Large-scale replicated field study of maize rhizosphere identifies heritable microbes.

Soil microbes that colonize plant roots and are responsive to differences in plant genotype remain to be ascertained for agronomically important crops. From a very large-scale longitudinal field study of 27 maize inbred lines planted in three fields, with partial replication 5 y later, we identify root-associated microbiota exhibiting reproducible associations with plant genotype. Analysis of 4,866 samples identified 143 operational taxonomic units (OTUs) whose variation in relative abundances across the samples was significantly regulated by plant genotype, and included five of seven core OTUs present in all samples. Plant genetic effects were significant amid the large effects of plant age on the rhizosphere microbiome, regardless of the specific community of each field, and despite microbiome responses to climate events. Seasonal patterns showed that the plant root microbiome is locally seeded, changes with plant growth, and responds to weather events. However, against this background of variation, specific taxa responded to differences in host genotype. If shown to have beneficial functions, microbes may be considered candidate traits for selective breeding.

Genetic basis of transcriptome diversity in Drosophila melanogaster.

Understanding how DNA sequence variation is translated into variation for complex phenotypes has remained elusive but is essential for predicting adaptive evolution, for selecting agriculturally important animals and crops, and for personalized medicine. Gene expression may provide a link between variation in DNA sequence and organismal phenotypes, and its abundance can be measured efficiently and accurately. Here we quantified genome-wide variation in gene expression in the sequenced inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP), increasing the annotated Drosophila transcriptome by 11%, including thousands of novel transcribed regions (NTRs). We found that 42% of the Drosophila transcriptome is genetically variable in males and females, including the NTRs, and is organized into modules of genetically correlated transcripts. We found that NTRs often were negatively correlated with the expression of protein-coding genes, which we exploited to annotate NTRs functionally. We identified regulatory variants for the mean and variance of gene expression, which have largely independent genetic control. Expression quantitative trait loci (eQTLs) for the mean, but not for the variance, of gene expression were concentrated near genes. Notably, the variance eQTLs often interacted epistatically with local variants in these genes to regulate gene expression. This comprehensive characterization of population-scale diversity of transcriptomes and its genetic basis in the DGRP is critically important for a systems understanding of quantitative trait variation.

Genetic architecture of natural variation in Drosophila melanogaster aggressive behavior.

Aggression is an evolutionarily conserved complex behavior essential for survival and the organization of social hierarchies. With the exception of genetic variants associated with bioamine signaling, which have been implicated in aggression in many species, the genetic basis of natural variation in aggression is largely unknown. Drosophila melanogaster is a favorable model system for exploring the genetic basis of natural variation in aggression. Here, we performed genome-wide association analyses using the inbred, sequenced lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and replicate advanced intercross populations derived from the most and least aggressive DGRP lines. We identified genes that have been previously implicated in aggressive behavior as well as many novel loci, including gustatory receptor 63a (Gr63a), which encodes a subunit of the receptor for CO2, and genes associated with development and function of the nervous system. Although genes from the two association analyses were largely nonoverlapping, they mapped onto a genetic interaction network inferred from an analysis of pairwise epistasis in the DGRP. We used mutations and RNAi knock-down alleles to functionally validate 79% of the candidate genes and 75% of the candidate epistatic interactions tested. Epistasis for aggressive behavior causes cryptic genetic variation in the DGRP that is revealed by changing allele frequencies in the outbred populations derived from extreme DGRP lines. This phenomenon may pertain to other fitness traits and species, with implications for evolution, applied breeding, and human genetics.

Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines.

The Drosophila melanogaster Genetic Reference Panel (DGRP) is a community resource of 205 sequenced inbred lines, derived to improve our understanding of the effects of naturally occurring genetic variation on molecular and organismal phenotypes. We used an integrated genotyping strategy to identify 4,853,802 single nucleotide polymorphisms (SNPs) and 1,296,080 non-SNP variants. Our molecular population genomic analyses show higher deletion than insertion mutation rates and stronger purifying selection on deletions. Weaker selection on insertions than deletions is consistent with our observed distribution of genome size determined by flow cytometry, which is skewed toward larger genomes. Insertion/deletion and single nucleotide polymorphisms are positively correlated with each other and with local recombination, suggesting that their nonrandom distributions are due to hitchhiking and background selection. Our cytogenetic analysis identified 16 polymorphic inversions in the DGRP. Common inverted and standard karyotypes are genetically divergent and account for most of the variation in relatedness among the DGRP lines. Intriguingly, variation in genome size and many quantitative traits are significantly associated with inversions. Approximately 50% of the DGRP lines are infected with Wolbachia, and four lines have germline insertions of Wolbachia sequences, but effects of Wolbachia infection on quantitative traits are rarely significant. The DGRP complements ongoing efforts to functionally annotate the Drosophila genome. Indeed, 15% of all D. melanogaster genes segregate for potentially damaged proteins in the DGRP, and genome-wide analyses of quantitative traits identify novel candidate genes. The DGRP lines, sequence data, genotypes, quality scores, phenotypes, and analysis and visualization tools are publicly available.

