Correction: Incomplete dominance of deleterious alleles contributes substantially to trait variation and heterosis in maize.

[This corrects the article DOI: 10.1371/journal.pgen.1007019.].

Dominance Effects and Functional Enrichments Improve Prediction of Agronomic Traits in Hybrid Maize.

Single-cross hybrids have been critical to the improvement of maize (Zea mays L.), but the characterization of their genetic architectures remains challenging. Previous studies of hybrid maize have shown the contribution of within-locus complementation effects (dominance) and their differential importance across functional classes of loci. However, they have generally considered panels of limited genetic diversity, and have shown little benefit from genomic prediction based on dominance or functional enrichments. This study investigates the relevance of dominance and functional classes of variants in genomic models for agronomic traits in diverse populations of hybrid maize. We based our analyses on a diverse panel of inbred lines crossed with two testers representative of the major heterotic groups in the U.S. (1106 hybrids), as well as a collection of 24 biparental populations crossed with a single tester (1640 hybrids). We investigated three agronomic traits: days to silking (DTS), plant height (PH), and grain yield (GY). Our results point to the presence of dominance for all traits, but also among-locus complementation (epistasis) for DTS and genotype-by-environment interactions for GY. Consistently, dominance improved genomic prediction for PH only. In addition, we assessed enrichment of genetic effects in classes defined by genic regions (gene annotation), structural features (recombination rate and chromatin openness), and evolutionary features (minor allele frequency and evolutionary constraint). We found support for enrichment in genic regions and subsequent improvement of genomic prediction for all traits. Our results suggest that dominance and gene annotations improve genomic prediction across diverse populations in hybrid maize.

Optimal Designs for Genomic Selection in Hybrid Crops.

Improved capacity of genomics and biotechnology has greatly enhanced genetic studies in different areas. Genomic selection exploits the genotype-to-phenotype relationship at the whole-genome level and is being implemented in many crops. Here we show that design-thinking and data-mining techniques can be leveraged to optimize genomic prediction of hybrid performance. We phenotyped a set of 276 maize hybrids generated by crossing founder inbreds of nested association mapping populations for flowering time, ear height, and grain yield. With 10 296 310 SNPs available from the parental inbreds, we explored the patterns of genomic relationships and phenotypic variation to establish training samples based on clustering, graphic network analysis, and genetic mating scheme. Our analysis showed that training set designs outperformed random sampling and earlier methods that either minimize the mean of prediction error variance or maximize the mean of generalized coefficient of determination. Additional analyses of 2556 wheat hybrids from an early-stage hybrid breeding system and 1439 rice hybrids from an established hybrid breeding system validated the approaches. Together, we demonstrated that effective genomic prediction models can be established with a training set 2%-13% of the size of the whole set, enabling an efficient exploration of enormous inference space of genetic combinations.

Incomplete dominance of deleterious alleles contributes substantially to trait variation and heterosis in maize.

Deleterious alleles have long been proposed to play an important role in patterning phenotypic variation and are central to commonly held ideas explaining the hybrid vigor observed in the offspring of a cross between two inbred parents. We test these ideas using evolutionary measures of sequence conservation to ask whether incorporating information about putatively deleterious alleles can inform genomic selection (GS) models and improve phenotypic prediction. We measured a number of agronomic traits in both the inbred parents and hybrids of an elite maize partial diallel population and re-sequenced the parents of the population. Inbred elite maize lines vary for more than 350,000 putatively deleterious sites, but show a lower burden of such sites than a comparable set of traditional landraces. Our modeling reveals widespread evidence for incomplete dominance at these loci, and supports theoretical models that more damaging variants are usually more recessive. We identify haplotype blocks using an identity-by-decent (IBD) analysis and perform genomic prediction analyses in which we weigh blocks on the basis of complementation for segregating putatively deleterious variants. Cross-validation results show that incorporating sequence conservation in genomic selection improves prediction accuracy for grain yield and other fitness-related traits as well as heterosis for those traits. Our results provide empirical support for an important role for incomplete dominance of deleterious alleles in explaining heterosis and demonstrate the utility of incorporating functional annotation in phenotypic prediction and plant breeding.

