A Kinesin-14 Motor Activates Neocentromeres to Promote Meiotic Drive in Maize.

Maize abnormal chromosome 10 (Ab10) encodes a classic example of true meiotic drive that converts heterochromatic regions called knobs into motile neocentromeres that are preferentially transmitted to egg cells. Here, we identify a cluster of eight genes on Ab10, called the Kinesin driver (Kindr) complex, that are required for both neocentromere motility and preferential transmission. Two meiotic drive mutants that lack neocentromere activity proved to be kindr epimutants with increased DNA methylation across the entire gene cluster. RNAi of Kindr induced a third epimutant and corresponding loss of meiotic drive. Kinesin gliding assays and immunolocalization revealed that KINDR is a functional minus-end-directed kinesin that localizes specifically to knobs containing 180 bp repeats. Sequence comparisons suggest that Kindr diverged from a Kinesin-14A ancestor ∼12 mya and has driven the accumulation of > 500 Mb of knob repeats and affected the segregation of thousands of genes linked to knobs on all 10 chromosomes.

Diamonds in the not-so-rough: Wild relative diversity hidden in crop genomes.

Crop production is becoming an increasing challenge as the global population grows and the climate changes. Modern cultivated crop species are selected for productivity under optimal growth environments and have often lost genetic variants that could allow them to adapt to diverse, and now rapidly changing, environments. These genetic variants are often present in their closest wild relatives, but so are less desirable traits. How to preserve and effectively utilize the rich genetic resources that crop wild relatives offer while avoiding detrimental variants and maladaptive genetic contributions is a central challenge for ongoing crop improvement. This Essay explores this challenge and potential paths that could lead to a solution.

Conflict over fertilization underlies the transient evolution of reinforcement.

When two species meet in secondary contact, the production of low fitness hybrids may be prevented by the adaptive evolution of increased prezygotic isolation, a process known as reinforcement. Theoretical challenges to the evolution of reinforcement are generally cast as a coordination problem, i.e., "how can statistical associations between traits and preferences be maintained in the face of recombination?" However, the evolution of reinforcement also poses a potential conflict between mates. For example, the opportunity costs to hybridization may differ between the sexes or species. This is particularly likely for reinforcement based on postmating prezygotic (PMPZ) incompatibilities, as the ability to fertilize both conspecific and heterospecific eggs is beneficial to male gametes, but heterospecific mating may incur a cost for female gametes. We develop a population genetic model of interspecific conflict over reinforcement inspired by "gametophytic factors", which act as PMPZ barriers among Zea mays subspecies. We demonstrate that this conflict results in the transient evolution of reinforcement-after females adaptively evolve to reject gametes lacking a signal common in conspecific gametes, this gamete signal adaptively introgresses into the other population. Ultimately, the male gamete signal fixes in both species, and isolation returns to pre-reinforcement levels. We interpret geographic patterns of isolation among Z. mays subspecies considering these findings and suggest when and how this conflict can be resolved. Our results suggest that sexual conflict over fertilization may pose an understudied obstacle to the evolution of reinforcement.

An adaptive teosinte mexicana introgression modulates phosphatidylcholine levels and is associated with maize flowering time.

Native Americans domesticated maize (Zea mays ssp. mays) from lowland teosinte parviglumis (Zea mays ssp. parviglumis) in the warm Mexican southwest and brought it to the highlands of Mexico and South America where it was exposed to lower temperatures that imposed strong selection on flowering time. Phospholipids are important metabolites in plant responses to low-temperature and phosphorus availability and have been suggested to influence flowering time. Here, we combined linkage mapping with genome scans to identify High PhosphatidylCholine 1 (HPC1), a gene that encodes a phospholipase A1 enzyme, as a major driver of phospholipid variation in highland maize. Common garden experiments demonstrated strong genotype-by-environment interactions associated with variation at HPC1, with the highland HPC1 allele leading to higher fitness in highlands, possibly by hastening flowering. The highland maize HPC1 variant resulted in impaired function of the encoded protein due to a polymorphism in a highly conserved sequence. A meta-analysis across HPC1 orthologs indicated a strong association between the identity of the amino acid at this position and optimal growth in prokaryotes. Mutagenesis of HPC1 via genome editing validated its role in regulating phospholipid metabolism. Finally, we showed that the highland HPC1 allele entered cultivated maize by introgression from the wild highland teosinte Zea mays ssp. mexicana and has been maintained in maize breeding lines from the Northern United States, Canada, and Europe. Thus, HPC1 introgressed from teosinte mexicana underlies a large metabolic QTL that modulates phosphatidylcholine levels and has an adaptive effect at least in part via induction of early flowering time.

