Differentially penalized regression to predict agronomic traits from metabolites and markers in wheat.

BACKGROUND: Genomic prediction of agronomic traits as targets for selection in plant breeding programmes is increasingly common. The methods employed can also be applied to predict traits from other sources of covariates, such as metabolomics. However, prediction combining sets of covariates can be less accurate than using the best of the individual sets. RESULTS: We describe a method, termed Differentially Penalized Regression (DiPR), which uses standard ridge regression software to combine sets of covariates while applying independent penalties to each. In a dataset of wheat varieties, field traits are better predicted, on average, by seed metabolites than by genetic markers, but DiPR using both sets of predictors is best. CONCLUSION: DiPR is a simple and accessible method of using existing software to combine multiple sets of covariates in trait prediction when there are more predictors than observations and the contribution to accuracy from each set differs.

Natural variation in grain composition of wheat and related cereals.

The wheat grain comprises three groups of major components, starch, protein, and cell wall polysaccharides (dietary fiber), and a range of minor components that may confer benefits to human health. Detailed analyses of dietary fiber and other bioactive components were carried out under the EU FP6 HEALTHGRAIN program on 150 bread wheat lines grown on a single site, 50 lines of other wheat species and other cereals grown on the same site, and 23-26 bread wheat lines grown in six environments. Principal component analysis allowed the 150 bread wheat lines to be classified on the basis of differences in their contents of bioactive components and wheat species (bread, durum, spelt, emmer, and einkorn wheats) to be clearly separated from related cereals (barley, rye, and oats). Such multivariate analyses could be used to define substantial equivalence when novel (including transgenic) cereals are considered.

Contents of dietary fibre components and their relation to associated bioactive components in whole grain wheat samples from the HEALTHGRAIN diversity screen.

A large and diverse material collection of whole grain wheat samples (n=129) was analysed for total dietary fibre (TDF) content and composition, including fructan (11.5-15.5%). Correlations between the dietary fibre components, associated bioactive components (e.g. tocols, sterols, phenolic acids and folates) and agronomic properties previously determined on the same samples were found with multivariate analysis (PCA). Samples from the same countries had similar characteristics. The first PC described variation in components concentrated in the starchy endosperm (e.g. starch, ß-glucan and fructan) and the dietary fibre components concentrated in the bran (e.g. TDF, arabinoxylan and cellulose). The second PC described the variation in kernel weight and other bran components such as alkylresorcinols, tocols and sterols. Interestingly, there was no correlation among these different groups of bran components, which reflected their concentration in different bran tissues. The results are of importance for plant breeders who wish to develop varieties with health-promoting effects.

Genotype and environment effects on the contents of vitamins B1, B2, B3, and B6 in wheat grain.

The total contents of thiamine (vitamin B1), riboflavin (B2), and pyridoxine (B6) and the bioavailable forms of niacin (B3) were determined on wholemeal flours of 24 winter wheat varieties grown on four sites (United Kingdom, Poland, France, and Hungary) in 2007 and of two spring varieties grown on the same sites with the exception of Poland. The contents of vitamins B1 (5.53-13.55 µg/g dw), B2 (0.77-1.40 µg/g dw), and B6 (1.27-2.97 µg/g dw) were within the ranges reported previously, while the content of bioavailable vitamin B3 (0.16-1.74 µg/g dw) was about 10-15% of the total contents of vitamin B3 reported in previous studies. Strong correlations were observed between the contents of vitamins B1, B3, and B6, and partitioning of the variance in the contents of these three B vitamins showed that between 48 and 70% was accounted for by the environment. By contrast, the content of vitamin B2 was not correlated with the contents of other B vitamins, and 73% of the variance was ascribed to the error term, which suggests that this trait may be influenced by genotype × environment interactions. Whereas the contents of vitamins B1, B3, and B6 were correlated positively with the mean temperature from heading to harvest (r > 0.8), the content of vitamin B2 was positively correlated with precipitation during the 3 months prior to heading. These results are discussed in relation to the development of new wheat varieties with enhanced health benefits.

Embryo and endosperm development in wheat (Triticum aestivum L.) kernels subjected to drought stress.

