Bread wheat, an allohexaploid cereal crop, possesses a very large genome with over 80% of repetitive sequences [14, 19]. Even though the whole genome sequence data of bread wheat and its two diploid progenitors (Ae. tauschii and T. urartu) are publicly available [14, 33,34,35], the accurate sequencing, assembly, and chromosomal localization of repetitive sequences remains challenging, especially for the unsequenced B genome donor. Moreover, the relationship between the S genome of Ae. speltoides and the B genome of durum and bread wheats is still being debated, even though Ae. speltoides is considered the most likely B donor [15, 36]. SSRs, which are abundant components present in wheat, is an efficient tool to study genomic alterations in eukaryotes [6, 17, 18, 20, 22, 30]. In this study, the distribution dynamics of SSRs on chromosomes of bread wheat and its progenitors were comprehensively investigated by FISH.
Short stretches of SSRs (20 ~ 250 bp) are normally undetectable by FISH, and the FISH detectable regions should be the regions enriched in long stretches of SSR sequences. In this study, 21 SSR sequences were labeled (Table 1) and hybridized with the chromosomes of bread wheat Chinese Spring, and only 6 SSR sequences, (AAC)n, (AAG)n, (ACA)n, (AG)n, (AGC)n and (ACG)n, displayed strong and stable FISH signals (Figs. 1, 2 and 3). This result suggested that these 6 SSRs were prone to forming long stretches of sequences in the wheat genome and could produce detectable FISH signals on chromosomes of bread wheat.
The selected SSRs, (AAC)n, (AAG)n, (AG)n and (ACG)n had already been used as FISH probes to study genetic diversity or genomic reconstruction either in wheat, wheat relatives or wheat progenitor species [17, 21, 23, 25,26,27, 29,30,31, 33, 37,38,39,40], but had not been comprehensively compared among wheat and its diploid and tetraploid progenitors. In this study, a systematic study was performed to investigate the distribution patterns of different SSRs on the chromosomes of wheat and its diploid and tetraploid progenitors, obvious SSR signal changes were observed from diploid donor to hexaploid wheat, which support the previous studies that the wheat genome has underwent extensive changes during its polyploidization and evolution [16, 36]. In addition, stronger and wider FISH signals than previous studies were detected in this study, which might be caused by the difference of probe labeling method. Using a PCR method in the absence of template, rather than random-primer labeling method or direct synthesized method [21,22,23, 25, 39], could produce longer probes and might reflect the actual location of large SSR clusters.
SSR sequences were not randomly distributed in wheat genomes, as their distribution on chromosomes depending on the motif, chromosome and genome, as demonstrated by Cuadrado et al. [21]. Although the size of SSR repeat units in wheat still needs to be confirmed, large SSR clusters could be located and compared in this study. In consistence with the studies that constitutive heterochromatic regions and SSRs are more abundant in the B genome chromosomes than those of A and D genomes [14, 36, 41, 42], most signals of (AAC)n, (AAG)n, (ACG)n and (AG)n were located on the B genome chromosome, followed by A genome chromosomes, and were least likely to be found on D genome chromosomes, and each SSR has its unique distribution patterns on different genomes and different chromosomes (Figs. 4, 5, 6 and 7). The results suggested that there would be a high heterogeneity of SSRs in wheat genome, especially B genome.
To integrate our SSR FISH results with their physical position in the genomes, the physical position of (AAC)n, (AAG)n, (AGC)n, and (AG)n were predicted using the web server B2DSC (http://mcgb.uestc.edu.cn/b2dsc) (Additional files 3, 4, 5 and 6) [27]. The prediction results indicated that high copy number SSR tandem repeats prone to cluster on chromosomes of wheat, especially the centromeric and pericentromeric regions of B genome chromosomes. Low copy number SSR repeats were more likely dispersed in the genomes of Ae. tauschii and T. urartu, which were not long enough to be detected by FISH, especially for the (AG)n sequence (Additional files 3, 4, 5 and 6). As the Ae. speltoides has not been sequenced yet, the physical location of SSR sequences in the genome was not analyzed here. As expected, the distribution of large SSR clusters in the predicted physical map was consistent with the results of FISH analysis, but the SSR sequences mapped by FISH were more concentrated, which showed the longer and abundant SSR sequence locations. In addition, more SSR repeats prone to form small clusters and widely distributed in genome, especially in genomes of Ae. tauschii and T. urartu. These small SSR clusters were not long enough to be detected by FISH (Additional files 3, 4, 5 and 6).