The genetic architecture of maize height.

Height is one of the most heritable and easily measured traits in maize (Zea mays L.). Given a pedigree or estimates of the genomic identity-by-state among related plants, height is also accurately predictable. But, mapping alleles explaining natural variation in maize height remains a formidable challenge. To address this challenge, we measured the plant height, ear height, flowering time, and node counts of plants grown in >64,500 plots across 13 environments. These plots contained >7300 inbreds representing most publically available maize inbreds in the United States and families of the maize Nested Association Mapping (NAM) panel. Joint-linkage mapping of quantitative trait loci (QTL), fine mapping in near isogenic lines (NILs), genome-wide association studies (GWAS), and genomic best linear unbiased prediction (GBLUP) were performed. The heritability of maize height was estimated to be >90%. Mapping NAM family-nested QTL revealed the largest explained 2.1 ± 0.9% of height variation. The effects of two tropical alleles at this QTL were independently validated by fine mapping in NIL families. Several significant associations found by GWAS colocalized with established height loci, including brassinosteroid-deficient dwarf1, dwarf plant1, and semi-dwarf2. GBLUP explained >80% of height variation in the panels and outperformed bootstrap aggregation of family-nested QTL models in evaluations of prediction accuracy. These results revealed maize height was under strong genetic control and had a highly polygenic genetic architecture. They also showed that multiple models of genetic architecture differing in polygenicity and effect sizes can plausibly explain a population's variation in maize height, but they may vary in predictive efficacy.

Exploring the maize rhizosphere microbiome in the field: A glimpse into a highly complex system.

Maize is one of the most economically important crops in the world. Understanding how the genetics and management of this staple crop interact with local field environments is vital to securing sustainable harvests. The interface zone between the plant root and its surrounding soil, or rhizosphere, supports essential interactions between roots and local soils. These interactions include the exchange of carbon for nutrients and are strongly influenced by the microbial constituents of the soil, or the microbiome. In a recent multi-environment study, we explored the diversity and heritability of the maize rhizosphere microbiome at flowering time. We assessed the bacterial diversity of the maize rhizosphere by pyrosequencing of 16S rRNA genes obtained from soil surrounding the roots of 27 genetically diverse maize inbreds grown in five field environments. We then partitioned variation in α- and ß-diversity of the microbiome across plant inbreds in the different fields. The results of this study revealed the heritability and significance of genotype-by-environment interactions on the maize rhizosphere microbiome at a single time point. Longitudinal studies detailing the maize rhizosphere throughout an entire growing season are currently underway and should provide a more detailed view of how plant genotypes interact with the environment to shape the microbiome. Future efforts will aim to incorporate these interactions into genetic models of economically important traits such as yield.

The genetic architecture of maize stalk strength.

