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.

A conserved genetic architecture among populations of the maize progenitor, teosinte, was radically altered by domestication.

Very little is known about how domestication was constrained by the quantitative genetic architecture of crop progenitors and how quantitative genetic architecture was altered by domestication. Yang et al. [C. J. Yang et al., Proc. Natl. Acad. Sci. U.S.A. 116, 5643-5652 (2019)] drew multiple conclusions about how genetic architecture influenced and was altered by maize domestication based on one sympatric pair of teosinte and maize populations. To test the generality of their conclusions, we assayed the structure of genetic variances, genetic correlations among traits, strength of selection during domestication, and diversity in genetic architecture within teosinte and maize. Our results confirm that additive genetic variance is decreased, while dominance genetic variance is increased, during maize domestication. The genetic correlations are moderately conserved among traits between teosinte and maize, while the genetic variance-covariance matrices (G-matrices) of teosinte and maize are quite different, primarily due to changes in the submatrix for reproductive traits. The inferred long-term selection intensities during domestication were weak, and the neutral hypothesis was rejected for reproductive and environmental response traits, suggesting that they were targets of selection during domestication. The G-matrix of teosinte imposed considerable constraint on selection during the early domestication process, and constraint increased further along the domestication trajectory. Finally, we assayed variation among populations and observed that genetic architecture is generally conserved among populations within teosinte and maize but is radically different between teosinte and maize. While selection drove changes in essentially all traits between teosinte and maize, selection explains little of the difference in domestication traits among populations within teosinte or maize.

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.

The genome-wide dynamics of purging during selfing in maize.

Self-fertilization (also known as selfing) is an important reproductive strategy in plants and a widely applied tool for plant genetics and plant breeding. Selfing can lead to inbreeding depression by uncovering recessive deleterious variants, unless these variants are purged by selection. Here we investigated the dynamics of purging in a set of eleven maize lines that were selfed for six generations. We show that heterozygous, putatively deleterious single nucleotide polymorphisms are preferentially lost from the genome during selfing. Deleterious single nucleotide polymorphisms were lost more rapidly in regions of high recombination, presumably because recombination increases the efficacy of selection by uncoupling linked variants. Overall, heterozygosity decreased more slowly than expected, by an estimated 35% to 40% per generation instead of the expected 50%, perhaps reflecting pervasive associative overdominance. Finally, three lines exhibited marked decreases in genome size due to the purging of transposable elements. Genome loss was more likely to occur for lineages that began with larger genomes with more transposable elements and chromosomal knobs. These three lines purged an average of 398 Mb from their genomes, an amount equivalent to three Arabidopsis thaliana genomes per lineage, in only a few generations.

TeoNAM: A Nested Association Mapping Population for Domestication and Agronomic Trait Analysis in Maize.

Recombinant inbred lines (RILs) are an important resource for mapping genes controlling complex traits in many species. While RIL populations have been developed for maize, a maize RIL population with multiple teosinte inbred lines as parents has been lacking. Here, we report a teosinte nested association mapping (TeoNAM) population, derived from crossing five teosinte inbreds to the maize inbred line W22. The resulting 1257 BC1S4 RILs were genotyped with 51,544 SNPs, providing a high-density genetic map with a length of 1540 cM. On average, each RIL is 15% homozygous teosinte and 8% heterozygous. We performed joint linkage mapping (JLM) and a genome-wide association study (GWAS) for 22 domestication and agronomic traits. A total of 255 QTL from JLM were identified, with many of these mapping near known genes or novel candidate genes. TeoNAM is a useful resource for QTL mapping for the discovery of novel allelic variation from teosinte. TeoNAM provides the first report that PROSTRATE GROWTH1, a rice domestication gene, is also a QTL associated with tillering in teosinte and maize. We detected multiple QTL for flowering time and other traits for which the teosinte allele contributes to a more maize-like phenotype. Such QTL could be valuable in maize improvement.

