al., 2016) suggests a massive amplification of these sequences in Ae. speltoides prior to emergence of ancient T. timopheevii.

Results of molecular cytogenetic analysis suggest that genome re-structuring process is still ongoing in natural populations of Ae. speltoides. This is exemplified by intraspecific Cbanding polymorphisms and diversity of labeling patterns of pAesp\_SAT86, Spelt-1 and Spelt-52 probes observed in this and other studies (Belyayev and Raskina, 2011, 2013; Raskina et al., 2011), fluctuation of copy number of retrotransposons and tandem repeats, and high number of chromosomal rearrangements (Belyayev and Raskina, 2013; Shams and Raskina, 2018).

The species of the Emarginata group are closely related to each other (Eig, 1929; Kihara, 1954; Friebe and Gill, 1996; Kilian et al., 2011; Gornicki et al., 2014; Feldman and Levy, 2015), which is supported by their similar karyotypes (Chennaveeraiah, 1960), C-banding and pSc119.2-labeling patterns (Badaeva et al., 1996a), distribution of rDNA probes (Yamamoto, 1992a,b; Badaeva et al., 1996b). Separation of Emarginata species from a common ancestor was associated with inactivation of major NORs on chromosome 1S∗ accompanied with the significant loss of 45S rDNA repeat copies. Despite similarity of pSc119.2 labeling patterns, there are obvious, but discontinuous changes in the patterns of other sequences. Our data show that most drastic changes occurred probably at the stage of radiation of Ae. sharonensis-Ae. longissima. These are massive amplification of Spelt-52 and CTT-repeats resulting in the gain of heterochromatin in Ae. sharonensis and Ae. longissima, leading to an approximately 12% increase of nuclear DNA content in Ae. sharonensis/ Ae. longissima as compared to Ae. searsii/ Ae. bicornis (Eilam et al., 2007). By contrast, the amount of the D-genome repeats pTa-535, pAs1 and especially pTa-s53 gradually decreased, and these sequences nearly disappeared in genomes of Ae. sharonensis and Ae. longissima. Spelt-52 patterns of Ae. sharonensis and Ae. longissima chromosomes are highly polymorphic. The similar distribution of all analyzed DNA sequences on chromosomes of Ae. sharonensis and Ae. longissima (**Figures 3**, **4**) point to a rather recent divergence of these species, which was accompanied by the species-specific translocation 4S∗ -7S∗ in Ae. longissima.

Formation of tetraploid Ae. peregrina and Ae. kotschyi did not cause significant alterations of the parental genomes. Considering the structure of chromosome 4S∗ , the Sp -genome of Ae. peregrina was donated by Ae. longissima, while Ae. sharonensis or the form preceding the split of these diploids could be the source of the S∗ -genome of Ae. kotschyi. These data are consistent with observations of other authors (Yu and Jahier, 1992; Zhang et al., 1992; Friebe et al., 1996), however they contradict the hypothesis about the possible ancestry of Ae. searsii in the origin of Ae. peregrina (Siregar et al., 1988). Merging of U and S∗ genomes in the tetraploid Ae. peregrina and Ae. kotschyi led to inactivation of 45S rDNA loci on the S ∗ -genome chromosomes (**Figure 7**). Similar was also recorded in the artificial allopolyploid Ae. umbellulata × Ae. sharonensis (Shcherban et al., 2008). Significantly smaller 45S rDNA sites on Ae. kotschyi chromosome 6Sk compared to the 6Sp of Ae. peregrina evidences in favor of a higher extent of gene loss at the respective locus, which can be due to earlier origin of Ae. kotschyi. The assumption that Ae. kotschyi is an older species is also supported by higher divergence of C-banding patterns relative to the parental species.

Interestingly, Ae. peregrina and Ae. kotschyi both possess only minor quantities of the Spelt-52 repeat, which is abundant in their diploid parents. According to the analyses of artificial wheat-Aegilops or Aegilops-Aegilops hybrids, the Spelt-52 was either amplified or retained at the same level upon polyploidization (Salina et al., 2004b). Considering these results we can expect massive amplification of the Spelt-52 sequence in Ae. peregrina and Ae. kotschyi genomes. However, this is not the case. Low amount of Spelt-52 in these species can be caused by the so-called "originator effect," if they obtained their S∗ genomes from genotype depleted with this repeat, or it can be caused by sequence elimination after formation of tetraploids.

The Sv -genome chromosomes of Ae. vavilovii are very similar to the Ss -genome chromosomes of Ae. searsii, which further supports their close relationships (Zhang and Dvorák, 1992; Dubkovsky and Dvorák, 1995). Our results strongly suggest that the Xcr genome of Ae. vavilovii is also the derivative of the S∗ genome of an unknown Emarginata species, but not of Ae. speltoides as proposed by Dubkovsky and Dvorák (1995); Edet et al. (2018). Significant differences between the Xcr and S s genomes, as well between Xcr and S∗ -genomes of all diploid Emarginata species in the C-banding and labeling patterns demonstrate that the Xcr genome was significantly modified during speciation.

