**4. Discussion**

With the recent development of next-generation sequencing technologies, an ever-increasing number of plant species have been studied and vast amounts of sequence data accumulated. Next-generation sequencing provides a convenient way to search for the presence of mini-satellite repeated sequences [14,15]. As a major class of repetitive DNA, mini-satellites consist of tandemly organized monomers. Zakrzewski et al. [16] isolated 517 novel repetitive sequences and used them for the identification of mini-satellite families in *Beta vulgaris*, and they revealed that mini-satellites are moderately to highly amplified by bioinformatic analysis and southern hybridization. Mogil et al. [15] identified five distinct, interleaved mini-satellite families in the pericentromeric regions of soybean (*Glycine max*) chromosomes, which were mediated by >3200 intact copies. The availability of a whole-genome assembly of wheat cv. Chinese Spring also provides opportunities to examine the distribution of retrotransposons [45] and of TRs [29] at a whole-genomic level. Our recent study predicted that TRs occupy about 3–5% of wheat chromosomes [29]. Overall, the total array number of TR mini-satellites (20–60 bp) was 1,453,457, and the total length of such mini-satellites reached 129,873,148 bp in the wheat genome by TRF [29]. Martienssen and Baulcombe [46] first identified mini-satellite arrays lying upstream of an alpha-amylase gene in hexaploid wheat. Somers et al. [24] amplified mini-satellite core sequences from wheat. However, these mini-satellite arrays in wheat represented moderately repeated families of less than 100 copies per array. Tang et al. [47] reported five probes with mini-satellite sequences among ND-FISH probes as predicted by TRF, and we predicted 120 to 260 copy numbers across specific chromosome regions with total lengths of under 20 kb by B2DSC web server. The present study found that Ta-3A1 represented the highest copy number of a mini-satellite ye<sup>t</sup> reported, and it covered a total length of over 2 Mb and was mainly located on five chromosomes of wheat (Figure 1). Two copies of Ta-3A1-like sequences were found in the intron 2 of Bradi1g11210.1 and the intron 4 of Bradi3g03878.1 in the *Brachypodium* genome. Ta-3A1 was absent from the sequenced barley genome [48]. With regard to the accumulation of Ta-3A1 in pericentromeric regions of chromosome 5D, the observed high number may have been related to centromeric specific retrotransposons (Figure 7). Just how the extensive duplication of the Ta-3A1 sequence on 5D occurred, which resulted in such grea<sup>t</sup> copy numbers, is as ye<sup>t</sup> unexplained and needs to be further investigated. A problem exists with the identification of the short tandem repeats, which are often reshuffled and diversified and are somehow able to escape computational detection. The quality of assembly of the wheat genome needs to be improved through the use of single-molecule sequencing, optical mapping, and chromosome conformation capture technologies [49,50]. The updated assembly of wheat genome will offer unprecedented insights into the detection of the structural features and relevant evolutionary characteristics of TRs, including mini-satellites.

**Figure 7.** The genome collinearity of pericentromeric regions of chromosome 5D between *Ae. tauschii* and wheat Chinese Spring (CS).

The sequence dynamics of a specific satDNA family may differ across genomic regions [51], populations [52], species, or higher taxonomic groups [53–56]. Consequently, detailed characterization of satDNA families has revealed that evolutionary patterns are more complex than previously anticipated. The study of mini-satellite sequence elements is essential to our understanding of the nature and consequences of genome size variation between different species and for studying the large-scale organization and evolution of grass genomes [5]. With regard to the ancestral species of *Hordeum* separated by a time period ranging from 11.6 to 15.6 Mya [48], the Ta-3A1 sequence might not have been amplified at all since no signals were detectable in wild and cultivated barley species. The *D. breviaristatum* species was also devoid of Ta-3A1 hybridization sites. It is likely that *D. breviaristatum* may be one of the ancestral Triticeae species after barley separation, which is consistent with data from our previous studies on seed gliadin evolution [57]. Bread wheat can strongly adapt to different climates, and one of the key factors of this characteristic is the allohexaploid genome structure, which originates from two distinct polyploidization events [58]. In the present study, we found that the copy number of Ta-3A1 was largely amplified from *T. urartu* (AA), *Ae. tauschii* (DD), and allotetraploid *T. turgidum* (AABB) to common wheat (AABBDD). Moreover, *Ae. speltoides* is cytogenetically distinct from the S genomes of other diploid and polyploid *Aegilops* species based on C-banding and FISH [59,60]. We also showed that the FISH patterns of Ta-3A1 appeared on the long arm of 5S in *Ae. speltoides* and showed close resemblance to those of the B genome in wheat. Therefore, the study of the accumulation of mini-satellite sequences can shed light on wheat genome organization and, specifically, on the role of repetitive elements by using Ta-3A1 as an example.

The satDNA family is characterized by complex features, including large variations in copy number and long-range organization of repeat units, genome location and distribution, as well as interchromosomal and intrachromosomal recombination rates [53,61–65]. Fluorescence in situ hybridization (FISH) using the probes of repetitive DNA sequences was used to determine their physical locations on individual chromosomes of common wheat and its relatives [66,67]. In this current study, we also found that *Secale*, *Dasypyrum*, and *Thinopyrum* species displayed varied FISH patterns of Ta-3A1 hybridization (Figure 6). It is possible to deduce trends in the complexity of Ta-3A1 organization during the evolution of the Triticeae tribe from wild species to cultivated species. The variability of Ta-3A1 representing mini-satellites has been used in numerous studies to identify plant chromosomes. The Ta-3A1 probe has been used to precisely identify the chromosomal breakage points in wheat—*Th. intermedium* introgression lines [68,69]. FISH specific hybridization sites by Ta-3A1 on *Secale*, *Dasypyrum*, and *Aegilops* chromosomes can also trace specific chromatin in wheat-alien introgression lines. The combination of both molecular cytogenetics and genomic research on TRs, including mini-satellites, will significantly benefit future wheat breeding activities, which focus on chromosome manipulation and engineering [29].