Hybrid Decay: A Transgenerational Epigenetic Decline in Vigor and Viability Triggered in Backcross Populations of Teosinte with Maize.

In the course of generating populations of maize with teosinte chromosomal introgressions, an unusual sickly plant phenotype was noted in individuals from crosses with two teosinte accessions collected near Valle de Bravo, Mexico. The plants of these Bravo teosinte accessions appear phenotypically normal themselves and the F1 plants appear similar to typical maize × teosinte F1s. However, upon backcrossing to maize, the BC1 and subsequent generations display a number of detrimental characteristics including shorter stature, reduced seed set, and abnormal floral structures. This phenomenon is observed in all BC individuals and there is no chromosomal segment linked to the sickly plant phenotype in advanced backcross generations. Once the sickly phenotype appears in a lineage, normal plants are never again recovered by continued backcrossing to the normal maize parent. Whole-genome shotgun sequencing reveals a small number of genomic sequences, some with homology to transposable elements, that have increased in copy number in the backcross populations. Transcriptome analysis of seedlings, which do not have striking phenotypic abnormalities, identified segments of 18 maize genes that exhibit increased expression in sickly plants. A de novo assembly of transcripts present in plants exhibiting the sickly phenotype identified a set of 59 upregulated novel transcripts. These transcripts include some examples with sequence similarity to transposable elements and other sequences present in the recurrent maize parent (W22) genome as well as novel sequences not present in the W22 genome. Genome-wide profiles of gene expression, DNA methylation, and small RNAs are similar between sickly plants and normal controls, although a few upregulated transcripts and transposable elements are associated with altered small RNA or methylation profiles. This study documents hybrid incompatibility and genome instability triggered by the backcrossing of Bravo teosinte with maize. We name this phenomenon "hybrid decay" and present ideas on the mechanism that may underlie it.

The genetic architecture of teosinte catalyzed and constrained maize domestication.

The process of evolution under domestication has been studied using phylogenetics, population genetics-genomics, quantitative trait locus (QTL) mapping, gene expression assays, and archaeology. Here, we apply an evolutionary quantitative genetic approach to understand the constraints imposed by the genetic architecture of trait variation in teosinte, the wild ancestor of maize, and the consequences of domestication on genetic architecture. Using modern teosinte and maize landrace populations as proxies for the ancestor and domesticate, respectively, we estimated heritabilities, additive and dominance genetic variances, genetic-by-environment variances, genetic correlations, and genetic covariances for 18 domestication-related traits using realized genomic relationships estimated from genome-wide markers. We found a reduction in heritabilities across most traits, and the reduction is stronger in reproductive traits (size and numbers of grains and ears) than vegetative traits. We observed larger depletion in additive genetic variance than dominance genetic variance. Selection intensities during domestication were weak for all traits, with reproductive traits showing the highest values. For 17 of 18 traits, neutral divergence is rejected, suggesting they were targets of selection during domestication. Yield (total grain weight) per plant is the sole trait that selection does not appear to have improved in maize relative to teosinte. From a multivariate evolution perspective, we identified a strong, nonneutral divergence between teosinte and maize landrace genetic variance-covariance matrices (G-matrices). While the structure of G-matrix in teosinte posed considerable genetic constraint on early domestication, the maize landrace G-matrix indicates that the degree of constraint is more unfavorable for further evolution along the same trajectory.

Stepwise cis-Regulatory Changes in ZCN8 Contribute to Maize Flowering-Time Adaptation.

