Potential genetic variance and the domestication of maize

Since Darwin, there has been a long and arduous struggle to understand the source and maintenance of natural genetic variation and its relationship to phenotype. The reason that this task is so difficult is that it requires integration of detailed, and as yet incomplete, knowledge from several biological disciplines, including evolutionary, population, and developmental genetics. In this 'post-genomic' era, it is relatively easy to identify differences in the DNA sequence between individuals. However, the task remains to delineate how this abundant genetic diversity actually contributes to phenotypic diversity. This necessitates tackling the problem of hidden genetic variation. Genetic polymorphisms can be conditionally cryptic, but have the potential to contribute to phenotypic variation in particular genetic backgrounds or under specific environmental conditions. A recent paper by Lauter and Doebley highlights the contribution of hidden genetic variation to traits characterizing the morphological evolution of modern maize from its wild grass-like progenitor teosinte.1 This work is the first to demonstrate hidden variance for selected (agronomically 'adaptive') traits in a well-characterized model for morphological evolution.     

Inheritance of the Morphological Differences between Maize and Teosinte: Comparison of Results for Two F(2) Populations

Molecular marker loci (MMLs) were employed to map quantitative trait loci (QTLs) in an F(2) population derived from a cross of maize (Zea mays ssp. mays) and its probable progenitor, teosinte (Z. mays ssp. parviglumis). A total of 50 significant associations (putative QTLs) between the MMLs and nine key traits that distinguish maize and teosinte were identified. Results from this analysis are compared with our previous analysis of an F(2) population derived from a cross of a different variety of maize and another subspecies of teosinte (Z. mays ssp. mexicana). For traits that measure the architectural differences between maize and teosinte, the two F(2) populations possessed similar suites of QTLs. For traits that measure components of yield, substantially different suites of QTLs were identified in the two populations. QTLs that control about 20% or more of the phenotypic variance for a trait in one population were detected in the other population 81% of the time, while QTLs that control less than 10% of the variance in one population were detected in the other population only 28% of the time. In our previously published analysis of the maize X ssp. mexicana population, we identified five regions of the genome that control most of the key morphological differences between maize and teosinte. These same five regions also control most of the differences in the maize X ssp. parviglumis population. Results from both populations support the hypothesis that a relatively small number of loci with large effects were involved in the early evolution of the key traits that distinguish maize and teosinte. It is suggested that loci with large effects on morphology may not be a specific feature of crop evolution, but rather a common phenomenon in plant evolution whenever a species invades a new niche with reduced competition.     

Evidence for a natural allelic series at the Maize domestication Locus teosinte branched1

Despite numerous quantitative trait loci and association mapping studies, our understanding of the extent to which natural allelic series contribute to the variation for complex traits is limited. In this study, we investigate the occurrence of a natural allelic series for complex traits at the teosinte branched1 (tb1) gene in natural populations of teosinte (Zea mays ssp. parviglumis, Z. mays ssp. mexicana, and Z. diploperennis). Previously, tb1 was shown to confer large effects on both plant architecture and ear morphology between domesticated maize and teosinte; however, the effect of tb1 on trait variation in natural populations of teosinte has not been investigated. We compare the effects of nine teosinte alleles of tb1 that were introgressed into an isogenic maize inbred background. Our results provide evidence for a natural allelic series at tb1 for several complex morphological traits. The teosinte introgressions separate into three distinct phenotypic classes, which correspond to the taxonomic origin of the alleles. The effects of the three allelic classes also correspond to known morphological differences between the teosinte taxa. Our results suggest that tb1 contributed to the morphological diversification of teosinte taxa as well as to the domestication of maize.     

