Structural variants in the soybean genome localize to clusters of biotic stress-response genes.

Genome-wide structural and gene content variations are hypothesized to drive important phenotypic variation within a species. Structural and gene content variations were assessed among four soybean (Glycine max) genotypes using array hybridization and targeted resequencing. Many chromosomes exhibited relatively low rates of structural variation (SV) among genotypes. However, several regions exhibited both copy number and presence-absence variation, the most prominent found on chromosomes 3, 6, 7, 16, and 18. Interestingly, the regions most enriched for SV were specifically localized to gene-rich regions that harbor clustered multigene families. The most abundant classes of gene families associated with these regions were the nucleotide-binding and receptor-like protein classes, both of which are important for plant biotic defense. The colocalization of SV with plant defense response signal transduction pathways provides insight into the mechanisms of soybean resistance gene evolution and may inform the development of new approaches to resistance gene cloning.

The composition and origins of genomic variation among individuals of the soybean reference cultivar Williams 82.

Soybean (Glycine max) is a self-pollinating species that has relatively low nucleotide polymorphism rates compared with other crop species. Despite the low rate of nucleotide polymorphisms, a wide range of heritable phenotypic variation exists. There is even evidence for heritable phenotypic variation among individuals within some cultivars. Williams 82, the soybean cultivar used to produce the reference genome sequence, was derived from backcrossing a Phytophthora root rot resistance locus from the donor parent Kingwa into the recurrent parent Williams. To explore the genetic basis of intracultivar variation, we investigated the nucleotide, structural, and gene content variation of different Williams 82 individuals. Williams 82 individuals exhibited variation in the number and size of introgressed Kingwa loci. In these regions of genomic heterogeneity, the reference Williams 82 genome sequence consists of a mosaic of Williams and Kingwa haplotypes. Genomic structural variation between Williams and Kingwa was maintained between the Williams 82 individuals within the regions of heterogeneity. Additionally, the regions of heterogeneity exhibited gene content differences between Williams 82 individuals. These findings show that genetic heterogeneity in Williams 82 primarily originated from the differential segregation of polymorphic chromosomal regions following the backcross and single-seed descent generations of the breeding process. We conclude that soybean haplotypes can possess a high rate of structural and gene content variation, and the impact of intracultivar genetic heterogeneity may be significant. This detailed characterization will be useful for interpreting soybean genomic data sets and highlights important considerations for research communities that are developing or utilizing a reference genome sequence.

Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases.

We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). A suite of ZFNs were engineered by the recently described context-dependent assembly platform--a rapid, open-source method for generating zinc-finger arrays. Specific ZFNs targeting dicer-like (DCL) genes and other genes involved in RNA silencing were cloned into a vector under an estrogen-inducible promoter. A hairy-root transformation system was employed to investigate the efficiency of ZFN mutagenesis at each target locus. Transgenic roots exhibited somatic mutations localized at the ZFN target sites for seven out of nine targeted genes. We next introduced a ZFN into soybean via whole-plant transformation and generated independent mutations in the paralogous genes DCL4a and DCL4b. The dcl4b mutation showed efficient heritable transmission of the ZFN-induced mutation in the subsequent generation. These findings indicate that ZFN-based mutagenesis provides an efficient method for making mutations in duplicate genes that are otherwise difficult to study due to redundancy. We also developed a publicly accessible Web-based tool to identify sites suitable for engineering context-dependent assembly ZFNs in the soybean genome.

Phenotypic and genomic analyses of a fast neutron mutant population resource in soybean.

