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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

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.
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MCM252F2: 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.

Mentions: Expression analysis of genes within the 2·5 Mb region containing the Ph1 locus had previously revealed that the overall level of transcription of the genes within this region showed no significant differences whether the 5B region was present or absent (Griffiths et al., 2006). Recently, more genomic sequences were obtained in this region. A more detailed analysis was therefore undertaken to assess the levels of transcription of the genes on 5B, 5D and 5A (three homoeologues) in the presence of Ph1, and any changes in the level of transcription of the genes located on the 5A and 5D genomes when the 5B chromosome region was deleted. The sequence analysis of the coding regions of the homoeologues enabled the design of conserved primers which could be used for all three homoeologues but which would yield PCR products carrying distinct SNPs for each homoeologue (see Materials and Methods). RT–PCR was carried out on the cDNA using these conserved primers. PCR products were cloned and 24 clones were sequenced for each gene. The transcript products were then assigned to the correct homoeologue based on the SNPs within the sequence. The relative contributions made by the homoeologues were calculated as a percentage (Fig. 2). No expression was detected for genes clk1, pep1, storage protein-like G1 (spG1), rlp1, rdr1 and raf1 from either wild-type wheat (CS) or the Ph1 mutant (ph1b) (Fig. 1). Analysis of the 2·5 Mb region revealed that the genes accounting for most of the transcription were located in the 5A chromosomal region (Fig. 2). This explains why there was apparently no major effect on the total level of transcription when the 5B region was deleted. However, there was a region where the expression was derived mainly from the 5B genes, namely the region containing the cdk-like and the spG1 clusters and hyp3 (Fig. 1). The Ph1 phenotype is specific to 5B and the Ph1 locus was therefore defined to this region containing specific expression from the 5B genes. In fact there was very little transcription from the genes on 5B flanking the Ph1 region, with mic1 being the only other gene which was transcribed to a significant level in the 2·5 Mb chromosomal region. Of the genes transcribed from 5B within the Ph1 region, hyp3 was not found in the equivalent region of the related grass Triticum timopheevi which has Ph1 activity, and hyp1 and hyp2 (Griffiths et al., 2006) are now known to encode storage-like proteins in wheat (Kawaura et al., 2005). Interestingly, when the 5B region was deleted, the loss of transcription of these genes was compensated by the homoeologues on the remaining genomes (5A and 5D) so that there was no apparent difference in the overall level of transcription in the presence or absence of Ph1 (Figs 2 and 3). In some cases, the compensation was provided by homoeologous genes lying on other chromosomes such as observed for transcription in both spike and seeds for hyp3 and in leaf for Slp1 genes. Thus, only the cdk-like gene cluster remained for more detailed analysis with respect to the Ph1 function.


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)

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

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

MCM252F2: 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.
Mentions: Expression analysis of genes within the 2·5 Mb region containing the Ph1 locus had previously revealed that the overall level of transcription of the genes within this region showed no significant differences whether the 5B region was present or absent (Griffiths et al., 2006). Recently, more genomic sequences were obtained in this region. A more detailed analysis was therefore undertaken to assess the levels of transcription of the genes on 5B, 5D and 5A (three homoeologues) in the presence of Ph1, and any changes in the level of transcription of the genes located on the 5A and 5D genomes when the 5B chromosome region was deleted. The sequence analysis of the coding regions of the homoeologues enabled the design of conserved primers which could be used for all three homoeologues but which would yield PCR products carrying distinct SNPs for each homoeologue (see Materials and Methods). RT–PCR was carried out on the cDNA using these conserved primers. PCR products were cloned and 24 clones were sequenced for each gene. The transcript products were then assigned to the correct homoeologue based on the SNPs within the sequence. The relative contributions made by the homoeologues were calculated as a percentage (Fig. 2). No expression was detected for genes clk1, pep1, storage protein-like G1 (spG1), rlp1, rdr1 and raf1 from either wild-type wheat (CS) or the Ph1 mutant (ph1b) (Fig. 1). Analysis of the 2·5 Mb region revealed that the genes accounting for most of the transcription were located in the 5A chromosomal region (Fig. 2). This explains why there was apparently no major effect on the total level of transcription when the 5B region was deleted. However, there was a region where the expression was derived mainly from the 5B genes, namely the region containing the cdk-like and the spG1 clusters and hyp3 (Fig. 1). The Ph1 phenotype is specific to 5B and the Ph1 locus was therefore defined to this region containing specific expression from the 5B genes. In fact there was very little transcription from the genes on 5B flanking the Ph1 region, with mic1 being the only other gene which was transcribed to a significant level in the 2·5 Mb chromosomal region. Of the genes transcribed from 5B within the Ph1 region, hyp3 was not found in the equivalent region of the related grass Triticum timopheevi which has Ph1 activity, and hyp1 and hyp2 (Griffiths et al., 2006) are now known to encode storage-like proteins in wheat (Kawaura et al., 2005). Interestingly, when the 5B region was deleted, the loss of transcription of these genes was compensated by the homoeologues on the remaining genomes (5A and 5D) so that there was no apparent difference in the overall level of transcription in the presence or absence of Ph1 (Figs 2 and 3). In some cases, the compensation was provided by homoeologous genes lying on other chromosomes such as observed for transcription in both spike and seeds for hyp3 and in leaf for Slp1 genes. Thus, only the cdk-like gene cluster remained for more detailed analysis with respect to the Ph1 function.

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