Expanding Maize Genetic Resources with Predomestication Alleles: Maize-Teosinte Introgression Populations.

Teosinte ( subsp. H. H. Iltis & Doebley) has greater genetic diversity than maize inbreds and landraces ( subsp. ). There are, however, limited genetic resources to efficiently evaluate and tap this diversity. To broaden resources for genetic diversity studies in maize, we developed and evaluated 928 near-isogenic introgression lines (NILs) from 10 teosinte accessions in the B73 background. Joint linkage analysis of the 10 introgression populations identified several large-effect quantitative trait loci (QTL) for days to anthesis (DTA), kernel row number (KRN), and 50-kernel weight (Wt50k). Our results confirm prior reports of kernel domestication loci and identify previously uncharacterized QTL with a range of allelic effects enabling future research into the genetic basis of these traits. Additionally, we used a targeted set of NILs to validate the effects of a KRN QTL located on chromosome 2. These introgression populations offer novel tools for QTL discovery and validation as well as a platform for initiating fine mapping.

Genetic Analysis of Kernel Traits in Maize-Teosinte Introgression Populations.

Seed traits have been targeted by human selection during the domestication of crop species as a way to increase the caloric and nutritional content of food during the transition from hunter-gather to early farming societies. The primary seed trait under selection was likely seed size/weight as it is most directly related to overall grain yield. Additional seed traits involved in seed shape may have also contributed to larger grain. Maize (Zea mays ssp. mays) kernel weight has increased more than 10-fold in the 9000 years since domestication from its wild ancestor, teosinte (Z. mays ssp. parviglumis). In order to study how size and shape affect kernel weight, we analyzed kernel morphometric traits in a set of 10 maize-teosinte introgression populations using digital imaging software. We identified quantitative trait loci (QTL) for kernel area and length with moderate allelic effects that colocalize with kernel weight QTL. Several genomic regions with strong effects during maize domestication were detected, and a genetic framework for kernel traits was characterized by complex pleiotropic interactions. Our results both confirm prior reports of kernel domestication loci and identify previously uncharacterized QTL with a range of allelic effects, enabling future research into the genetic basis of these traits.

Identification of QTL for resistance to Mediterranean corn borer in a maize tropical line to improve temperate germplasm.

BACKGROUND: A QTL mapping study for maize resistance to the Mediterranean corn borer (MCB) was performed with a RIL population derived from the cross B73 × CML103. To develop commercial inbreds of maize resistant to the MCB for use in Europe, it would be useful to transfer resistance from tropical germplasm like the subtropical inbred CML103 to temperate lines. The inbred B73 was chosen as representative of the Stiff Stock heterotic group, a major heterotic group used in hybrid grown in both North American and Europe. The objectives were to study the architecture of genetic factors for resistance to MCB and to check the feasibility of using marker-assisted selection (MAS) for transferring those genetic factors. RESULTS: Eight quantitative trait loci (QTL) were declared significant for resistance traits and eight QTL were located for agronomic traits. Alleles from CML103 at QTL significant for tunnel length could reduce tunnel length made for MCB in inbred B73 in more than 8 cm; favorable alleles for yield were also found in CML103 and no genetic correlation coefficient between tunnel length and yield was detected. CONCLUSIONS: MAS for transferring resistance genes to corn borer attack from CML103 to B73 could be successful based on cross validation results and a negative effect on yield would not be expected.

The Genomic Impacts of Drift and Selection for Hybrid Performance in Maize.