Stalk strength is an important trait in maize (Zea mays L.). Strong stalks reduce lodging and maximize harvestable yield. Studies show rind penetrometer resistance (RPR), or the force required to pierce a stalk rind with a spike, is a valid approximation of strength. We measured RPR across 4,692 recombinant inbreds (RILs) comprising the maize nested association mapping (NAM) panel derived from crosses of diverse inbreds to the inbred, B73. An intermated B73×Mo17 family (IBM) of 196 RILs and a panel of 2,453 diverse inbreds from the North Central Regional Plant Introduction Station (NCRPIS) were also evaluated. We measured RPR in three environments. Family-nested QTL were identified by joint-linkage mapping in the NAM panel. We also performed a genome-wide association study (GWAS) and genomic best linear unbiased prediction (GBLUP) in each panel. Broad sense heritability computed on a line means basis was low for RPR. Only 8 of 26 families had a heritability above 0.20. The NCRPIS diversity panel had a heritability of 0.54. Across NAM and IBM families, 18 family-nested QTL and 141 significant GWAS associations were identified for RPR. Numerous weak associations were also found in the NCRPIS diversity panel. However, few were linked to loci involved in phenylpropanoid and cellulose synthesis or vegetative phase transition. Using an identity-by-state (IBS) relationship matrix estimated from 1.6 million single nucleotide polymorphisms (SNPs) and RPR measures from 20% of the NAM panel, genomic prediction by GBLUP explained 64±2% of variation in the remaining RILs. In the NCRPIS diversity panel, an IBS matrix estimated from 681,257 SNPs and RPR measures from 20% of the panel explained 33±3% of variation in the remaining inbreds. These results indicate the high genetic complexity of stalk strength and the potential for genomic prediction to hasten its improvement.

Comprehensive genotyping of the USA national maize inbred seed bank.

BACKGROUND: Genotyping by sequencing, a new low-cost, high-throughput sequencing technology was used to genotype 2,815 maize inbred accessions, preserved mostly at the National Plant Germplasm System in the USA. The collection includes inbred lines from breeding programs all over the world. RESULTS: The method produced 681,257 single-nucleotide polymorphism (SNP) markers distributed across the entire genome, with the ability to detect rare alleles at high confidence levels. More than half of the SNPs in the collection are rare. Although most rare alleles have been incorporated into public temperate breeding programs, only a modest amount of the available diversity is present in the commercial germplasm. Analysis of genetic distances shows population stratification, including a small number of large clusters centered on key lines. Nevertheless, an average fixation index of 0.06 indicates moderate differentiation between the three major maize subpopulations. Linkage disequilibrium (LD) decays very rapidly, but the extent of LD is highly dependent on the particular group of germplasm and region of the genome. The utility of these data for performing genome-wide association studies was tested with two simply inherited traits and one complex trait. We identified trait associations at SNPs very close to known candidate genes for kernel color, sweet corn, and flowering time; however, results suggest that more SNPs are needed to better explore the genetic architecture of complex traits. CONCLUSIONS: The genotypic information described here allows this publicly available panel to be exploited by researchers facing the challenges of sustainable agriculture through better knowledge of the nature of genetic diversity.

Diversity and heritability of the maize rhizosphere microbiome under field conditions.

The rhizosphere is a critical interface supporting the exchange of resources between plants and their associated soil environment. Rhizosphere microbial diversity is influenced by the physical and chemical properties of the rhizosphere, some of which are determined by the genetics of the host plant. However, within a plant species, the impact of genetic variation on the composition of the microbiota is poorly understood. Here, we characterized the rhizosphere bacterial diversity of 27 modern maize inbreds possessing exceptional genetic diversity grown under field conditions. Randomized and replicated plots of the inbreds were planted in five field environments in three states, each with unique soils and management conditions. Using pyrosequencing of bacterial 16S rRNA genes, we observed substantial variation in bacterial richness, diversity, and relative abundances of taxa between bulk soil and the maize rhizosphere, as well as between fields. The rhizospheres from maize inbreds exhibited both a small but significant proportion of heritable variation in total bacterial diversity across fields, and substantially more heritable variation between replicates of the inbreds within each field. The results of this study should facilitate expanded studies to identify robust heritable plant-microbe interactions at the level of individual polymorphisms by genome wide association, so that plant-microbiome interactions can ultimately be incorporated into plant breeding.

GAPIT: genome association and prediction integrated tool.

SUMMARY: Software programs that conduct genome-wide association studies and genomic prediction and selection need to use methodologies that maximize statistical power, provide high prediction accuracy and run in a computationally efficient manner. We developed an R package called Genome Association and Prediction Integrated Tool (GAPIT) that implements advanced statistical methods including the compressed mixed linear model (CMLM) and CMLM-based genomic prediction and selection. The GAPIT package can handle large datasets in excess of 10 000 individuals and 1 million single-nucleotide polymorphisms with minimal computational time, while providing user-friendly access and concise tables and graphs to interpret results. AVAILABILITY: http://www.maizegenetics.net/GAPIT. CONTACT: zhiwu.zhang@cornell.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A first-generation haplotype map of maize.