# CONCLUSIONS

Analysis of the S-genomes of diploid and polyploid Triticum and Aegilops species using FISH with nine DNA probes, including 5S and 45S rDNA, two microsatellites and five tandem repeats showed an isolated position of Ae. speltoides among other Aegilops species.  The evolution of polyploid wheats was associated with different species-specific chromosome translocations and the amplification/ elimination of repeats, re-pattering or, possibly with an exchange of repetitive DNA families with the A-genome chromosomes. Evolutionary changes in the Ae. speltoides genome occurred independently from polyploid wheats.

Diploid Aegilops species of Emarginata group are similar, but are substantially different from Ae. speltoides based on Cbanding and FISH patterns. The genome evolution in this group was mainly associated with an increase of high copy DNA fraction due to amplification of CTT-repeat, re-distribution of C-bands, (CTT)n-, (GTT)n-, and pAesp\_SAT86-clusters, massive amplification of Spelt-52 and gradual elimination of the Dgenome-specific sequences pAs1, pTa-535 and pTa-s53. These changes were more profound at the stage of divergence of Ae. sharonensis/Ae. longissima. Tetraploid Ae. peregrina and Ae. kotschyi originated independently from hybridization of Ae. umbellulata with Ae. longissima (Ae. peregrina) or Ae. sharonensis or its immediate precursor (Ae. kotschyi). The S ∗ -genomes of both tetraploids show little differences to the parental species. The Sk -genome is characterized by more modifications than the Sp -genome, suggesting that Ae. kotschyi is older than Ae. peregrina. Chromosome introgressions recorded in some accessions of both species (Badaeva et al., 2004) can be explained by gene flow between Ae. peregrina and Ae. kotschyi.

Our study confirmed that Ae. vavilovii is a natural hybrid between tetraploid Ae. crassa and Ae. searsii. The similarity of Cbanding and FISH patterns of Ae. vavilovii and corresponding parental species points to rather recent origin of this hexaploid. The assumption that the Xcr genome is an additional derivative of the S∗ genome obtained from an unknown or extinct species of the Emarginata group, which was substantially modified over the course of evolution is supported.

#### AUTHOR CONTRIBUTIONS

EB planned the research, performed and coordinate the analysis. EB and AR performed the research and wrote the paper.

#### ACKNOWLEDGMENTS

The authors thank Drs. O.M. Raskina and A.A. Belyayev (Institute of Evolution, University of Haifa, Israel) for providing seeds and for the opportunity to collect seed materials from their natural habitat. Additional material was obtained from gene banks of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK, Gatersleben, Germany); VIR Collection of Plant Genetic Resources (Saint Petersburg, Russia); Institute of Evolution, University of Haifa (Haifa, Israel); Weizmann Institute of Science (Rehovot, Israel); USDA-ARS (Aberdeen, Idaho, USA); Wheat Genetics and Genomic Resource Center, Kansas State University (Manhattan, Kansas, USA); ICARDA (Aleppo, Syria). We thank Dr. A. Houben [Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany] for critical reading and valuable comments. We thank Dr. S. Surzhikov (Engehardt Institute of Molecular Biology, Russian Academy of Sciences) for

#### REFERENCES

the synthesis of oligo-probes for analysis. This work was supported by grant from Russian State Foundation of Basic Research (project No 17-04-00087). The publication of this article was funded by the Open Access Fund of the Leibniz Association.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018. 01756/full#supplementary-material

Figure S1 | Diversity of the C-banding patterns in Aegilops speltoides accessions: (a), No1, from Turkey (provided by Dr. N. Aminov); (b–f), genotypes collected from Israeli populations; (b,c), C1, Technion park, Haifa; (d), C2, Nahal Mearot; (e), G2.46, Ramat haNadiv; (f), C14, Keshon; (g), no 2734 (unknown provided by Dr. B. Kilian); (h), No 2 from Iran (provided by Dr. N. Aminov); (i), i-570060; (j), TS89, Katzir, Israel; (k), PI 487233, and (l), PI 487231 (from Syria); (m), PI 542269 (Turkey). Arrows show unbalanced chromosome modifications.

Figure S2 | Interspecific and intraspecific variation of the C-banding patterns in Ae. bicornis (a1–a3), Ae. searsii (b1–b4), Ae. sharonensis (c1–c8), and Ae. longissima (d1–d10). Accession codes: (a1), TA1942; (a2), TB04-3; (a3), TB10-2; (b1), G.7.15; (b2), TE01-1; (b3), G7.12; (b4), IG 47619; (c1,c2), C6, Keshon; (c3), C5, Caesaria; (c4), TH04; (c5), TH01; (c6), TH02; (c7), C4, Atlit; (c8), C7, HaBonim; (d1), TL06; (d2), TL01; (d3), G6.77 (Sa'ad); (d4), G6.58 (Tel Akko); (d5), TL03; (d6), C3 (HaBonim); (d7), G6.32 (Nizzanim); (d8), G6.55 (Zomet Shoked); (d9), G17-3; (d10), TL05.

