Comparative Systems Biology Reveals Allelic Variation Modulating Tocochromanol Profiles in Barley (Hordeum vulgare L.)

Tocochromanols are recognized for nutritional content, plant stress response, and seed longevity. Here we present a systems biological approach to characterize and develop predictive assays for genes affecting tocochromanol variation in barley. Major QTL, detected in three regions of a SNP linkage map, affected multiple tocochromanol forms. Candidate genes were identified through barley/rice orthology and sequenced in genotypes with disparate tocochromanol profiles. Gene-specific markers, designed based on observed polymorphism, mapped to the originating QTL, increasing R2 values at the respective loci. Polymorphism within promoter regions corresponded to motifs known to influence gene expression. Quantitative PCR analysis revealed a trend of increased expression in tissues grown at cold temperatures. These results demonstrate utility of a novel method for rapid gene identification and characterization, and provide a resource for efficient development of barley lines with improved tocochromanol profiles

Predicted secondary structure (A) and three-dimensional structure (B) of HGGT proteins in barley cultivars Falcon and Azhul.

<p>Sequence polymorphism contributing to folding changes is indicated by the green consensus guide (A). Blue curved arrows represent turns, orange arrows represent beta strands, pink cylinders represent alpha helices, and gray wavy lines represent coils. Pink labels within ribbon diagrams correspond to the predicted active site.</p

Quantitative PCR analysis of <i>VTE</i>4 (top) and <i>HGGT</i> (bottom) in Falcon and Azhul barley cultivars.

<p>Tissue samples used for cDNA preparation were taken from shoots and embryos grown at room temperature and at 4°C.</p

Motifs affected by promoter sequence polymorphism between Falcon and Azhul.

<p>Motifs affected by promoter sequence polymorphism between Falcon and Azhul.</p

Major QTL regions influencing tocochromanol biosynthesis in the barley Falcon x Azhul RIL population.

<p>QTL common to all environments are indicated by a colored bar. Black extensions indicate regions affected by at least one environment. Marker names and map positions are colored by additive effect of parental alleles. Blue indicates a greater influence of the Falcon allele; red a greater influence of Azhul.</p

Tocochromanol biosynthesis.

<p>Dotted lines represent double bonds in the tocotrienol prenyl side chain. Dual labels correspond to tocopherol and tocotrienol, respectively.</p

Sequence alignments for gene and promoter regions of VTE4 (A) and HGGT (B) in barley cultivars Falcon and Azhul.

<p>Polymorphisms are indicated by black interruptions within the gray bars, with narrow sections corresponding to indels. Annotations are indicated by colored bars above the consensus guide. Within the promoter region, blue indicates a promoter sequence, light green a TSS, purple a TATA or CAAT box, and gray all other motifs. The transcription start codon is indicated in green, and the stop codon in red. White bars indicate introns.</p

Tocochromanol means of Falcon and Azhul parents and two barley checks grown over four location years.

<p>Means and heritability estimates were calculated for Falcon x Azhul (FA) RILs using combined years at each location.</p>a<p>Concentrations given in µg/g. Values followed by the same letter or number of asterisks are not significantly different within a column (p<0.05). For means of genotypes within an form, a single asterisk is equivalent to A, two asterisks to B, and three asterisks to C.</p>+<p>indicates an intermediate value.</p>b<p>Irrigated field trial in Aberdeen.</p>c<p>Non-irrigated field trial at Tetonia.</p>d<p>Broad sense heritability calculated as genotype variance divided by cumulative variance including error.</p

Tocochromanol quantitative trait locus analysis summary for Falcon x Azhul RILs across four location years.

a<p>Indicates the tocochromanol form. T – Tocopherol; T3 – Tocotrienol.</p>b<p>AB indicates the irrigated field trial in Aberdeen; TE indicates the non-irrigated field trial in Tetonia.</p>c<p>Flanking marker to the left of the QTL peak.</p>d<p>Interval based on one LOD to each side of the peak. Peak and interval positions are in cM.</p>e<p>QTL detection was based on a LOD threshold of 2.5 (1000 permutations and type I error of 5%).</p>f<p>Total phenotypic variation explained by all QTL.</p>g<p>Percentage of phenotypic variation accounted for by QTL.</p