A naturally occurring variant of Hsp90 that is associated with decanalization

The heat shock protein Hsp90 has been the focus of many studies since it was suggested that it acts to mediate the buffering of phenotypic variation. Hsp90-mediated buffering may result in the accumulation of cryptic genetic variation that, when released either as a consequence of environmental or genetic stress, increases the evolvability of a population. Recent studies using laboratory-induced mutations of Hsp90 and/or chemical inhibition to disrupt Hsp90 function confirm that Hsp90 can buffer cryptic genetic variation. We have previously identified a naturally occurring variant in the charged linker region of the Hsp90 gene, and now examine whether this variant is associated with altered levels of trait variability. The variant is associated with the release of cryptic genetic variation for canalized morphological (bristle) traits, but not for uncanalized morphological (wing and bristle) traits, and the effect on canalized traits depends on culture temperature. This suggests that natural genetic variation in Hsp90 may mediate the evolution of canalized morphological traits even if it does not influence the expression of variation for uncanalized traits.     

The evolutionary genetics of canalization

Evolutionary genetics has recently made enormous progress in understanding how genetic variation maps into phenotypic variation. However why some traits are phenotypically invariant despite apparent genetic and environmental changes has remained a major puzzle. In the 1940s, Conrad Hal Waddington coined the concept and term "canalization" to describe the robustness of phenotypes to perturbation; a similar concept was proposed by Waddington's contemporary Ivan Ivanovich Schmalhausen. This paper reviews what has been learned about canalization since Waddington. Canalization implies that a genotype's phenotype remains relatively invariant when individuals of a particular genotype are exposed to different environments (environmental canalization) or when individuals of the same single- or multilocus genotype differ in their genetic background (genetic canalization). Consequently, genetic canalization can be viewed as a particular kind of epistasis, and environmental canalization and phenotypic plasticity are two aspects of the same phenomenon. Canalization results in the accumulation of phenotypically cryptic genetic variation, which can be released after a "decanalizing" event. Thus, canalized genotypes maintain a cryptic potential for expressing particular phenotypes, which are only uncovered under particular decanalizing environmental or genetic conditions. Selection may then act on this newly released genetic variation. The accumulation of cryptic genetic variation by canalization may therefore increase evolvability at the population level by leading to phenotypic diversification under decanalizing conditions. On the other hand, under canalizing conditions, a major part of the segregating genetic variation may remain phenotypically cryptic; canalization may therefore, at least temporarily, constrain phenotypic evolution. Mechanistically, canalization can be understood in terms of transmission patterns, such as epistasis, pleiotropy, and genotype by environment interactions, and in terms of genetic redundancy, modularity, and emergent properties of gene networks and biochemical pathways. While different forms of selection can favor canalization, the requirements for its evolution are typically rather restrictive. Although there are several methods to detect canalization, there are still serious problems with unambiguously demonstrating canalization, particularly its adaptive value.     

Adaptive phenotypic divergence in an annual grass differs across biotic contexts*

Climate is a powerful force shaping adaptation within species, yet adaptation to climate occurs against a biotic background: species interactions can filter fitness consequences of genetic variation by altering phenotypic expression of genotypes. We investigated this process using populations of teosinte, a wild annual grass related to maize (Zea mays ssp. mexicana), sampling plants from 10 sites along an elevational gradient as well as rhizosphere biota from three of those sites. We grew half‐sibling teosinte families in each biota to test whether trait divergence among teosinte populations reflects adaptation or drift, and whether rhizosphere biota affect expression of diverged traits. We further assayed the influence of rhizosphere biota on contemporary additive genetic variation. We found that adaptation across environment shaped divergence of some traits, particularly flowering time and root biomass. We also observed that different rhizosphere biota shifted expressed values of these traits within teosinte populations and families and altered within‐population genetic variance and covariance. In sum, our results imply that changes in trait expression and covariance elicited by rhizosphere communities could have played a historical role in teosinte adaptation to environments and that they are likely to play a role in the response to future selection.     