Following polyploidization, the wheat genome has undergone massive genomic rearrangements, including chromosome variation, sequence amplification and elimination [7, 11,12,13, 16]. To trace its genome evolution dynamics during its formation and evolution, karyotypes of wheat and its diploid and tetraploid progenitors based on our four SSR FISH results were constructed, and differences were analysed in terms of the abundance and localization of SSR motifs between different genomes and chromosomes. During wheat formation, compared with its progenitors, more SSR FISH signal changes were detected as SSR sequence expansion and/or elimination on B genome chromosomes (judged by the rough strength and number of FISH signals). Briefly and importantly, during wheat formation, the main distribution changes of our studied four SSRs from diploid progenitors to the bread wheat genome should be the sequence expansion on chromosomes 1B, 2B, 4B, 6B and 7B for (AAC)n; the sequence expansion on chromosomes 1B, 2B, 3B, 4B, 6B, 7B and 4A and the sequence elimination on chromosomes 5B, 2D, 3D and 4D for (AAG)n; the sequence expansion on chromosomes 1B-7B (especially 4B, 6B and 7B), 4A, 6A and 7A and both expansion and elimination on chromosomes 4B and 5B for (AGC)n and the sequence elimination on chromosomes 2B, 4B, 6B, 7B, 2D, 3D and 4D; and both expansion and elimination on chromosomes 3B, 5B, and 6B for (AG)n. These results suggested that SSR sequences preferred to move and/or amplify around their original locations rather than different genomes or chromosomes during wheat formation. This phenomenon could be explained because variability of SSRs in the genome is primarily the result of slipped-strand mispairing followed by replication and recombination, or repair errors, which could change the lengths of microsatellites [43]. Among the four SSR probes, signal changes of (AAG)n was the biggest across the B genome chromosomes, followed by (AAC)n, (ACG)n and (AG)n (Figs. 4, 5, 6 and 7), which suggested that different SSRs evolved at different speed across the B genome chromosomes. For (AAC)n, signal changes on 1B, 4B, 6B and 7B were larger than those on 2B, 3B and 5B; For (AAG)n, signal changes on 1B, 4B, 5B and 7B were larger than those on 2B, 3B and 6B; For (ACG)n, signal changes on 3B, 4B, 5B, 6B and 7B were larger than those on 1B and 2B. These results is consistent with the view that genome differentiation during wheat allopolyploidization from S to B proceeds at different speeds over the chromosomes, which was revealed by genome-wide exon sequencing and resultant phylogenetic analysis [36].
The origin of the B genome of wheat had been debated by researchers for a very long time, as it evolved “at a higher rate of evolution” than the A and D genome, the B genome of wheat is now more different from S genome of Ae. speltoides [15, 36], which was also elucidated in this study. But even so, most SSR FISH signals on chromosomes of Ae. speltoides could be found in chromosomes of the bread wheat (Figs. 4, 5, 6 and 7), which supports the viewpoint that Ae. speltoides is likely to be the direct donor of all B genome chromosomes of wheat [15, 36]. Moreover, more sequence changes were detected between wheat and its diploid progenitors rather than between wheat and its tetraploid progenitors, and the result suggested that the genome shock brought by the first hybridization event might be larger than the second hybridization event during wheat formation.
Revealed by phylogenetic analysis and FISH, centromeric satellites in wheat genome have undergone rapid changes in the three subgenomes and satellite signals decreased from diploid to hexaploid wheat [44]. In this study, it is obvious that the SSR sequence expansion occurred predominately in the centromeric and pericentromeric regions of B genome chromosomes during wheat formation, despite the SSR copy number variation in the centromeric region was not precisely calculated. These results suggested that the wheat centromeric and pericentromeric regions were sensitive to “genome shock” and evolved rapidly during the evolution of wheat. All of these findings support the idea that the wheat genome is a dynamic system with a high level of plasticity [44], and a changing sequence repertoire shaped by sequence losses and expansion.
Our SSR FISH results indicated that the genome of wheat has evolved substantially following its polyploidization, and the rearrangement of SSRs might be important for facilitating wheat genome evolution and stabilizing chromosomes of different subgenomes. To address this, more work needs to be performed, such as the investigation of newly formed wheat species or relatives by more SSR FISH. Moreover, new studies might uncover the underlying mechanisms responsible for the widescale genome rearrangement in polyploids.