Shawn M. Kaeppler

Comparative population genomics of maize domestication and improvement

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

Maize HapMap2 identifies extant variation from a genome in flux

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

Genome-wide atlas of transcription during maize development

Maize is an important model species and a major constituent of human and animal diets. It has also emerged as a potential feedstock and model system for bioenergy research due to recent worldwide interest in developing plant biomass-based, carbon-neutral liquid fuels. To understand how the underlying genome sequence results in specific plant phenotypes, information on the temporal and spatial transcription patterns of genes is crucial. Here we present a comprehensive atlas of global transcription profiles across developmental stages and plant organs. We used a NimbleGen microarray containing 80,301 probe sets to profile transcription patterns in 60 distinct tissues representing 11 major organ systems of inbred line B73. Of the 30,892 probe sets representing the filtered B73 gene models, 91.4% were expressed in at least one tissue. Interestingly, 44.5% of the probe sets were expressed in all tissues, indicating a substantial overlap of gene expression among plant organs. Clustering of maize tissues based on global gene expression profiles resulted in formation of groups of biologically related tissues. We utilized this dataset to examine the expression of genes that encode enzymes in the lignin biosynthetic pathway, and found that expansion of distinct gene families was accompanied by divergent, tissue-specific transcription patterns of the paralogs. This comprehensive expression atlas represents a valuable resource for gene discovery and functional characterization in maize.

Lax leaf maize: cell wall composition and nutritional value

Forage nutritive value, which comprises traits such as digestibility, fibre, lignin and protein content, is an important criterion for maize (Zea mays L) harvested as silage. Lines with a characteristic phenotype (‘lax leaf’) could be useful sources of genes for improved nutritive value in maize. A study was conducted to characterise the cell wall composition of the lax leaf line. Lax leaf inbreds and inbreds representing ‘normal’ maize were evaluated for cell wall neutral sugars, uronic acids, Klason lignin and phenolic acids in five tissues from the ear node and the internode above it. Acid detergent fibre (ADF) and neutral detergent fibre (NDF) and 48 h in vitro true digestibility (IVTD) were predicted using near-infrared reflectance spectrophotometry (NIRS) calibrated with a subset of the scanned samples. Lax leaf inbred tissues had lower levels of ADF, NDF, lignin and xylose and were more digestible than tissues from the inbreds representing ‘normal’ maize. It was not known whether the lax leaf phenotype resulted from alterations in nutritive value traits or whether laxness and nutritive value traits are independent from one another. A second study was conducted to determine the nature of genetic control of the lax leaf character and to determine the genotypic relation between the lax leaf character and nutritive value. A recombinant inbred mapping population was developed from a cross between the lax leaf line and an inbred line with stiff upright leaves. Whole-plant samples from each recombinant inbred line were evaluated for ADF, NDF, acid detergent lignin (ADL) and IVTD of dry matter using NIRS. Laxness, measured by number of broken leaves, was associated with lower nutritive value in this population (genetic correlations 0.16–0.34), which was contrary to expectation. Amplified restriction fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers were used to identify linkage groups associated with the lax leaf character, digestibility and fibre content. Several linkage groups were associated with both the lax leaf character and nutritive value. Where these characters were associated with the same linkage group, the lax leaf parent allele was associated with greater laxness but reduced nutritive value. The lax leaf parent allele was associated with increased nutritive value in linkage groups unassociated with the lax leaf character. While the lax leaf line may be a good source for alleles for improved nutritive value, selection for laxness will not likely be accompanied by improvement in forage quality. © 2000 Society of Chemical Industry

Mapping of QTLs for lateral root branching and length in maize (Zea mays L.) under differential phosphorus supply

