Screening of FISH positive SSR probes in bread wheat
After analysis with Tandem Repeats Finder version 4.09 [32], 21 SSR motifs (Table 1) repeated more than 5 times in the wheat genome were labelled and used for FISH positive SSR probes screening. After hybridization with mitotic chromosomes of bread wheat using SSR sequences as the probes for FISH, a total of 6 SSR sequences, (AAC)n, (AAG)n, (ACA)n, (AG)n, (AGC)n and (ACG)n, displayed strong and stable FISH signals and 3 SSR sequences, (AGG)n, (ATC)n and (ACC)n showed weak FISH signals on chromosomes of bread wheat (Figs. 1 to 3). Among these 9 FISH positive SSR sequences, (ACA)n and (AAC)n showed similar signal distribution (Figs. 3A and 3B), and (AGC)n and (ACG)n showed similar signal distribution (Figs. 3C and 3D). (AAC)n, (AAG)n, (AGC)n and (AG)n were selected for extensive investigation.
Table 1
SSR primers for probe labeling
SSR motifs
|
Primers for probe labelling
|
FISH signals on
wheat chromosomes
|
Forward primer (5´-´3)
|
Reverse primer (5´-´3)
|
AAC
|
AACAACAACAACAACAACAACAACAACAAC
|
GTTGTTGTTGTTGTTGTTGTTGTTGTTGTT
|
Strong
|
AAG
|
AAGAAGAAGAAGAAGAAGAAGAAGAAGAAG
|
CTTCTTCTTCTTCTTCTTCTTCTTCTTCTT
|
Strong
|
AGC
|
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGC
|
GCTGCTGCTGCTGCTGCTGCTGCTGCTGCT
|
Strong
|
ACG
|
ACGACGACGACGACGACGACGACGACGACG
|
CGTCGTCGTCGTCGTCGTCGTCGTCGTCGT
|
Strong
|
ACA
|
ACAACAACAACAACAACAACAACAACAACA
|
TGTTGTTGTTGTTGTTGTTGTTGTTGTTGT
|
Strong
|
AG
|
AGAGAGAGAGAGAGAGAGAG
|
CTCTCTCTCTCTCTCTCTCT
|
Strong
|
AGG
|
AGGAGGAGGAGGAGGAGGAGGAGGAGGAGG
|
CCTCCTCCTCCTCCTCCTCCTCCTCCTCCT
|
Weak
|
ATC
|
ATCATCATCATCATCATCATCATCATCATC
|
GATGATGATGATGATGATGATGATGATGAT
|
Weak
|
ACC
|
ACCACCACCACCACCACCACCACCACCACC
|
GGTGGTGGTGGTGGTGGTGGTGGTGGTGGT
|
Weak
|
GT
|
GTGTGTGTGTGTGTGTGTGT
|
ACACACACACACACACACAC
|
None
|
TA
|
TATATATATATATATATATA
|
TATATATATATATATATATA
|
None
|
GC
|
GCGCGCGCGCGCGCGCGCGC
|
GCGCGCGCGCGCGCGCGCGC
|
None
|
CGG
|
CGGCGGCGGCGGCGGCGGCGGCGGCGGCGG
|
CCGCCGCCGCCGCCGCCGCCGCCGCCGCCG
|
None
|
GTC
|
GTCGTCGTCGTCGTCGTCGTCGTCGTCGTC
|
GACGACGACGACGACGACGACGACGACGAC
|
None
|
GGA
|
GGAGGAGGAGGAGGAGGAGGAGGAGGAGGA
|
CCTCCTCCTCCTCCTCCTCCTCCTCCTCCT
|
None
|
TCC
|
TTCTTCTTCTTCTTCTTCTTCTTCTTCTTC
|
GAAGAAGAAGAAGAAGAAGAAGAAGAAGAA
|
None
|
GCG
|
GCGGCGGCGGCGGCGGCGGCGGCGGCGGCG
|
CGCCGCCGCCGCCGCCGCCGCCGCCGCCGC
|
None
|
TCA
|
TCATCATCATCATCATCATCATCATCATCA
|
TGATGATGATGATGATGATGATGATGATGA
|
None
|
GAG
|
GAGGAGGAGGAGGAGGAGGAGGAGGAGGAG
|
CTCCTCCTCCTCCTCCTCCTCCTCCTCCTC
|
None
|
GCAC
|
GCACGCACGCACGCACGCACGCACGCACGCACGCACGCAC
|
GTGCGTGCGTCGTGCGTGCGTGCGTGCGTGCGTGCGTGC
|
None
|
TAAT
|
TAATTAATTAATTAATTAATTAATTAATTAATTAATTAAT
|
ATTAATTAATTAATTAATTAATTAATTAATTAATTAATTA
|
None
|
The distribution patterns of (AAC) n on chromosomes of bread wheat and its progenitors.