Figure S3 | C-banding polymorphism of Ae. peregrina (a–f) and Ae. kotschyi (g–j) chromosomes: (a), TA1888; (b), C11 (Nahal Mearot, Israel); (c), K-61; (d), C12 (Caesaria, Israel); (e), C13 (Natufia, Israel); (f), i-570632; (g), PI 487279; (h), K-91; (i), TA2206; (j), K-201; (k) K-2905. 1–7, homoeologous groups.

Table S1 | List of material and their origins.

Table S2 | Distribution of Spelt-52 probe on chromosomes of Ae. longissima and Ae. sharonensis.

of the U-genome cluster. Plant Syst. Evol. 246, 45–76. doi: 10.1128/AEM.02 052-08

Zoshchuk, S. A., Zoshchuk, N. V., Amosova, A. V., Dedkova, O. S., and Badaeva, E. D. (2009). Intraspecific divergence in wheats of the Emmer group using in situ hybridization with the Spelt-1 family of tandem repeats. Rus. J. Genet. 45, 1376–1384. doi: 10.1134/S102279540911012X

**Conflict of Interest Statement:** 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.

Copyright © 2018 Ruban and Badaeva. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Repetitive DNA in the Architecture, Repatterning, and Diversification of the Genome of Aegilops speltoides Tausch (Poaceae, Triticeae)

Yulia Pollak1,2, Einat Zelinger1 and Olga Raskina3 \*

1 The CSI Center for Scientific Imaging, The Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, Israel, 2 The Electron Microscopy Unit, Faculty of Natural Science, University of Haifa, Haifa, Israel, 3 Institute of Evolution, University of Haifa, Haifa, Israel

#### Edited by:

Eva Darko, Hungarian Academy of Sciences (MTA), Hungary

#### Reviewed by:

Adél Sepsi, Hungarian Academy of Sciences (MTA), Hungary Hieronim Golczyk, The John Paul II Catholic University of Lublin, Poland

\*Correspondence:

Olga Raskina<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Breeding, a section of the journal Frontiers in Plant Science

Received: 29 August 2018 Accepted: 15 November 2018 Published: 04 December 2018

#### Citation:

Pollak Y, Zelinger E and Raskina O (2018) Repetitive DNA in the Architecture, Repatterning, and Diversification of the Genome of Aegilops speltoides Tausch (Poaceae, Triticeae). Front. Plant Sci. 9:1779. doi: 10.3389/fpls.2018.01779 The genome's adaptability to environmental changes, especially during rapid climatic fluctuations, underlies the existence and evolution of species. In the wild, genetic and epigenetic genomic changes are accompanied by significant alterations in the complex nuclear repetitive DNA fraction. Current intraspecific polymorphism of repetitive DNA is closely related to ongoing chromosomal rearrangements, which typically result from erroneous DNA repair and recombination. In this study, we addressed tandem repeat patterns and interaction/reshuffling both in pollen mother cell (PMC) development and somatogenesis in the wild diploid cereal Aegilops speltoides, with a focus on genome repatterning and stabilization. Individual contrasting genotypes were investigated using the fluorescent in situ hybridization (FISH) approach by applying correlative fluorescence and electron microscopy. Species-specific Spelt1 and tribe-specific Spelt52 tandem repeats were used as the markers for monitoring somatic and meiotic chromosomal interactions and dynamics in somatic interphase nuclei. We found that, the number of tandem repeat clusters in nuclei is usually lower than the number on chromosomes due to the associations of clusters of the same type in common blocks. In addition, tightly associated Spelt1–Spelt52 clusters were revealed in different genotypes. The frequencies of nonhomologous/ectopic associations between tandem repeat clusters were revealed in a genotype-/populationspecific manner. An increase in the number of tandem repeat clusters in the genome causes an increase in the frequencies of their associations. The distal/terminal regions of homologous chromosomes are separated in nuclear space, and nonhomologous chromosomes are often involved in somatic recombination as seen by frequently formed interchromosomal chromatin bridges. In both microgametogenesis and somatogenesis, inter- and intrachromosomal associations are likely to lead to DNA breaks during chromosome disjunction in the anaphase stage. Uncondensed/improperly packed DNA fibers, mainly in heterochromatic regions, were revealed in both the meiotic and somatic prophases that might be a result of broken associations. Altogether, the data obtained showed that intraorganismal dynamics of repetitive DNA under the conditions of natural out-crossing and artificial intraspecific hybridization mirrors the structural plasticity of the Ae. speltoides genome, which is interlinked with genetic diversity through the species distribution area in contrasting ecogeographical environments in and around the Fertile Crescent.

