Bacterium-enabled transient gene activation by artificial transcription factors for resolving gene regulation in maize.

Understanding gene regulatory networks is essential to elucidate developmental processes and environmental responses. Here, we studied regulation of a maize (Zea mays) transcription factor gene using designer transcription activator-like effectors (dTALes), which are synthetic Type III TALes of the bacterial genus Xanthomonas and serve as inducers of disease susceptibility gene transcription in host cells. The maize pathogen Xanthomonas vasicola pv. vasculorum was used to introduce 2 independent dTALes into maize cells to induced expression of the gene glossy3 (gl3), which encodes a MYB transcription factor involved in biosynthesis of cuticular wax. RNA-seq analysis of leaf samples identified, in addition to gl3, 146 genes altered in expression by the 2 dTALes. Nine of the 10 genes known to be involved in cuticular wax biosynthesis were upregulated by at least 1 of the 2 dTALes. A gene previously unknown to be associated with gl3, Zm00001d017418, which encodes aldehyde dehydrogenase, was also expressed in a dTALe-dependent manner. A chemically induced mutant and a CRISPR-Cas9 mutant of Zm00001d017418 both exhibited glossy leaf phenotypes, indicating that Zm00001d017418 is involved in biosynthesis of cuticular waxes. Bacterial protein delivery of dTALes proved to be a straightforward and practical approach for the analysis and discovery of pathway-specific genes in maize.

Cold mediates maize root hair developmental plasticity via epidermis-specific transcriptomic responses.

Cold stress during early development limits maize (Zea mays L.) production in temperate zones. Low temperatures restrict root growth and reprogram gene expression. Here, we provide a systematic transcriptomic landscape of maize primary roots, their tissues, and cell types in response to cold stress. The epidermis exhibited a unique transcriptomic cold response, and genes involved in root hair formation were dynamically regulated in this cell type by cold. Consequently, activation of genes involved in root hair tip growth contributed to root hair recovery under moderate cold conditions. The maize root hair defective mutants roothair defective 5 (rth5) and roothair defective 6 (rth6) displayed enhanced cold tolerance with respect to primary root elongation. Furthermore, dehydration response element-binding protein 2.1 (dreb2.1) was the only member of the dreb subfamily of AP2/EREB transcription factor genes upregulated in primary root tissues and cell types but exclusively downregulated in root hairs upon cold stress. Plants overexpressing dreb2.1 significantly suppressed root hair elongation after moderate cold stress. Finally, the expression of rth3 was regulated by dreb2.1 under cold conditions, while rth6 transcription was regulated by dreb2.1 irrespective of the temperature regime. We demonstrated that dreb2.1 negatively regulates root hair plasticity at low temperatures by coordinating the expression of root hair defective genes in maize.

Spatiotemporal transcriptomic plasticity in barley roots: unravelling water deficit responses in distinct root zones.

BACKGROUND: Drought poses a major threat to agricultural production and thus food security. Understanding the processes shaping plant responses to water deficit is essential for global food safety. Though many studies examined the effect of water deficit on the whole-root level, the distinct functions of each root zone and their specific stress responses remain masked by this approach. RESULTS: In this study, we investigated the effect of water deficit on root development of the spring barley (Hordeum vulgare L.) cultivar Morex and examined transcriptomic responses at the level of longitudinal root zones. Water deficit significantly reduced root growth rates after two days of treatment. RNA-sequencing revealed root zone and temporal gene expression changes depending on the duration of water deficit treatment. The majority of water deficit-regulated genes were unique for their respective root zone-by-treatment combination, though they were associated with commonly enriched gene ontology terms. Among these, we found terms associated with transport, detoxification, or cell wall formation affected by water deficit. Integration of weighted gene co-expression analyses identified differential hub genes, that highlighted the importance of modulating energy and protein metabolism and stress response. CONCLUSION: Our findings provide new insights into the highly dynamic and spatiotemporal response cascade triggered by water deficit and the underlying genetic regulations on the level of root zones in the barley cultivar Morex, providing potential targets to enhance plant resilience against environmental constraints. This study further emphasizes the importance of considering spatial and temporal resolution when examining stress responses.

MuWU: Mutant-seq library analysis and annotation.