In Chinese Spring, (AAC)n displayed a strong clustered hybridization pattern on all chromosomes of the B genome, and some of the adjacent signals tended to be coalesced and appeared as a condensed large band (Figs. 4F and 4J). Sporadically weak signals were detected on chromosomes 2A, 4A and 7A (Figs. 4F and 4N). No obvious signals were observed on D genome chromosomes (Fig. 4F). In T. urartu, the (AAC)n motif produced several strong spot-like signals in the pericentromeric regions of both arms of chromosome 1A, the centromeric region of chromosome 4A, a signal on proximal third part of long arm of chromosome 5A, a weak signal in the centromeric region of chromosome 6A and a strong signal in the pericentromeric region of the long arm of chromosome 7A (Figs. 4A and 4K). In Ae. tauschii, (AAC)n only produced weaker spot-like signals in centromeric regions of chromosome 4D (Fig. 4C). In Ae. speltoides, strong signals were observed in the centromeric and pericentromeric regions of all the chromosomes (S genome), which are weaker and fewer than those on chromosomes of the B genome in Chinese Spring (Figs. 4B and 4G). In wild emmer wheat (T. turgidum ssp. dicoccoides), the wild relative of durum wheat, strong condensed broad band signals were observed on both arms of chromosomes 1B, 2B, 4B, 6B and 7B, short arms of chromosome 5B and pericentromeric regions of chromosome 3B; in addition to band-like signals, dot-like signals could also be observed on both arms of chromosomes 1B, 2B, 3B, 4B and 4A, on long arms of chromosomes 5B, 6B and 7A, and on short arms of 7B and 2A chromosomes (Figs. 4D, 4H and 4L). In domesticated emmer wheat (T. turgidum ssp. dicoccum), similar signal distribution patterns to those of wild emmer wheat were observed, except chromosomes 2B and 2A, which showed narrower band signals and fewer dot-like signals (Figs. 4E, 4I and 4M).
Comparing the chromosomal distribution patterns of (AAC)n between bread wheat and its diploid progenitors, obvious (AAC)n sequence expansion was observed on chromosomes 1B, 2B, 7B and 2A; sequence elimination was detected on chromosomes 1A, 5A, 6A and 4D; and both expansion and elimination were observed on chromosomes 2B, 3B, 6B, 4A and 7A in bread wheat (Fig. 4). However, the distribution changes of (AAC)n from progenitors to the bread wheat genome was mainly the sequence expansion in the B genome (especially in chromosomes 1B, 2B, 4B, 6B and 7B). Comparing the chromosomal distribution patterns of (AAC)n between bread wheat and its tetraploid progenitors, sequence elimination was observed on 1B, 3B, 4B, 5B and 7B chromosomes; whereas, both sequence expansion and elimination were detected on 2B chromosomes in bread wheat (Fig. 4).
The distribution patterns of (AAG) n on chromosomes of bread wheat and its progenitors.
In Chinese Spring, (AAG)n showed strong signal distribution in centromeric and pericentromeric regions of all B genome chromosomes. Compared with the distribution of (AAC)n, (AAG)n were more distally located. In addition, unlike (AAC)n, obvious subtelomeric signals of (AAG)n were present on chromosomes 1B, 2B and 3B, and a strong intercalary signal band could also be found on 5BS (Figs. 5F and 5J). There were sporadically weak (AAG)n signals in pericentromeric regions of chromosomes 2A, 3A, and 4A and the centromeric region of chromosome 5A and interstitial region of the chromosome 1A arm; apparent signals in the subtelomeric regions of chromosome 4A; and obvious telomeric signals on chromosome 7A (Fig. 5F and 5N). (AAG)n showed weak signals in subtelomeric regions of chromosomes 1D and 7D, and pericentromeric regions of chromosome 2D (Figs. 5F and 5P).
In T. urartu, the (AAG)n motif produced two dot-like signals in the pericentromeric regions of 4A and 5A (Figs. 5A and 5K). In Ae. speltoides, the FISH signals of (AAG)n were observed in the centromeric and pericentromeric regions of all the chromosomes, and they were slightly weaker than those of (AAC)n, except chromosome 5S (Figs. 5B and 5G), while most signals were weaker than those in Chinese Spring. In Ae. tauschii, (AAG)n displayed signals in the pericentromeric regions of chromosomes 2D, 3D and 4D (Figs. 5C and 5O). In wild and emmer wheats, strong condensed band-like signals were observed in the pericentromeric regions of all chromosomes; dot-like signals could be detected on both arms of chromosome 3B, short arms of chromosomes 2B and 7B, and long arms of chromosomes 3B, 4B, 5B, 2A and 4A (Figs. 5D, 5H and 5L). In domesticated emmer wheat, broader band-like signals than wild emmer wheat were observed in the pericentromeric regions of all chromosomes; more dot-like signals than those of wild emmer wheat were observed on both arms of chromosomes 1B, 2B, 3B and 7B, short arm of chromosome 6B, and long arms of chromosomes 4B, 5B, 2A and 4A (Figs. 5E, 5I and 5M).
