Importance of genetic architecture in marker selection decisions for genomic prediction.

KEY MESSAGE: We demonstrate potential for improved multi-environment genomic prediction accuracy using structural variant markers. However, the degree of observed improvement is highly dependent on the genetic architecture of the trait. Breeders commonly use genetic markers to predict the performance of untested individuals as a way to improve the efficiency of breeding programs. These genomic prediction models have almost exclusively used single nucleotide polymorphisms (SNPs) as their source of genetic information, even though other types of markers exist, such as structural variants (SVs). Given that SVs are associated with environmental adaptation and not all of them are in linkage disequilibrium to SNPs, SVs have the potential to bring additional information to multi-environment prediction models that are not captured by SNPs alone. Here, we evaluated different marker types (SNPs and/or SVs) on prediction accuracy across a range of genetic architectures for simulated traits across multiple environments. Our results show that SVs can improve prediction accuracy, but it is highly dependent on the genetic architecture of the trait and the relative gain in accuracy is minimal. When SVs are the only causative variant type, 70% of the time SV predictors outperform SNP predictors. However, the improvement in accuracy in these instances is only 1.5% on average. Further simulations with predictors in varying degrees of LD with causative variants of different types (e.g., SNPs, SVs, SNPs and SVs) showed that prediction accuracy increased as linkage disequilibrium between causative variants and predictors increased regardless of the marker type. This study demonstrates that knowing the genetic architecture of a trait in deciding what markers to use in large-scale genomic prediction modeling in a breeding program is more important than what types of markers to use.

Linking genetic and environmental factors through marker effect networks to understand trait plasticity.

Understanding how plants adapt to specific environmental changes and identifying genetic markers associated with phenotypic plasticity can help breeders develop plant varieties adapted to a rapidly changing climate. Here, we propose the use of marker effect networks as a novel method to identify markers associated with environmental adaptability. These marker effect networks are built by adapting commonly used software for building gene coexpression networks with marker effects across growth environments as the input data into the networks. To demonstrate the utility of these networks, we built networks from the marker effects of ∼2,000 nonredundant markers from 400 maize hybrids across 9 environments. We demonstrate that networks can be generated using this approach, and that the markers that are covarying are rarely in linkage disequilibrium, thus representing higher biological relevance. Multiple covarying marker modules associated with different weather factors throughout the growing season were identified within the marker effect networks. Finally, a factorial test of analysis parameters demonstrated that marker effect networks are relatively robust to these options, with high overlap in modules associated with the same weather factors across analysis parameters. This novel application of network analysis provides unique insights into phenotypic plasticity and specific environmental factors that modulate the genome.

Quantitative Genetic Analysis of Hydroxycinnamic Acids in Maize (Zea mays L.) for Plant Improvement and Production of Health-Promoting Compounds.

Hydroxycinnamic acids, including ferulic acid and p-coumaric acid, have been tied to multiple positive health and agronomic benefits. However, little work has been done to improve the concentration of hydroxycinnamic acids in maize. We evaluated a set of 12 commercially important maize (Zea mays L.) inbred lines and 66 hybrids derived from their crosses for hydroxycinnamic acid concentration in the grain, grain yield, and test weight. The grain was obtained from replicated field experiments, which were conducted for 3 years. Both ferulic acid and p-coumaric acid were found to be highly heritable, and most of the genetic variation was additive. Grain yield and test weight were not correlated with hydroxycinnamic acid concentration. These findings suggest that breeding maize for improved hydroxycinnamic acid concentration is feasible. Maize hybrids with high hydroxycinnamic acid concentrations in the grain could be useful for the production of dietary supplements or all-natural food additives while imparting enhanced resistance to biotic and abiotic stresses during the growing season and grain storage.

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (Zea mays L.).

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole grain composition and processing characteristics vary among maize hybrids and can impact the quality of the final processed product. Therefore, in order to produce healthier processed food products from maize, it is necessary to know how to optimize processing parameters for particular sets of germplasm to account for these differences in grain composition and processing characteristics. This includes a better understanding of how current processing techniques impact the nutritional quality of the final processed food product. Here, we describe a microscale protocol that both simulates the processing pipeline to produce cornflakes from large flaking grits and allows for the processing of multiple grain samples simultaneously. The flaking grits, the intermediate processed products, or final processed product, as well as the corn grain itself, can be analyzed for nutritional content as part of a high-throughput analytical pipeline. This procedure was developed specifically for incorporation into a maize breeding research program, and it can be modified for other grain crops. We provide an example of the analysis of insoluble-bound ferulic acid and p-coumaric acid content in maize. Samples were taken at five different processing stages. We demonstrate that sampling can take place at multiple stages during microscale processing, that the processing technique can be utilized in the context of a specialized maize breeding program, and that, in our example, most of the nutritional content was lost during food product processing.

