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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

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.
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MCM252F1: 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.

Mentions: Prior to the present study, the Ph1 locus had been defined to a 2·5 Mb region on chromosome 5B of hexaploid wheat (Griffiths et al., 2006). This region had been characterized at the molecular level and found to contain approx. 36 genes including a cdk-like gene cluster containing a segment of sub-telomeric heterochromatin as shown in Fig. 1. By exploiting the genomic information gained from the analysis of this region, a systematic screen of mutagenized wheat populations was undertaken to assess the efficiency of generating deletions localized within the 2·5 Mb region. Wheat seeds were treated with either 5, 10, 20, 30, 50 or 100 Gy of X-ray or 150, 200 or 250 Gy of γ-irradiation. The mutagenized populations derived from the seed were then screened using a multiplex PCR-based assay with markers derived from the Ph1 region. The multiplex PCR assay had been previously described by Griffiths et al. (2006). No deletion mutants were identified from screening 500 individuals of M2 populations irradiated with 5–150 Gy. However, two and three plants carrying a deletion in the Ph1 region were identified after screening 500 and 900 individuals from the 200 and 250 Gy treatments, respectively. One of the five plants (γ250-19) revealed a wild-type chromosome pairing phenotype and carried the smallest deletion of approx. 500 kb in size (Fig. 1). It eliminated eight genes as being responsible for the Ph1 phenotype (hyp5, ugg1, cmt1, hsp20-1, at1, plp1, wdb1 and hyp6) (Fig. 1). Another mutant plant (γ250-214) carried a deletion of approx. 1 Mb in size which overlapped with the γ250-19 deletion (Fig. 1). This deletion encompassed a further 11 genes in this region which flanked the sub-telomeric insertion (Fig. 1). However, it was not possible to score the pairing phenotype of this plant accurately because it did not survive. The other three remaining mutants carried larger deletions than those described above, ranging from 1 to >10 Mb. This work clearly demonstrated that wheat seeds treated with 200–250 Gy of γ-irradiation can yield a range of deletion sizes with relatively high frequency. The deletion analysis still defined the Ph1 locus to the region containing the cdk-like (locus) cluster and the segment of heterochromatin (Fig. 1).


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)

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

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

MCM252F1: 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.
Mentions: Prior to the present study, the Ph1 locus had been defined to a 2·5 Mb region on chromosome 5B of hexaploid wheat (Griffiths et al., 2006). This region had been characterized at the molecular level and found to contain approx. 36 genes including a cdk-like gene cluster containing a segment of sub-telomeric heterochromatin as shown in Fig. 1. By exploiting the genomic information gained from the analysis of this region, a systematic screen of mutagenized wheat populations was undertaken to assess the efficiency of generating deletions localized within the 2·5 Mb region. Wheat seeds were treated with either 5, 10, 20, 30, 50 or 100 Gy of X-ray or 150, 200 or 250 Gy of γ-irradiation. The mutagenized populations derived from the seed were then screened using a multiplex PCR-based assay with markers derived from the Ph1 region. The multiplex PCR assay had been previously described by Griffiths et al. (2006). No deletion mutants were identified from screening 500 individuals of M2 populations irradiated with 5–150 Gy. However, two and three plants carrying a deletion in the Ph1 region were identified after screening 500 and 900 individuals from the 200 and 250 Gy treatments, respectively. One of the five plants (γ250-19) revealed a wild-type chromosome pairing phenotype and carried the smallest deletion of approx. 500 kb in size (Fig. 1). It eliminated eight genes as being responsible for the Ph1 phenotype (hyp5, ugg1, cmt1, hsp20-1, at1, plp1, wdb1 and hyp6) (Fig. 1). Another mutant plant (γ250-214) carried a deletion of approx. 1 Mb in size which overlapped with the γ250-19 deletion (Fig. 1). This deletion encompassed a further 11 genes in this region which flanked the sub-telomeric insertion (Fig. 1). However, it was not possible to score the pairing phenotype of this plant accurately because it did not survive. The other three remaining mutants carried larger deletions than those described above, ranging from 1 to >10 Mb. This work clearly demonstrated that wheat seeds treated with 200–250 Gy of γ-irradiation can yield a range of deletion sizes with relatively high frequency. The deletion analysis still defined the Ph1 locus to the region containing the cdk-like (locus) cluster and the segment of heterochromatin (Fig. 1).

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