Nathan M. Springer

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 inbreds exhibit high levels of copy number variation (CNV) and presence/absence variation (PAV) in genome content

Following the domestication of maize over the past ∼10,000 years, breeders have exploited the extensive genetic diversity of this species to mold its phenotype to meet human needs. The extent of structural variation, including copy number variation (CNV) and presence/absence variation (PAV), which are thought to contribute to the extraordinary phenotypic diversity and plasticity of this important crop, have not been elucidated. Whole-genome, array-based, comparative genomic hybridization (CGH) revealed a level of structural diversity between the inbred lines B73 and Mo17 that is unprecedented among higher eukaryotes. A detailed analysis of altered segments of DNA conservatively estimates that there are several hundred CNV sequences among the two genotypes, as well as several thousand PAV sequences that are present in B73 but not Mo17. Haplotype-specific PAVs contain hundreds of single-copy, expressed genes that may contribute to heterosis and to the extraordinary phenotypic diversity of this important crop.

Genome-wide patterns of genetic variation among elite maize inbred lines

We have resequenced a group of six elite maize inbred lines, including the parents of the most productive commercial hybrid in China. This effort uncovered more than 1,000,000 SNPs, 30,000 indel polymorphisms and 101 low-sequence-diversity chromosomal intervals in the maize genome. We also identified several hundred complete genes that show presence/absence variation among these resequenced lines. We discuss the potential roles of complementation of presence/absence variations and other deleterious mutations in contributing to heterosis. High-density SNP and indel polymorphism markers reported here are expected to be a valuable resource for future genetic studies and the molecular breeding of this important crop.

Discovery of induced point mutations in maize genes by TILLING

BackgroundGoing from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community.ResultsWe demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious.ConclusionsOur findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project.

Differentiation of the maize subgenomes by genome dominance and both ancient and ongoing gene loss

Ancient tetraploidies are found throughout the eukaryotes. After duplication, one copy of each duplicate gene pair tends to be lost (fractionate). For all studied tetraploidies, the loss of duplicated genes, known as homeologs, homoeologs, ohnologs, or syntenic paralogs, is uneven between duplicate regions. In maize, a species that experienced a tetraploidy 5–12 million years ago, we show that in addition to uneven ancient gene loss, the two complete genomes contained within maize are differentiated by ongoing fractionation among diverse inbreds as well as by a pattern of overexpression of genes from the genome that has experienced less gene loss. These expression differences are consistent over a range of experiments quantifying RNA abundance in different tissues. We propose that the universal bias in gene loss between the genomes of this ancient tetraploid, and perhaps all tetraploids, is the result of selection against loss of the gene responsible for the majority of total expression for a duplicate gene pair. Although the tetraploidy of maize is ancient, biased gene loss and expression continue today and explain, at least in part, the remarkable genetic diversity found among modern maize cultivars.

Cis-transcriptional variation in maize inbred lines B73 and Mo17 leads to additive expression patterns in the F1 hybrid.

Microarray analysis of gene expression patterns in immature ear, seedling, and embryo tissues from the maize inbred lines B73 and Mo17 identified numerous genes with variable expression. Some genes had detectable expression in only one of the two inbreds; most of these genes were detected in the genomic DNA of both inbreds, indicating that the expression differences are likely caused by differential regulation rather than by differences in gene content. Gene expression was also monitored in the reciprocal F1 hybrids B73 × Mo17 and Mo17 × B73. The reciprocal F1 hybrid lines did not display parental effects on gene expression levels. Approximately 80% of the differentially expressed genes displayed additive expression patterns in the hybrids relative to the inbred parents. The ∼20% of genes that display nonadditive expression patterns tend to be expressed at levels within the parental range, with minimal evidence for novel expression levels greater than the high parent or less than the low parent. Analysis of allele-specific expression patterns in the hybrid suggested that intraspecific variation in gene expression levels is largely attributable to cis-regulatory variation in maize. Collectively, our data suggest that allelic cis-regulatory variation between B73 and Mo17 dictates maintenance of inbred allelic expression levels in the F1 hybrid, resulting in additive expression patterns.