MOTIVATION: Insertional mutagenesis allows for the creation of loss-of-function mutations on a genome-wide scale. In theory, every gene can be 'knocked out' via the insertion of an additional DNA sequence. Resources of sequence-indexed mutants of plant and animal model organisms are instrumental for functional genomics studies. Such repositories significantly speed up the acquisition of interesting genotypes and allow for the validation of hypotheses regarding phenotypic consequences in reverse genetics. To create such resources, comprehensive sequencing of flanking sequence tags using protocols such as Mutant-seq requires various downstream computational tasks, and these need to be performed in an efficient and reproducible manner. RESULTS: Here, we present MuWU, an automated Mutant-seq workflow utility initially created for the identification of Mutator insertion sites of the BonnMu resource, representing a reverse genetics mutant collection for functional genetics in maize (Zea mays). MuWU functions as a fast, one-stop downstream processing pipeline of Mutant-seq reads. It takes care of all complex bioinformatic tasks, such as identifying tagged genes and differentiating between germinal and somatic mutations/insertions. Furthermore, MuWU automatically assigns insertions to the corresponding mutated seed stocks. We discuss the implementation and how parameters can easily be adapted to use MuWU for other species/transposable elements. AVAILABILITY AND IMPLEMENTATION: MuWU is a Snakemake-based workflow and freely available at https://github.com/tgstoecker/MuWU. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BonnMu: A Sequence-Indexed Resource of Transposon-Induced Maize Mutations for Functional Genomics Studies.

Sequence-indexed insertional libraries in maize (Zea mays) are fundamental resources for functional genetics studies. Here, we constructed a Mutator (Mu) insertional library in the B73 inbred background designated BonnMu A total of 1,152 Mu-tagged F2-families were sequenced using the Mu-seq approach. We detected 225,936 genomic Mu insertion sites and 41,086 high quality germinal Mu insertions covering 16,392 of the annotated maize genes (37% of the B73v4 genome). On average, each F2-family of the BonnMu libraries captured 37 germinal Mu insertions in genes of the Filtered Gene Set (FGS). All BonnMu insertions and phenotypic seedling photographs of Mu-tagged F2-families can be accessed via MaizeGDB.org Downstream examination of 137,410 somatic and germinal insertion sites revealed that 50% of the tagged genes have a single hotspot, targeted by Mu By comparing our BonnMu (B73) data to the UniformMu (W22) library, we identified conserved insertion hotspots between different genetic backgrounds. Finally, the vast majority of BonnMu and UniformMu transposons was inserted near the transcription start site of genes. Remarkably, 75% of all BonnMu insertions were in closer proximity to the transcription start site (distance: 542 bp) than to the start codon (distance: 704 bp), which corresponds to open chromatin, especially in the 5' region of genes. Our European sequence-indexed library of Mu insertions provides an important resource for functional genetics studies of maize.

Stability of Single-Parent Gene Expression Complementation in Maize Hybrids upon Water Deficit Stress.

Heterosis is the superior performance of F1 hybrids compared with their homozygous, genetically distinct parents. In this study, we monitored the transcriptomic divergence of the maize (Zea mays) inbred lines B73 and Mo17 and their reciprocal F1 hybrid progeny in primary roots under control and water deficit conditions simulated by polyethylene glycol treatment. Single-parent expression (SPE) of genes is an extreme instance of gene expression complementation, in which genes are active in only one of two parents but are expressed in both reciprocal hybrids. In this study, 1,997 genes only expressed in B73 and 2,024 genes only expressed in Mo17 displayed SPE complementation under control and water deficit conditions. As a consequence, the number of active genes in hybrids exceeded the number of active genes in the parental inbred lines significantly independent of treatment. SPE patterns were substantially more stable to expression changes by water deficit treatment than other genotype-specific expression profiles. While, on average, 75% of all SPE patterns were not altered in response to polyethylene glycol treatment, only 17% of the remaining genotype-specific expression patterns were not changed by water deficit. Nonsyntenic genes that lack syntenic orthologs in other grass species, and thus evolved late in the grass lineage, were significantly overrepresented among SPE genes. Hence, the significant overrepresentation of nonsyntenic genes among SPE patterns and their stability under water limitation might suggest a function of these genes during the early developmental manifestation of heterosis under fluctuating environmental conditions in hybrid progeny of the inbred lines B73 and Mo17.

Nonsyntenic Genes Drive Tissue-Specific Dynamics of Differential, Nonadditive, and Allelic Expression Patterns in Maize Hybrids.

