Dysregulation of expression correlates with rare-allele burden and fitness loss in maize.

Here we report a multi-tissue gene expression resource that represents the genotypic and phenotypic diversity of modern inbred maize, and includes transcriptomes in an average of 255 lines in seven tissues. We mapped expression quantitative trait loci and characterized the contribution of rare genetic variants to extremes in gene expression. Some of the new mutations that arise in the maize genome can be deleterious; although selection acts to keep deleterious variants rare, their complete removal is impeded by genetic linkage to favourable loci and by finite population size. Modern maize breeders have systematically reduced the effects of this constant mutational pressure through artificial selection and self-fertilization, which have exposed rare recessive variants in elite inbred lines. However, the ongoing effect of these rare alleles on modern inbred maize is unknown. By analysing this gene expression resource and exploiting the extreme diversity and rapid linkage disequilibrium decay of maize, we characterize the effect of rare alleles and evolutionary history on the regulation of expression. Rare alleles are associated with the dysregulation of expression, and we correlate this dysregulation to seed-weight fitness. We find enrichment of ancestral rare variants among expression quantitative trait loci mapped in modern inbred lines, which suggests that historic bottlenecks have shaped regulation. Our results suggest that one path for further genetic improvement in agricultural species lies in purging the rare deleterious variants that have been associated with crop fitness.

Genome-wide analysis of deletions in maize population reveals abundant genetic diversity and functional impact.

KEY MESSAGE: Two read depth methods were jointly used in next-generation sequencing data to identify deletions in maize population. GWAS by deletions were analyzed for gene expression pattern and classical traits, respectively. Many studies have confirmed that structural variation (SV) is pervasive throughout the maize genome. Deletion is one type of SV that may impact gene expression and cause phenotypic changes in quantitative traits. In this study, two read count approaches were used to analyze the deletions in the whole-genome sequencing data of 270 maize inbred lines. A total of 19,754 deletion windows overlapped 12,751 genes, which were unevenly distributed across the genome. The deletions explained population structure well and correlated with genomic features. The deletion proportion of genes was determined to be negatively correlated with its expression. The detection of gene expression quantitative trait loci (eQTL) indicated that local eQTL were fewer but had larger effects than distant ones. The common associated genes were related to basic metabolic processes, whereas unique associated genes with eQTL played a role in the stress or stimulus responses in multiple tissues. Compared with the eQTL detected by SNPs derived from the same sequencing data, 89.4% of the associated genes could be detected by both markers. The effect of top eQTL detected by SNPs was usually larger than that detected by deletions for the same gene. A genome-wide association study (GWAS) on flowering time and plant height illustrated that only a few loci could be consistently captured by SNPs, suggesting that combining deletion and SNP for GWAS was an excellent strategy to dissect trait architecture. Our findings will provide insights into characteristic and biological function of genome-wide deletions in maize.

Coregulation of ribosomal RNA with hundreds of genes contributes to phenotypic variation.

Ribosomal repeats occupy 5% of a plant genome, yet there has been little study of their diversity in the modern age of genomics. Ribosomal copy number and expression variation present an opportunity to tap a novel source of diversity. In the present study, we estimated the ribosomal DNA (rDNA) copy number and ribosomal RNA (rRNA) expression for a population of maize inbred lines and investigated the potential role of rDNA and rRNA dosage in regulating global gene expression. Extensive variation was found in both ribosomal DNA copy number and ribosomal RNA expression among maize inbred lines. However, rRNA abundance was not consistent with the copy number of the rDNA. We have not found that the rDNA gene dosage has a regulatory role in gene expression; however, thousands of genes are identified to be coregulated with rRNA expression, including genes participating in ribosome biogenesis and other functionally relevant pathways. We further investigated the potential roles of copy number and the expression level of rDNA on agronomic traits and found that both correlated with flowering time but through different regulatory mechanisms. This comprehensive analysis suggested that rRNA expression variation is a valuable source of functional diversity that affects gene expression variation and field-based phenotypic changes.

Changes at the 3'-untranslated region stabilize Rubisco activase transcript levels during heat stress in Arabidopsis.