Although maize is naturally an outcrossing organism, modern breeding utilizes highly inbred lines in controlled crosses to produce hybrids. The U.S. Department of Agriculture's reciprocal recurrent selection experiment between the Iowa Stiff Stalk Synthetic (BSSS) and the Iowa Corn Borer Synthetic No. 1 (BSCB1) populations represents one of the longest running experiments to understand the response to selection for hybrid performance. To investigate the genomic impact of this selection program, we genotyped the progenitor lines and >600 individuals across multiple cycles of selection using a genome-wide panel of ∼40,000 SNPs. We confirmed previous results showing a steady temporal decrease in genetic diversity within populations and a corresponding increase in differentiation between populations. Thanks to detailed historical information on experimental design, we were able to perform extensive simulations using founder haplotypes to replicate the experiment in the absence of selection. These simulations demonstrate that while most of the observed reduction in genetic diversity can be attributed to genetic drift, heterozygosity in each population has fallen more than expected. We then took advantage of our high-density genotype data to identify extensive regions of haplotype fixation and trace haplotype ancestry to single founder inbred lines. The vast majority of regions showing such evidence of selection differ between the two populations, providing evidence for the dominance model of heterosis. We discuss how this pattern is likely to occur during selection for hybrid performance and how it poses challenges for dissecting the impacts of modern breeding and selection on the maize genome.

Prolonged expression of the BX1 signature enzyme is associated with a recombination hotspot in the benzoxazinoid gene cluster in Zea mays.

Benzoxazinoids represent preformed protective and allelopathic compounds. The main benzoxazinoid in maize (Zea mays L.) is 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA). DIMBOA confers resistance to herbivores and microbes. Protective concentrations are found predominantly in young plantlets. We made use of the genetic diversity present in the maize nested association mapping (NAM) panel to identify lines with significant benzoxazinoid concentrations at later developmental stages. At 24 d after imbibition (dai), only three lines, including Mo17, showed effective DIMBOA concentrations of 1.5mM or more; B73, by contrast, had low a DIMBOA content. Mapping studies based on Mo17 and B73 were performed to reveal mechanisms that influence the DIMBOA level in 24 dai plants. A major quantitative trait locus mapped to the Bx gene cluster located on the short arm of chromosome 4, which encodes the DIMBOA biosynthetic genes. Mo17 was distinguished from all other NAM lines by high transcriptional expression of the Bx1 gene at later developmental stages. Bx1 encodes the signature enzyme of the pathway. In Mo17×B73 hybrids at 24 dai, only the Mo17 Bx1 allele transcript was detected. A 3.9kb cis-element, termed DICE (distal cis-element), that is located in the Bx gene cluster approximately 140 kb upstream of Bx1, was required for high Bx1 transcript levels during later developmental stages in Mo17. The DICE region was a hotspot of meiotic recombination. Genetic analysis revealed that high 24 dai DIMBOA concentrations were not strictly dependent on high Bx1 transcript levels. However, constitutive expression of Bx1 in transgenics increased DIMBOA levels at 24 dai, corroborating a correlation between DIMBOA content and Bx1 transcription.

Insights into the effects of long-term artificial selection on seed size in maize.

Grain produced from cereal crops is a primary source of human food and animal feed worldwide. To understand the genetic basis of seed-size variation, a grain yield component, we conducted a genome-wide scan to detect evidence of selection in the maize Krug Yellow Dent long-term divergent seed-size selection experiment. Previous studies have documented significant phenotypic divergence between the populations. Allele frequency estimates for ∼3 million single nucleotide polymorphisms (SNPs) in the base population and selected populations were estimated from pooled whole-genome resequencing of 48 individuals per population. Using FST values across sliding windows, 94 divergent regions with a median of six genes per region were identified. Additionally, 2729 SNPs that reached fixation in both selected populations with opposing fixed alleles were identified, many of which clustered in two regions of the genome. Copy-number variation was highly prevalent between the selected populations, with 532 total regions identified on the basis of read-depth variation and comparative genome hybridization. Regions important for seed weight in natural variation were identified in the maize nested association mapping population. However, the number of regions that overlapped with the long-term selection experiment did not exceed that expected by chance, possibly indicating unique sources of variation between the two populations. The results of this study provide insights into the genetic elements underlying seed-size variation in maize and could also have applications for other cereal crops.

The genetic architecture of maize height.