Unraveling Prevalence and Effects of Deleterious Mutations in Maize Elite Lines across Decades of Modern Breeding.

Future breeding is likely to involve the detection and removal of deleterious alleles, which are mutations that negatively affect crop fitness. However, little is known about the prevalence of such mutations and their effects on phenotypic traits in the context of modern crop breeding. To address this, we examined the number and frequency of deleterious mutations in 350 elite maize inbred lines developed over the past few decades in China and the United States. Our findings reveal an accumulation of weakly deleterious mutations and a decrease in strongly deleterious mutations, indicating the dominant effects of genetic drift and purifying selection for the two types of mutations, respectively. We also discovered that slightly deleterious mutations, when at lower frequencies, were more likely to be heterozygous in the developed hybrids. This is consistent with complementation as a potential explanation for heterosis. Subsequently, we found that deleterious mutations accounted for more of the variation in phenotypic traits than nondeleterious mutations with matched minor allele frequencies, especially for traits related to leaf angle and flowering time. Moreover, we detected fewer deleterious mutations in the promoter and gene body regions of differentially expressed genes across breeding eras than in nondifferentially expressed genes. Overall, our results provide a comprehensive assessment of the prevalence and impact of deleterious mutations in modern maize breeding and establish a useful baseline for future maize improvement efforts.

Conserved noncoding sequences provide insights into regulatory sequence and loss of gene expression in maize.

Thousands of species will be sequenced in the next few years; however, understanding how their genomes work, without an unlimited budget, requires both molecular and novel evolutionary approaches. We developed a sensitive sequence alignment pipeline to identify conserved noncoding sequences (CNSs) in the Andropogoneae tribe (multiple crop species descended from a common ancestor ∼18 million years ago). The Andropogoneae share similar physiology while being tremendously genomically diverse, harboring a broad range of ploidy levels, structural variation, and transposons. These contribute to the potential of Andropogoneae as a powerful system for studying CNSs and are factors we leverage to understand the function of maize CNSs. We found that 86% of CNSs were comprised of annotated features, including introns, UTRs, putative cis-regulatory elements, chromatin loop anchors, noncoding RNA (ncRNA) genes, and several transposable element superfamilies. CNSs were enriched in active regions of DNA replication in the early S phase of the mitotic cell cycle and showed different DNA methylation ratios compared to the genome-wide background. More than half of putative cis-regulatory sequences (identified via other methods) overlapped with CNSs detected in this study. Variants in CNSs were associated with gene expression levels, and CNS absence contributed to loss of gene expression. Furthermore, the evolution of CNSs was associated with the functional diversification of duplicated genes in the context of maize subgenomes. Our results provide a quantitative understanding of the molecular processes governing the evolution of CNSs in maize.

The genomic ecosystem of transposable elements in maize.

Transposable elements (TEs) constitute the majority of flowering plant DNA, reflecting their tremendous success in subverting, avoiding, and surviving the defenses of their host genomes to ensure their selfish replication. More than 85% of the sequence of the maize genome can be ascribed to past transposition, providing a major contribution to the structure of the genome. Evidence from individual loci has informed our understanding of how transposition has shaped the genome, and a number of individual TE insertions have been causally linked to dramatic phenotypic changes. Genome-wide analyses in maize and other taxa have frequently represented TEs as a relatively homogeneous class of fragmentary relics of past transposition, obscuring their evolutionary history and interaction with their host genome. Using an updated annotation of structurally intact TEs in the maize reference genome, we investigate the family-level dynamics of TEs in maize. Integrating a variety of data, from descriptors of individual TEs like coding capacity, expression, and methylation, as well as similar features of the sequence they inserted into, we model the relationship between attributes of the genomic environment and the survival of TE copies and families. In contrast to the wholesale relegation of all TEs to a single category of junk DNA, these differences reveal a diversity of survival strategies of TE families. Together these generate a rich ecology of the genome, with each TE family representing the evolution of a distinct ecological niche. We conclude that while the impact of transposition is highly family- and context-dependent, a family-level understanding of the ecology of TEs in the genome can refine our ability to predict the role of TEs in generating genetic and phenotypic diversity.

