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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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Related in: MedlinePlus

Map of gene specific PCR fragments by using wheat NT- and deletion lines.In this figure only wheat chromosomes are shown harbouring mapped PCR fragments. The white bar is the chromosome, the constriction symbolised the centromere, on the left side of chromosomes deletion break points are listed and the black bars are the regions of mapped PCR fragments with appended candidate gene.
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pone.0142746.g003: Map of gene specific PCR fragments by using wheat NT- and deletion lines.In this figure only wheat chromosomes are shown harbouring mapped PCR fragments. The white bar is the chromosome, the constriction symbolised the centromere, on the left side of chromosomes deletion break points are listed and the black bars are the regions of mapped PCR fragments with appended candidate gene.

Mentions: Chromosomal localisation via Nulli-tetrasomic (NT)-lines of Chinese Spring [43] of a set of 86 single band PCR amplicons revealed that 65 fragments were located on expected chromosomes according to the literature. Out of these 65 fragments, six were products of combination of already published and newly designed primers. The remaining 19 fragments showed an incorrect localisation (literature vs. NT-lines) or no localisation was possible as all NT-lines showed a fragment. Correctly assigned amplicons originated from 19 genes and were located on 11 wheat chromosomes (Table 3, Fig 3). A set of 10 out of 19 analysed genes were located on wheat chromosome group 5, out of 119 PCR fragments 65 single bands were correctly localised. That is equivalent to a success rate of 54.6%. These 65 amplicons represent 19 frost tolerance genes, are gene specific and were therefore selected for further studies (Table 4, S2 Table).


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)

Map of gene specific PCR fragments by using wheat NT- and deletion lines.In this figure only wheat chromosomes are shown harbouring mapped PCR fragments. The white bar is the chromosome, the constriction symbolised the centromere, on the left side of chromosomes deletion break points are listed and the black bars are the regions of mapped PCR fragments with appended candidate gene.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142746.g003: Map of gene specific PCR fragments by using wheat NT- and deletion lines.In this figure only wheat chromosomes are shown harbouring mapped PCR fragments. The white bar is the chromosome, the constriction symbolised the centromere, on the left side of chromosomes deletion break points are listed and the black bars are the regions of mapped PCR fragments with appended candidate gene.
Mentions: Chromosomal localisation via Nulli-tetrasomic (NT)-lines of Chinese Spring [43] of a set of 86 single band PCR amplicons revealed that 65 fragments were located on expected chromosomes according to the literature. Out of these 65 fragments, six were products of combination of already published and newly designed primers. The remaining 19 fragments showed an incorrect localisation (literature vs. NT-lines) or no localisation was possible as all NT-lines showed a fragment. Correctly assigned amplicons originated from 19 genes and were located on 11 wheat chromosomes (Table 3, Fig 3). A set of 10 out of 19 analysed genes were located on wheat chromosome group 5, out of 119 PCR fragments 65 single bands were correctly localised. That is equivalent to a success rate of 54.6%. These 65 amplicons represent 19 frost tolerance genes, are gene specific and were therefore selected for further studies (Table 4, S2 Table).

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
Related in: MedlinePlus