Distantly related maize (Zea mays) inbred lines display an exceptional degree of genomic diversity. F1 progeny of such inbred lines are often more vigorous than their parents, a phenomenon known as heterosis. In this study, we investigated how the genetic divergence of the maize inbred lines B73 and Mo17 and their F1 hybrid progeny is reflected in differential, nonadditive, and allelic expression patterns in primary root tissues. In pairwise comparisons of the four genotypes, the number of differentially expressed genes between the two parental inbred lines significantly exceeded those of parent versus hybrid comparisons in all four tissues under analysis. No differentially expressed genes were detected between reciprocal hybrids, which share the same nuclear genome. Moreover, hundreds of nonadditive and allelic expression ratios that were different from the expression ratios of the parents were observed in the reciprocal hybrids. The overlap of both nonadditive and allelic expression patterns in the reciprocal hybrids significantly exceeded the expected values. For all studied types of expression - differential, nonadditive, and allelic - substantial tissue-specific plasticity was observed. Significantly, nonsyntenic genes that evolved after the last whole genome duplication of a maize progenitor from genes with synteny to sorghum (Sorghum bicolor) were highly overrepresented among differential, nonadditive, and allelic expression patterns compared with the fraction of these genes among all expressed genes. This observation underscores the role of nonsyntenic genes in shaping the transcriptomic landscape of maize hybrids during the early developmental manifestation of heterosis in root tissues of maize hybrids.

Root Type-Specific Reprogramming of Maize Pericycle Transcriptomes by Local High Nitrate Results in Disparate Lateral Root Branching Patterns.

The adaptability of root system architecture to unevenly distributed mineral nutrients in soil is a key determinant of plant performance. The molecular mechanisms underlying nitrate dependent plasticity of lateral root branching across the different root types of maize are only poorly understood. In this study, detailed morphological and anatomical analyses together with cell type-specific transcriptome profiling experiments combining laser capture microdissection with RNA-seq were performed to unravel the molecular signatures of lateral root formation in primary, seminal, crown, and brace roots of maize (Zea mays) upon local high nitrate stimulation. The four maize root types displayed divergent branching patterns of lateral roots upon local high nitrate stimulation. In particular, brace roots displayed an exceptional architectural plasticity compared to other root types. Transcriptome profiling revealed root type-specific transcriptomic reprogramming of pericycle cells upon local high nitrate stimulation. The alteration of the transcriptomic landscape of brace root pericycle cells in response to local high nitrate stimulation was most significant. Root type-specific transcriptome diversity in response to local high nitrate highlighted differences in the functional adaptability and systemic shoot nitrogen starvation response during development. Integration of morphological, anatomical, and transcriptomic data resulted in a framework underscoring similarity and diversity among root types grown in heterogeneous nitrate environments.

Nonsyntenic genes drive highly dynamic complementation of gene expression in maize hybrids.

Maize (Zea mays) displays an exceptional level of structural genomic diversity, which is likely unique among higher eukaryotes. In this study, we surveyed how the genetic divergence of two maize inbred lines affects the transcriptomic landscape in four different primary root tissues of their F1-hybrid progeny. An extreme instance of complementation was frequently observed: genes that were expressed in only one parent but in both reciprocal hybrids. This single-parent expression (SPE) pattern was detected for 2341 genes with up to 1287 SPE patterns per tissue. As a consequence, the number of active genes in hybrids exceeded that of their parents in each tissue by >400. SPE patterns are highly dynamic, as illustrated by their excessive degree of tissue specificity (80%). The biological significance of this type of complementation is underpinned by the observation that a disproportionally high number of SPE genes (75 to 82%) is nonsyntenic, as opposed to all expressed genes (36%). These genes likely evolved after the last whole-genome duplication and are therefore younger than the syntenic genes. In summary, SPE genes shape the remarkable gene expression plasticity between root tissues and complementation in maize hybrids, resulting in a tissue-specific increase of active genes in F1-hybrids compared with their inbred parents.

A high-resolution tissue-specific proteome and phosphoproteome atlas of maize primary roots reveals functional gradients along the root axes.

A high-resolution proteome and phosphoproteome atlas of four maize (Zea mays) primary root tissues, the cortex, stele, meristematic zone, and elongation zone, was generated. High-performance liquid chromatography coupled with tandem mass spectrometry identified 11,552 distinct nonmodified and 2,852 phosphorylated proteins across the four root tissues. Two gradients reflecting the abundance of functional protein classes along the longitudinal root axis were observed. While the classes RNA, DNA, and protein peaked in the meristematic zone, cell wall, lipid metabolism, stress, transport, and secondary metabolism culminated in the differentiation zone. Functional specialization of tissues is underscored by six of 10 cortex-specific proteins involved in flavonoid biosynthesis. Comparison of this data set with high-resolution seed and leaf proteome studies revealed 13% (1,504/11,552) root-specific proteins. While only 23% of the 1,504 root-specific proteins accumulated in all four root tissues, 61% of all 11,552 identified proteins accumulated in all four root tissues. This suggests a much higher degree of tissue-specific functionalization of root-specific proteins. In summary, these data illustrate the remarkable plasticity of the proteomic landscape of maize primary roots and thus provide a starting point for gaining a better understanding of their tissue-specific functions.