Keywords: Aegilops speltoides, interphase nuclei, nonhomologous recombination, repetitive DNA, somatic recombination, tandem repeats

#### INTRODUCTION

fpls-09-01779 November 30, 2018 Time: 15:13 # 2

In the wild, the genome's ability to adapt to changing environments, especially in a period of significant climatic changes, underlies the existence of the species and its evolution (Grant, 1981; Tchernov, 1988). In plant populations, genetic changes and epigenetic modifications are accompanied by significant diversification in the abundances and patterns of repetitive DNA, which is the prevailing genomic fraction in cereals (Bennetzen, 1996; Feschotte et al., 2002). Transposable elements (TEs) and tandem repeats compose the largest portion of the genome of wild diploid grass species Aegilops speltoides (sect. Sitopsis; 2n = 2x = 14) (Middleton et al., 2013). Ae. speltoides is a dimorphic species; the differences in spike morphology are controlled through a block of closely linked genes encoding dominant ssp. ligustica and recessive ssp. aucheri morphotypes, which coexist in mixed panmictic populations with different ratios (Zohary and Imber, 1963; Kimber and Feldman, 1987). Plants with intermediate ligustica/aucheri phenotypes have also been observed in the wild, suggesting genetic changes in the linked group of genes (Belyayev and Raskina, 2013). Ae. speltoides is only out-crossing species among the five diploid species of the sect. Sitopsis; however, under a changing environment, specifically, in drought conditions, Ae. speltoides transits to self-pollination, which is an extremely rare phenomenon in the plant kingdom (Zohary and Imber, 1963). The plasticity of the genome underlies a wide range of Ae. speltoides distribution and adaptability to contrasting ecogeographical environments in and around the Fertile Crescent (Zohary and Imber, 1963; Kimber and Feldman, 1987). Thus, at the northern periphery of the species distribution area, Turkish winter-type populations have a long vegetative cycle and exhibit a specific morphology, which significantly distinguishes them from the peripheral/marginal southern Israeli populations (Belyayev and Raskina, 2013). In parallel, the Ae. speltoides genome is characterized by high intraspecific polymorphism in abundance and patterns of different types of repetitive DNA, specifically, TEs (Middleton et al., 2013; Yaakov et al., 2013) and tandem repeats (Badaeva et al., 1996; Raskina et al., 2011; Molnár et al., 2014; Raskina, 2017), which underlie permanent intraorganismal and intraspecific genome reshuffling (Belyayev et al., 2010; Shams and Raskina, 2018). The current intraspecific polymorphism and intraorganismal dynamics of the highly repetitive DNA fraction in the genome of Ae. speltoides is largely caused by ongoing chromosomal rearrangements, which are typical results of erroneous DNA repair and recombination (Andersen and Sekelsky, 2010; Knoll et al., 2014; Zeman and Cimprich, 2015).

In the present research, we addressed repetitive DNA dynamics in the Ae. speltoides genome, both during pollen mother cell (PMC) development and in somatogenesis, with a focus on genome repatterning and stabilization. We traced tandem repeats' reshuffling/interactions during the cell cycle using fluorescent in situ hybridization (FISH), applying correlative fluorescence and electron microscopy. Speciesspecific Spelt1 (Salina et al., 1998) and tribe-specific Spelt52 (Anamthawat-Jónsson and Heslop-Harrison, 1993) tandem repeats were used as the markers for monitoring somatic and meiotic chromosomal interactions and dynamics in somatic interphase nuclei. We found that Spelt1 and Spelt52 demonstrated sequence-specific and genotype-/populationspecific abundances and dynamics in interphase nuclei. The number of tandem repeat clusters in nuclei is usually lower than the number on chromosomes due to the associations of clusters of the same type in common blocks. In addition, tightly associated Spelt1–Spelt52 clusters were revealed in different genotypes. An increase in the number of tandem repeat clusters in the genome causes an increase in the frequencies of their associations in common blocks in interphase nuclei. Frequent cell-specific interchromosomal somatic associations and nonhomologous recombination in microsporogenesis were revealed. It is speculated that significant number of nonhomologous chromosomal associations detected in microgametogenesis might be the consequences of cell-specific ectopic recombination events that occurred in premeiotic cell lineages.

#### MATERIALS AND METHODS

#### Plant Material

Original plants of Ae. speltoides from contrasting allopatric populations Cankiri (Turkey; PI 573448, USDA), Ankara (Turkey; PI 573452, USDA), Katzir (Israel; 2.93, Institute of Evolution University of Haifa), Ramat haNadiv (Israel; 2.46, Institute of Evolution University of Haifa), and artificial F1–F2 intraspecific hybrids (Raskina, 2017), were analyzed.

#### Preparation of Chromosomal Spreads

Anthers containing PMC cells at the pachytene-diakinesis stages and seedling shoot apical meristems of individual plants were used for meiotic and mitotic chromosome spreads, respectively. Seeds were germinated on moist filter paper at 24◦C in the dark. Seedlings 5–7 mm length were transferred to ice water for 24–26 h to accumulate metaphases and then fixed in 3:1 (v/v) 100% ethanol:acetic acid. The procedure of chromosome

spread preparation has previously been described (Raskina et al., 2004). Specifically, the fixed seedlings and anthers were washed (3 × 5 min) in water and then incubated in an enzyme buffer (10 mM citrate buffer at pH 4.6) and partially digested (meristem–for 50 min, anthers–for 30 min) in 6% pectinase plus 0.5% cellulase (NBC Biomedicals, United Kingdom) plus 5% cellulase "Onozuka" R-10 (Yakult Honsha Co., Ltd.) followed by washes in enzyme buffer (3 × 5 min) and distilled water (3 × 5 min). The material, in a drops of water, was transferred onto a grease-free microscope slide, and the cells were spread with a metal stainless needle in the drop of 60% acetic acid at 45–47◦C on the hot plate, then fixed in 3:1 (v/v) 100% ethanol : acetic acid, and then immersed in absolute ethanol for 3–5 s. Dry chromosome spreads were used for in situ hybridization.