Similar to (AAC)n, (AAG)n was mainly dispersed on B genome chromosomes. The main changes in (AAG)n distribution from diploid progenitors to the bread wheat genome were the sequence expansions on chromosomes 1B, 2B, 3B, 4B, 6B, 7B and 4A and the sequence elimination of chromosomes 5B, 2D, 3D and 4D during wheat formation. The main changes of (AAG)n distribution from tetraploid progenitors to the bread wheat genome were the sequence expansions on all B and A genome chromosomes (Fig. 5).
The distribution patterns of the (AGC) n motif in bread wheat and its progenitors.
In Chinese Spring, the FISH pattern of (AGC)n on B genome chromosomes was more similar to (AAC)n, but with lower signal density, especially on chromosomes 1B and 2B (Figs. 6F and 6J); dot-like signals were detected in the pericentromeric regions of chromosomes 4A, 6A and 7A, and subtelomeric regions of chromosome 7A (Figs. 6F and 6J), and no signals were detectable on D genome chromosomes (Fig. 6F). In Ae. speltoides, similar to the distribution patterns of (AAC)n and (AAG)n, the (AGC)n motif signals were also concentrated in the pericentromeric regions, but with lower intensity (Figs. 6C and 6G). No (AGC)n signals were detectable in the diploid species T. urartu and Ae. tauschii (Figs. 6A and 6B). In wild emmer wheat, band-like signals were observed on the pericentromeric regions of chromosomes 2B, 3B, 5B, 6B and 7B; dot-like signals could be detected on both arms of chromosomes 1B and 4B, long arms of chromosomes 2B, 3B and 6B, short arms of chromosome 7B, and pericentromeric regions of chromosomes 4B and 7A (Figs. 6D, 6H and 6K). In domesticated emmer wheat, band-like signals were observed on the pericentromeric regions of all B genome chromosomes, except chromosome 4B; dot-like signals were observed on both arms of chromosome 1B, 3B, and 7B, short arms of chromosome 4A, long arms of chromosomes 2B, 6B, 6A and 7A, and pericentromeric regions of chromosomes 4B, 6B, 4A and 7A (Figs. 6E, 6I and 6L).
By comparison of the chromosomal distribution patterns of (AGC)n between bread wheat and its progenitors, obvious sequence expansion of (AGC)n was observed on chromosomes 1B-7B (especially 4B, 6B and 7B), 4A, 6A and 7A; both expansion and elimination of (AGC)n sequences were observed on chromosomes on 4B and 5B of bread wheat (Fig. 6). In the progenitors of bread wheat, similar to (AAC)n and (AAG)n, (AGC)n FISH signals were predominantly detected on B genome chromosomes and rarely detected on A and D genome chromosomes. The expansion of (AGC)n along B genome chromosomes was also the main change during wheat formation.
The distribution patterns of the (AG) n motif in bread wheat and its progenitors.
Different from other SSRs, (AG)n signals were only clustered in the pericentromeric regions of chromosomes 3B, 5B and 6B in Chinese Spring (Figs. 7F and 7J), and no detectable signals were observed on the other chromosomes. In Ae. speltoides, (AG)n signals were observed in the pericentromeric or near the pericentromeric regions of chromosomes 2S to 7S, and the distal regions of long arms of chromosomes 3S and 5S, but there was no detectable signal on chromosome 1S (Figs. 7B and 7G); in addition, spot-like signals were observed near the pericentromeric regions of chromosomes 2D, 3D and 4D in Ae. tauschii (Figs. 7C and 7K), even though no D genome chromosome located signals were observed in Chinese Spring (Figs. 7F and 7J). In wild and emmer wheat, spot-like signals were observed on the pericentromeric regions of chromosomes 1B, 5B and 6B, and short arms of chromosome 3B (Figs. 7D and 7H). In domesticated emmer wheat, a similar signal distribution pattern to those of wild emmer wheat was observed except on chromosome 6B (Figs. 7E and 7I).
In the comparison of the chromosomal distribution patterns of (AG)n between bread wheat and its progenitors, (AG)n sequence elimination was observed on chromosomes 2B, 4B, 6B, 7B, 2D, 3D and 4D in bread wheat; and both expansion and elimination were observed on 3B, 5B, and 6B chromosomes of wheat (Fig. 7). The elimination of the (AG)n sequence in the B genome was the main changes during wheat formation.