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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 for an intron-exon structure, intron length differences, exon SNPs and primer position of the three copies of the Vrn1 gene.(I) A, B and D on the left border stand for the three different wheat sub-genomes and I1 to I7 on the top for the seven introns of Vrn1. The arrows which bend to the right are forward primers and the arrows which bend to the left are reverse primers. The red vertical lines show SNPs between the three gene copies. A I1 has a length of 8518 bp, B I1 2821 bp, D I1 8625bp, A I2 1475 bp, B I2 1246 bp, D I2 1504 bp, A I3 90 bp, B I3 92 bp, D I3 90 bp, A I4 192 bp, B I4 196 bp, D I4 188 bp, A I5 152 bp, B I5 156 bp, D I5 156 bp, A I6 93 bp, B I6 91 bp, D I6 91 bp, A I7 166 bp, B I7 168 bp and D I7 168 bp. (II) This figure shows the SNPs between the three sub-genomes and their coding sequences (CDS) position.
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pone.0142746.g002: Example for an intron-exon structure, intron length differences, exon SNPs and primer position of the three copies of the Vrn1 gene.(I) A, B and D on the left border stand for the three different wheat sub-genomes and I1 to I7 on the top for the seven introns of Vrn1. The arrows which bend to the right are forward primers and the arrows which bend to the left are reverse primers. The red vertical lines show SNPs between the three gene copies. A I1 has a length of 8518 bp, B I1 2821 bp, D I1 8625bp, A I2 1475 bp, B I2 1246 bp, D I2 1504 bp, A I3 90 bp, B I3 92 bp, D I3 90 bp, A I4 192 bp, B I4 196 bp, D I4 188 bp, A I5 152 bp, B I5 156 bp, D I5 156 bp, A I6 93 bp, B I6 91 bp, D I6 91 bp, A I7 166 bp, B I7 168 bp and D I7 168 bp. (II) This figure shows the SNPs between the three sub-genomes and their coding sequences (CDS) position.

Mentions: For all of the 27 candidate genes we were able to re-construct the gene structure or at least a part of it. A set of 119 PCR products was obtained from 157 primers pairs designed in this study. 13 of them have recently been published in Keilwagen et al. [53]. Additional 12 primers from literature were used for the amplification of targeted genes. By combining the primers from this study and the 12 primers from literature a total of 169 primers were analysed. As an example the reconstruction of the three copies of the Vrn1 gene structure, primer positions, intron length differences and exon SNPs are shown in Fig 2.


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 for an intron-exon structure, intron length differences, exon SNPs and primer position of the three copies of the Vrn1 gene.(I) A, B and D on the left border stand for the three different wheat sub-genomes and I1 to I7 on the top for the seven introns of Vrn1. The arrows which bend to the right are forward primers and the arrows which bend to the left are reverse primers. The red vertical lines show SNPs between the three gene copies. A I1 has a length of 8518 bp, B I1 2821 bp, D I1 8625bp, A I2 1475 bp, B I2 1246 bp, D I2 1504 bp, A I3 90 bp, B I3 92 bp, D I3 90 bp, A I4 192 bp, B I4 196 bp, D I4 188 bp, A I5 152 bp, B I5 156 bp, D I5 156 bp, A I6 93 bp, B I6 91 bp, D I6 91 bp, A I7 166 bp, B I7 168 bp and D I7 168 bp. (II) This figure shows the SNPs between the three sub-genomes and their coding sequences (CDS) position.
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Related In: Results  -  Collection

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pone.0142746.g002: Example for an intron-exon structure, intron length differences, exon SNPs and primer position of the three copies of the Vrn1 gene.(I) A, B and D on the left border stand for the three different wheat sub-genomes and I1 to I7 on the top for the seven introns of Vrn1. The arrows which bend to the right are forward primers and the arrows which bend to the left are reverse primers. The red vertical lines show SNPs between the three gene copies. A I1 has a length of 8518 bp, B I1 2821 bp, D I1 8625bp, A I2 1475 bp, B I2 1246 bp, D I2 1504 bp, A I3 90 bp, B I3 92 bp, D I3 90 bp, A I4 192 bp, B I4 196 bp, D I4 188 bp, A I5 152 bp, B I5 156 bp, D I5 156 bp, A I6 93 bp, B I6 91 bp, D I6 91 bp, A I7 166 bp, B I7 168 bp and D I7 168 bp. (II) This figure shows the SNPs between the three sub-genomes and their coding sequences (CDS) position.
Mentions: For all of the 27 candidate genes we were able to re-construct the gene structure or at least a part of it. A set of 119 PCR products was obtained from 157 primers pairs designed in this study. 13 of them have recently been published in Keilwagen et al. [53]. Additional 12 primers from literature were used for the amplification of targeted genes. By combining the primers from this study and the 12 primers from literature a total of 169 primers were analysed. As an example the reconstruction of the three copies of the Vrn1 gene structure, primer positions, intron length differences and exon SNPs are shown in Fig 2.

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