Low phosphorus availability is a primary constraint for plant growth in terrestrial ecosystems. Lateral root initiation and elongation may play an important role in the uptake of immobile nutrients such as phosphorus by increasing soil exploration and phosphorus acquisition. The objective of this study was to identify quantitative trait loci (QTLs) controlling lateral root length (LRL), number (LRN), and plasticity of the primary seedling root of maize under varying phosphorus availability. Using a cigar roll culture in a controlled environment, we evaluated primary root LRL and LRN at low and high phosphorus availability in 160 recombinant inbred lines (RILs) derived from a cross between maize genotypes B73 and Mo17, which have contrasting adaptation to low phosphorus availability in the field. Low phosphorus availability increased LRL by 19% in Mo17, the phosphorus-efficient parent, but significantly decreased LRL in B73, the phosphorus-inefficient genotype. Substantial genetic variation and transgressive segregation for LRL and LRN existed in the population. The plasticity of LRL ranged from −100% to 146.3%, with a mean of 30.4%, and the plasticity of LRN ranged from −82.2% to 164.1%, with a mean of 18.5%. On the basis of composite interval mapping with a LOD threshold of 3.27, one QTL was associated with LRN plasticity, five QTLs were associated with LRL and one QTL was associated with LRN under high fertility. Under low fertility, six QTLs were associated with LRL and one QTL with LRN. No QTLs were detected for plasticity of LRL. A number of RILs exceeded Mo17, the phosphorus-efficient parent, for LRL, LRN, and plasticity. The detection of QTLs for these traits, in combination with the observation of transgressive segregants in our population, indicates that favorable alleles can be combined to increase seedling lateral root growth in maize.

Enhanced maize productivity by inoculation with diazotrophic bacteria

The objective of this work over the last 3 years was to identify maize-endophyte associations with increased plant productivity compared with uninoculated controls. We have used a collection of endophytes isolated by several groups. The experiments were done under field and greenhouse conditions in the presence or absence of added fixed nitrogen (N). Significant yield enhancements of N-fertilized maize were obtained with bacterial endophytes that we have isolated from N-efficient lines of maize (such as Klebsiella pneumoniae 342) or switchgrass (Pantoea agglomerans P101 and P102). Several other strains from other groups were also tested with our best yield enhancements from two Brazilian strains, Gluconacetobacter diazotrophicus PA15 and Herbaspirillum seropedicae Z152. Field experiments in Wisconsin were conducted in 1998, 1999 and 2000 and in an additional four states (Illinois, Iowa, Indiana and Nebraska) in 2000, with a minimum of two elite lines of maize at each site, each year. No strains were capable of relieving the N-deficiency symptoms of unfertilized maize in either the field or the greenhouse.

Topsoil foraging and phosphorus acquisition efficiency in maize (Zea mays)

In soybean and common bean, enhanced topsoil foraging permitted by shallow root architectures is advantageous for phosphorus acquisition from stratified soils. The importance of this phenomenon in graminaceous crops, which have different root architecture and morphology from legumes, is unclear. In this study we evaluated the importance of shallow roots for phosphorus acquisition in maize (Zea mays L.). In a field study, maize genotypes with shallower roots had greater growth in low phosphorus soil than deep-rooted genotypes. For physiological analysis, four maize genotypes differing in root shallowness in the field were grown in solid media with stratified phosphorus availability in a controlled environment. Of the four genotypes, one shallow and one deep genotype were also inoculated with arbuscular mycorrhiza (AM). Shallower genotypes had significantly greater growth and phosphorus accumulation compared with deeper genotypes at low phosphorus availability. Mycorrhizal colonisation altered root shallowness under low phosphorus conditions, increasing shallowness substantially in a deep-rooted genotype but slightly decreasing shallowness in a shallow-rooted genotype. Mycorrhizal colonisation increased phosphorus acquisition under low phosphorus availability. Respiration costs of roots and shoots of phosphorus-efficient genotypes were significantly lower under low phosphorus conditions compared with inefficient genotypes. The physiological efficiency of phosphorus acquisition, expressed as root respiration per unit of phosphorus acquisition, was greater in shallow rooted genotypes. Our results demonstrate that genetic variation for root shallowness exists in maize, that phosphorus and AM can modulate root shallowness independently, and that a shallower root system is beneficial for plant performance in maize at low phosphorus availability. We propose that root architectural traits that enhance topsoil foraging are important traits for improved phosphorus acquisition efficiency of annual grain crops such as maize in addition to legumes.