#### In situ Hybridization Procedures

For the FISH experiments, cytological slides of individual anthers and seedling shoot apical meristems containing well-spread chromosomal plates were used. The FISH procedures were conducted as previously described (Shams and Raskina, 2018).

Cells were treated with 1 µg/mL of DNase-free RNase A in 2 × SSC (0.3 M NaCl plus 30 mM trisodium citrate) for 60 min at 37◦C followed by 3 × 3 min washes with 2 × SSC at 37◦C. The preparations were then dehydrated in an ethanol series (70, 90, and 100%, 3 min each) at room temperature, washed 2 × 2 min with 2 × SSC, and then allowed to airdry. The hybridization mixture (20 µL per slide under the glass coverslip 22 mm × 22 mm) contained 10% dextran sulfate, 2 × SSC, and 50 ng each of DNA probe. DNA probes and chromosome spreads were simultaneously denatured at 95◦C for 3 min and hybridized using ThermoBrite StatSpin System (Abbott, United States). Hybridization was carried out at 63◦C for 2 h. After removal of the coverslips in 2 × SSC at 63◦C, the slides were washed for 2 × 5 min in 2 × SSC at 63◦C, additionally once in 0.1 × SSC for 5 min at 63◦C to increase stringency, then cooled to 37◦C and washed for 2 × 5 min in 0.1 × SSC; cooled to room temperature, washed in distilled water for 1 min, allowed to air-dry for 20 min, and mounted in VECTASHIELD antifade mounting medium (Vector Laboratories).

Tandem repeats Spelt1 (Salina et al., 1998), Spelt52 (Anamthawat-Jónsson and Heslop-Harrison, 1993), pSc119.2 (Bedbrook et al., 1980), pTa71 (for the localization of 45S rDNA) (Taketa et al., 2000), and As5SDNAE (for the localization of 5S rDNA) (Baum and Bailey, 2001) were used as the DNA probes for FISH. The DNA probes were directly labeled with Cy-3, Fluorescein-12-dUTP, and ATTO-425 (Jena Bioscience, Germany). AT-specific 40 ,6-diamidino-2-phenylindole (DAPI) fluorochrome was used for differential staining.

#### Epi-Fluorescence Imaging

The slides were examined on a Leica DMR fluorescent microscope equipped with a DFC300 FX CCD color camera using following filter sets: A for DAPI, I3 for Fluorescein-12-dUTP, N2.1 for Cy3, FI/RH for Fluorescein-12-dUTP/Cy3, and B/G/R for blue/green/red fluorescence.

# Confocal Image Acquisition and Analysis

Confocal imaging was done using a LEICA SP8 (CTR6000) microscope with a Leica HC PL APO CS2 × 63 N.A 1.4 oil objective. Lasers used: EX 405 EM 430–470 for DAPI, EX 488 EM 500–540 for Fluorescein-12-dUTP, and EX 552 EM 560–590 for Cy3. All images were collected with the HyD (hybrid detector). Image z-stacks of 15–25 × 0.2 µm slices per specimen were acquired and reconstructed by the 3D built-in module of LEICA SP8 in the LAS AF software (**Supplementary Figures S1–S3**). 3D Movies were created from the z-stacks by the LEICA SP8 3D module (**Supplementary Movies S1–S4**). For statistical analysis and 3D reconstruction the ImageJ software1 was used (**Supplementary Figure S3B**).

For 3D reconstruction modeling, the images were processed using the Imaris surface reconstruction tool with module "Imaris cell" (Bitplane Scientific Software, Zurich, Switzerland) (**Supplementary Figure S4**).

#### Scanning Electron Microscopy

The scanning electron microscopy (SEM) images were recorded using Field emission scanning electron microscopes (i) Sigma-HD, ZEISS, and (ii) JEOL, JSM-7800F, accelerating voltage of 1.5–2.0 kV. Slides were carbon coated using Quorum Q150T.

## RESULTS

### Patterns of Tandem Repeats on Chromosomes and in Somatic Interphase Nuclei in Plants From the Cankiri Population

There are nine Spelt52 clusters of different sizes and fluorescence intensity, two large Spelt1 clusters on the long arms of the both homologs of chromosome 7, and two small Spelt1 clusters on the long arms of both homologs of chromosomes 5 in the diploid genome(s) of plant(s) from the Cankiri population (**Figure 1A**). The number of Spelt52 clusters in the interphase nuclei varied between six and nine. Among 300 interphase nuclei of different sizes and varying degrees of chromatin compactness analyzed on two slides from the seedling shoot apical meristems, two large Spelt1 clusters were observed in most cases (**Figures 1A,B**) and only in eight nuclei (2.7%) was a single Spelt1 cluster revealed (**Figure 1C**). Thus, these data point to separation of the homologous chromosomes in nuclear space; associations of the distal/terminal chromosomal regions and the formation of complex tandem repeat clusters rarely occurred.