The aim of the present work was to reveal the histological alterations triggered in developing wheat kernels by soil drought stress during early seed development resulting in yield losses at harvest. For this purpose, observations were made on the effect of drought stress, applied in a controlled environment from the 5th to the 9th day after pollination, on the kernel morphology, starch content and grain yield of the drought-sensitive Cappelle Desprez and drought-tolerant Plainsman V winter wheat (Triticum aestivum L.) varieties. As a consequence of water withdrawal, there was a decrease in the size of the embryos and the number of A-type starch granules deposited in the endosperm, while the development of aleurone cells and the degradation of the cell layers surrounding the ovule were significantly accelerated in both genotypes. In addition, the number of B-type starch granules per cell was significantly reduced. Drought stress affected the rate of grain filling shortened the grain-filling and ripening period and severely reduced the yield. With respect to the recovery of vegetative tissues, seed set and yield, the drought-tolerant Plainsman V responded significantly better to drought stress than Cappelle Desprez. The reduction in the size of the mature embryos was significantly greater in the sensitive genotype. Compared to Plainsman V, the endosperm cells of Cappelle Desprez accumulated significantly fewer B-type starch granules. In stressed kernels of the tolerant genotype, the accumulation of protein bodies occurred significantly earlier than in the sensitive variety.

The HEALTHGRAIN wheat diversity screen: effects of genotype and environment on phytochemicals and dietary fiber components.

Analysis of the contents of bioactive components (tocols, sterols, alkylresorcinols, folates, phenolic acids, and fiber components) in 26 wheat cultivars grown in six site x year combinations showed that the extent of variation due to variety and environment differed significantly between components. The total contents of tocols, sterols, and arabinoxylan fiber were highly heritable and hence an appropriate target for plant breeding. However, significant correlations between the contents of bioactive components and environmental factors (precipitation and temperature) during grain development also occurred, with even highly heritable components differing in amount between grain samples grown in different years on different sites.

Effects of genotype and environment on the content and composition of phytochemicals and dietary fiber components in rye in the HEALTHGRAIN diversity screen.

The effects of genotype and environment on the content of bioactive components in rye were determined with four varieties being grown on one site for three years and on three additional sites in the third year and a fourth variety being included in all trials except year 1. Clear differences were observed in the extent to which the contents of dietary fiber components (arabinoxylan, beta-glucan, total dietary fiber) and phytochemicals (folates, alkylresorcinols, sterols, tocols, phenolic acids) varied between varieties and between the same varieties grown in different sites (United Kingdom, France, Hungary, Poland) and years (2005-2007 in Hungary), with sterols being the most stable and phenolic acids the least. However, no single variety could be selected as having the highest overall level of bioactive components or as being more stable in comparison across environments.

Environment and genotype effects on the content of dietary fiber and its components in wheat in the HEALTHGRAIN diversity screen.

Within the HEALTHGRAIN diversity screen, the variability of the contents of dietary fiber (DF) and components thereof was studied in wheat. Furthermore, the contribution of genotype and environment to this variability was estimated. The levels of total DF (TDF), total nonstarch polysaccharide (TOTNSP), water-extractable nonstarch polysaccharide (WENSP), total arabinoxylan (TOTAX), lignin, and beta-glucan in whole meal, flour, and/or bran varied approximately 1.8-fold. The highest variability was observed for the water-extractable arabinoxylan (WEAX) level in flour and bran (approximately 3.7-fold). Genotype and environment contributed to a similar extent to the variability in TDF, TOTNSP, and TOTAX content in wheat. The observed relatively high impact of genotype-environment interaction suggests that the levels of these constituents are weak breeding parameters. The WENSP level is a more stable parameter as the effect of the interaction term was much less than the impact of genotype. For TOTAX and WEAX in flour, WEAX in bran, beta-glucan in whole meal, and extract viscosity, wheat genotype determined approximately 50% or higher of the variation observed, whereas the impact of the genotype-environment interaction was relatively low. These findings suggest that the health-related and technological functionality of wheat can be directed to a certain extent by selection of appropriate wheat varieties.

Variability in xylanase and xylanase inhibition activities in different cereals in the HEALTHGRAIN diversity screen and contribution of environment and genotype to this variability in common wheat.