Pervasive gene content variation and copy number variation in maize and its undomesticated progenitor

Individuals of the same species are generally thought to have very similar genomes. However, there is growing evidence that structural variation in the form of copy number variation (CNV) and presence-absence variation (PAV) can lead to variation in the genome content of individuals within a species. Array comparative genomic hybridization (CGH) was used to compare gene content and copy number variation among 19 diverse maize inbreds and 14 genotypes of the wild ancestor of maize, teosinte. We identified 479 genes exhibiting higher copy number in some genotypes (UpCNV) and 3410 genes that have either fewer copies or are missing in the genome of at least one genotype relative to B73 (DownCNV/PAV). Many of these DownCNV/PAV are examples of genes present in B73, but missing from other genotypes. Over 70% of the CNV/PAV examples are identified in multiple genotypes, and the majority of events are observed in both maize and teosinte, suggesting that these variants predate domestication and that there is not strong selection acting against them. Many of the genes affected by CNV/PAV are either maize specific (thus possible annotation artifacts) or members of large gene families, suggesting that the gene loss can be tolerated through buffering by redundant functions encoded elsewhere in the genome. While this structural variation may not result in major qualitative variation due to genetic buffering, it may significantly contribute to quantitative variation.

Reciprocal Silencing, Transcriptional Bias and Functional Divergence of Homeologs in Polyploid Cotton (Gossypium)

Polyploidy is an important force in the evolution of flowering plants. Genomic merger and doubling induce an extensive array of genomic effects, including immediate and long-term alterations in the expression of duplicate genes (“homeologs”). Here we employed a novel high-resolution, genome-specific, mass-spectrometry technology and a well-established phylogenetic framework to investigate relative expression levels of each homeolog for 63 gene pairs in 24 tissues in naturally occurring allopolyploid cotton (Gossypium L.), a synthetic allopolyploid of the same genomic composition, and models of the diploid progenitor species. Results from a total of 2177 successful expression assays permitted us to determine the extent of expression evolution accompanying genomic merger of divergent diploid parents, genome doubling, and genomic coevolution in a common nucleus subsequent to polyploid formation. We demonstrate that 40% of homeologs are transcriptionally biased in at least one stage of cotton development, that genome merger per se has a large effect on relative expression of homeologs, and that the majority of these alterations are caused by cis-regulatory divergence between the diploid progenitors. We describe the scope of transcriptional subfunctionalization and 15 cases of probable neofunctionalization among 8 tissues. To our knowledge, this study represents the first characterization of transcriptional neofunctionalization in an allopolyploid. These results provide a novel temporal perspective on expression evolution of duplicate genomes and add to our understanding of the importance of polyploidy in plants.

Progress Toward Understanding Heterosis in Crop Plants

Although heterosis, or hybrid vigor, is widely exploited in agriculture, a complete description of its molecular underpinnings has remained elusive despite extensive investigation. It appears that there is not a single, simple explanation for heterosis. Instead, it is likely that heterosis arises in crosses between genetically distinct individuals as a result of a diversity of mechanisms. Heterosis generally results from the action of multiple loci, and different loci affect heterosis for different traits and in different hybrids. Hence, multigene models are likely to prove most informative for understanding heterosis. Complementation of allelic variation, as well as complementation of variation in gene content and gene expression patterns, is likely to be an important contributor to heterosis. Epigenetic variation has the potential to interact in hybrid genotypes via novel mechanisms. Several other intriguing hypotheses are also under investigation. In crops, heterosis must be considered within the context of the genomic impacts of prior selection for agronomic traits.

Parent-of-Origin Effects on Gene Expression and DNA Methylation in the Maize Endosperm

This work uses deep sequencing of RNAs from maize endosperm and embryo to identify 54 maternally expressed genes and 46 paternally expressed genes and then examines genome-wide DNA methylation and gene expression, finding hypomethylation of the maternal allele and endosperm-specific expression for many of the imprinted genes. Imprinting describes the differential expression of alleles based on their parent of origin. Deep sequencing of RNAs from maize (Zea mays) endosperm and embryo tissue 14 d after pollination was used to identify imprinted genes among a set of ~12,000 genes that were expressed and contained sequence polymorphisms between the B73 and Mo17 genotypes. The analysis of parent-of-origin patterns of expression resulted in the identification of 100 putative imprinted genes in maize endosperm, including 54 maternally expressed genes (MEGs) and 46 paternally expressed genes (PEGs). Three of these genes have been previously identified as imprinted, while the remaining 97 genes represent novel imprinted maize genes. A genome-wide analysis of DNA methylation identified regions with reduced endosperm DNA methylation in, or near, 19 of the 100 imprinted genes. The reduced levels of DNA methylation in endosperm are caused by hypomethylation of the maternal allele for both MEGs and PEGs in all cases tested. Many of the imprinted genes with reduced DNA methylation levels also show endosperm-specific expression patterns. The imprinted maize genes were compared with imprinted genes identified in genome-wide screens of rice (Oryza sativa) and Arabidopsis thaliana, and at least 10 examples of conserved imprinting between maize and each of the other species were identified.