Fertility of CMS wheat is restored by two Rf loci located on a recombined acrocentric chromosome

Abstract

Cytoplasmic male sterility (CMS) results from incompatibility between nuclear and cytoplasmic genomes, and is characterized by the inability to produce viable pollen. The restoration of male fertility generally involves the introgression of nuclear genes, termed restorers of fertility (Rf). CMS has been widely used for hybrid seed production in many crops but not in wheat, partly owing to the complex genetics of fertility restoration. In this study, an acrocentric chromosome that restores pollen fertility of CMS wheat in Hordeum chilense cytoplasm (msH1 system) is studied. The results show that this chromosome, of H. chilense origin and named H(ch)ac, originated from a complex reorganization of the short arm of chromosomes 1H(ch) (1H(ch)S) and 6H(ch) (6H(ch)S). Diversity arrays technology (DArT) markers and cytological analysis indicate that H(ch)ac is a kind of `zebra-like' chromosome composed of chromosome 1H(ch)S and alternate fragments of interstitial and distal regions of chromosome 6H(ch)S. PCR-based markers together with FISH, GISH, and meiotic pairing analysis support this result. A restorer of fertility gene, named Rf6H(ch)S, has been identified on the short arm of chromosome 6H(ch)S. Moreover, restoration by the addition of chromosome 1H(ch)S has been observed at a very low frequency and under certain environmental conditions. Therefore, the results indicate the presence of two Rf genes on the acrocentric chromosome: Rf6H(ch)S and Rf1H(ch)S, the restoration potential of Rf6H(ch)S being greater. The stable and high restoration of pollen fertility in the msH1 system is therefore the result of the interaction between these two restorer genes.

Keywords: Acrocentric chromosome; Hordeum chilense; Triticum aestivum; cytoplasmic male sterility; restorer gene; zebra-like chromosome..

Fig. 1.

Visualization of markers present in the acrocentric chromosome in the map of H. chilense (partial view of chromosomes 1H^ch and 6H^ch). Markers in red were absent in the acrocentric chromosome. The centromeric region, as estimated in previous works, is shown in green. Markers in green are located in the centromeric region.

Fig. 2.

PCR amplification products using (A) primer pairs designed in the AK362725 gene from H. vulgare that amplifies specifically the 1H^chS chromosome in H. chilense and does not produce an amplification product in wheat; (B) SSR marker Bmac 316 that amplifies specifically the 6H^chS chromosome in H. chilense. T749, T21-H. chilense disomic addition of H^chac in H1 cytoplasm; T528, T26-H. chilense disomic addition of H^chac in H1 cytoplasm; T26, T. aestivum carrying the translocation T1RS·1BL; T700, T21-H. chilense monosomic addition of H^chac in H1 cytoplasm; T21, T. aestivum cv. Chinese Spring; H1, H. chilense accession H1; T21A1H₁S, T21 ditelosomic addition of 1H^chS; and T21A6H₁S, T21 ditelosomic addition of 6H^chS.

Fig. 3.

In situ hybridization to root-tip metaphase cells from restored line T749. (A) Double FISH signals using H. chilense genomic DNA detected with streptavidin–Cy3 (magenta) and a telomere repeat sequence probe detected with FITC (green). Blue DAPI staining shows wheat chromosomes. The acrocentric chromosome H^chac displays magenta colour indicating its pure barley origin. Telomere sequences can be observed at both ends of the H^chac chromosome. (B) FISH signal using the repetitive probe pAs1 detected with FITC. pAs1 shows a characteristic hybridization pattern in H. chilense that allows for the identification of the different chromosome arms. The H^chac chromosome shows hybridization sites in both arms.

Fig. 4.

Graphical representation showing the locations of telomere repeat sequences (TRS), pSc119.2, pTa71 rDNA, and pAs1 repetitive probes in 1H^chS, 6H^chS, and the acrocentric chromosome H^chac.

Fig. 5.

Meiotic pairing analysis of the acrocentric chromosome with 1H^ch and 1H^chS chromosomes. (A) In situ hybridization to a pachytene cell of the double monosomic H^chac-1H^ch line. Double FISH signals were observed using H. chilense genomic DNA detected with streptavidin–Cy3 (magenta) and a telomere repeat sequence probe detected with FITC (green). The acrocentric chromosome H^chac is perfectly paired with the 1H^ch chromosome except for one of its distal parts. (B) Meiotic anaphase I of a plant double monosomic for H^chac and 1H^chS stained with carmine. Tension is observed (indicated by an arrow) between the short arm of the acrocentic chromosome and the 1H^chS as a result of their pairing.

Fig. 6.

In situ hybridization to a pachytene cell from the line double monosomic for H^chac and 6H^chS. Double FISH signals were observed using H. chilense genomic DNA detected with streptavidin–Cy3 (magenta) and a telomere repeat sequence probe detected with FITC (green). Blue DAPI staining shows wheat chromosomes. The acrocentric chromosome H^chac and the 6H^chS chromosome pair along one of their distal parts. The centromeric part of the 6H^chS is indicated by an arrow.

Fig. 7.

Male fertility restoration of the alloplasmic line Chinese Spring in H. chilense cytoplasm, with different chromosome additions: 6H^chS, 1H^chS, 6H^chS+1H^chS, and H^chac. (H1)CS-H^ch MtA6H^chS, CS-H. chilense monotelosomic addition of 6H^chS; (H1)CS-H^ch MtA1H^chS, CS-H. chilense monotelosomic addition of 1H^chS; (H1)CS-H^ch MtA1H^chS-MtA6H^chS, CS-H. chilense monotelosomic addition of 1H^chS-monotelosomic addition of 6H^chS; (H1)CS-H^ch MAH^chac, CS-H. chilense monosomic addition of H^chac in H1 cytoplasm.