Selective sorting of ancestral introgression in maize and teosinte along an elevational cline.

While often deleterious, hybridization can also be a key source of genetic variation and pre-adapted haplotypes, enabling rapid evolution and niche expansion. Here we evaluate these opposing selection forces on introgressed ancestry between maize (Zea mays ssp. mays) and its wild teosinte relative, mexicana (Zea mays ssp. mexicana). Introgression from ecologically diverse teosinte may have facilitated maize's global range expansion, in particular to challenging high elevation regions (> 1500 m). We generated low-coverage genome sequencing data for 348 maize and mexicana individuals to evaluate patterns of introgression in 14 sympatric population pairs, spanning the elevational range of mexicana, a teosinte endemic to the mountains of Mexico. While recent hybrids are commonly observed in sympatric populations and mexicana demonstrates fine-scale local adaptation, we find that the majority of mexicana ancestry tracts introgressed into maize over 1000 generations ago. This mexicana ancestry seems to have maintained much of its diversity and likely came from a common ancestral source, rather than contemporary sympatric populations, resulting in relatively low FST between mexicana ancestry tracts sampled from geographically distant maize populations. Introgressed mexicana ancestry in maize is reduced in lower-recombination rate quintiles of the genome and around domestication genes, consistent with pervasive selection against introgression. However, we also find mexicana ancestry increases across the sampled elevational gradient and that high introgression peaks are most commonly shared among high-elevation maize populations, consistent with introgression from mexicana facilitating adaptation to the highland environment. In the other direction, we find patterns consistent with adaptive and clinal introgression of maize ancestry into sympatric mexicana at many loci across the genome, suggesting that maize also contributes to adaptation in mexicana, especially at the lower end of its elevational range. In sympatric maize, in addition to high introgression regions we find many genomic regions where selection for local adaptation maintains steep gradients in introgressed mexicana ancestry across elevation, including at least two inversions: the well-characterized 14 Mb Inv4m on chromosome 4 and a novel 3 Mb inversion Inv9f surrounding the macrohairless1 locus on chromosome 9. Most outlier loci with high mexicana introgression show no signals of sweeps or local sourcing from sympatric populations and so likely represent ancestral introgression sorted by selection, resulting in correlated but distinct outcomes of introgression in different contemporary maize populations.

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.].

Domestication reshaped the genetic basis of inbreeding depression in a maize landrace compared to its wild relative, teosinte.

Inbreeding depression is the reduction in fitness and vigor resulting from mating of close relatives observed in many plant and animal species. The extent to which the genetic load of mutations contributing to inbreeding depression is due to large-effect mutations versus variants with very small individual effects is unknown and may be affected by population history. We compared the effects of outcrossing and self-fertilization on 18 traits in a landrace population of maize, which underwent a population bottleneck during domestication, and a neighboring population of its wild relative teosinte. Inbreeding depression was greater in maize than teosinte for 15 of 18 traits, congruent with the greater segregating genetic load in the maize population that we predicted from sequence data. Parental breeding values were highly consistent between outcross and selfed offspring, indicating that additive effects determine most of the genetic value even in the presence of strong inbreeding depression. We developed a novel linkage scan to identify quantitative trait loci (QTL) representing large-effect rare variants carried by only a single parent, which were more important in teosinte than maize. Teosinte also carried more putative juvenile-acting lethal variants identified by segregation distortion. These results suggest a mixture of mostly polygenic, small-effect partially recessive effects in linkage disequilibrium underlying inbreeding depression, with an additional contribution from rare larger-effect variants that was more important in teosinte but depleted in maize following the domestication bottleneck. Purging associated with the maize domestication bottleneck may have selected against some large effect variants, but polygenic load is harder to purge and overall segregating mutational burden increased in maize compared to teosinte.

Allele-specific Expression Reveals Multiple Paths to Highland Adaptation in Maize.

