Stable barley chromosomes without centromeric repeats

Abstract

The satellite sequences (AGGGAG)(n) and Ty3/gypsy-like retrotransposons are known to localize at the barley centromeres. Using a gametocidal system, which induces chromosomal mutations in barley chromosomes added to common wheat, we obtained an isochromosome for the short arm of barley chromosome 7H (7HS) that lacked the barley-specific satellite sequence (AGGGAG)(n). Two telocentric derivatives of the isochromosome arose in the progeny: 7HS* with and 7HS** without the pericentromeric C-band. FISH analysis demonstrated that both telosomes lacked not only the barley-specific centromeric (AGGGAG)(n) repeats and retroelements but also any of the known wheat centromeric tandem repeats, including the 192-bp, 250-bp, and TaiI sequences. Although they lacked these centromeric repeats, 7HS* and 7HS** both showed normal mitotic and meiotic transmission. Translocation of barley centromeric repeats to a wheat chromosome 4A did not generate a dicentric chromosome. Indirect immunostaining revealed that all tested centromere-specific proteins (rice CENH3, maize CENP-C, and putative barley homologues of the yeast kinetochore proteins CBF5 and SKP1) and histone H3 phosphorylated at serines 10 and 28 localized at the centromeric region of 7HS*. We conclude that the barley centromeric repeats are neither sufficient nor obligatory to assemble kinetochores, and we discuss the possible formation of a novel centromere in a barley chromosome.

Fig. 1.

Characterization of barley centromeres in regular and altered chromosomal positions. (A) Partial mitotic metaphase cell of barley cv. Shinebis after N-banding (Left) and FISH using the pGP7 probe (green signal) counterstained with propidium iodide (Right). (B–D) Chromosomes 7H (B), 7HS (C), and 7HL (D) after C-banding (Left) and FISH using the (AGGGAG)_n probe (Right). (E) Robertsonian translocation between 7HS and a wheat chromosome segment after C-banding (Left), FISH using the (AGGGAG)_n probe (green, Center), and FISH/GISH (Right) using the HvT01 (green) and genomic barley DNA (red, Right). (F) The 7HS isochromosome after FISH with (AGGGAG)_n and GISH with genomic barley DNA (red, Inset). (G) The 7HS isochromosome after C-banding (Left) and subsequent FISH/GISH with HvT01 (green) and barley genomic DNA (red, Right). (H) 7HS* after C-banding (Left) and subsequent FISH/GISH with HvT01 (green) and barley genomic DNA (red, Right). (I) 7HS** after C-banding (Left) and subsequent FISH/GISH with HvT01 (green) and barley genomic DNA (red, Right). (J) The 7HS* bivalent at meiotic metaphase I after FISH with HvT01 (green) and GISH (red). (K) Mitotic chromosomes 7HS* and normal 7HS in the same cell after FISH with HvT01 (green) and (AGGGAG)_n (red). (L) 7HS* and normal 7HS after FISH with LTR sequences of cereba (green), 7HS* after subsequent FISH with HvT01 (green) and GISH (red) (Inset Left), and 7HS after subsequent FISH with (AGGGAG)_n (red, Right). (M) 7HS** after FISH with LTR (green) and after subsequent FISH/GISH with HvT01 (green) and genomic barley DNA (red, Inset). (N) 7HS* after FISH with the 192-bp centromeric repeat of wheat (green) and after subsequent FISH/GISH with HvT01 (green) and genomic barley DNA (red, Inset). (O) 7HS* after FISH with 250-bp centromeric repeat of wheat (green) and after subsequent FISH/GISH with HvT01 (green) and genomic barley DNA (red, Inset). (P) 7HS* after FISH with TaiI (green) and after subsequent FISH/GISH with HvT01 (green) and genomic barley DNA (red, Inset). (Q) 7HS* (counterstained red) after GISH with wheat labeled genomic DNA (green) and an excess of unlabeled barley DNA. (R) C-banding (Center) and FISH (Left and Right) with (AGGGAG)_n (red) on chromosomes of the 4A+ line; note that much longer exposure was required to detect the minor signals on chromosomes 3B and 5B (Left) than to detect those of 4A+ (Right). (S) Partial metaphase of wheat including chromosome 7HS* after immunostaining with antibodies against histone H3 phosphorylated at serines 10 (red, Lower) and 28 (green, Upper). (T) Isolated telosomes 7HS* after immunostaining with antibodies (green) against rice CENH3 (Upper Left), ZmCENP-C (Upper Second), CBF5 (Upper Third), and SKP1 (Upper Right). Red signals of subsequent FISH with HvT01 are shown in Lower, demonstrating that the telosomes were 7HS*. (Scale bars, 5 μm. The magnification is the same for A–P, except for Insets.)

Fig. 2.

C-bands (solid bars) and the presence (+) or absence (–) of FISH signals in chromosomes 7H, 7HS, 7HS*, and 7HS**.

Fig. 3.

PCR amplification of the regions between cereba LTR and adjacent (AGGGAG)_n sequences. Primer combinations used were cerebaLTR-R/(AGGGAG)₃-F (Left) and cerebaLTR-R/(AGGGAG)₃-RG (Right). The barley-specific amplification products are indicated with arrows. Opposite orientations of cereba LTR and (AGGGAG)_n repeats inferred from the different amplification products are shown below the gel.