Comparative Analysis of SET Domain Proteins in Maize and Arabidopsis Reveals Multiple Duplications Preceding the Divergence of Monocots and Dicots

Histone proteins play a central role in chromatin packaging, and modification of histones is associated with chromatin accessibility. SET domain [Su(var)3-9, Enhancer-of-zeste, Trithorax] proteins are one class of proteins that have been implicated in regulating gene expression through histone methylation. The relationships of 22 SET domain proteins from maize (Zea mays) and 32 SET domain proteins from Arabidopsis were evaluated by phylogenetic analysis and domain organization. Our analysis reveals five classes of SET domain proteins in plants that can be further divided into 19 orthology groups. In some cases, such as the Enhancer of zeste-like and trithorax-like proteins, plants and animals contain homologous proteins with a similar organization of domains outside of the SET domain. However, a majority of plant SET domain proteins do not have an animal homolog with similar domain organization, suggesting that plants have unique mechanisms to establish and maintain chromatin states. Although the domains present in plant and animal SET domain proteins often differ, the domains found in the plant proteins have been generally implicated in protein-protein interactions, indicating that most SET domain proteins operate in complexes. Combined analysis of the maize and Arabidopsis SET domain proteins reveals that duplication of SET domain proteins in plants is extensive and has occurred via multiple mechanisms that preceded the divergence of monocots and dicots.

Insights into the Maize Pan-Genome and Pan-Transcriptome

Transcriptome sequencing of diverse maize inbreds provided insights into the nature of the maize pan-genome, including identification of 8681 loci absent in the B73 reference sequence. Genome-wide association studies using single nucleotide polymorphisms and transcript abundance variants in the maize pan-genome identified loci associated with traits important for fitness and adaptation. Genomes at the species level are dynamic, with genes present in every individual (core) and genes in a subset of individuals (dispensable) that collectively constitute the pan-genome. Using transcriptome sequencing of seedling RNA from 503 maize (Zea mays) inbred lines to characterize the maize pan-genome, we identified 8681 representative transcript assemblies (RTAs) with 16.4% expressed in all lines and 82.7% expressed in subsets of the lines. Interestingly, with linkage disequilibrium mapping, 76.7% of the RTAs with at least one single nucleotide polymorphism (SNP) could be mapped to a single genetic position, distributed primarily throughout the nonpericentromeric portion of the genome. Stepwise iterative clustering of RTAs suggests, within the context of the genotypes used in this study, that the maize genome is restricted and further sampling of seedling RNA within this germplasm base will result in minimal discovery. Genome-wide association studies based on SNPs and transcript abundance in the pan-genome revealed loci associated with the timing of the juvenile-to-adult vegetative and vegetative-to-reproductive developmental transitions, two traits important for fitness and adaptation. This study revealed the dynamic nature of the maize pan-genome and demonstrated that a substantial portion of variation may lie outside the single reference genome for a species.

Maize Chromomethylase Zea methyltransferase2 Is Required for CpNpG Methylation

A cytosine DNA methyltransferase containing a chromodomain, Zea methyltransferase2 (Zmet2), was cloned from maize. The sequence of ZMET2 is similar to that of the Arabidopsis chromomethylases CMT1 and CMT3, with C-terminal motifs characteristic of eukaryotic and prokaryotic DNA methyltransferases. We used a reverse genetics approach to determine the function of the Zmet2 gene. Plants homozygous for a Mutator transposable element insertion into motif IX had a 13% reduction in methylated cytosines. DNA gel blot analysis of these plants with methylation-sensitive restriction enzymes and bisulfite sequencing of a 180-bp knob sequence showed reduced methylation only at CpNpG sites. No reductions in methylation were observed at CpG or asymmetric sites in heterozygous or homozygous mutant plants. Our research shows that chromomethylase Zmet2 is required for in vivo methylation of CpNpG sequences.