Endo-1,4-beta-d-xylanases (EC 3.2.1.8, xylanases) and xylanase inhibitors, that is, TAXI (Triticum aestivum xylanase inhibitor), XIP (xylanase inhibiting protein), and TLXI (thaumatin-like xylanase inhibitor) type xylanase inhibitors, which naturally occur in cereals, are believed to be at the basis of a significant part of the variability in biotechnological functional properties of cereals. Xylanase inhibitors in particular affect grain functionality during processing and in animal feeds when xylanases are used to improve processing parameters and product quality. In the present study the variability of xylanase, TAXI, and XIP activities was quantified in different cereals, including different wheat types [common wheat (Triticum aestivum L.), durum wheat (Triticum durum Desf.), spelt wheat (Triticum spelta L.), einkorn wheat (Triticum monococcum L.), and emmer wheat (Triticum dicoccum Schübler)], barley (Hordeum vulgare L.), rye (Secale cereale L.), and oat (Avena sativa L.), and the contribution of genotype and environment to this variability in common wheat was estimated. Substantial differences in xylanase, TAXI, and XIP activities exist between the different cereal types and varieties. Under the experimental conditions of this study, the durum wheat samples show very high xylanase activities compared to the other cereals. High TAXI and XIP activities were measured in, for example, common wheat, spelt wheat, and rye, whereas low activities occur in barley and oat. For wheat, a significant part of the variability in inhibitor levels can be explained by genotype, whereas xylanase activity is most strongly determined by environment. The results obtained suggest that plant breeders and industry to certain extent can select for wheat varieties with high or low xylanase inhibition activities, but the relatively high contribution of the genotype-environment interaction term to the total variability in inhibition activities indicates that TAXI and XIP activities are not very stable breeding parameters.

Free amino acids and sugars in rye grain: implications for acrylamide formation.

Acrylamide forms from free asparagine and sugars during cooking, and products derived from the grain of cereals, including rye, contribute a large proportion of total dietary intake. In this study, free amino acid and sugar concentrations were measured in the grain of a range of rye varieties grown at locations in Hungary, France, Poland, and the United Kingdom and harvested in 2005, 2006, and 2007. Genetic and environmental (location and harvest year) effects on the levels of acrylamide precursors were assessed. The data showed free asparagine concentration to be the main determinant of acrylamide formation in heated rye flour, as it is in wheat. However, in contrast to wheat, sugar, particularly sucrose, concentration also correlated both with asparagine concentration and with acrylamide formed. Free asparagine concentration was shown to be under genetic (G), environmental (E), and integrated (G x E) control. The same was true for glucose, whereas maltose and fructose were affected mainly by environmental factors and sucrose was largely under genetic control. The ratio of variation due to varieties (genotype) to the total variation (a measure of heritability) for free asparagine concentration in the grain was 23%. Free asparagine concentration was closely associated with bran yield, whereas sugar concentration was associated with low Hagberg falling number. Rye grain was found to contain much higher concentrations of free proline than wheat grain, and less acrylamide formed per unit of asparagine in rye than in wheat flour.

Mutation discovery for crop improvement.

Increasing crop yields to ensure food security is a major challenge. Mutagenesis is an important tool in crop improvement and is free of the regulatory restrictions imposed on genetically modified organisms. The forward genetic approach enables the identification of improved or novel phenotypes that can be exploited in conventional breeding programmes. Powerful reverse genetic strategies that allow the detection of induced point mutations in individuals of the mutagenized populations can address the major challenge of linking sequence information to the biological function of genes and can also identify novel variation for plant breeding. This review briefly discusses recent advances in the detection of mutants and the potential of mutagenesis for crop improvement.

Transgenic approach to improve wheat (Triticum aestivum L.) nutritional quality.