Height is one of the most heritable and easily measured traits in maize (Zea mays L.). Given a pedigree or estimates of the genomic identity-by-state among related plants, height is also accurately predictable. But, mapping alleles explaining natural variation in maize height remains a formidable challenge. To address this challenge, we measured the plant height, ear height, flowering time, and node counts of plants grown in >64,500 plots across 13 environments. These plots contained >7300 inbreds representing most publically available maize inbreds in the United States and families of the maize Nested Association Mapping (NAM) panel. Joint-linkage mapping of quantitative trait loci (QTL), fine mapping in near isogenic lines (NILs), genome-wide association studies (GWAS), and genomic best linear unbiased prediction (GBLUP) were performed. The heritability of maize height was estimated to be >90%. Mapping NAM family-nested QTL revealed the largest explained 2.1 ± 0.9% of height variation. The effects of two tropical alleles at this QTL were independently validated by fine mapping in NIL families. Several significant associations found by GWAS colocalized with established height loci, including brassinosteroid-deficient dwarf1, dwarf plant1, and semi-dwarf2. GBLUP explained >80% of height variation in the panels and outperformed bootstrap aggregation of family-nested QTL models in evaluations of prediction accuracy. These results revealed maize height was under strong genetic control and had a highly polygenic genetic architecture. They also showed that multiple models of genetic architecture differing in polygenicity and effect sizes can plausibly explain a population's variation in maize height, but they may vary in predictive efficacy.

The genetic architecture of maize stalk strength.

Stalk strength is an important trait in maize (Zea mays L.). Strong stalks reduce lodging and maximize harvestable yield. Studies show rind penetrometer resistance (RPR), or the force required to pierce a stalk rind with a spike, is a valid approximation of strength. We measured RPR across 4,692 recombinant inbreds (RILs) comprising the maize nested association mapping (NAM) panel derived from crosses of diverse inbreds to the inbred, B73. An intermated B73×Mo17 family (IBM) of 196 RILs and a panel of 2,453 diverse inbreds from the North Central Regional Plant Introduction Station (NCRPIS) were also evaluated. We measured RPR in three environments. Family-nested QTL were identified by joint-linkage mapping in the NAM panel. We also performed a genome-wide association study (GWAS) and genomic best linear unbiased prediction (GBLUP) in each panel. Broad sense heritability computed on a line means basis was low for RPR. Only 8 of 26 families had a heritability above 0.20. The NCRPIS diversity panel had a heritability of 0.54. Across NAM and IBM families, 18 family-nested QTL and 141 significant GWAS associations were identified for RPR. Numerous weak associations were also found in the NCRPIS diversity panel. However, few were linked to loci involved in phenylpropanoid and cellulose synthesis or vegetative phase transition. Using an identity-by-state (IBS) relationship matrix estimated from 1.6 million single nucleotide polymorphisms (SNPs) and RPR measures from 20% of the NAM panel, genomic prediction by GBLUP explained 64±2% of variation in the remaining RILs. In the NCRPIS diversity panel, an IBS matrix estimated from 681,257 SNPs and RPR measures from 20% of the panel explained 33±3% of variation in the remaining inbreds. These results indicate the high genetic complexity of stalk strength and the potential for genomic prediction to hasten its improvement.

Comprehensive genotyping of the USA national maize inbred seed bank.

BACKGROUND: Genotyping by sequencing, a new low-cost, high-throughput sequencing technology was used to genotype 2,815 maize inbred accessions, preserved mostly at the National Plant Germplasm System in the USA. The collection includes inbred lines from breeding programs all over the world. RESULTS: The method produced 681,257 single-nucleotide polymorphism (SNP) markers distributed across the entire genome, with the ability to detect rare alleles at high confidence levels. More than half of the SNPs in the collection are rare. Although most rare alleles have been incorporated into public temperate breeding programs, only a modest amount of the available diversity is present in the commercial germplasm. Analysis of genetic distances shows population stratification, including a small number of large clusters centered on key lines. Nevertheless, an average fixation index of 0.06 indicates moderate differentiation between the three major maize subpopulations. Linkage disequilibrium (LD) decays very rapidly, but the extent of LD is highly dependent on the particular group of germplasm and region of the genome. The utility of these data for performing genome-wide association studies was tested with two simply inherited traits and one complex trait. We identified trait associations at SNPs very close to known candidate genes for kernel color, sweet corn, and flowering time; however, results suggest that more SNPs are needed to better explore the genetic architecture of complex traits. CONCLUSIONS: The genotypic information described here allows this publicly available panel to be exploited by researchers facing the challenges of sustainable agriculture through better knowledge of the nature of genetic diversity.