Mutagenized populations have become indispensable resources for introducing variation and studying gene function in plant genomics research. In this study, fast neutron (FN) radiation was used to induce deletion mutations in the soybean (Glycine max) genome. Approximately 120,000 soybean seeds were exposed to FN radiation doses of up to 32 Gray units to develop over 23,000 independent M2 lines. Here, we demonstrate the utility of this population for phenotypic screening and associated genomic characterization of striking and agronomically important traits. Plant variation was cataloged for seed composition, maturity, morphology, pigmentation, and nodulation traits. Mutants that showed significant increases or decreases in seed protein and oil content across multiple generations and environments were identified. The application of comparative genomic hybridization (CGH) to lesion-induced mutants for deletion mapping was validated on a midoleate x-ray mutant, M23, with a known FAD2-1A (for fatty acid desaturase) gene deletion. Using CGH, a subset of mutants was characterized, revealing deletion regions and candidate genes associated with phenotypes of interest. Exome resequencing and sequencing of PCR products confirmed FN-induced deletions detected by CGH. Beyond characterization of soybean FN mutants, this study demonstrates the utility of CGH, exome sequence capture, and next-generation sequencing approaches for analyses of mutant plant genomes. We present this FN mutant soybean population as a valuable public resource for future genetic screens and functional genomics research.

Disruption of imprinting by mutator transposon insertions in the 5' proximal regions of the Zea mays Mez1 locus.

Imprinting is a form of epigenetic gene regulation in which alleles are differentially regulated according to the parent of origin. The Mez1 gene in maize is imprinted such that the maternal allele is expressed in the endosperm while the paternal allele is not expressed. Three novel Mez1 alleles containing Mutator transposon insertions within the promoter were identified. These mez1-mu alleles do not affect vegetative expression levels or result in morphological phenotypes. However, these alleles can disrupt imprinted expression of Mez1. Maternal inheritance of the mez-m1 or mez1-m4 alleles results in activation of the normally silenced paternal allele of Mez1. Paternal inheritance of the mez1-m2 or mez1-m4 alleles can also result in a loss of silencing of the paternal Mez1 allele. The paternal disruption of imprinting by transposon insertions may reflect a requirement for sequence elements involved in targeting silencing of the paternal allele. The maternal disruption of imprinting by transposon insertions within the Mez1 promoter suggests that maternally produced MEZ1 protein may be involved in silencing of the paternal Mez1 allele. The endosperms with impaired imprinting did not exhibit phenotypic consequences associated with bi-allelic Mez1 expression.

Maternal and paternal alleles exhibit differential histone methylation and acetylation at maize imprinted genes.

Imprinting is an epigenetically controlled form of gene regulation in which the expression of a gene is based on its parent of origin. This epigenetic regulation is likely to involve allele-specific DNA or histone modifications. The relative abundance of eight different histone modifications was tested at various regions in several imprinted maize (Zea mays) genes using a chromatin immunoprecipitation protocol coupled with quantitative allele-specific single nucleotide polymorphism assays. Histone H3 lysine-27 di- and tri-methylation are paternally enriched at the imprinted loci Mez1, ZmFie1 and Nrp1. In contrast, acetylation of histones H3 and H4 and H3K4 dimethylation are enriched at the maternal alleles of these genes. Di- and tri-methylation of H3 lysine-9, which is generally associated with constitutively silenced chromatin, was not enriched at either allele of imprinted loci. These patterns of enrichment were specific to tissues that exhibit imprinting. In addition, the enrichment of these modifications was dependent upon the parental origin of an allele and not sequence differences between the alleles, as demonstrated by reciprocal crosses. This study presents a detailed view of the chromatin modifications that are associated with the maternal and paternal alleles at imprinted loci and provides evidence for common histone modifications at multiple imprinted loci.

Gene expression analyses in maize inbreds and hybrids with varying levels of heterosis.