An amaranth (Amaranthus hypochondriacus) albumin gene, encoding the 35-kDa AmA1 protein of the seed, with a high content of essential amino acids, was used in the biolistic transformation of bread wheat (Triticum aestivum L.) variety Cadenza. The transformation cassette carried the ama1 gene under the control of a powerful wheat endosperm-specific promoter (1Bx17 HMW-GS). Southern-blot analysis of T(1) lines confirmed the integration of the foreign gene, while RT-PCR and Western-blot analyses of the samples confirmed the transcription and translation of the transgene. The effects of the extra albumin protein on the properties of flour, produced from bulked T(2) seeds, were calculated using total protein and essential amino acid content analysis, polymeric/monomeric protein and HMW/LMW glutenin subunit ratio measurements. The results indicated that not only can essential amino acid content be increased, but some parameters associated with functional quality may also be improved because of the expression of the AmA1 protein.

Design and management of field trials of transgenic cereals.

The development of gene transformation systems has allowed the introgression of alien genes into plant genomes, thus providing a mechanism for broadening the genetic resources available to plant breeders. The design and the management of field trials vary according to the purpose for which transgenic cereals are developed. Breeders study the phenotypic and genotypic stability of transgenic plants, monitor the increase in homozygosity of transgenic genotypes under field conditions, and develop backcross generations to transfer the introduced genes into secondary transgenic cereal genotypes. For practical purposes, they may also multiply seed of the transgenic lines to produce sufficient amounts of grain for the detailed analysis of trait(s) of interest, to determine the field performance of transgenic lines, and to compare them with the non-transformed parental genotypes. Prior to variety registration, the Distinctness, Uniformity and Stability (DUS) tests and Value for Cultivation and Use (VCU) experiments are carried out in field trials. Field testing includes specific requirements for transgenic cereals to assess potential environmental risks. The capacity of the pollen to survive, establish and disseminate in the field test environment, the potential for gene transfer, the effects of products expressed by the introduced sequences and phenotypic and genotypic instability that might cause deleterious effects must all be specifically monitored, as required by EU Directives 2003/701/EC (1) on the release of genetically modified higher plants in the environment.

The HEALTHGRAIN Cereal Diversity Screen: concept, results, and prospects.

One hundred and fifty bread wheat lines and 50 other lines of small-grain cereals (spelt, durum wheat, Triticum monococcum, Triticum dicoccum, oats, rye, and barley) were selected for diversity in their geographical origin, age, and characteristics. They were grown on a single site in Hungary in 2004-2005, harvested, milled, and analyzed for a range of phytochemicals (tocols, sterols, phenolic acids, folates, alkylresorcinols) and fiber components that are considered to have health benefits. Detailed analyses of these components in the different species are reported in a series of accompanying papers. The present paper discusses the comparative levels of the bioactive components in the different species, showing differences in both ranges and mean amounts. Furthermore, detailed comparisons of the bread wheat lines show that it is possible to identify lines in which high levels of phytochemicals and dietary fiber components are combined with good yield and processing quality. This means that commercially competitive lines with high levels of bioactive components are a realistic goal for plant breeders.

Composition and end-use quality of 150 wheat lines selected for the HEALTHGRAIN Diversity Screen.

The HEALTHGRAIN program is focused on developing new healthy food products based on wholegrains of wheat and other cereals, by combining enhanced nutritional quality with good agronomic performance and processing quality. A sample set comprising 130 winter and 20 spring wheat varieties was therefore selected to identify the range of variation in a number of phytochemical and dietary fiber components. These lines were also analyzed for their technological properties (protein and gluten contents, Zeleny sedimentation, bran yield, kernel hardness, etc.), using samples grown on adjacent sites for two successive seasons (2004-2005, 2005-2006). On the basis of the frequency distribution and principal component analysis it was concluded that significant variation for technological quality traits is present in the 150 wheat lines and that it is possible to combine enhanced nutritional quality with good agronomic performance and processing properties.

Phytochemicals and dietary fiber components in rye varieties in the HEALTHGRAIN Diversity Screen.

Ten rye varieties grown in one location were analyzed for their contents of dietary fiber (arabinoxylan and beta-glucan) and phytochemicals (folate, tocols, phenolic acids, alkylresorcinols, and sterols). The varieties included old and modern varieties from five European countries. Significant differences were observed in the contents of all phytochemicals in whole grains and in the fiber contents in the flour and bran. The old French varieties Haute Loire and Queyras had high contents of most phytochemicals, whereas the Polish varieties Dankowskie-Zlote and Warko were relatively poor in phytochemicals. The varieties with a high content of folate tended to have low alkylresorcinol contents and vice versa. Furthermore, high contents of arabinoxylans were associated with high contents in tocols and sterols. The 10 selected rye samples comprising old populations and old and modern varieties from different ecological regions of Europe demonstrate high natural variation in their composition and show that landraces and old populations are useful genetic resources for plant breeding. The contents of single phytochemicals can likely be affected by breeding, and they may be adjusted by the right selection of genotype.