Comparative population genomics of maize domestication and improvement.

Domestication and plant breeding are ongoing 10,000-year-old evolutionary experiments that have radically altered wild species to meet human needs. Maize has undergone a particularly striking transformation. Researchers have sought for decades to identify the genes underlying maize evolution, but these efforts have been limited in scope. Here, we report a comprehensive assessment of the evolution of modern maize based on the genome-wide resequencing of 75 wild, landrace and improved maize lines. We find evidence of recovery of diversity after domestication, likely introgression from wild relatives, and evidence for stronger selection during domestication than improvement. We identify a number of genes with stronger signals of selection than those previously shown to underlie major morphological changes. Finally, through transcriptome-wide analysis of gene expression, we find evidence both consistent with removal of cis-acting variation during maize domestication and improvement and suggestive of modern breeding having increased dominance in expression while targeting highly expressed genes.

Maize HapMap2 identifies extant variation from a genome in flux.

Whereas breeders have exploited diversity in maize for yield improvements, there has been limited progress in using beneficial alleles in undomesticated varieties. Characterizing standing variation in this complex genome has been challenging, with only a small fraction of it described to date. Using a population genetics scoring model, we identified 55 million SNPs in 103 lines across pre-domestication and domesticated Zea mays varieties, including a representative from the sister genus Tripsacum. We find that structural variations are pervasive in the Z. mays genome and are enriched at loci associated with important traits. By investigating the drivers of genome size variation, we find that the larger Tripsacum genome can be explained by transposable element abundance rather than an allopolyploid origin. In contrast, intraspecies genome size variation seems to be controlled by chromosomal knob content. There is tremendous overlap in key gene content in maize and Tripsacum, suggesting that adaptations from Tripsacum (for example, perennialism and frost and drought tolerance) can likely be integrated into maize.

ZmCCT and the genetic basis of day-length adaptation underlying the postdomestication spread of maize.

Teosinte, the progenitor of maize, is restricted to tropical environments in Mexico and Central America. The pre-Columbian spread of maize from its center of origin in tropical Southern Mexico to the higher latitudes of the Americas required postdomestication selection for adaptation to longer day lengths. Flowering time of teosinte and tropical maize is delayed under long day lengths, whereas temperate maize evolved a reduced sensitivity to photoperiod. We measured flowering time of the maize nested association and diverse association mapping panels in the field under both short and long day lengths, and of a maize-teosinte mapping population under long day lengths. Flowering time in maize is a complex trait affected by many genes and the environment. Photoperiod response is one component of flowering time involving a subset of flowering time genes whose effects are strongly influenced by day length. Genome-wide association and targeted high-resolution linkage mapping identified ZmCCT, a homologue of the rice photoperiod response regulator Ghd7, as the most important gene affecting photoperiod response in maize. Under long day lengths ZmCCT alleles from diverse teosintes are consistently expressed at higher levels and confer later flowering than temperate maize alleles. Many maize inbred lines, including some adapted to tropical regions, carry ZmCCT alleles with no sensitivity to day length. Indigenous farmers of the Americas were remarkably successful at selecting on genetic variation at key genes affecting the photoperiod response to create maize varieties adapted to vastly diverse environments despite the hindrance of the geographic axis of the Americas and the complex genetic control of flowering time.

Genetic architecture of maize kernel composition in the nested association mapping and inbred association panels.