Changes in Phenolic Acid Content in Maize during Food Product Processing.

The notion that many nutrients and beneficial phytochemicals in maize are lost due to food product processing is common, but this has not been studied in detail for the phenolic acids. Information regarding changes in phenolic acid content throughout processing is highly valuable because some phenolic acids are chemopreventive agents of aging-related diseases. It is unknown when and why these changes in phenolic acid content might occur during processing, whether some maize genotypes might be more resistant to processing induced changes in phenolic acid content than other genotypes, or if processing affects the bioavailability of phenolic acids in maize-based food products. For this study, a laboratory-scale processing protocol was developed and used to process whole maize kernels into toasted cornflakes. High-throughput microscale wet-lab analyses were applied to determine the concentrations of soluble and insoluble-bound phenolic acids in samples of grain, three intermediate processing stages, and toasted cornflakes obtained from 12 ex-PVP maize inbreds and seven hybrids. In the grain, insoluble-bound ferulic acid was the most common phenolic acid, followed by insoluble-bound p-coumaric acid and soluble cinnamic acid, a precursor to the phenolic acids. Notably, the ferulic acid content was approximately 1950 µg/g, more than ten-times the concentration of many fruits and vegetables. Processing reduced the content of the phenolic acids regardless of the genotype. Most changes occurred during dry milling due to the removal of the bran. The concentration of bioavailable soluble ferulic and p-coumaric acid increased negligibly due to thermal stresses. Therefore, the current dry milling based processing techniques used to manufacture many maize-based foods, including breakfast cereals, are not conducive for increasing the content of bioavailable phenolics in processed maize food products. This suggests that while maize is an excellent source of phenolics, alternative or complementary processing methods must be developed before this nutritional resource can be utilized.

Quantitative Trait Loci Mapping of Western Corn Rootworm (Coleoptera: Chrysomelidae) Host Plant Resistance in Two Populations of Doubled Haploid Lines in Maize (Zea mays L.).

Over the last 70 yr, more than 12,000 maize accessions have been screened for their level of resistance to western corn rootworm, Diabrotica virgifera virgifera (LeConte; Coleoptera: Chrysomelidae), larval feeding. Less than 1% of this germplasm was selected for initiating recurrent selection or other breeding programs. Selected genotypes were mostly characterized by large root systems and superior root regrowth after root damage caused by western corn rootworm larvae. However, no hybrids claiming native (i.e., host plant) resistance to western corn rootworm larval feeding are currently commercially available. We investigated the genetic basis of western corn rootworm resistance in maize materials with improved levels of resistance using linkage disequilibrium mapping approaches. Two populations of topcrossed doubled haploid maize lines (DHLs) derived from crosses between resistant and susceptible maize lines were evaluated for their level of resistance in three to four different environments. For each DHL topcross an average root damage score was estimated and used for quantitative trait loci (QTL) analysis. We found genomic regions contributing to western corn rootworm resistance on all maize chromosomes, except for chromosome 4. Models fitting all QTL simultaneously explained about 30 to 50% of the genotypic variance for root damage scores in both mapping populations. Our findings confirm the complex genetic structure of host plant resistance against western corn rootworm larval feeding in maize. Interestingly, three of these QTL regions also carry genes involved in ascorbate biosynthesis, a key compound we hypothesize is involved in the expression of western corn rootworm resistance.

Concentration of Beneficial Phytochemicals in Harvested Grain of U.S. Yellow Dent Maize (Zea mays L.) Germplasm.

Although previous studies have examined the concentration of various nutritional compounds in maize, little focus has been devoted to the study of commercial maize hybrids or their inbred parents. In this study, a genetically and phenotypically diverse set of maize hybrids and inbreds relevant to U.S. commercial maize germplasm was evaluated for its variability in phytochemical content. Total protein, unsaturated fatty acids, tocopherols, soluble phenolics, and insoluble-bound phenolics were evaluated in this study. Of these compounds, only soluble and insoluble-bound phenolic acids exhibited means and variances that were at least as large as the means and variances reported for other sets of germplasm. This suggests that selection for high phenolic acid content is possible in current U.S. commercial germplasm. In contrast, while the total protein, unsaturated fatty acid, or tocopherol content could possibly be improved using current U.S. commercial germplasm, the results of this study indicate that the incorporation of more diverse sources of germplasm would most likely result in quicker genetic gains.

Genomic Prediction of Single Crosses in the Early Stages of a Maize Hybrid Breeding Pipeline.

