Fig 1.
Stable and novel chromosomes induced by universal centromere variations in wheat and wide hybrids.
Centromere alterations occurred in aneuploid wheat and in wheat hybrids. Reduction or even deletion of the centromeric sequences did not affect the transmission of the chromosomes or chromosome fragments to the next generation because of de novo centromere formation. Centromere expansion and breakage led to novel alien chromosomes in wheat and wide hybrids.
Table 1.
Centromere variation and de novo centromere formation in wheat aneuploids and derivatives of wheat wild hybrids.
Fig 2.
Centromeric sequence reduction in the ditelosomic lines 5DL, 5DS and 1BS.
(A), (E) and (I). FISH analysis of 5DL, 5DS, and 1BS, respectively. The CRW sequences are labeled in red, and DAPI staining is labeled in blue. The insets show high-magnification images of chromosomes 5DL, 5DS and 1BS. (B), (F) and (J). Intensity of CRW fluorescence in 5DL, 5DS and 1BS, respectively, compared with the controls in Chinese Spring. The Y-axis represents the relative CRW fluorescence intensity. The error bars show the standard deviation (s.d.). The double asterisks denote significant differences at P<0.001 (two-tailed Student’s t-test). P = 3.4E-16 (B), P = 8.22E-18 (F), P = 1.97E-13 (J). (C), (G) and (J). Multi-color FISH analysis of 5DL, 5DS, and 1BS, respectively. The DNA for the wheat A genome is labeled in green, the DNA for the D genome is labeled in red, and the DNA for the B genome is used as a block. (D), (H) and (L). Immunostaining of 5DL, 5DS, and 1BS, respectively, with antibodies against CENH3 (green). The arrows indicate CENH3 on the 5DL, 5DS and 1BS chromosomes. Bar = 10 μm.
Fig 3.
Centromeric sequence deletion in the ditelosomic line 4DS.
(A). FISH on 4DS with CRW (red). DAPI staining is labeled in blue. (B). Intensity of CRW fluorescence in 4DS compared with the controls in Chinese Spring. The Y-axis represents the relative CRW fluorescence intensity. The error bars show the standard deviation (s.d.). The double asterisks denote significant differences at P<0.001 (two-tailed Student’s t-test). P = 3.59E-15. (C). Multi-color FISH on 4DS. The DNA for the wheat A genome is labeled in green, the DNA for the D genome is labeled in red, and the DNA for the B genome is used as a block. (D)-(F). Immunostaining results of 4DS with antibodies against CENH3 (D), H3 phosphorylation at Ser-10 (E), and H2A phosphorylation at Thr-133 (F) are shown in green. The insets show high-magnification images of chromosome 4DS. Bar = 10 μm.
Fig 4.
Deletion of centromeric sequences in the wheat-Th. intermedium addition line TAI-14.
(A). Genomic in situ hybridization (GISH) and FISH of somatic metaphase chromosomes in TAI-14. The genomic DNA of Th. intermedium is labeled in green, the CRW sequences are labeled in red, and DAPI staining is labeled in blue. (B). Immunostaining of TAI-14 with antibodies against CENH3 (green). (C) and (D). FISH analysis of the novel centromeric sequences TAI-14-1 (C) and TAI-14-2 (D), which are labeled in green in TAI-14. The CRW sequences are labeled in red. The insets show high-magnification images of the alien chromosomes in TAI-14. Bar = 10 μm.
Fig 5.
Deletion of centromeric sequences in the wheat-Th. elongatum addition lines.
The genomic DNA of Th. elongatum is labeled in green, the CRW sequences are labeled in red, and DAPI staining is labeled in blue. (A) FISH analysis of a ditelosomic addition line from the hybrid of T. durum and 8802. There are no detectable FISH signals in the new telosomic chromosomes when CRW is used as a probe. (B) FISH analysis of an addition line from the hybrid of N6AT6B×8802 that shows weak CRW FISH signals. The insets show high-magnification images of the alien chromosomes with centromere changes from the addition lines. Bar = 10 μm.
Fig 6.
ChIP-seq mapping results of the ditelosomic line 4DS with the CENH3 antibody.
The top box shows the ChIP-seq results of the control Chinese Spring (CS) and 4DS by mapping to the CS genome. A 994-kb region was detected as a difference between CS and 4DS and is involved in 4DS neocentromere formation. 4DS Scaffold 2287721 (3,661 bp, indicated by the magenta box) was selected as a probe for FISH confirmation. The scaffold was enriched with transposable elements, as shown in the middle box. In the wheat D genome, Scaffold 33994 (~68 kb, anchored to chromosome 4D) has high homology with Scaffold 2287721 and shows a mapping difference between CS and 4DS. The 994-kb region, Scaffold 2287721 and Scaffold 33994 had high GC contents.
Fig 7.
FISH of a novel centromeric sequence in 4DS identified by ChIP-seq mapping and dot-blot hybridization.
(A). Scaffold 2287721 (labeled in green), which was obtained from ChIP-seq, contained sequences of the de novo centromere in 4DS. (B). 4DS-1 (labeled in green), which was obtained from the dot-blot, was detected as a novel centromere sequence in 4DS. The CRW sequences are labeled in red. The insets show high-magnification images of 4DS. Bar = 10 μm. (C). RT-qPCR analysis of 4DS-1-1 and 4DS-1-2. (D). The RNA-ChIP-qPCR results for 4DS-1-1 and 4DS-1-2 suggest that transcription levels of both sequences at the 4DS centromeres were increased.
Fig 8.
Two- and three-locus centromeres in the F5 generation of hybrids between nulli-tetrasomic N6AT6B× 8802.
(A) and (B). FISH and GISH of chromosomes with two- and three-locus centromeres, respectively. The genomic DNAs of Th. elongatum are labeled in green, the CRW sequences are labeled in red. DAPI staining is labeled in blue. (C) and (D). Karyotype analysis of chromosomes with two- and three-locus centromeres using probes pAsI (green) and pSc119.2 (red). (E) and (F). Immunostaining-FISH analysis of chromosomes with two- and three-locus centromeres. The CRW sequences are labeled in green, and CENH3 is labeled in red. The insets show high-magnification images of the chromosomes with two- and three-locus centromeres. Bar = 10 μm.
Fig 9.
Centromere expansion in the wheat-rye addition lines.
(A)-(C). FISH indicates centromere expansion in the 2R-2R, novel 2R and 6R addition lines using the pAWRC.1 (green) and CRW (red) probes. (D)-(F). Karyotype analysis of centromere expansion in the 2R-2R (D), novel 2R (E) and 6R (F) addition lines using the AAC (red) and pSc119.2 (green) probes. The insets show high-magnification images of the chromosomes with expanded centromeres. Bar = 10 μm.