Maize (Zea mays ssp. mays) was domesticated in southwestern Mexico ∼9,000 years ago from its wild ancestor, teosinte (Zea mays ssp. parviglumis) [1]. From its center of origin, maize experienced a rapid range expansion and spread over 90° of latitude in the Americas [2-4], which required a novel flowering-time adaptation. ZEA CENTRORADIALIS 8 (ZCN8) is the maize florigen gene and has a central role in mediating flowering [5, 6]. Here, we show that ZCN8 underlies a major quantitative trait locus (QTL) (qDTA8) for flowering time that was consistently detected in multiple maize-teosinte experimental populations. Through association analysis in a large diverse panel of maize inbred lines, we identified a SNP (SNP-1245) in the ZCN8 promoter that showed the strongest association with flowering time. SNP-1245 co-segregated with qDTA8 in maize-teosinte mapping populations. We demonstrate that SNP-1245 is associated with differential binding by the flowering activator ZmMADS1. SNP-1245 was a target of selection during early domestication, which drove the pre-existing early flowering allele to near fixation in maize. Interestingly, we detected an independent association block upstream of SNP-1245, wherein the early flowering allele that most likely originated from Zea mays ssp. mexicana introgressed into the early flowering haplotype of SNP-1245 and contributed to maize adaptation to northern high latitudes. Our study demonstrates how independent cis-regulatory variants at a gene can be selected at different evolutionary times for local adaptation, highlighting how complex cis-regulatory control mechanisms evolve. Finally, we propose a polygenic map for the pre-Columbian spread of maize throughout the Americas.

Construction of the third-generation Zea mays haplotype map.

Background: Characterization of genetic variations in maize has been challenging, mainly due to deterioration of collinearity between individual genomes in the species. An international consortium of maize research groups combined resources to develop the maize haplotype version 3 (HapMap 3), built from whole-genome sequencing data from 1218 maize lines, covering predomestication and domesticated Zea mays varieties across the world. Results: A new computational pipeline was set up to process more than 12 trillion bp of sequencing data, and a set of population genetics filters was applied to identify more than 83 million variant sites. Conclusions: We identified polymorphisms in regions where collinearity is largely preserved in the maize species. However, the fact that the B73 genome used as the reference only represents a fraction of all haplotypes is still an important limiting factor.

Defining the Role of the MADS-Box Gene, Zea Agamous-like1, a Target of Selection During Maize Domestication.

Genomic scans for genes that show the signature of past selection have been widely applied to a number of species and have identified a large number of selection candidate genes. In cultivated maize (Zea mays ssp. mays) selection scans have identified several hundred candidate domestication genes by comparing nucleotide diversity and differentiation between maize and its progenitor, teosinte (Z. mays ssp. parviglumis). One of these is a gene called zea agamous-like1 (zagl1), a MADS-box transcription factor, that is known for its function in the control of flowering time. To determine the trait(s) controlled by zagl1 that was (were) the target(s) of selection during maize domestication, we created a set of recombinant chromosome isogenic lines that differ for the maize versus teosinte alleles of zagl1 and which carry cross-overs between zagl1 and its neighbor genes. These lines were grown in a randomized trial and scored for flowering time and domestication related traits. The results indicated that the maize versus teosinte alleles of zagl1 affect flowering time as expected, as well as multiple traits related to ear size with the maize allele conferring larger ears with more kernels. Our results suggest that zagl1 may have been under selection during domestication to increase the size of the maize ear.

Genome-wide Analysis of Transcriptional Variability in a Large Maize-Teosinte Population.

Gene expression regulation plays an important role in controlling plant phenotypes and adaptation. Here, we report a comprehensive assessment of gene expression variation through the transcriptome analyses of a large maize-teosinte experimental population. Genome-wide mapping identified 25 660 expression quantitative trait loci (eQTL) for 17 311 genes, capturing an unprecedented range of expression variation. We found that local eQTL were more frequently mapped to adjacent genes, displaying a mode of expression piggybacking, which consequently created co-regulated gene clusters. Genes within the co-regulated gene clusters tend to have relevant functions and shared chromatin modifications. Distant eQTL formed 125 significant distant eQTL hotspots with their targets significantly enriched in specific functional categories. By integrating different sources of information, we identified putative trans- regulators for a variety of metabolic pathways. We demonstrated that the bHLH transcription factor R1 and hexokinase HEX9 might act as crucial regulators for flavonoid biosynthesis and glycolysis, respectively. Moreover, we showed that domestication or improvement has significantly affected global gene expression, with many genes targeted by selection. Of particular interest, the Bx genes for benzoxazinoid biosynthesis may have undergone coordinated cis-regulatory divergence between maize and teosinte, and a transposon insertion that inactivates Bx12 was under strong selection as maize spread into temperate environments with a distinct herbivore community.