Variation in the content of dietary fiber and components thereof in wheats in the HEALTHGRAIN Diversity Screen.

Within the HEALTHGRAIN diversity screening program, the variation in the content of dietary fiber and components thereof in different types of wheat was studied. The wheat types were winter (131 varieties) and spring (20 varieties) wheats (both Triticum aestivum L., also referred to as common wheats), durum wheat (Triticum durum Desf., 10 varieties), spelt wheat (Triticum spelta L., 5 varieties), einkorn wheat (T. monococcum L., 5 varieties), and emmer wheat (Triticum dicoccum Schubler, 5 varieties). Common wheats contained, on average, the highest level of dietary fiber [11.5-18.3% of dry matter (dm)], whereas einkorn and emmer wheats contained the lowest level (7.2-12.8% of dm). Intermediate levels were measured in durum and spelt wheats (10.7-15.5% of dm). Also, on the basis of the arabinoxylan levels in bran, the different wheat types could be divided this way, with ranges of 12.7-22.1% of dm for common wheats, 6.1-14.4% of dm for einkorn and emmer wheats, and 10.9-13.9% of dm for durum and spelt wheats. On average, bran arabinoxylan made up ca. 29% of the total dietary fiber content of wheat. In contrast to what was the case for bran, the arabinoxylan levels in flour were comparable between the different types of wheat. For wheat, in general, they varied between 1.35 and 2.75% of dm. Einkorn, emmer, and durum wheats contained about half the level of mixed-linkage beta-glucan (0.25-0.45% of dm) present in winter, spring, and spelt wheats (0.50-0.95% of dm). All wheat types had Klason lignin, the levels of which varied from 1.40 to 3.25% of dm. The arabinoxylan contents in bran and the dietary fiber contents in wholemeal were inversely and positively related with bran yield, respectively. Aqueous wholemeal extract viscosity, a measure for the level of soluble dietary fiber, was determined to large extent by the level of water-extractable arabinoxylan. In conclusion, the present study revealed substantial variation in the contents of dietary fiber and constituents thereof between different wheat types and varieties.

Phytochemical and dietary fiber components in barley varieties in the HEALTHGRAIN Diversity Screen.

Ten different barley varieties grown in one location were studied for their content of tocols, folate, plant sterols, alkylresorcinols, and phenolic acids, as well as dietary fiber components (arabinoxylan and beta-glucan). The samples included hulled and hull-less barley types and types with normal, high-amylose, and waxy starch. The aim was to study the composition of raw materials, and therefore the hulls were not removed from the hulled barleys. A large variation was observed in the contents of all phytochemicals and dietary fibers. Two varieties from the INRA Clermont Ferrand barley program in France (CFL93-149 and CFL98-398) had high content of tocopherols and alkylresorcinols, whereas the variety Dicktoo was highest in dietary fiber content and phenolics. Positive correlations were found between 1000 kernel weight, alkylresorcinols, and tocols, as well as between dietary fiber content and phenolic compounds. The results demonstrate that the levels of phytochemicals in barley can likely be affected by breeding and that the contents of single phytochemicals may easily be adjusted by a right selection of a genotype.

Phytochemical and fiber components in oat varieties in the HEALTHGRAIN Diversity Screen.

The levels and compositions of a range of phytochemicals (sterols, tocols, avenanthramides, folates, phenolic acids) and dietary fiber components were determined in five oat cultivars (four husked and one naked) grown on a single site in 2005. The total levels of tocols, phenolic acids, and avenanthramides varied by over 2-fold between cultivars, but less variation occurred in total sterols and total folates. Limited variation was also observed in the dietary fiber content and composition of the four husked lines. These results indicate that it may be possible to selectively breed for lines with high contents of dietary fiber and specific groups of phytochemicals.