## Patterns of Tandem Repeats on Chromosomes and in Somatic Interphase Nuclei in Plants From the Katzir Population

In contrast to the genotypes from the Cankiri population, plants from the Katzir population are enriched with Spelt1

1https://imagej.net/Fiji

FIGURE 1 | Patterns of tandem repeats in the Ae. speltoides contrasting genotypes. Fluorescent in situ hybridization (FISH) on somatic chromosomes and interphase nuclei from the seedling shoot apical meristems of the plants from Cankiri (A–C) and Katzir (D,E) populations. DNA probes for FISH: Spelt1 (in green), Spelt52 (in red), pSc119.2 (in blue), 5S rDNA (in blue, red), and 45S rDNA (in green). (A) Somatic chromosomes (top) and interphase nuclei (bottom) of the plant from the Cankiri population. Two large terminal Spelt1 clusters on the long arms of homologous chromosome 7 and two small terminal clusters on the long arms of homologous chromosome 5 were revealed (arrows). Nine Spelt52 clusters were detected in the diploid genome. In the interphase nuclei, Spelt52 and two large (Continued)

#### FIGURE 1 | Continued

fpls-09-01779 November 30, 2018 Time: 15:13 # 5

Spelt1 (arrows) clusters are separated from each other. Four separated 45S rDNA clusters are revealed in the nuclei. (B) Two large Spelt1 clusters in the interphase nuclei of different sizes are indicated with arrows. (C) Single Spelt1 cluster (arrow) and separated Spelt52 clusters are observed in the interphase nucleus of Cankiri plant. (D) Tandem repeats Spelt1 and pSc119.2 form complex clusters in chromosome termini (top left). There are 26 Spelt1 clusters in the diploid genome; 12 chromosomes carry the Spelt1 cluster in both arms and two chromosomes carry Spelt1 in one arm, with terminal pSc119.2 clusters shown in the other arm with arrowheads. In the interphase nucleus (bottom), 14 Spelt1 clusters are revealed due to the association of individual clusters. In addition, Spelt1 and pSc119.2 tandem repeats compose common clusters (arrows). Two 5S rDNA clusters are separated in the nuclear space. Re-probing with Spelt52 and 45S rDNA (middle) revealed 14 Spelt52 clusters on the chromosomes and in the nucleus. Ectopic chromatin fibers between nonhomologous chromosomes (arrows; enlargement in the small boxes) are indicated with arrows (right). (E) Interphase nucleus of the Katzir genotype. Spelt52 clusters are separated in the nuclear space, while the number of Spelt1 clusters is almost half that on the chromosomes. Tightly associated Spelt1 and Spelt52 clusters are indicated with arrow. Scale bar = 10 µm.

tandem repeats (**Figures 1D,E**). There are 14 Spelt52 and 26 large terminal Spelt1 clusters in the diploid genome; only two chromosome arms do not carry Spelt1 clusters (**Figure 1D**, top); instead, clusters of the pSc119.2 tandem repeat are detected in these positions. The genome of Ae. speltoides is enriched with the pSc119.2 tandem repeat, which forms numerous clusters in the distal/terminal and intercalary chromosomal regions (Badaeva et al., 1996; Molnár et al., 2014). In the interphase nuclei, the Spelt1 and pSc119.2 tandem repeats form common clusters (**Figure 1D**). The number of Spelt1 clusters is reduced almost by half due to associations, while the number of Spelt52 clusters equals the number of clusters on the chromosomes, and 5S rDNA clusters are separated in the nuclear space. Among the 130 nuclei from the seedling shoot apical meristems analyzed on this cytological slide, the number of Spelt1 clusters was reduced in most cases, and one to three tightly associated Spelt1–Spelt52 clusters were revealed in 123 (94.6%, n = 130) nuclei (**Figures 1D,E**); and in other genotype, colocalization of the Spelt1–Spelt52 clusters was revealed in most cases (78.5%, n = 250).

#### Patterns of Tandem Repeats on Chromosomes and in Somatic Interphase Nuclei in Plants From the Ramat haNadiv Population