ZmCCT9 enhances maize adaptation to higher latitudes.

From its tropical origin in southwestern Mexico, maize spread over a wide latitudinal cline in the Americas. This feat defies the rule that crops are inhibited from spreading easily across latitudes. How the widespread latitudinal adaptation of maize was accomplished is largely unknown. Through positional cloning and association mapping, we resolved a flowering-time quantitative trait locus to a Harbinger-like transposable element positioned 57 kb upstream of a CCT transcription factor (ZmCCT9). The Harbinger-like element acts in cis to repress ZmCCT9 expression to promote flowering under long days. Knockout of ZmCCT9 by CRISPR/Cas9 causes early flowering under long days. ZmCCT9 is diurnally regulated and negatively regulates the expression of the florigen ZCN8, thereby resulting in late flowering under long days. Population genetics analyses revealed that the Harbinger-like transposon insertion at ZmCCT9 and the CACTA-like transposon insertion at another CCT paralog, ZmCCT10, arose sequentially following domestication and were targeted by selection for maize adaptation to higher latitudes. Our findings help explain how the dynamic maize genome with abundant transposon activity enabled maize to adapt over 90° of latitude during the pre-Columbian era.

Selection During Maize Domestication Targeted a Gene Network Controlling Plant and Inflorescence Architecture.

Selection during evolution, whether natural or artificial, acts through the phenotype. For multifaceted phenotypes such as plant and inflorescence architecture, the underlying genetic architecture is comprised of a complex network of interacting genes rather than single genes that act independently to determine the trait. As such, selection acts on entire gene networks. Here, we begin to define the genetic regulatory network to which the maize domestication gene, teosinte branched1 (tb1), belongs. Using a combination of molecular methods to uncover either direct or indirect regulatory interactions, we identified a set of genes that lie downstream of tb1 in a gene network regulating both plant and inflorescence architecture. Additional genes, known from the literature, also act in this network. We observed that tb1 regulates both core cell cycle genes and another maize domestication gene, teosinte glume architecture1 (tga1). We show that several members of the MADS-box gene family are either directly or indirectly regulated by tb1 and/or tga1, and that tb1 sits atop a cascade of transcriptional regulators controlling both plant and inflorescence architecture. Multiple members of the tb1 network appear to have been the targets of selection during maize domestication. Knowledge of the regulatory hierarchies controlling traits is central to understanding how new morphologies evolve.

A Gene for Genetic Background in Zea mays: Fine-Mapping enhancer of teosinte branched1.2 to a YABBY Class Transcription Factor.

The effects of an allelic substitution at a gene often depend critically on genetic background, i.e., the genotypes at other genes in the genome. During the domestication of maize from its wild ancestor (teosinte), an allelic substitution at teosinte branched (tb1) caused changes in both plant and ear architecture. The effects of tb1 on phenotype were shown to depend on multiple background loci, including one called enhancer of tb1.2 (etb1.2). We mapped etb1.2 to a YABBY class transcription factor (ZmYAB2.1) and showed that the maize alleles of ZmYAB2.1 are either expressed at a lower level than teosinte alleles or disrupted by insertions in the sequences. tb1 and etb1.2 interact epistatically to control the length of internodes within the maize ear, which affects how densely the kernels are packed on the ear. The interaction effect is also observed at the level of gene expression, with tb1 acting as a repressor of ZmYAB2.1 expression. Curiously, ZmYAB2.1 was previously identified as a candidate gene for another domestication trait in maize, nonshattering ears. Consistent with this proposed role, ZmYAB2.1 is expressed in a narrow band of cells in immature ears that appears to represent a vestigial abscission (shattering) zone. Expression in this band of cells may also underlie the effect on internode elongation. The identification of ZmYAB2.1 as a background factor interacting with tb1 is a first step toward a gene-level understanding of how tb1 and the background within which it works evolved in concert during maize domestication.