Prediction of single-cross performance has been a major goal of plant breeders since the beginning of hybrid breeding. Recently, genomic prediction has shown to be a promising approach, but only limited studies have examined the accuracy of predicting single-cross performance. Moreover, no studies have examined the potential of predicting single crosses among random inbreds derived from a series of biparental families, which resembles the structure of germplasm comprising the initial stages of a hybrid maize breeding pipeline. The main objectives of this study were to evaluate the potential of genomic prediction for identifying superior single crosses early in the hybrid breeding pipeline and optimize its application. To accomplish these objectives, we designed and analyzed a novel population of single crosses representing the Iowa Stiff Stalk synthetic/non-Stiff Stalk heterotic pattern commonly used in the development of North American commercial maize hybrids. The performance of single crosses was predicted using parental combining ability and covariance among single crosses. Prediction accuracies were estimated using cross-validation and ranged from 0.28 to 0.77 for grain yield, 0.53 to 0.91 for plant height, and 0.49 to 0.94 for staygreen, depending on the number of tested parents of the single cross and genomic prediction method used. The genomic estimated general and specific combining abilities showed an advantage over genomic covariances among single crosses when one or both parents of the single cross were untested. Overall, our results suggest that genomic prediction of single crosses in the early stages of a hybrid breeding pipeline holds great potential to redesign hybrid breeding and increase its efficiency.

Characterization of mature maize (Zea mays L.) root system architecture and complexity in a diverse set of Ex-PVP inbreds and hybrids.

The mature root system is a vital plant organ, which is critical to plant performance. Commercial maize (Zea mays L.) breeding has resulted in a steady increase in plant performance over time, along with noticeable changes in above ground vegetative traits, but the corresponding changes in the root system are not presently known. In this study, roughly 2500 core root systems from field trials of a set of 10 diverse elite inbreds formerly protected by Plant Variety Protection plus B73 and Mo17 and the 66 diallel intercrosses among them were evaluated for root traits using high throughput image-based phenotyping. Overall root architecture was modeled by root angle (RA) and stem diameter (SD), while root complexity, the amount of root branching, was quantified using fractal analysis to obtain values for fractal dimension (FD) and fractal abundance (FA). For each trait, per se line effects were highly significant and the most important contributor to trait performance. Mid-parent heterosis and specific combining ability was also highly significant for FD, FA, and RA, while none of the traits showed significant general combining ability. The interaction between the environment and the additive line effect was also significant for all traits. Within the inbred and hybrid generations, FD and FA were highly correlated (rp ≥ 0.74), SD was moderately correlated to FD and FA (0.69 ≥ rp ≥ 0.48), while the correlation between RA and other traits was low (0.13 ≥ rp ≥ -0.40). Inbreds with contrasting effects on complexity and architecture traits were observed, suggesting that root complexity and architecture traits are inherited independently. A more comprehensive understanding of the maize root system and the way it interacts with the environment will be useful for defining adaptation to nutrient acquisition and tolerance to stress from drought and high plant densities, critical factors in the yield gains of modern hybrids.

The role of root architecture in foraging behavior of entomopathogenic nematodes.

As obligate parasites, entomopathogenic nematodes (EPN) rely on insect hosts to complete their development. In insect pest management, EPN infectiousness has varied a lot. A better understanding of their host-finding behavior in the rhizosphere is therefore crucial to enhance EPN potential in biological control. As previously demonstrated, roots can be used as a pathway to insect hosts by EPN, but this interaction and its impact on EPN foraging remain poorly documented. Three artificial model-roots with different degrees of complexity and connectivity were designed to investigate the impact of root architecture on foraging behavior of the EPN Heterorhabditis megidis. Insect baits were placed at the bottom of each model-root that was subsequently buried in moist sand. After injection of the EPN, the number of EPN-infected baits as well as the number of mature nematodes inside each individual carcass was recorded. The influence of insect-induced root volatiles was also evaluated by spiking the baits with a synthetic version of a natural insect-induced root cue. The ecological relevance of the results was tested in soil with two maize genotypes each exhibiting broadly different root architectures. H. megidi performed better in presence of model-roots. Foraging performances of H. megidis declined with the increasing model-root complexity. Adding the synthetic root volatile dramatically changed this pattern and favored the EPN on the most complex model-roots. H. megidis also moved in the vicinity of maize roots to find the insect baits in soil, and natural root architecture also tended to shape H. megidis foraging behavior. This study adds to the scarce body of literature characterizing physical and chemical interactions between EPN and roots. The present data illustrate that root architecture not only modifies plant quality but also shapes upper trophic levels' ecology.

Heritable variation in the inflorescence replacement program of Arabidopsis thaliana.