Maize is a staple food of smallholder farmers living in highland regions up to 4,000 m above sea level worldwide. Mexican and South American highlands are two major highland maize growing regions, and population genetic data suggest the maize's adaptation to these regions occurred largely independently, providing a case study for convergent evolution. To better understand the mechanistic basis of highland adaptation, we crossed maize landraces from 108 highland and lowland sites of Mexico and South America with the inbred line B73 to produce F1 hybrids and grew them in both highland and lowland sites in Mexico. We identified thousands of genes with divergent expression between highland and lowland populations. Hundreds of these genes show patterns of convergent evolution between Mexico and South America. To dissect the genetic architecture of the divergent gene expression, we developed a novel allele-specific expression analysis pipeline to detect genes with divergent functional cis-regulatory variation between highland and lowland populations. We identified hundreds of genes with divergent cis-regulation between highland and lowland landrace alleles, with 20 in common between regions, further suggesting convergence in the genes underlying highland adaptation. Further analyses suggest multiple mechanisms contribute to this convergence in gene regulation. Although the vast majority of evolutionary changes associated with highland adaptation were region specific, our findings highlight an important role for convergence at the gene expression and gene regulation levels as well.

Improved maize reference genome with single-molecule technologies.

Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation. These resources facilitate the determination of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions. Here we report the assembly and annotation of a reference genome of maize, a genetic and agricultural model species, using single-molecule real-time sequencing and high-resolution optical mapping. Relative to the previous reference genome, our assembly features a 52-fold increase in contig length and notable improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed more than 130,000 intact transposable elements, allowing us to identify transposable element lineage expansions that are unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by single-molecule real-time sequencing. In addition, comparative optical mapping of two other inbred maize lines revealed a prevalence of deletions in regions of low gene density and maize lineage-specific genes.

The genetic architecture of the maize progenitor, teosinte, and how it was altered during maize domestication.

The genetics of domestication has been extensively studied ever since the rediscovery of Mendel's law of inheritance and much has been learned about the genetic control of trait differences between crops and their ancestors. Here, we ask how domestication has altered genetic architecture by comparing the genetic architecture of 18 domestication traits in maize and its ancestor teosinte using matched populations. We observed a strongly reduced number of QTL for domestication traits in maize relative to teosinte, which is consistent with the previously reported depletion of additive variance by selection during domestication. We also observed more dominance in maize than teosinte, likely a consequence of selective removal of additive variants. We observed that large effect QTL have low minor allele frequency (MAF) in both maize and teosinte. Regions of the genome that are strongly differentiated between teosinte and maize (high FST) explain less quantitative variation in maize than teosinte, suggesting that, in these regions, allelic variants were brought to (or near) fixation during domestication. We also observed that genomic regions of high recombination explain a disproportionately large proportion of heritable variance both before and after domestication. Finally, we observed that about 75% of the additive variance in both teosinte and maize is "missing" in the sense that it cannot be ascribed to detectable QTL and only 25% of variance maps to specific QTL. This latter result suggests that morphological evolution during domestication is largely attributable to very large numbers of QTL of very small effect.

Molecular Parallelism Underlies Convergent Highland Adaptation of Maize Landraces.

Convergent phenotypic evolution provides some of the strongest evidence for adaptation. However, the extent to which recurrent phenotypic adaptation has arisen via parallelism at the molecular level remains unresolved, as does the evolutionary origin of alleles underlying such adaptation. Here, we investigate genetic mechanisms of convergent highland adaptation in maize landrace populations and evaluate the genetic sources of recurrently selected alleles. Population branch excess statistics reveal substantial evidence of parallel adaptation at the level of individual single-nucleotide polymorphism (SNPs), genes, and pathways in four independent highland maize populations. The majority of convergently selected SNPs originated via migration from a single population, most likely in the Mesoamerican highlands, while standing variation introduced by ancient gene flow was also a contributor. Polygenic adaptation analyses of quantitative traits reveal that alleles affecting flowering time are significantly associated with elevation, indicating the flowering time pathway was targeted by highland adaptation. In addition, repeatedly selected genes were significantly enriched in the flowering time pathway, indicating their significance in adapting to highland conditions. Overall, our study system represents a promising model to study convergent evolution in plants with potential applications to crop adaptation across environmental gradients.

Genetic architecture and selective sweeps after polygenic adaptation to distant trait optima.