The maize (Zea mays) kernel plays a critical role in feeding humans and livestock around the world and in a wide array of industrial applications. An understanding of the regulation of kernel starch, protein, and oil is needed in order to manipulate composition to meet future needs. We conducted joint-linkage quantitative trait locus mapping and genome-wide association studies (GWAS) for kernel starch, protein, and oil in the maize nested association mapping population, composed of 25 recombinant inbred line families derived from diverse inbred lines. Joint-linkage mapping revealed that the genetic architecture of kernel composition traits is controlled by 21-26 quantitative trait loci. Numerous GWAS associations were detected, including several oil and starch associations in acyl-CoA:diacylglycerol acyltransferase1-2, a gene that regulates oil composition and quantity. Results from nested association mapping were verified in a 282 inbred association panel using both GWAS and candidate gene association approaches. We identified many beneficial alleles that will be useful for improving kernel starch, protein, and oil content.

A large maize (Zea mays L.) SNP genotyping array: development and germplasm genotyping, and genetic mapping to compare with the B73 reference genome.

SNP genotyping arrays have been useful for many applications that require a large number of molecular markers such as high-density genetic mapping, genome-wide association studies (GWAS), and genomic selection. We report the establishment of a large maize SNP array and its use for diversity analysis and high density linkage mapping. The markers, taken from more than 800,000 SNPs, were selected to be preferentially located in genes and evenly distributed across the genome. The array was tested with a set of maize germplasm including North American and European inbred lines, parent/F1 combinations, and distantly related teosinte material. A total of 49,585 markers, including 33,417 within 17,520 different genes and 16,168 outside genes, were of good quality for genotyping, with an average failure rate of 4% and rates up to 8% in specific germplasm. To demonstrate this array's use in genetic mapping and for the independent validation of the B73 sequence assembly, two intermated maize recombinant inbred line populations - IBM (B73×Mo17) and LHRF (F2×F252) - were genotyped to establish two high density linkage maps with 20,913 and 14,524 markers respectively. 172 mapped markers were absent in the current B73 assembly and their placement can be used for future improvements of the B73 reference sequence. Colinearity of the genetic and physical maps was mostly conserved with some exceptions that suggest errors in the B73 assembly. Five major regions containing non-colinearities were identified on chromosomes 2, 3, 6, 7 and 9, and are supported by both independent genetic maps. Four additional non-colinear regions were found on the LHRF map only; they may be due to a lower density of IBM markers in those regions or to true structural rearrangements between lines. Given the array's high quality, it will be a valuable resource for maize genetics and many aspects of maize breeding.

Distinct genetic architectures for male and female inflorescence traits of maize.

We compared the genetic architecture of thirteen maize morphological traits in a large population of recombinant inbred lines. Four traits from the male inflorescence (tassel) and three traits from the female inflorescence (ear) were measured and studied using linkage and genome-wide association analyses and compared to three flowering and three leaf traits previously studied in the same population. Inflorescence loci have larger effects than flowering and leaf loci, and ear effects are larger than tassel effects. Ear trait models also have lower predictive ability than tassel, flowering, or leaf trait models. Pleiotropic loci were identified that control elongation of ear and tassel, consistent with their common developmental origin. For these pleiotropic loci, the ear effects are larger than tassel effects even though the same causal polymorphisms are likely involved. This implies that the observed differences in genetic architecture are not due to distinct features of the underlying polymorphisms. Our results support the hypothesis that genetic architecture is a function of trait stability over evolutionary time, since the traits that changed most during the relatively recent domestication of maize have the largest effects.

Genome-wide association study of leaf architecture in the maize nested association mapping population.

US maize yield has increased eight-fold in the past 80 years, with half of the gain attributed to selection by breeders. During this time, changes in maize leaf angle and size have altered plant architecture, allowing more efficient light capture as planting density has increased. Through a genome-wide association study (GWAS) of the maize nested association mapping panel, we determined the genetic basis of important leaf architecture traits and identified some of the key genes. Overall, we demonstrate that the genetic architecture of the leaf traits is dominated by small effects, with little epistasis, environmental interaction or pleiotropy. In particular, GWAS results show that variations at the liguleless genes have contributed to more upright leaves. These results demonstrate that the use of GWAS with specially designed mapping populations is effective in uncovering the basis of key agronomic traits.