The inheritance and evolution of leaf pigmentation and pubescence in teosinte

To investigate the genetic mechanisms that underlie morphological evolution in natural populations, we employed QTL mapping to dissect the inheritance of leaf sheath characters that distinguish Chalco from Balsas teosinte. Abundant macrohairs (trichomes) and intense anthocyanin accumulation are found in Chalco teosinte sheaths whereas Balsas teosinte leaf sheaths are green and glabrous. These character states may represent adaptations to the cooler highland (Chalco) vs. warmer middle-elevation (Balsas) climates. QTL mapping in multiple populations revealed a mix of major- and minor-effect QTL affecting both sheath color (anthocyanin) and macrohair abundance. The major QTL for macrohairs accounts for 52% of the parental difference. Epistatic interactions were detected between the major-effect QTL and multiple other QTL for both traits, accounting for substantial portions of phenotypic variance. Developmental analyses suggest that regulatory program changes underlie the phenotypic differences. Sheath anthocyanin QTL are clearly associated with b1 and a3, both of which are regulators of anthocyanin biosynthesis. Our findings suggest that changes in a small number of QTL can lead to morphological evolution by modulating existing developmental programs.     

Compensatory Evolution and the Origins of Innovations

Cryptic genetic sequences have attenuated effects on phenotypes. In the classic view, relaxed selection allows cryptic genetic diversity to build up across individuals in a population, providing alleles that may later contribute to adaptation when co-opted—e.g., following a mutation increasing expression from a low, attenuated baseline. This view is described, for example, by the metaphor of the spread of a population across a neutral network in genotype space. As an alternative view, consider the fact that most phenotypic traits are affected by multiple sequences, including cryptic ones. Even in a strictly clonal population, the co-option of cryptic sequences at different loci may have different phenotypic effects and offer the population multiple adaptive possibilities. Here, we model the evolution of quantitative phenotypic characters encoded by cryptic sequences and compare the relative contributions of genetic diversity and of variation across sites to the phenotypic potential of a population. We show that most of the phenotypic variation accessible through co-option would exist even in populations with no polymorphism. This is made possible by a history of compensatory evolution, whereby the phenotypic effect of a cryptic mutation at one site was balanced by mutations elsewhere in the genome, leading to a diversity of cryptic effect sizes across sites rather than across individuals. Cryptic sequences might accelerate adaptation and facilitate large phenotypic changes even in the absence of genetic diversity, as traditionally defined in terms of alternative alleles.     

Teosinte Branched1 and the Origin of Maize: Evidence for Epistasis and the Evolution of Dominance

Two quantitative trait loci (QTL) controlling differences in plant and inflorescence architecture between maize and its progenitor (teosinte) were analyzed. Complementation tests indicate that one of these, which is on chromosome arm 1L, is the locus for the maize mutant teosinte branched1 (tb1). This QTL has effects on inflorescence sex and the number and length of internodes in the lateral branches and inflorescences. This QTL has strong phenotypic effects in teosinte background but reduced effects in maize background. The second QTL, which is on chromosome arm 3L, affects the same traits as the QTL on 1L. We identify two candidate loci for this QTL. The effects of this QTL on several traits are reduced in both maize and teosinte background as compared to a maize-teosinte F(2) population. Genetic background appears to affect gene action for both QTL. Analysis of a population in which both QTL were segregating revealed that they interact epistatically. Together, these two QTL substantially transform both plant and inflorescence architecture. We propose that tb1 is involved in the teosinte plant's response to local environment to produce either long or short branches and that maize evolution involved a change at this locus to produce short branches under all environments.     

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.     

The genetic architecture of complex traits in teosinte (Zea mays ssp. parviglumis): new evidence from association mapping

Previous association analyses showed that variation at major regulatory genes contributes to standing variation for complex traits in Balsas teosinte, the progenitor of maize. This study expands our previous association mapping effort in teosinte by testing 123 markers in 52 candidate genes for association with 31 traits in a population of 817 individuals. Thirty-three significant associations for markers from 15 candidate genes and 10 traits survive correction for multiple testing. Our analyses suggest several new putative causative relationships between specific genes and trait variation in teosinte. For example, two ramosa genes (ra1 and ra2) associate with ear structure, and the MADS-box gene, zagl1, associates with ear shattering. Since zagl1 was previously shown to be a target of selection during maize domestication, we suggest that this gene was under selection for its effect on the loss of ear shattering, a key domestication trait. All observed effects were relatively small in terms of the percentage of phenotypic variation explained (<10%). We also detected several epistatic interactions between markers in the same gene that associate with the same trait. Candidate-gene-based association mapping appears to be a promising method for investigating the inheritance of complex traits in teosinte.     

