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

Al-Kaff N, Knight E, Bertin I, Foote T, Hart N, Griffiths S, Moore G - Ann. Bot. (2007)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: John Innes Centre, Norwich Research Park, Colney Lane, Norwich, Norfolk NR4 7UH, UK.

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.

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Related in: MedlinePlus

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.
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MCM252F4: 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.

Mentions: Before undertaking a detailed analysis of the expression of the cdk-like genes in euploid wheat (CS) in comparison with the Ph1 mutant (ph1b), the CS BAC library was screened with specific probes for cdk-like and storage protein-like genes. DNA was prepared from >60 BACs identified. Southern blot analyses were carried out, with blots being probed with the conserved regions of the cdk-like and the storage protein-like genes. Primers were designed in the most conserved domains of all known cdk-like genes already identified in the Ph1 region and corresponding regions in 5A and 5D. These primers were used to amplify DNA from euploid wheat, ph1b, CS isomic/tetrasomic lines N5AT5B, N5BT5D and N5DT5A, and all the BACs identified as containing cdk-like genes. The PCR products were then cloned and sequenced. Sequence analysis revealed that the cdk loci on 5B and 5A were more complex than initially thought. There were higher numbers of both cdk-like and storage protein-like genes within this region. BAC and genomic analyses showed that seven, five and two cdk-like genes were present in the corresponding regions of 5B, 5A and 5D chromosomes, respectively (Figs 1 and 4). The number of storage protein-like genes was also more than initially predicted, and at least four genes on each chromosome were identified (Figs 1 and 4). The main finding was that there had been gene duplication of both cdk-like and storage protein-like genes (Figs 1 and 4). This made BAC analysis more complex because of similar size fragments containing cdk-like or storage protein-like genes, requiring them to be distinguished by sequencing. Interestingly, the gene duplication differed between 5B, 5A and 5D, indicating that these clusters arose at different times.


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

Al-Kaff N, Knight E, Bertin I, Foote T, Hart N, Griffiths S, Moore G - Ann. Bot. (2007)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2710213&req=5

MCM252F4: 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.
Mentions: Before undertaking a detailed analysis of the expression of the cdk-like genes in euploid wheat (CS) in comparison with the Ph1 mutant (ph1b), the CS BAC library was screened with specific probes for cdk-like and storage protein-like genes. DNA was prepared from >60 BACs identified. Southern blot analyses were carried out, with blots being probed with the conserved regions of the cdk-like and the storage protein-like genes. Primers were designed in the most conserved domains of all known cdk-like genes already identified in the Ph1 region and corresponding regions in 5A and 5D. These primers were used to amplify DNA from euploid wheat, ph1b, CS isomic/tetrasomic lines N5AT5B, N5BT5D and N5DT5A, and all the BACs identified as containing cdk-like genes. The PCR products were then cloned and sequenced. Sequence analysis revealed that the cdk loci on 5B and 5A were more complex than initially thought. There were higher numbers of both cdk-like and storage protein-like genes within this region. BAC and genomic analyses showed that seven, five and two cdk-like genes were present in the corresponding regions of 5B, 5A and 5D chromosomes, respectively (Figs 1 and 4). The number of storage protein-like genes was also more than initially predicted, and at least four genes on each chromosome were identified (Figs 1 and 4). The main finding was that there had been gene duplication of both cdk-like and storage protein-like genes (Figs 1 and 4). This made BAC analysis more complex because of similar size fragments containing cdk-like or storage protein-like genes, requiring them to be distinguished by sequencing. Interestingly, the gene duplication differed between 5B, 5A and 5D, indicating that these clusters arose at different times.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: John Innes Centre, Norwich Research Park, Colney Lane, Norwich, Norfolk NR4 7UH, UK.

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.

Show MeSH
Related in: MedlinePlus