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An Efficient Approach for the Development of Locus Specific Primers in Bread Wheat (Triticum aestivum L.) and Its Application to Re-Sequencing of Genes Involved in Frost Tolerance.

Babben S, Perovic D, Koch M, Ordon F - PLoS ONE (2015)

Bottom Line: Out of these a set of 35 fragments was selected for validation via Sanger's amplicon re-sequencing.All fragments, with the exception of one, could be assigned to the original reference sequence.The approach presented here showed a much higher specificity in primer development in comparison to techniques used so far in bread wheat and can be applied to other polyploid species with a known draft sequence.

View Article: PubMed Central - PubMed

Affiliation: Julius Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants, Institute for Resistance Research and Stress Tolerance, Quedlinburg, Sachsen-Anhalt, Germany.

ABSTRACT
Recent declines in costs accelerated sequencing of many species with large genomes, including hexaploid wheat (Triticum aestivum L.). Although the draft sequence of bread wheat is known, it is still one of the major challenges to developlocus specific primers suitable to be used in marker assisted selection procedures, due to the high homology of the three genomes. In this study we describe an efficient approach for the development of locus specific primers comprising four steps, i.e. (i) identification of genomic and coding sequences (CDS) of candidate genes, (ii) intron- and exon-structure reconstruction, (iii) identification of wheat A, B and D sub-genome sequences and primer development based on sequence differences between the three sub-genomes, and (iv); testing of primers for functionality, correct size and localisation. This approach was applied to single, low and high copy genes involved in frost tolerance in wheat. In summary for 27 of these genes for which sequences were derived from Triticum aestivum, Triticum monococcum and Hordeum vulgare, a set of 119 primer pairs was developed and after testing on Nulli-tetrasomic (NT) lines, a set of 65 primer pairs (54.6%), corresponding to 19 candidate genes, turned out to be specific. Out of these a set of 35 fragments was selected for validation via Sanger's amplicon re-sequencing. All fragments, with the exception of one, could be assigned to the original reference sequence. The approach presented here showed a much higher specificity in primer development in comparison to techniques used so far in bread wheat and can be applied to other polyploid species with a known draft sequence.

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Example of fragment localisation from Cbf1 via NT- and deletion-lines.(A) The missing PCR fragment on NT-line N5D-T5B indicated the location on wheat chromosome 5D. (B) The missing PCR fragment on Csdel 5DL-1 indicated the location on wheat long arm of chromosome 5D between the deletion segments 1 and 5.
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pone.0142746.g004: Example of fragment localisation from Cbf1 via NT- and deletion-lines.(A) The missing PCR fragment on NT-line N5D-T5B indicated the location on wheat chromosome 5D. (B) The missing PCR fragment on Csdel 5DL-1 indicated the location on wheat long arm of chromosome 5D between the deletion segments 1 and 5.

Mentions: Specificity of primers is the non-recurring binding in the target genome. This is reflected in a single PCR and a correct or syntenically localised amplicon. Fig 4 shows an example of the Cbf1 amplicon localisation via NT- and deletion-lines.


An Efficient Approach for the Development of Locus Specific Primers in Bread Wheat (Triticum aestivum L.) and Its Application to Re-Sequencing of Genes Involved in Frost Tolerance.

Babben S, Perovic D, Koch M, Ordon F - PLoS ONE (2015)

Example of fragment localisation from Cbf1 via NT- and deletion-lines.(A) The missing PCR fragment on NT-line N5D-T5B indicated the location on wheat chromosome 5D. (B) The missing PCR fragment on Csdel 5DL-1 indicated the location on wheat long arm of chromosome 5D between the deletion segments 1 and 5.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643983&req=5

pone.0142746.g004: Example of fragment localisation from Cbf1 via NT- and deletion-lines.(A) The missing PCR fragment on NT-line N5D-T5B indicated the location on wheat chromosome 5D. (B) The missing PCR fragment on Csdel 5DL-1 indicated the location on wheat long arm of chromosome 5D between the deletion segments 1 and 5.
Mentions: Specificity of primers is the non-recurring binding in the target genome. This is reflected in a single PCR and a correct or syntenically localised amplicon. Fig 4 shows an example of the Cbf1 amplicon localisation via NT- and deletion-lines.

Bottom Line: Out of these a set of 35 fragments was selected for validation via Sanger's amplicon re-sequencing.All fragments, with the exception of one, could be assigned to the original reference sequence.The approach presented here showed a much higher specificity in primer development in comparison to techniques used so far in bread wheat and can be applied to other polyploid species with a known draft sequence.

View Article: PubMed Central - PubMed

Affiliation: Julius Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants, Institute for Resistance Research and Stress Tolerance, Quedlinburg, Sachsen-Anhalt, Germany.

ABSTRACT
Recent declines in costs accelerated sequencing of many species with large genomes, including hexaploid wheat (Triticum aestivum L.). Although the draft sequence of bread wheat is known, it is still one of the major challenges to developlocus specific primers suitable to be used in marker assisted selection procedures, due to the high homology of the three genomes. In this study we describe an efficient approach for the development of locus specific primers comprising four steps, i.e. (i) identification of genomic and coding sequences (CDS) of candidate genes, (ii) intron- and exon-structure reconstruction, (iii) identification of wheat A, B and D sub-genome sequences and primer development based on sequence differences between the three sub-genomes, and (iv); testing of primers for functionality, correct size and localisation. This approach was applied to single, low and high copy genes involved in frost tolerance in wheat. In summary for 27 of these genes for which sequences were derived from Triticum aestivum, Triticum monococcum and Hordeum vulgare, a set of 119 primer pairs was developed and after testing on Nulli-tetrasomic (NT) lines, a set of 65 primer pairs (54.6%), corresponding to 19 candidate genes, turned out to be specific. Out of these a set of 35 fragments was selected for validation via Sanger's amplicon re-sequencing. All fragments, with the exception of one, could be assigned to the original reference sequence. The approach presented here showed a much higher specificity in primer development in comparison to techniques used so far in bread wheat and can be applied to other polyploid species with a known draft sequence.

Show MeSH