Identification and fine mapping of quantitative trait loci for the number of vascular bundle in maize stem

Studies that investigated the genetic basis of source and sink related traits have been widely conducted. However, the vascular system that links source and sink received much less attention. When maize was domesticated from its wild ancestor, teosinte, the external morphology has changed dramatically; however, less is known for the internal anatomy changes. In this study, using a large maize‐teosinte experimental population, we performed a high‐resolution quantitative trait locus (QTL) mapping for the number of vascular bundle in the uppermost internode of maize stem. The results showed that vascular bundle number is dominated by a large number of small‐effect QTLs, in which a total of 16 QTLs that jointly accounts for 52.2% of phenotypic variation were detected, with no single QTL explaining more than 6% of variation. Different from QTLs for typical domestication traits, QTLs for vascular bundle number might not be under directional selection following domestication. Using Near Isogenic Lines (NILs) developed from heterogeneous inbred family (HIF), we further validated the effect of one QTL qVb9‐2 on chromosome 9 and fine mapped the QTL to a 1.8‐Mb physical region. This study provides important insights for the genetic architecture of vascular bundle number in maize stem and sets basis for cloning of qVb9‐2.     

Wide variability in kernel composition, seed characteristics, and zein profiles among diverse maize inbreds, landraces, and teosinte

All crop species have been domesticated from their wild relatives, and geneticists are just now beginning to understand the consequences of artificial (human) selection on agronomic traits that are relevant today. The primary consequence is a basal loss of diversity across the genome, and an additional reduction in diversity for genes underlying traits targeted by selection. An understanding of attributes of the wild relatives may provide insight into target traits and valuable allelic variants for modern agriculture. This is especially true for maize (Zea mays ssp. mays), where its wild ancestor, teosinte (Z. mays ssp. parviglumis), is so strikingly different than modern maize. One obvious target of selection is the size and composition of the kernel. We evaluated kernel characteristics, kernel composition, and zein profiles for a diverse set of modern inbred lines, teosinte accessions, and landraces, the intermediate between inbreds and teosinte. We found that teosinte has very small seeds, but twice the protein content of landraces and inbred lines. Teosinte has a higher average alpha zein content (nearly 89% of total zeins as compared to 72% for inbred lines and 76% for landraces), and there are many novel alcohol-soluble proteins in teosinte relative to the other two germplasm groups. Nearly every zein protein varied in abundance among the germplasm groups, especially the methionine-rich delta zein protein, and the gamma zeins. Teosinte and landraces harbor phenotypic variation that will facilitate genetic dissection of kernel traits and grain quality, ultimately leading to improvement via traditional plant breeding and/or genetic engineering.     

Pleiotropic effects of the duplicate maize FLORICAULA/LEAFY genes zfl1 and zfl2 on traits under selection during maize domestication

Phenotypic variation on which selection can act during evolution may be caused by variation in activity level of developmental regulatory genes. In many cases, however, such genes affect multiple traits. This situation can lead to co-evolution of traits, or evolutionary constraint if some pleiotropic effects are detrimental. Here, we present an analysis of quantitative traits associated with gene copy number of two important maize regulatory genes, the duplicate FLORICAULA/LEAFY orthologs zfl1 and zfl2. We found statistically significant associations between several quantitative traits and copy number of both zfl genes in several maize genetic backgrounds. Despite overlap in traits associated with these duplicate genes, zfl1 showed stronger associations with flowering time, while zfl2 associated more strongly with branching and inflorescence structure traits, suggesting some divergence of function. Since zfl2 associates with quantitative variation for ear rank and also maps near a quantitative trait locus (QTL) on chromosome 2 controlling ear rank differences between maize and teosinte, we tested whether zfl2 might have been involved in the evolution of this trait using a QTL complementation test. The results suggest that zfl2 activity is important for the QTL effect, supporting zfl2 as a candidate gene for a role in morphological evolution of maize.     