Transcriptomic and anatomical complexity of primary, seminal, and crown roots highlight root type-specific functional diversity in maize (Zea mays L.).

Maize develops a complex root system composed of embryonic and post-embryonic roots. Spatio-temporal differences in the formation of these root types imply specific functions during maize development. A comparative transcriptomic study of embryonic primary and seminal, and post-embryonic crown roots of the maize inbred line B73 by RNA sequencing along with anatomical studies were conducted early in development. Seminal roots displayed unique anatomical features, whereas the organization of primary and crown roots was similar. For instance, seminal roots displayed fewer cortical cell files and their stele contained more meta-xylem vessels. Global expression profiling revealed diverse patterns of gene activity across all root types and highlighted the unique transcriptome of seminal roots. While functions in cell remodeling and cell wall formation were prominent in primary and crown roots, stress-related genes and transcriptional regulators were over-represented in seminal roots, suggesting functional specialization of the different root types. Dynamic expression of lignin biosynthesis genes and histochemical staining suggested diversification of cell wall lignification among the three root types. Our findings highlight a cost-efficient anatomical structure and a unique expression profile of seminal roots of the maize inbred line B73 different from primary and crown roots.

Conserved and unique features of the homeologous maize Aux/IAA proteins ROOTLESS WITH UNDETECTABLE MERISTEM 1 and RUM1-like 1.

The maize (Zea mays L.) Aux/IAA protein RUM1 (ROOTLESS WITH UNDETECTABLE MERISTEM 1) is a key regulator of lateral and seminal root formation. An ancient maize genome duplication resulted in the emergence of its homeolog rum1-like1 (rul1), which displays 92% amino acid sequence identity with RUM1. Both, RUL1 and RUM1 exhibit the canonical four domain structure of Aux/IAA proteins. Moreover, both are localized to the nucleus, are instable and have similar short half-lives of ~23min. Moreover, RUL1 and RUM1 can be stabilized by specific mutations in the five amino acid degron sequence of domain II. In addition, proteins encoded by both genes interact in vivo with auxin response factors (ARFs) such as ZmARF25 and ZmARF34 in protoplasts. Although it was demonstrated that RUL1 and RUM1 can homo and heterodimerize in vivo, rul1 expression is independent of rum1. Moreover, on average rul1 expression is ~84-fold higher than rum1 in the 12 tested tissues and developmental stages, although the relative expression levels in different root tissues are very similar. While RUM1 and RUL1 display conserved biochemical properties, yeast-two-hybrid in combination with BiFC experiments identified a RUM1-associated protein 1 (RAP1) that specifically interacts with RUM1 but not with RUL1. This suggests that RUM1 and RUL1 are at least in part interwoven into different molecular networks.

Extensive tissue-specific transcriptomic plasticity in maize primary roots upon water deficit.

Water deficit is the most important environmental constraint severely limiting global crop growth and productivity. This study investigated early transcriptome changes in maize (Zea mays L.) primary root tissues in response to moderate water deficit conditions by RNA-Sequencing. Differential gene expression analyses revealed a high degree of plasticity of the water deficit response. The activity status of genes (active/inactive) was determined by a Bayesian hierarchical model. In total, 70% of expressed genes were constitutively active in all tissues. In contrast, <3% (50 genes) of water deficit-responsive genes (1915) were consistently regulated in all tissues, while >75% (1501 genes) were specifically regulated in a single root tissue. Water deficit-responsive genes were most numerous in the cortex of the mature root zone and in the elongation zone. The most prominent functional categories among differentially expressed genes in all tissues were 'transcriptional regulation' and 'hormone metabolism', indicating global reprogramming of cellular metabolism as an adaptation to water deficit. Additionally, the most significant transcriptomic changes in the root tip were associated with cell wall reorganization, leading to continued root growth despite water deficit conditions. This study provides insight into tissue-specific water deficit responses and will be a resource for future genetic analyses and breeding strategies to develop more drought-tolerant maize cultivars.