Meiotic chromosomes and interphase nuclei from the somatic anther tissues were analyzed on the same slide(s) for different genotypes from the Ramat haNadiv population (**Figure 2**). At the early stages of anther' development, specifically, at the pachytene–diakinesis–metaphase I stages, the following cell layers, namely epidermis, endothecium, middle layer, tapetum, and connective tissues are distinguished (Browne et al., 2018). The nuclei of different sizes, shapes, and chromatin compactness at different stages of the interphase from the anther's somatic tissues vary significantly in the numbers of Spelt1 and Spelt52 clusters (**Figures 2A–A8**, **Supplementary Figures S1,S2,** and **Supplementary Movies S1,S2**). Colocalization of the tandem repeat clusters of the same type is observed in a cell-specific manner; in addition, Spelt1 and Spelt52 tandem repeats form tightly associated/common clusters (100%; n = 400; three slides/genotypes), which are condensed or partially decondensed at different interphase stages and in different anther somatic tissues. Numerous polyploid (**Figure 2A3**) and amitotically dividing tapetal nuclei (**Figure 2A6**) are revealed on the same cytological slide(s). Typically, partial decondensation of tandem repeat clusters and the occurrence of large common clusters are observed in small compact nuclei (**Figures 2A4– A6,C,E, Supplementary Figures S3,S4,** and **Supplementary Movies S3,S4**). Somatic recombination in the interphase nucleus was documented using a SEM (**Figure 2D**). Specifically, two chromatin forks/two different chromosomes are recombined in their terminal regions, which form continuous chromatin fiber. At the end of the interphase/in the early somatic prophase stage, as condensed chromatin fibers become visible, numerous associations between clusters of the same type and between Spelt1 and Spelt52 clusters are still observed (**Figures 2A7,A8**, **G**,**H**).

#### Nonhomologous Chromosome Associations in Microsporogenesis in Different Genotypes From the Ramat haNadiv Population

Along with somatic interphase nuclei from the anther tissues, meiotic cells were analyzed on the same slide(s). A wide spectrum of cell-specific structural alterations and chromosomal rearrangements at the meiosis I stages were revealed in different genotypes (**Figures 2I–K**). Thus, alterations in Spelt1 and Spelt52 clusters' condensation and the appearance of tightly associated clusters were observed in late meiotic prophase I (**Figure 2I**); chromatin fibers appear damaged and uncondensed. At the stages of diakinesis–metaphase I, cell-specific chromosomal rearrangements and numerous nonhomologous associations were revealed in all plants (**Figures 2J,K**). Interchromosomal fibers were documented using both fluorescent and scanning electron microscopy.

#### Tandem Repeat Cluster Associations in Somatic Interphase Nuclei in Parental Genotype From the Katzir Population and Intraspecific Hybrids of Aegilops speltoides

In the maternal genotype from the population Katzir, there are 12 Spelt52 and 22 Spelt1 clusters on 14 individual chromosomes, or 14 Spelt1 and 7 Spelt52 clusters per seven bivalents, that is, when homologs are paired (**Figure 3A**, on the left). In somatic nuclei (**Figure 3A**, on the right), the number of both types of clusters vary and often is smaller than the number on 14 chromosomes (statistical analysis was not performed).

In the F1 hybrid genotype (**Figure 3B**; Raskina, 2017), maternal and paternal chromosomes are identified according

FIGURE 2 | Patterning of Spelt1 and Spelt52 tandem repeats on meiotic chromosomes and in interphase nuclei from the anther' somatic tissues in individual genotypes of Ae. speltoides from the Ramat haNadiv population. DNA probes for FISH: Spelt1 (in green) and Spelt52 (in red); counterstaining with DAPI (in blue). (A) Interphase nuclei at different interphase stages belonging to different anther somatic tissues from the same cytological slide. (A–A8) (Supplementary Figures S1,S2 and Supplementary Movies S1,S2) In nuclei of different sizes and shapes, various numbers of Spelt1 and Spelt52 clusters are observed. The nuclei differ in number and sizes of associated clusters of the same types and Spelt1–Spelt52 colocalized clusters. Condensed and undercondensed single (Continued)

#### FIGURE 2 | Continued

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and associated Spelt1 and Spelt52 tandem repeat clusters are revealed in different nuclei. A polyploid nucleus is shown in A3. (A6) Amitotically dividing nuclei. (A7,A8) Nuclei at the late interphase–early prophase stages. Spelt1 and Spelt52 clusters are partially decondensed and interconnected with extended fibers; due to associations, the cluster number is less than on meiotic chromosomes (J,K). (B–D) Nuclei from the same cytological slide. (B) Number of condensed Spelt1 and Spelt52 clusters of different sizes and fluorescent intensities are 11 and 17, respectively. Most Spelt1 and Spelt52 clusters associate in common clusters. (C) Large Spelt1 and Spelt52 clusters aggregate in complex blocks. (D) In the image of the interphase nucleus obtained using scanning electron microscopy, two interconnected Y-shaped chromatin structures are revealed. Chromatids of two different chromosomes form a double-strand chain (red dashed line and arrow in the scheme on the right). (E–H) Nuclei of the other genotype from the same cytological slide. (E) (Supplementary Figures S3,S4 and Supplementary Movies S3,S4) Large Spelt1 and Spelt52 partially decondensed clusters aggregate in complex blocks. (F) Highly condensed clusters of Spelt1 and Spelt52 form common blocks. (G,H) Large nuclei at the late interphase–early prophase stages; condensed chromatid fibers are revealed. Spelt1 and Spelt52 clusters interconnected with extended fibers. (I) Meiotic prophase I, pachytene stage: homologous synapsis is completed. Associated Spelt1 and Spelt52 clusters form common blocks, and extended fibers between clusters are observed; improper condensation and damaged chromatin fibers are indicated with dashed lines and arrows on the image obtained via scanning electron microscopy (right). (J,K) Meiotic chromosomes and nuclei of different genotypes. Numbers of Spelt52 vary from 11 to 12 clusters and Spelt1 from 13 to 14 clusters per seven bivalents. (J) Three bivalents are involved in nonhomologous recombination. Extended Spelt1 fibers connect two homologs of one bivalent with single chromosomes of two other bivalents. In two small boxes, enlargement of this region obtained with confocal fluorescent (left) and scanning electron microscopy (right), respectively, is shown. (K) Four meiotic chromosomal plates at the diakinesis–metaphase I stages and dividing polyploid cell of tapetum at the anaphase stage with chromosomal bridges (red arrow). Images were taken consistently using fluorescent and scanning electron microscopes. Nonhomologous chromosome associations are indicated with arrows; the extended thin Spelt1 fiber between two bivalents is indicated with a pink dashed line. Scale bar = 10 µm.