Inhibition of photosynthesis by heat stress is accompanied by functional impairment of Rubisco's chaperone, activase (RCA), resulting in deactivation of Rubisco. Since activase is extremely sensitive to thermal denaturation, changes in expression of RCA at the transcript or protein level could provide a mechanism for acclimation of photosynthesis to prolonged heat stress. Using quantitative real-time PCR (qPCR) we show steady-state RCA transcript levels in Arabidopsis thaliana are stabilized during prolonged exposure to moderate heat (35  °C). A survey of RCA transcripts indicates heat stress did not alter the relative abundance of transcripts encoding α and ß-isoforms of activase that are produced by alternative splicing of the pre-mRNA. Instead, mRNA stabilization in heat-stressed plants coincided with a significant reduction in the average length of activase 3'-untranslated regions, and was associated with enrichment of an uncharacterized activase mRNA splice variant, AtRCAß2. Transcript-specific qPCR revealed AtRCAß2 mRNA was more stable than AtRCAα and AtRCAß mRNA in heat-stressed plants. Using an inducible transgenic system, we found that RCA transcripts lacking their native 3'-untranslated region were significantly more stable than their full-length counterparts in vivo. Using this system, stability of the RCA protein was examined over 24 h in vivo, in the absence of RCA transcription. At both optimal and elevated temperatures, RCA protein levels remained stable in plants lacking RCA mRNA, but increased when RCA mRNA was present, particularly in heat-stressed plants. This study reveals a possible mechanism, involving post-transcriptional regulation of an important photosynthesis regulatory gene, for acclimation of photosynthesis to heat stress.

Local adaptation contributes to gene expression divergence in maize.

Gene expression links genotypes to phenotypes, so identifying genes whose expression is shaped by selection will be important for understanding the traits and processes underlying local adaptation. However, detecting local adaptation for gene expression will require distinguishing between divergence due to selection and divergence due to genetic drift. Here, we adapt a QST-FST framework to detect local adaptation for transcriptome-wide gene expression levels in a population of diverse maize genotypes. We compare the number and types of selected genes across a wide range of maize populations and tissues, as well as selection on cold-response genes, drought-response genes, and coexpression clusters. We identify a number of genes whose expression levels are consistent with local adaptation and show that genes involved in stress response show enrichment for selection. Due to its history of intense selective breeding and domestication, maize evolution has long been of interest to researchers, and our study provides insight into the genes and processes important for in local adaptation of maize.

Transcriptome-Wide Association Supplements Genome-Wide Association in Zea mays.

Modern improvement of complex traits in agricultural species relies on successful associations of heritable molecular variation with observable phenotypes. Historically, this pursuit has primarily been based on easily measurable genetic markers. The recent advent of new technologies allows assaying and quantifying biological intermediates (hereafter endophenotypes) which are now readily measurable at a large scale across diverse individuals. The usefulness of endophenotypes for delineating the regulatory landscape of the genome and genetic dissection of complex trait variation remains underexplored in plants. The work presented here illustrated the utility of a large-scale (299-genotype and seven-tissue) gene expression resource to dissect traits across multiple levels of biological organization. Using single-tissue- and multi-tissue-based transcriptome-wide association studies (TWAS), we revealed that about half of the functional variation acts through altered transcript abundance for maize kernel traits, including 30 grain carotenoid abundance traits, 20 grain tocochromanol abundance traits, and 22 field-measured agronomic traits. Comparing the efficacy of TWAS with genome-wide association studies (GWAS) and an ensemble approach that combines both GWAS and TWAS, we demonstrated that results of TWAS in combination with GWAS increase the power to detect known genes and aid in prioritizing likely causal genes. Using a variance partitioning approach in the largely independent maize Nested Association Mapping (NAM) population, we also showed that the most strongly associated genes identified by combining GWAS and TWAS explain more heritable variance for a majority of traits than the heritability captured by the random genes and the genes identified by GWAS or TWAS alone. This not only improves the ability to link genes to phenotypes, but also highlights the phenotypic consequences of regulatory variation in plants.

Cytogenetic and Sequence Analyses of Mitochondrial DNA Insertions in Nuclear Chromosomes of Maize.

The transfer of mitochondrial DNA (mtDNA) into nuclear genomes is a regularly occurring process that has been observed in many species. Few studies, however, have focused on the variation of nuclear-mtDNA sequences (NUMTs) within a species. This study examined mtDNA insertions within chromosomes of a diverse set of Zea mays ssp. mays (maize) inbred lines by the use of fluorescence in situ hybridization. A relatively large NUMT on the long arm of chromosome 9 (9L) was identified at approximately the same position in four inbred lines (B73, M825, HP301, and Oh7B). Further examination of the similarly positioned 9L NUMT in two lines, B73 and M825, indicated that the large size of these sites is due to the presence of a majority of the mitochondrial genome; however, only portions of this NUMT (~252 kb total) were found in the publically available B73 nuclear sequence for chromosome 9. Fiber-fluorescence in situ hybridization analysis estimated the size of the B73 9L NUMT to be ~1.8 Mb and revealed that the NUMT is methylated. Two regions of mtDNA (2.4 kb and 3.3 kb) within the 9L NUMT are not present in the B73 mitochondrial NB genome; however, these 2.4-kb and 3.3-kb segments are present in other Zea mitochondrial genomes, including that of Zea mays ssp. parviglumis, a progenitor of domesticated maize.