BACKGROUND: Heterosis is the superior performance of F1 hybrid progeny relative to the parental phenotypes. Maize exhibits heterosis for a wide range of traits, however the magnitude of heterosis is highly variable depending on the choice of parents and the trait(s) measured. We have used expression profiling to determine whether the level, or types, of non-additive gene expression vary in maize hybrids with different levels of genetic diversity or heterosis. RESULTS: We observed that the distributions of better parent heterosis among a series of 25 maize hybrids generally do not exhibit significant correlations between different traits. Expression profiling analyses for six of these hybrids, chosen to represent diversity in genotypes and heterosis responses, revealed a correlation between genetic diversity and transcriptional variation. The majority of differentially expressed genes in each of the six different hybrids exhibited additive expression patterns, and approximately 25% exhibited statistically significant non-additive expression profiles. Among the non-additive profiles, approximately 80% exhibited hybrid expression levels between the parental levels, approximately 20% exhibited hybrid expression levels at the parental levels and ~1% exhibited hybrid levels outside the parental range. CONCLUSION: We have found that maize inbred genetic diversity is correlated with transcriptional variation. However, sampling of seedling tissues indicated that the frequencies of additive and non-additive expression patterns are very similar across a range of hybrid lines. These findings suggest that heterosis is probably not a consequence of higher levels of additive or non-additive expression, but may be related to transcriptional variation between parents. The lack of correlation between better parent heterosis levels for different traits suggests that transcriptional diversity at specific sets of genes may influence heterosis for different traits.

Natural variation for alleles under epigenetic control by the maize chromomethylase zmet2.

The contribution of epigenetic alterations to natural variation for gene transcription levels remains unclear. In this study, we investigated the functional targets of the maize chromomethylase ZMET2 in multiple inbred lines to determine whether epigenetic changes conditioned by this chromomethylase are conserved or variable within the species. Gene expression microarrays were hybridized with RNA samples from the inbred lines B73 and Mo17 and from near-isogenic derivatives containing the loss-of-function allele zmet2-m1. A set of 126 genes that displayed statistically significant differential expression in zmet2 mutants relative to wild-type plants in at least one of the two genetic backgrounds was identified. Analysis of the transcript levels in both wild-type and mutant individuals revealed that only 10% of these genes were affected in zmet2 mutants in both B73 and Mo17 genetic backgrounds. Over 80% of the genes with expression patterns affected by zmet2 mutations display variation for gene expression between wild-type B73 and Mo17 plants. Further analysis was performed for 7 genes that were transcriptionally silent in wild-type B73, but expressed in B73 zmet2-m1, wild-type Mo17, and Mo17 zmet2-m1 lines. Mapping experiments confirmed that the expression differences in wild-type B73 relative to Mo17 inbreds for these genes were caused by cis-acting regulatory variation. Methylation-sensitive PCR and bisulfite sequencing demonstrated that for 5 of these genes the CpNpG methylation in the wild-type B73 genetic background was substantially decreased in the B73 zmet2-m1 mutant and in wild-type Mo17. A survey of eight maize inbreds reveals that each of these 5 genes exhibit transcriptionally silent and methylated states in some inbred lines and unmethylated, expressed states in other inbreds, providing evidence for natural variation in epigenetic states for some maize genes.

Genomic imprinting, methylation and molecular evolution of maize Enhancer of zeste (Mez) homologs.

Imprinted gene expression refers to differential transcription of alleles depending on their parental origin. To date, most examples of imprinted gene expression in plants occur in the triploid endosperm tissue. The Arabidopsis gene MEDEA displays an imprinted pattern of gene expression and has homology to the Drosophila Polycomb group (PcG) protein Enhancer-of-zeste (E(z)). We have tested the allele-specific expression patterns of the three maize E(z)-like genes Mez1, Mez2 and Mez3. The expression of Mez2 and Mez3 is not imprinted, with a bi-allelic pattern of transcription for both genes in both the endosperm and embryonic tissue. In contrast, Mez1 displays a bi-allelic expression pattern in the embryonic tissue, and a mono-allelic expression pattern in the developing endosperm tissue. We demonstrate that mono-allelic expression of the maternal Mez1 allele occurs throughout endosperm development. We have identified a 556 bp differentially methylated region (DMR) located approximately 700 bp 5' of the Mez1 transcription start site. This region is heavily methylated at CpG and CpNpG nucleotides on the non-expressed paternal allele but has low levels of methylation on the expressed maternal allele. Molecular evolutionary analysis indicates that conserved domains of all three Mez genes are under purifying selection. The common imprinted expression of Mez1 and MEDEA, in concert with their likely evolutionary origins, suggests that there may be a requirement for imprinting of at least one E(z)-like gene in angiosperms.