Owing to their sessile habits and trophic position within global ecosystems, higher plants display a sundry assortment of adaptations to the threat of predation. Unlike animals, nearly all higher plants can replace reproductive structures lost to predators by activating reserved growing points called axillary meristems. As the first step in a program aimed at defining the genetic architecture of the inflorescence replacement program (IRP) of Arabidopsis thaliana, we describe the results of a quantitative germplasm survey of developmental responses to loss of the primary reproductive axis. Eighty-five diverse accessions were grown in a replicated common garden and assessed for six life history traits and four IRP traits, including the number and lengths of axillary inflorescences present on the day that the first among them re-flowered after basal clipping of the primary inflorescence. Significant natural variation and high heritabilities were observed for all measured characters. Pairwise correlations among the 10 focal traits revealed a multi-dimensional phenotypic space sculpted by ontogenic and plastic allometries as well as apparent constraints and outliers of genetic interest. Cluster analysis of the IRP traits sorted the 85 accessions into 5 associations, a topology that establishes the boundaries within which the evolving Arabidopsis genome extends and restricts the species' IRP repertoire to that observable worldwide.

Conventional screening overlooks resistance sources: rootworm damage of diverse inbred lines and their B73 hybrids is unrelated.

The western corn rootworm, Diabrotica virgifera virgifera (LeConte), is a major pest of maize, Zea mays L., in the United States and Europe, and it is likely to increase in importance as a trend toward increased nonrotated maize favors larger rootworm populations. Options for rootworm management in nonrotated maize in Europe and in nontransgenic "refuge" areas in countries that permit transgenic maize are limited to insecticides. Development of additional options for growers would be helpful. Screening maize germplasm (e.g., landraces, populations, inbreds) for native resistance to western corn rootworm is labor-intensive and is usually conducted on unfinished germplasm and not on hybrid materials. However, we have recently observed that topcrossed (hybrid) materials tend to have reduced western corn rootworm damage. To formally test whether rootworm damage to inbreds and associated hybrids were correlated, we evaluated 25 diverse inbred lines and their B73 hybrids for western corn rootworm damage in seven environments. Overall, hybrids had significantly less damage than inbreds, but unfortunately, the correlation between inbreds and hybrids was not significant. These findings have important implications regarding screening germplasm for western corn rootworm resistance, namely, that inbred materials and perhaps populations should be topcrossed to form hybrid materials before screening for western corn rootworm damage to ensure that valuable sources of resistance to western corn rootworm are not missed during the screening process.

Native resistance to western corn rootworm (Coleoptera: Chrysomelidae) larval feeding: characterization and mechanisms.

Seven maize, Zea mays L., genotypes selected for native resistance to western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), larval feeding damage (SUM2068, SUM2162, CRW3(S1)C6, NSS1 x CRW3(S1)C6, PI583927, CRW2(C5), and AR17056-16) were evaluated along with three control maize genotypes in the field for plant damage, larval recovery, adult emergence, root size, and root regrowth. Larvae recovered were further analyzed for head capsule width and dry weight and adults for dry weight. All factors evaluated with the exception of adult dry weight varied significantly among maize genotypes. Control genotypes included a highly susceptible hybrid, B37 x H84, a transgenic rootworm-resistant hybrid expressing the modified Cry3A protein (MIR604), and the untransformed modern hybrid with the same genetic background as the MIR604 we used (isoline) as a second susceptible control. In general, the genotypes previously selected for resistance to western corn rootworm larval feeding had less damage, fewer larvae recovered, smaller larvae recovered, and fewer adults recovered than the susceptible controls. SUM2162 was significantly less damaged than all other native sources of resistance. Western corn rootworm larvae recovered from SUM2162 and SUM2068 were significantly smaller in terms of head capsule width and average weight than larvae recovered from all other maize genotypes, indicating that antibiosis is a mechanism of resistance for these two hybrids.

Adaptation and invasiveness of western corn rootworm: intensifying research on a worsening pest.

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is an established insect pest of maize (Zea mays L.) in North America. The rotation of maize with another crop, principally soybeans, Glycine max (L.), was the primary management strategy utilized by North American producers and remained highly effective until the mid-1990s. In 1995, widespread and severe root injury occurred in east-central Illinois and northern Indiana maize fields that had been annually rotated with soybeans on a regular basis for several decades. The failure of this cultural tactic from a pest management perspective was attributed to a behavioral adaptation by a variant western corn rootworm that had lost fidelity to maize for egg laying. In 1992, an infestation of western corn rootworm was found within a small maize field near the Belgrade Airport. By 2007, the presence of this insect pest had been confirmed in 20 European countries. More recent molecular studies have confirmed that at least three separate invasions (until 2004) of western corn rootworms have occurred in Europe, increasing the risk that rotation-resistant western corn rootworms will be introduced into a new continent. Although biological control and use of conventional resistant maize hybrids have not achieved widespread success in the management of western corn rootworms in North America, these tactics are being evaluated in Europe.