Detailed dissection of the chromosomal region containing the Ph1 locus in wheat Triticum aestivum: with deletion mutants and expression profiling

Abstract

Background and aims: Understanding Ph1, a dominant homoeologous chromosome pairing suppressor locus on the long arm of chromosome 5B in wheat Triticum aestivum L., is the core of the investigation in this article. The Ph1 locus restricts chromosome pairing and recombination at meiosis to true homologues. The importance of wheat as a crop and the need to exploit its wild relatives as donors for economically important traits in wheat breeding programmes is the main drive to uncover the mechanism of the Ph1 locus and regulate its activity.

Methods: Following the molecular genetic characterization of the Ph1 locus, five additional deletion mutants covering the region have been identified. In addition, more bacterial artificial chromosomes (BACs) were sequenced and analysed to elucidate the complexity of this locus. A semi-quantitative RT-PCR was used to compare the expression profiles of different genes in the 5B region containing the Ph1 locus with their homoeologues on 5A and 5D. PCR products were cloned and sequenced to identify the gene from which they were derived.

Key results: Deletion mutants and expression profiling of genes in the region containing the Ph1 locus on 5B has further restricted Ph1 to a cluster of cdk-like genes. Bioinformatic analysis of the cdk-like genes revealed their close homology to the checkpoint kinase Cdk2 from humans. Cdk2 is involved in the initiation of replication and is required in early meiosis. Expression profiling has revealed that the cdk-like gene cluster is unique within the region analysed on 5B in that these genes are transcribed. Deletion of the cdk-like locus on 5B results in activation of transcription of functional cdk-like copies on 5A and 5D. Thus the cdk locus on 5B is dominant to those on 5A and 5D in determining the overall activity, which will be dependent on a complex interplay between transcription from non-functional and functional cdk-like genes.

Conclusions: The Ph1 locus has been defined to a cdk-like gene cluster related to Cdk2 in humans, a master checkpoint gene involved in the initiation of replication and required for early meiosis.

Fig. 1.

Schematic diagram of the deletion mutants and annotated genes in the region containing the Ph1 locus on chromosome 5B compared with the equivalent regions on chromosomes 5A and 5D. The three horizontal blue bars represent part of chromosome 5B and its homoeologous regions on chromosome 5A and 5D. The yellow box within the 5B chromosome bar represents the inserted sub-telomeric repeats. The two rectangles with red dotted lines represent the regions deleted in the two γ-irradiated mutant lines. Grey dots represent genes outside the Ph1 locus. The magenta dots represent cdk-like genes. The green dots represent the storage protein-like genes (SP) where G1, G2 and G3 indicate that there are differences at the protein level. The stars represent the expression pattern of different genes: uncoloured stars represent genes that are mainly expressed from 5A or 5D, magenta stars represent genes expressed from 5B and genes lacking stars are not expressed. Apart from the cdk-like locus, the gene content presented is as described in Griffiths et al. (2006). A more detailed analysis of the cdk-like loci is provided in Fig. 4.

Fig. 2.

Expression pattern of genes within the 2·5 Mb region covering the Ph1 locus on 5B and equivalent regions on 5A and 5D. Total RNA was extracted from leaf, spike and seeds of Chinese Spring and Ph1 mutant (ph1b). For each gene, RT–PCR products were cloned and 24 clones were sequenced. SNPs enabled a semi-quantification of the expression from the genes on chromosomes B, A and D. Results are presented in the form of pie charts where the sizes of the blue, green and orange sectors correspond to the proportion of transcription contributed by genes on chromosomes 5B, A and D, respectively. A white sector indicates that the sequenced transcript did not correspond to any of these genes.

Fig. 3.

Transcription of specific genes in the presence or absence of the Ph1 locus. Semi-quantitated RT–PCR products were amplified from total RNA extracted from the spikes of Chinese Spring (CS) and the Ph1 mutant. Primers were designed in the conserved regions of elp1, mic1 and the storage protein-like gene spG3b. The glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene was used as a semi-quantitative positive control.

Fig. 4.

Detailed schematic diagram of the cdk cluster on chromosome 5B and homoeologous regions on chromosomes 5A and 5D. The homoeologous regions of 5B, 5A and 5D are defined in three white boxes. Pale pink blocks represent the BACs. Blocks in magenta represent the cdk-like genes, large green cylinders represent storage protein-like genes (sp) where G1, G2 and G3 indicate that there are differences at the protein level; smaller green cylinders represent fragments of storage protein-like genes. The solid blue horizontal line indicates that the genes are found on the same BAC sequence contig. Sub-telomeric heterochromatin is shown inserted between cdk-like genes 6 and 7 on 5B.

Fig. 5.

Sequence alignment of cdk-like genes on 5B, 5A and 5D. The sequences are in order from top to bottom: cdk-like B1–B7, cdk-like A1–A5 and cdk-like D1 and D2. The CACTA transposon has been removed from the sequence of cdk-like B7 for this comparison. As explained within the text, 5A cdk-like gene sequences may contain errors.

Fig. 6.

Protein alignment of cdk-like genes on 5B, 5A and 5D. The protein sequences are in order from top to bottom: cdk-like B1–B7, cdk-like A1–5 and cdk-like D1 and D2.

Fig. 7.

Protein alignment of cdk-like D2 with kinase Cdk2 from humans. Protein alignment of the meiotic checkpoint gene in humans (Debondt et al., 1993) with the cdk-like protein D2 gene. The shaded areas represent the overall homology to these two known genes. Eleven of 15 functional domains are conserved between kinases (underlined).

Fig. 8.

Expression pattern of cdk-like genes from spikes of wild-type wheat (Chinese Spring; CS) and the Ph1 mutant. Total RNA was extracted from spikes of CS and the Ph1 mutant and used in a semi-quantitative RT–PCR amplification. All PCR components in the master-mix remained constant except for the primers. The primers were designed specifically for each cdk-like gene in the three homoeologous regions from B, A and D genomes. The glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene was used as a semi-quantitative positive control. A DNA marker from Invitrogen was used to size the PCR product.