Understanding the genetic basis of phenotypic adaptation to changing environments is an essential goal of population and quantitative genetics. While technological advances now allow interrogation of genome-wide genotyping data in large panels, our understanding of the process of polygenic adaptation is still limited. To address this limitation, we use extensive forward-time simulation to explore the impacts of variation in demography, trait genetics, and selection on the rate and mode of adaptation and the resulting genetic architecture. We simulate a population adapting to an optimum shift, modeling sequence variation for 20 QTL for each of 12 different demographies for 100 different traits varying in the effect size distribution of new mutations, the strength of stabilizing selection, and the contribution of the genomic background. We then use random forest regression approaches to learn the relative importance of input parameters in determining a number of aspects of the process of adaptation, including the speed of adaptation, the relative frequency of hard sweeps and sweeps from standing variation, or the final genetic architecture of the trait. We find that selective sweeps occur even for traits under relatively weak selection and where the genetic background explains most of the variation. Though most sweeps occur from variation segregating in the ancestral population, new mutations can be important for traits under strong stabilizing selection that undergo a large optimum shift. We also show that population bottlenecks and expansion impact overall genetic variation as well as the relative importance of sweeps from standing variation and the speed with which adaptation can occur. We then compare our results to two traits under selection during maize domestication, showing that our simulations qualitatively recapitulate differences between them. Overall, our results underscore the complex population genetics of individual loci in even relatively simple quantitative trait models, but provide a glimpse into the factors that drive this complexity and the potential of these approaches for understanding polygenic adaptation.

Parallel altitudinal clines reveal trends in adaptive evolution of genome size in Zea mays.

While the vast majority of genome size variation in plants is due to differences in repetitive sequence, we know little about how selection acts on repeat content in natural populations. Here we investigate parallel changes in intraspecific genome size and repeat content of domesticated maize (Zea mays) landraces and their wild relative teosinte across altitudinal gradients in Mesoamerica and South America. We combine genotyping, low coverage whole-genome sequence data, and flow cytometry to test for evidence of selection on genome size and individual repeat abundance. We find that population structure alone cannot explain the observed variation, implying that clinal patterns of genome size are maintained by natural selection. Our modeling additionally provides evidence of selection on individual heterochromatic knob repeats, likely due to their large individual contribution to genome size. To better understand the phenotypes driving selection on genome size, we conducted a growth chamber experiment using a population of highland teosinte exhibiting extensive variation in genome size. We find weak support for a positive correlation between genome size and cell size, but stronger support for a negative correlation between genome size and the rate of cell production. Reanalyzing published data of cell counts in maize shoot apical meristems, we then identify a negative correlation between cell production rate and flowering time. Together, our data suggest a model in which variation in genome size is driven by natural selection on flowering time across altitudinal clines, connecting intraspecific variation in repetitive sequence to important differences in adaptive phenotypes.

Transposable elements contribute to dynamic genome content in maize.

Transposable elements (TEs) are ubiquitous components of eukaryotic genomes and can create variation in genome organization and content. Most maize genomes are composed of TEs. We developed an approach to define shared and variable TE insertions across genome assemblies and applied this method to four maize genomes (B73, W22, Mo17 and PH207) with uniform structural annotations of TEs. Among these genomes we identified approximately 400 000 TEs that are polymorphic, encompassing 1.6 Gb of variable TE sequence. These polymorphic TEs include a combination of recent transposition events as well as deletions of older TEs. There are examples of polymorphic TEs within each of the superfamilies of TEs and they are found distributed across the genome, including in regions of recent shared ancestry among individuals. There are many examples of polymorphic TEs within or near maize genes. In addition, there are 2380 gene annotations in the B73 genome that are located within variable TEs, providing evidence for the role of TEs in contributing to the substantial differences in annotated gene content among these genotypes. TEs are highly variable in our survey of four temperate maize genomes, highlighting the major contribution of TEs in driving variation in genome organization and gene content. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://github.com/SNAnderson/maizeTE_variation; https://mcstitzer.github.io/maize_TEs.

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.

Gene Fractionation and Function in the Ancient Subgenomes of Maize.

The maize genome experienced an ancient whole genome duplication ∼10 MYA and the duplicate subgenomes have since experienced reciprocal gene loss such that many genes have returned to single-copy status. This process has not affected the subgenomes equally; reduced gene expression in one of the subgenomes mitigates the consequences of mutations and gene deletions and is thought to drive higher rates of fractionation. Here, we use published data to show that, in accordance with predictions of this model, paralogs with greater expression contribute more to phenotypic variation compared with their lowly expressed counterparts. Furthermore, paralogous genes in the least-fractionated subgenome account for a greater degree of phenotypic diversity than those resident on the more-fractionated subgenome. Intriguingly, analysis of singleton genes reveals this difference persists even after fractionation is complete. Additionally, we show that the two subgenomes of maize may differ in their epigenetic profiles.