to contrasting Spelt1 and Spelt52 patterning, as detailed in the small box. Maternal chromosomes carry 12 Spelt1 and 2 Spelt52 clusters; paternal chromosomes carry 3 Spelt1 and 5 Spelt52 clusters. In terms of the seven bivalents, there are 13 Spelt1 and 5 Spelt52 clusters in the genome. Somatic interphase nuclei from the anther tissues on the same cytological slide differ in the number of Spelt1 and Spelt52 clusters. Two nuclei (on the left and in the middle) are similar and contain 12 Spelt1 and 8 Spelt52 clusters, while in the third nucleus (on the right), the numbers of clusters are reduced due to associations.

In the F2 hybrid genotype (**Figure 3C**), the Spelt1 fibers between the homologous chromosomes at the stage of anaphase I are the consequences of meiotic recombination in the terminal chromosome regions; interchromosomal connections will be broken, and homologs will move to opposite pools with altered termini.

#### Ectopic Associations of Somatic Chromosomes in Different Genotypes

Frequent cell-specific interchromosomal somatic associations in apical shoot meristem were revealed in plants from different populations. Thus, in the Katzir genotype, among 32 metaphase plates analyzed on the same cytological slide, in 21 cases (65.6%), ectopic associations between chromosomes were detected in a cell-specific manner, regardless of the presence or absence of any tandem repeat clusters in the points of chromosomal connections (**Figure 1D**). Interchromatid somatic associations between three chromosomes were documented for the other genotype from the Katzir population (**Figure 4A**). In this instance, two chromatids of one chromosome are connected with single chromatids of two other chromosomes. Clusters of Spelt1 and Spelt52 are involved in one association and form interchromosomal fibers; in other association, the fiber between chromatids of two different chromosomes was revealed with DAPI staining.

No associations between the Spelt1 clusters were observed for homologous chromosomes 7 in the Cankiri plants. Partially decondensed Spelt1 clusters were documented in somatic nuclei (**Figure 4B**). Cell-specific alterations in Spelt1 clusters' condensation/decondensation was observed in somatic prophase cells (**Figure 4C**).

In the genotype from the Ramat haNadiv population, intrachromosomal ectopic recombination was revealed (**Figure 4D**). Here, the Spelt1 fiber is observed as a dotted line, which may be an indication of intercalation/alternation of Spelt1 subclusters with some other type of uncolored DNA sequences or decondensation of uncolored regions. Ectopic associations between chromosomes 1 and 6, which carry 45S rDNA clusters, and chromosome 5, carrying 5S rDNA clusters, were documented for the genotype from the Ankara population (**Figure 4E**). Ectopic interchromatid fibers, which are revealed with DAPI staining, connect the short and/or long arms of somatic chromosomes, and the rDNA clusters appear intact.

In the F2 hybrid (♀Cankiri × ♂Katzir) genotype, at different interphase stages, 12 Spelt1 clusters were typically interconnected when decondensed (**Figure 4F**), and they apparently formed two groups of six clusters each when condensed, in the diploid nucleus (**Figure 4G**). On the prometaphase chromosomes (**Figure 4H**), partial decondensation and interconnections between five Spelt1 clusters were observed. In this genotype, two chromosomes carried Spelt1 clusters on both the short and long arms (**Figure 4I**). Therefore, the fibers between the four clusters in the prometaphase could be consequences of cell-specific ectopic associations between the short and long arms in the interphase, with the fifth cluster belonging to some other chromosome; alternatively, all five clusters could belong to different/nonhomologous chromosomes. However, in the other F2 hybrid genotype (**Figures 4J–L**) containing 10 Spelt1 clusters in the diploid genome, all chromosomes carry single Spelt1 clusters in one arm; while in the nuclei, associations of two and three clusters were revealed (**Figures 4J,L**). Two clusters may have belonged to homologous chromosomes, while three undoubtedly pointed to nonhomologous associations. In addition, ectopic chromatin fibers were detected between the chromosomal regions, and these did not carry tandem repeat