Mapping Prolificacy QTL in Maize and Teosinte.

Teosinte, the ancestor of maize, possesses multiple ears at each node along its main stalk, whereas maize has only a single ear at each node. With its greater ear number, teosinte is referred to as being more prolific. The grassy tillers 1 (gt1) gene has been identified as a large-effect quantitative trait locus underlying this prolificacy difference between maize and teosinte, and the causal polymorphism for the difference was mapped to a 2.7kb control region 5' of the gt1 ORF. The most common maize haplotype (M1) at the gt1 control region confers low prolificacy. A prior study reported that 29% of maize varieties possess the teosinte haplotype (T) for the control region, although these varieties are nonprolific. This observation suggested that these maize lines might possess an additional factor, other than gt1, suppressing prolificacy in maize. We discovered that the factor suppressing prolificacy in maize varieties with the gt1 T haplotype mapped to a 3.20 cM interval, which includes gt1 Subsequent DNA sequence analysis revealed that the maize varieties with the apparent T haplotype actually possess a distinct maize haplotype (M2) that is similar, but not identical, to the T haplotype in sequence but is associated with a nonprolific phenotype similar to the M1 haplotype. Our data indicate that the M2 haplotype or a closely linked factor confers a nonprolific phenotype. Our data suggest that 2 different alleles or haplotypes (M1 and M2) of gt1 were selected during domestication, and that nonprolificacy in all maize varieties is likely a result of allele substitutions at gt1.

Fine Mapping of a QTL Associated with Kernel Row Number on Chromosome 1 of Maize.

The genetic factors underlying changes in ear morphology, and particularly the inheritance of kernel row number (KRN), have been broadly investigated in diverse mapping populations in maize (Zea mays L.). In this study, we mapped a region on the long arm of chromosome 1 containing a QTL for KRN. This work was performed using a set of recombinant chromosome nearly isogenic lines (RCNILs) derived from a BC2S3 population produced using the inbred maize line W22 and teosinte (Zea mays ssp. parviglumis) as the parents. A set of 48 RCNILs was evaluated in the field during the summer of 2013 in order to perform the mapping. A QTL for KRN was found that explained approximately 51% of the phenotypic variance and had a 1.5-LOD confidence interval of 203 kb. Seven genes are described in this interval. One of these candidate genes may have been the target of domestication processes in maize and contributed to the shift from two kernel row ears in teosinte to a highly polystichous ear in maize.

Evidence That the Origin of Naked Kernels During Maize Domestication Was Caused by a Single Amino Acid Substitution in tga1.

teosinte glume architecture1 (tga1), a member of the SBP-box gene family of transcriptional regulators, has been identified as the gene conferring naked kernels in maize vs. encased kernels in its wild progenitor, teosinte. However, the identity of the causative polymorphism within tga1 that produces these different phenotypes has remained unknown. Using nucleotide diversity data, we show that there is a single fixed nucleotide difference between maize and teosinte in tga1, and this difference confers a Lys (teosinte allele) to Asn (maize allele) substitution. This substitution transforms TGA1 into a transcriptional repressor. While both alleles of TGA1 can bind a GTAC motif, maize-TGA1 forms more stable dimers than teosinte-TGA1. Since it is the only fixed difference between maize and teosinte, this alteration in protein function likely underlies the differences in maize and teosinte glume architecture. We previously reported a difference in TGA1 protein abundance between maize and teosinte based on relative signal intensity of a Western blot. Here, we show that this signal difference is not due to tga1 but to a second gene, neighbor of tga1 (not1). Not1 encodes a protein that has 92% amino acid similarity to TGA1 and that is recognized by the TGA1 antibody. Genetic mapping and phenotypic data show that tga1, without a contribution from not1, controls the difference in covered vs. naked kernels. No trait differences could be associated with the maize vs. teosinte alleles of not1. Our results document how morphological evolution can be driven by a simple nucleotide change that alters protein function.