Roothairless5, which functions in maize (Zea mays L.) root hair initiation and elongation encodes a monocot-specific NADPH oxidase.

Root hairs are instrumental for nutrient uptake in monocot cereals. The maize (Zea mays L.) roothairless5 (rth5) mutant displays defects in root hair initiation and elongation manifested by a reduced density and length of root hairs. Map-based cloning revealed that the rth5 gene encodes a monocot-specific NADPH oxidase. RNA-Seq, in situ hybridization and qRT-PCR experiments demonstrated that the rth5 gene displays preferential expression in root hairs but also accumulates to low levels in other tissues. Immunolocalization detected RTH5 proteins in the epidermis of the elongation and differentiation zone of primary roots. Because superoxide and hydrogen peroxide levels are reduced in the tips of growing rth5 mutant root hairs as compared with wild-type, and Reactive oxygen species (ROS) is known to be involved in tip growth, we hypothesize that the RTH5 protein is responsible for establishing the high levels of ROS in the tips of growing root hairs required for elongation. Consistent with this hypothesis, a comparative RNA-Seq analysis of 6-day-old rth5 versus wild-type primary roots revealed significant over-representation of only two gene ontology (GO) classes related to the biological functions (i.e. oxidation/reduction and carbohydrate metabolism) among 893 differentially expressed genes (FDR <5%). Within these two classes the subgroups 'response to oxidative stress' and 'cellulose biosynthesis' were most prominently represented.

Complementation contributes to transcriptome complexity in maize (Zea mays L.) hybrids relative to their inbred parents.

Typically, F(1)-hybrids are more vigorous than their homozygous, genetically distinct parents, a phenomenon known as heterosis. In the present study, the transcriptomes of the reciprocal maize (Zea mays L.) hybrids B73×Mo17 and Mo17×B73 and their parental inbred lines B73 and Mo17 were surveyed in primary roots, early in the developmental manifestation of heterotic root traits. The application of statistical methods and a suitable experimental design established that 34,233 (i.e., 86%) of all high-confidence maize genes were expressed in at least one genotype. Nearly 70% of all expressed genes were differentially expressed between the two parents and 42%-55% of expressed genes were differentially expressed between one of the parents and one of the hybrids. In both hybrids, ∼10% of expressed genes exhibited nonadditive gene expression. Consistent with the dominance model (i.e., complementation) for heterosis, 1124 genes that were expressed in the hybrids were expressed in only one of the two parents. For 65 genes, it could be shown that this was a consequence of complementation of genomic presence/absence variation. For dozens of other genes, alleles from the inactive inbred were activated in the hybrid, presumably via interactions with regulatory factors from the active inbred. As a consequence of these types of complementation, both hybrids expressed more genes than did either parental inbred. Finally, in hybrids, ∼14% of expressed genes exhibited allele-specific expression (ASE) levels that differed significantly from the parental-inbred expression ratios, providing further evidence for interactions of regulatory factors from one parental genome with target genes from the other parental genome.

Transcriptomic complexity in young maize primary roots in response to low water potentials.

BACKGROUND: Widespread and more frequently occurring drought conditions are a consequence of global warming and increase the demand for tolerant crop varieties to feed the growing world population. A better understanding of the molecular mechanisms underlying the water deficit response of crops will enable targeted breeding strategies to develop robust cultivars. RESULTS: In the present study, the transcriptional response of maize (Zea mays L.) primary roots to low water potentials was monitored by RNA sequencing (RNA-Seq) experiments. After 6 h and 24 h of mild (-0.2 MPa) and severe (-0.8 MPa) water deficit conditions, the primary root transcriptomes of seedlings grown under water deficit and control conditions were compared. The number of responsive genes was dependent on and increased with intensification of water deficit treatment. After short-term mild and severe water deficit 249 and 3,000 genes were differentially expressed, respectively. After a 24 h treatment the number of affected genes increased to 7,267 and 12,838 for mild and severe water deficit, respectively, including more than 80% of the short-term responsive genes. About half of the differentially expressed genes were up-regulated and maximal fold-changes increased with treatment intensity to more than 300-fold. A consensus set of 53 genes was differentially regulated independently of the nature of deficit treatment. Characterization revealed an overrepresentation of the Gene Ontology (GO) categories "oxidoreductase activity" and "heme binding" among regulated genes connecting the water deficit response to ROS metabolism. CONCLUSION: This study gives a comprehensive insight in water deficit responsive genes in young maize primary roots and provides a set of candidate genes that merit further genetic analyses in the future.