Genetic Analysis of the Morphological Differences between Maize and Teosinte

Molecular marker loci were used to investigate the inheritance of morphological traits that distinguish maize (Zea mays ssp. mays) from a closely related wild relative, teosinte (Z. mays ssp. mexicana). Regression and interval mapping analyses gave largely congruent results concerning the numbers of loci controlling the morphological traits and the magnitudes of their effects; however, interval mapping tended to give larger estimates for the magnitudes of the effects of the morphological trait loci. This tendency was exaggerated for traits that were non-normally distributed. Variation for most inflorescence traits is controlled by one or two regions of the genome with large effects plus several other regions with relatively small effects. As such, the data are congruent with a mode of inheritance for most traits involving one or two major loci plus several minor loci. Regions of the genome with large effects on one trait consistently had smaller effects on several other traits, possibly as a result of pleiotropy. Most of the variation for the dramatic differences in inflorescence morphology between maize and teosinte is explained by five restricted regions of the genome. One of these regions encompasses a previously described gene, tb1 (teosinte branched), and the effects of this region on inflorescence architecture are similar to the known effects of tb1. Implications of this work for the genetic basis of morphological evolution in plants are discussed.     

Inflorescence development in a new teosinte: Zea nicaraguensis (Poaceae)

Inflorescence development in a newly discovered teosinte, Zea nicaraguensis (Poaceae), from Nicaragua has been investigated using scanning electron microscopy (SEM). The SEM examination revealed that the pattern of both male and female inflorescence development was similar to previously described inflorescence in other Zea taxa. Branch primordia were initiated acropetally in a distichous pattern along the rachis of male and female inflorescences. Spikelet pair primordia bifurcated into pedicellate and sessile spikelet primordia. Predictably, pedicellate spikelet development was arrested and aborted in the female teosinte inflorescence. Organogenesis of functional spikelets and florets was similar to that previously described in maize and teosintes. The results were consistent with our hypothesis that both femininity and masculinity share a common mechanism of inflorescence development in Zea and Tripsacum and are in accord with a putative common mechanism of sex determination in the Andropogoneae. Interestingly, this population of teosinte, unique in its ability to grow in water-logged soils, showed a stable pattern of early inflorescence development. Our results also revealed the uncharacteristic presence of inflorescence polystichy in this population of Zea nicaraguensis. We propose this novel phenotypic variation raises the possibility that a domestic evolution of polystichy in maize was enabled by an occasional polystichous phenotypic in teosinte.     

Developmental analysis of teosinte glume architecture1: A key locus in the evolution of maize (Poaceae)

A key event in the evolution of maize from teosinte was a reduction in the cupulate fruitcase and softening of the glumes, which increased the accessibility of kernels for harvest. The teosinte glume architecture1 (tga1) locus largely controls this difference between maize and teosinte, and thus may have played a pivotal role in maize evolution. The teosinte allele (tga1+teosinte) lengthens inflorescence internodes, shortens rachillae, and makes glumes longer, thicker, and harder. Developmental characterization of morphometric traits reveals that differences among genotypes are apparent early in female inflorescence development. Increased hardening in glumes homozygous for tga1+teosinte is correlated with a thicker abaxial mesoderm of lignified cells. Silica deposition in the abaxial epidermal cells of the glumes is also affected. In the maize background, glumes homozygous for tga1+teosinte deposit silica in both the short and long cells of the glume epidermis, whereas glumes homozygous for the maize allele (Tga1+Maize) concentrate silica only in the short cells. Silica deposition also appears to be affected by genetic background. The effects of tga1 appear largely to explain the differences in glume induration between maize and teosinte. The diverse pleiotropic effects of tga1 suggest that it is regulatory in nature.     

Using Association Mapping in Teosinte to Investigate the Function of Maize Selection-Candidate Genes

Background

Large-scale screens of the maize genome identified 48 genes that show the putative signature of artificial selection during maize domestication or improvement. These selection-candidate genes may act as quantitative trait loci (QTL) that control the phenotypic differences between maize and its progenitor, teosinte. The selection-candidate genes appear to be located closer in the genome to domestication QTL than expected by chance.