Maize is an important crop species of high genetic diversity. We identified and genotyped several million sequence polymorphisms among 27 diverse maize inbred lines and discovered that the genome was characterized by highly divergent haplotypes and showed 10- to 30-fold variation in recombination rates. Most chromosomes have pericentromeric regions with highly suppressed recombination that appear to have influenced the effectiveness of selection during maize inbred development and may be a major component of heterosis. We found hundreds of selective sweeps and highly differentiated regions that probably contain loci that are key to geographic adaptation. This survey of genetic diversity provides a foundation for uniting breeding efforts across the world and for dissecting complex traits through genome-wide association studies.

Association mapping: critical considerations shift from genotyping to experimental design.

The goal of many plant scientists' research is to explain natural phenotypic variation in terms of simple changes in DNA sequence. Traditionally, linkage mapping has been the most commonly employed method to reach this goal: experimental crosses are made to generate a family with known relatedness, and attempts are made to identify cosegregation of genetic markers and phenotypes within this family. In vertebrate systems, association mapping (also known as linkage disequilibrium mapping) is increasingly being adopted as the mapping method of choice. Association mapping involves searching for genotype-phenotype correlations in unrelated individuals and often is more rapid and cost-effective than traditional linkage mapping. We emphasize here that linkage and association mapping are complementary approaches and are more similar than is often assumed. Unlike in vertebrates, where controlled crosses can be expensive or impossible (e.g., in humans), the plant scientific community can exploit the advantages of both controlled crosses and association mapping to increase statistical power and mapping resolution. While the time and money required for the collection of genotype data were critical considerations in the past, the increasing availability of inexpensive DNA sequencing and genotyping methods should prompt researchers to shift their attention to experimental design. This review provides thoughts on finding the optimal experimental mix of association mapping using unrelated individuals and controlled crosses to identify the genes underlying phenotypic variation.

The genetic architecture of maize flowering time.

Flowering time is a complex trait that controls adaptation of plants to their local environment in the outcrossing species Zea mays (maize). We dissected variation for flowering time with a set of 5000 recombinant inbred lines (maize Nested Association Mapping population, NAM). Nearly a million plants were assayed in eight environments but showed no evidence for any single large-effect quantitative trait loci (QTLs). Instead, we identified evidence for numerous small-effect QTLs shared among families; however, allelic effects differ across founder lines. We identified no individual QTLs at which allelic effects are determined by geographic origin or large effects for epistasis or environmental interactions. Thus, a simple additive model accurately predicts flowering time for maize, in contrast to the genetic architecture observed in the selfing plant species rice and Arabidopsis.

A spatial dissection of the Arabidopsis floral transcriptome by MPSS.

BACKGROUND: We have further characterized floral organ-localized gene expression in the inflorescence of Arabidopsis thaliana by comparison of massively parallel signature sequencing (MPSS) data. Six libraries of RNA sequence tags from immature inflorescence tissues were constructed and matched to their respective loci in the annotated Arabidopsis genome. These signature libraries survey the floral transcriptome of wild-type tissue as well as the floral homeotic mutants, apetala1, apetala3, agamous, a superman/apetala1 double mutant, and differentiated ovules dissected from the gynoecia of wild-type inflorescences. Comparing and contrasting these MPSS floral expression libraries enabled demarcation of transcripts enriched in the petals, stamens, stigma-style, gynoecia, and those with predicted enrichment within the sepal/sepal-petals, petal-stamens, or gynoecia-stamens. RESULTS: By comparison of expression libraries, a total of 572 genes were found to have organ-enriched expression within the inflorescence. The bulk of characterized organ-enriched transcript diversity was noted in the gynoecia and stamens, whereas fewer genes demonstrated sepal or petal-localized expression. Validation of the computational analyses was performed by comparison with previously published expression data, in situ hybridizations, promoter-reporter fusions, and reverse transcription PCR. A number of well-characterized genes were accurately delineated within our system of transcript filtration. Moreover, empirical validations confirm MPSS predictions for several genes with previously uncharacterized expression patterns. CONCLUSION: This extensive MPSS analysis confirms and supplements prior microarray floral expression studies and illustrates the utility of sequence survey-based expression analysis in functional genomics. Spatial floral expression data accrued by MPSS and similar methods will be advantageous in the elucidation of more comprehensive genetic regulatory networks governing floral development.