The role of cis regulatory evolution in maize domestication.

Gene expression differences between divergent lineages caused by modification of cis regulatory elements are thought to be important in evolution. We assayed genome-wide cis and trans regulatory differences between maize and its wild progenitor, teosinte, using deep RNA sequencing in F1 hybrid and parent inbred lines for three tissue types (ear, leaf and stem). Pervasive regulatory variation was observed with approximately 70% of ∼17,000 genes showing evidence of regulatory divergence between maize and teosinte. However, many fewer genes (1,079 genes) show consistent cis differences with all sampled maize and teosinte lines. For ∼70% of these 1,079 genes, the cis differences are specific to a single tissue. The number of genes with cis regulatory differences is greatest for ear tissue, which underwent a drastic transformation in form during domestication. As expected from the domestication bottleneck, maize possesses less cis regulatory variation than teosinte with this deficit greatest for genes showing maize-teosinte cis regulatory divergence, suggesting selection on cis regulatory differences during domestication. Consistent with selection on cis regulatory elements, genes with cis effects correlated strongly with genes under positive selection during maize domestication and improvement, while genes with trans regulatory effects did not. We observed a directional bias such that genes with cis differences showed higher expression of the maize allele more often than the teosinte allele, suggesting domestication favored up-regulation of gene expression. Finally, this work documents the cis and trans regulatory changes between maize and teosinte in over 17,000 genes for three tissues.

Genetic dissection of a genomic region with pleiotropic effects on domestication traits in maize reveals multiple linked QTL.

The domesticated crop maize and its wild progenitor, teosinte, have been used in numerous experiments to investigate the nature of divergent morphologies. This study examines a poorly understood region on the fifth chromosome of maize associated with a number of traits under selection during domestication, using a quantitative trait locus (QTL) mapping population specific to the fifth chromosome. In contrast with other major domestication loci in maize where large-effect, highly pleiotropic, single genes are responsible for phenotypic effects, our study found the region on chromosome five fractionates into multiple-QTL regions, none with singularly large effects. The smallest 1.5-LOD support interval for a QTL contained 54 genes, one of which was a MADS MIKC(C) transcription factor, a family of proteins implicated in many developmental programs. We also used simulated trait data sets to investigate the power of our mapping population to identify QTL for which there is a single underlying causal gene. This analysis showed that while QTL for traits controlled by single genes can be accurately mapped, our population design can detect no more than ∼4.5 QTL per trait even when there are 100 causal genes. Thus when a trait is controlled by ≥5 genes in the simulated data, the number of detected QTL can represent a simplification of the underlying causative factors. Our results show how a QTL region with effects on several domestication traits may be due to multiple linked QTL of small effect as opposed to a single gene with large and pleiotropic effects.

Defining the Role of prolamin-box binding factor1 Gene During Maize Domestication.

The prolamin-box binding factor1 (pbf1) gene encodes a transcription factor that controls the expression of seed storage protein (zein) genes in maize. Prior studies show that pbf1 underwent selection during maize domestication although how it affected trait change during domestication is unknown. To assay how pbf1 affects phenotypic differences between maize and teosinte, we compared nearly isogenic lines (NILs) that differ for a maize versus teosinte allele of pbf1 Kernel weight for the teosinte NIL (162mg) is slightly but significantly greater than that for the maize NIL (156mg). RNAseq data for developing kernels show that the teosinte allele of pbf1 is expressed at about twice the level of the maize allele. However, RNA and protein assays showed no difference in zein profile between the two NILs. The lower expression for the maize pbf1 allele suggests that selection may have favored this change; however, how reduced pbf1 expression alters phenotype remains unknown. One possibility is that pbf1 regulates genes other than zeins and thereby is a domestication trait. The observed drop in seed weight associated with the maize allele of pbf1 is counterintuitive but could represent a negative pleiotropic effect of selection on some other aspect of kernel composition.