Methods and Findings

As a step toward defining the traits controlled by these genes, we performed phenotype-genotype association mapping in teosinte for 32 of the 48 plus three other selection-candidate genes. Our analyses assayed 32 phenotypic traits, many of which were altered during maize domestication or improvement. We observed several significant associations between SNPs in the selection-candidate genes and trait variation in teosinte. These included two associations that surpassed the Bonferroni correction and five instances where a gene significantly associated with the same trait in both of our association mapping panels. Despite these significant associations, when compared as a group the selection-candidate genes performed no better than randomly chosen genes.

Conclusions

Our results suggest association analyses can be helpful for identifying traits under the control of selection-candidate genes. Indeed, we present evidence for new functions for several selection-candidate genes. However, with the current set of selection-candidate genes and our association mapping strategy, we found very few significant associations overall and no more than we would have found with randomly chosen genes. We discuss possible reasons that a large number of significant genotype-phenotype associations were not discovered.     

Investigating the genetic architecture of conditional strategies using the environmental threshold model

The threshold expression of dichotomous phenotypes that are environmentally cued or induced comprise the vast majority of phenotypic dimorphisms in colour, morphology, behaviour and life history. Modelled as conditional strategies under the framework of evolutionary game theory, the quantitative genetic basis of these traits is a challenge to estimate. The challenge exists firstly because the phenotypic expression of the trait is dichotomous and secondly because the apparent environmental cue is separate from the biological signal pathway that induces the switch between phenotypes. It is the cryptic variation underlying the translation of cue to phenotype that we address here. With a ‘half-sib common environment’ and a ‘family-level split environment’ experiment, we examine the environmental and genetic influences that underlie male dimorphism in the earwig Forficula auricularia. From the conceptual framework of the latent environmental threshold (LET) model, we use pedigree information to dissect the genetic architecture of the threshold expression of forceps length. We investigate for the first time the strength of the correlation between observable and cryptic ‘proximate’ cues. Furthermore, in support of the environmental threshold model, we found no evidence for a genetic correlation between cue and the threshold between phenotypes. Our results show strong correlations between observable and proximate cues and less genetic variation for thresholds than previous studies have suggested. We discuss the importance of generating better estimates of the genetic variation for thresholds when investigating the genetic architecture and heritability of threshold traits. By investigating genetic architecture by means of the LET model, our study supports several key evolutionary ideas related to conditional strategies and improves our understanding of environmentally cued decisions.     

Developmental plasticity and the origin of novel forms: unveiling cryptic genetic variation via "use and disuse"

Natural selection eliminates phenotypic variation from populations, generation after generation-an observation that haunted Darwin. So, how does new phenotypic variation arise, and is it always random with respect to fitness? Repeated behavioral responses to a novel environment-particularly those that are learned-are typically advantageous. If those behaviors yield more extreme or novel morphological variants via developmental plasticity, then previously cryptic genetic variation may be exposed to natural selection. Significantly, because the mean phenotypic effect of "use and disuse" is also typically favorable, previously cryptic genetic variation can be transformed into phenotypic variation that is both visible to selection and biased in an adaptive direction. Therefore, use-induced developmental plasticity in a very real sense "creates" new phenotypic variation that is nonrandom with respect to fitness, in contrast to the random phenotypic effects of mutation, recombination, and "direct effects" of environment (stress, nutrition). I offer here (a) a brief review of the immense literature on the effects of "use and disuse" on morphology, (b) a simple yet general model illustrating how cryptic genetic variation may be exposed to selection by developmentally plastic responses that alter trait performance in response to "use and disuse," and (c) a more detailed model of a positive feedback loop between learning (handed behavior) and morphological plasticity (use-induced morphological asymmetry) that may rapidly generate novel phenotypic variation and facilitate the evolution of conspicuous morphological asymmetries. Evidence from several sources suggests that handed behaviors played an important role both in the origin of novel forms (asymmetries) and in their subsequent evolution.