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.

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

Primer sequences used for amplification of candidate genes.

<p>Primer names with ^† are developed in course of this work but published from Keilwagen et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142746#pone.0142746.ref053" target="_blank">53</a>].</p><p>Primer names with * as already published were used in combination with primers with ^† and without labels.</p><p>¹[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142746#pone.0142746.ref086" target="_blank">86</a>]</p><p>²[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142746#pone.0142746.ref073" target="_blank">73</a>]</p><p>³[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142746#pone.0142746.ref087" target="_blank">87</a>]</p><p>⁴[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142746#pone.0142746.ref081" target="_blank">81</a>]</p><p>⁵[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142746#pone.0142746.ref037" target="_blank">37</a>]</p><p>⁶ [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142746#pone.0142746.ref053" target="_blank">53</a>]</p><p>Primer sequences used for amplification of candidate genes.</p

Map of gene specific PCR fragments by using wheat NT- and deletion lines.

<p>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.</p

Plant material for PCR amplification and re-sequencing.

<p>Complete set of 24 genotypes (without `Chinese Spring`) were used for sequencing.</p><p>* Genotypes for primer testing</p><p>Plant material for PCR amplification and re-sequencing.</p

Overview of chromosome localisation of candidate genes (PCR fragment) via NT-lines, deletion-lines and literature.

<p>The table shows the analysed frost tolerance candidate gene, their chromosomal localisation and fine mapping via NT and deletion-lines. The column deletion-line localisation section shows the approximate chromosomal position of respective genes based on deletion break points.</p><p>Overview of chromosome localisation of candidate genes (PCR fragment) via NT-lines, deletion-lines and literature.</p

Workflow of development gene specific primers and PCR fragments in wheat.

<p>The method comprises 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 on sequence differences between the three sub-genomes, and (iv); primer and PCR fragment testing for functionality, correct size and localisation. The dashed lines show optional applications.</p

Nucleotide polymorphisms of coding and noncoding candidate gene regions.

<p>h haplotypes</p><p>Hd haplotype diversity</p><p>k average number of nucleotide differences</p><p>π nucleotide diversity</p><p>(i) InDel</p><p>Nucleotide polymorphisms of coding and noncoding candidate gene regions.</p

Genome wide association study of frost tolerance in wheat

Winter wheat growing areas in the Northern hemisphere are regularly exposed to heavy frost. Due to the negative impact on yield, the identification of genetic factors controlling frost tolerance (FroT) and development of tools for breeding is of prime importance. Here, we detected QTL associated with FroT by genome wide association studies (GWAS) using a diverse panel of 276 winter wheat genotypes that was phenotyped at five locations in Germany and Russia in three years. The panel was genotyped using the 90 K iSelect array and SNPs in FroT candidate genes. In total, 17,566 SNPs were used for GWAS resulting in the identification of 53 markers significantly associated (LOD &gt;/= 4) to FroT, corresponding to 23 QTL regions located on 11 chromosomes (1A, 1B, 2A, 2B, 2D, 3A, 3D, 4A, 5A, 5B and 7D). The strongest QTL effect confirmed the importance of chromosome 5A for FroT. In addition, to our best knowledge, eight FroT QTLs were discovered for the first time in this study comprising one QTL on chromosomes 3A, 3D, 4A, 7D and two on chromosomes 1B and 2D. Identification of novel FroT candidate genes will help to better understand the FroT mechanism in wheat and to develop more effective combating strategies

Auxin-dependent regulation of cell division rates governs root thermomorphogenesis

Roots are highly plastic organs enabling plants to adapt to a changing below-ground environment. In addition to abiotic factors like nutrients or mechanical resistance, plant roots also respond to temperature variation. Below the heat stress threshold, Arabidopsis thaliana seedlings react to elevated temperature by promoting primary root growth, possibly to reach deeper soil regions with potentially better water saturation. While above-ground thermomorphogenesis is enabled by thermo-sensitive cell elongation, it was unknown how temperature modulates root growth. We here show that roots are able to sense and respond to elevated temperature independently of shoot-derived signals. This response is mediated by a yet unknown root thermosensor that employs auxin as a messenger to relay temperature signals to the cell cycle. Growth promotion is achieved primarily by increasing cell division rates in the root apical meristem, depending on de novo local auxin biosynthesis and temperature-sensitive organization of the polar auxin transport system. Hence, the primary cellular target of elevated ambient temperature differs fundamentally between root and shoot tissues, while the messenger auxin remains the same.ISSN:0261-4189ISSN:1460-207

Additional file 4: of Association genetics studies on frost tolerance in wheat (Triticum aestivum L.) reveal new highly conserved amino acid substitutions in CBF-A3, CBF-A15, VRN3 and PPD1 genes

Figure S1. Scatter plot of mean winter survival and number of days under − 15 °C from ten environments. (PDF 24 kb