The Aux/IAA gene rum1 involved in seminal and lateral root formation controls vascular patterning in maize (Zea mays L.) primary roots.

The maize (Zea mays L.) Aux/IAA protein RUM1 (ROOTLESS WITH UNDETECTABLE MERISTEMS 1) controls seminal and lateral root initiation. To identify RUM1-dependent gene expression patterns, RNA-Seq of the differentiation zone of primary roots of rum1 mutants and the wild type was performed in four biological replicates. In total, 2 801 high-confidence maize genes displayed differential gene expression with Fc ≥2 and FDR ≤1%. The auxin signalling-related genes rum1, like-auxin1 (lax1), lax2, (nam ataf cuc 1 nac1), the plethora genes plt1 (plethora 1), bbm1 (baby boom 1), and hscf1 (heat shock complementing factor 1) and the auxin response factors arf8 and arf37 were down-regulated in the mutant rum1. All of these genes except nac1 were auxin-inducible. The maize arf8 and arf37 genes are orthologues of Arabidopsis MP/ARF5 (MONOPTEROS/ARF5), which controls the differentiation of vascular cells. Histological analyses of mutant rum1 roots revealed defects in xylem organization and the differentiation of pith cells around the xylem. Moreover, histochemical staining of enlarged pith cells surrounding late metaxylem elements demonstrated that their thickened cell walls displayed excessive lignin deposition. In line with this phenotype, rum1-dependent mis-expression of several lignin biosynthesis genes was observed. In summary, RNA-Seq of RUM1-dependent gene expression in maize primary roots, in combination with histological and histochemical analyses, revealed the specific regulation of auxin signal transduction components by RUM1 and novel functions of RUM1 in vascular development.

BonnMu: A Resource for Functional Genomics in Maize (Zea mays L.).

The BonnMu resource is a public transposon-tagged population designed for reverse and forward genetics studies in maize (Zea mays L.). The resource was created by crossing an active Mutator (Mu) transposon line into different inbred lines to induce insertional mutations. The resulting F1 generation was self-pollinated to generate segregating BonnMu F2 stocks. The Mu-tagged BonnMu F2 stocks have insertions in 83% of all annotated maize gene models, and Mu insertion positions and photos of the seedling phenotypes of the segregating BonnMu F2 stocks are deposited in the Maize Genetics and Genomics Database (MaizeGDB), with seeds available to the community. Here, we discuss the creation, expansion, and application of the BonnMu resource for identifying and characterizing mutations induced by Mu transposons, which represents a useful tool for functional genomics studies in maize.

Identification of Transposon Insertion Sites in Maize Mu-Tagged Mutants Using Mu-Seq.

Mutator (Mu) transposons facilitate untargeted insertional mutagenesis in maize by moving within the genome and disrupting genes. Such an approach has been used to generate collections such as the BonnMu resource, a Mu-tagged maize population for functional genomics studies. Mutant-Seq (Mu-Seq) is a sequencing-based method for the high-throughput identification and mapping of Mu insertion sites. The approach involves the construction of multiplexed sequencing libraries (known as Mu-Seq libraries) from Mu-tagged populations, followed by high-throughput sequencing and data processing using the Mu-Seq Workflow Utility (MuWU) tool, to determine the location of Mu insertions. Here, we provide a detailed protocol for Mu-Seq, from the generation of the maize Mu-tagged mutant population to data analysis. Researchers can use this approach to develop mutant collections customized to specific genetic backgrounds of interest, which can aid in characterizing genotype-specific mutations and identifying candidate genes linked to visible mutant phenotypes.

Use of Maize (Zea mays L.) Mutator Transposon-Induced Mutants of the BonnMu Resource for Forward and Reverse Genetics Studies.

The BonnMu resource represents a tagged collection of maize (Zea mays L.) Mutator (Mu) transposon-induced mutants, designed for functional genomics studies. Here, we describe the use of the BonnMu collection for identifying and characterizing mutations. Specifically, we describe workflows for use in both reverse and forward genetics strategies in maize. For reverse genetics, users first acquire a BonnMu F2 stock of interest based on data accessible at the Maize Genetics and Genomics Database (MaizeGDB). We provide details here for their subsequent propagation and for the confirmation of Mu insertions by genotyping via PCR, with the ultimate goal of establishing genotype-phenotype relationships of interest. For forward genetics studies, we describe a workflow that involves a combined approach of Mutant-Seq (Mu-Seq) and bulked segregant RNA-seq (BSR-Seq), to identify the causal gene underlying a mutant phenotype of interest.