Charles T. Hunter

Genetic Resources for Maize Cell Wall Biology

Grass species represent a major source of food, feed, and fiber crops and potential feedstocks for biofuel production. Most of the biomass is contributed by cell walls that are distinct in composition from all other flowering plants. Identifying cell wall-related genes and their functions underpins a fundamental understanding of growth and development in these species. Toward this goal, we are building a knowledge base of the maize (Zea mays) genes involved in cell wall biology, their expression profiles, and the phenotypic consequences of mutation. Over 750 maize genes were annotated and assembled into gene families predicted to function in cell wall biogenesis. Comparative genomics of maize, rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana) sequences reveal differences in gene family structure between grass species and a reference eudicot species. Analysis of transcript profile data for cell wall genes in developing maize ovaries revealed that expression within families differed by up to 100-fold. When transcriptional analyses of developing ovaries before pollination from Arabidopsis, rice, and maize were contrasted, distinct sets of cell wall genes were expressed in grasses. These differences in gene family structure and expression between Arabidopsis and the grasses underscore the requirement for a grass-specific genetic model for functional analyses. A UniformMu population proved to be an important resource in both forward- and reverse-genetics approaches to identify hundreds of mutants in cell wall genes. A forward screen of field-grown lines by near-infrared spectroscopic screen of mature leaves yielded several dozen lines with heritable spectroscopic phenotypes. Pyrolysis-molecular beam mass spectrometry confirmed that several nir mutants had altered carbohydrate-lignin compositions.

Regulation of assimilate import into sink organs: update on molecular drivers of sink strength

Recent developments have altered our view of molecular mechanisms that determine sink strength, defined here as the capacity of non-photosynthetic structures to compete for import of photoassimilates. We review new findings from diverse systems, including stems, seeds, flowers, and fruits. An important advance has been the identification of new transporters and facilitators with major roles in the accumulation and equilibration of sugars at a cellular level. Exactly where each exerts its effect varies among systems. Sugarcane and sweet sorghum stems, for example, both accumulate high levels of sucrose, but may do so via different paths. The distinction is central to strategies for targeted manipulation of sink strength using transporter genes, and shows the importance of system-specific analyses. Another major advance has been the identification of deep hypoxia as a feature of normal grain development. This means that molecular drivers of sink strength in endosperm operate in very low oxygen levels, and under metabolic conditions quite different than previously assumed. Successful enhancement of sink strength has nonetheless been achieved in grains by up-regulating genes for starch biosynthesis. Additionally, our understanding of sink strength is enhanced by awareness of the dual roles played by invertases (INVs), not only in sucrose metabolism, but also in production of the hexose sugar signals that regulate cell cycle and cell division programs. These contributions of INV to cell expansion and division prove to be vital for establishment of young sinks ranging from flowers to fruit. Since INV genes are themselves sugar-responsive “feast genes,” they can mediate a feed-forward enhancement of sink strength when assimilates are abundant. Greater overall productivity and yield have thus been attained in key instances, indicating that even broader enhancements may be achievable as we discover the detailed molecular mechanisms that drive sink strength in diverse systems.

Sequence-indexed mutations in maize using the UniformMu transposon-tagging population

BackgroundGene knockouts are a critical resource for functional genomics. In Arabidopsis, comprehensive knockout collections were generated by amplifying and sequencing genomic DNA flanking insertion mutants. These Flanking Sequence Tags (FSTs) map each mutant to a specific locus within the genome. In maize, FSTs have been generated using DNA transposons. Transposable elements can generate unstable insertions that are difficult to analyze for simple knockout phenotypes. Transposons can also generate somatic insertions that fail to segregate in subsequent generations.ResultsTransposon insertion sites from 106 UniformMu FSTs were tested for inheritance by locus-specific PCR. We confirmed 89% of the FSTs to be germinal transposon insertions. We found no evidence for somatic insertions within the 11% of insertion sites that were not confirmed. Instead, this subset of insertion sites had errors in locus-specific primer design due to incomplete or low-quality genomic sequences. The locus-specific PCR assays identified a knockout of a 6-phosphogluconate dehydrogenase gene that co-segregates with a seed mutant phenotype. The mutant phenotype linked to this knockout generates novel hypotheses about the role for the plastid-localized oxidative pentose phosphate pathway during grain-fill.ConclusionWe show that FSTs from the UniformMu population identify stable, germinal insertion sites in maize. Moreover, we show that these sequence-indexed mutations can be readily used for reverse genetic analysis. We conclude from these data that the current collection of 1,882 non-redundant insertion sites from UniformMu provide a genome-wide resource for reverse genetics.

Environmental DNA (eDNA) Sampling Improves Occurrence and Detection Estimates of Invasive Burmese Pythons

Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors. Generic sampling design and terminology are proposed to standardize and clarify interpretations of eDNA-based occupancy models.

Cellulose Synthase-Like D1 is integral to normal cell division, expansion, and leaf development in maize

The Cellulose Synthase-Like D (CslD) genes have important, although still poorly defined, roles in cell wall formation. Here, we show an unexpected involvement of CslD1 from maize (Zea mays) in cell division. Both division and expansion were altered in the narrow-organ and warty phenotypes of the csld1 mutants. Leaf width was reduced by 35%, due mainly to a 47% drop in the number of cell files across the blade. Width of other organs was also proportionally reduced. In leaf epidermis, the deficiency in lateral divisions was only partially compensated by a modest, uniform increase in cell width. Localized clusters of misdivided epidermal cells also led to the formation of warty lesions, with cell clusters bulging from the epidermal layer, and some cells expanding to volumes 75-fold greater than normal. The decreased cell divisions and localized epidermal expansions were not associated with detectable changes in the cell wall composition of csld1 leaf blades or epidermal peels, yet a greater abundance of thin, dense walls was indicated by high-resolution x-ray tomography of stems. Cell-level defects leading to wart formation were traced to sites of active cell division and expansion at the bases of leaf blades, where cytokinesis and cross-wall formation were disrupted. Flow cytometry confirmed a greater frequency of polyploid cells in basal zones of leaf blades, consistent with the disruption of cytokinesis and/or the cell cycle in csld1 mutants. Collectively, these data indicate a previously unrecognized role for CSLD activity in plant cell division, especially during early phases of cross-wall formation.

Mu-seq: Sequence-Based Mapping and Identification of Transposon Induced Mutations

Mutations tagged by transposon insertions can be readily mapped and identified in organisms with sequenced genomes. Collections of such mutants allow a systematic analysis of gene function, and can be sequence-indexed to build invaluable resources. Here we present Mu-seq (Mutant-seq), a high-throughput NextGen sequencing method for harnessing high-copy transposons. We illustrate the efficacy of Mu-seq by applying it to the Robertson’s Mutator system in a large population of maize plants. A single Mu-seq library, for example, constructed from 576 different families (2304 plants), enabled 4, 723 novel, germinal, transposon insertions to be detected, identified, and mapped with single base-pair resolution. In addition to the specificity, efficiency, and reproducibility of Mu-seq, a key feature of this method is its adjustable scale that can accomodate simultaneous profiling of transposons in thousands of individuals. We also describe a Mu-seq bioinformatics framework tailored to high-throughput, genome-wide, and population-wide analysis of transposon insertions.

Phenotype to genotype using forward-genetic Mu-seq for identification and functional classification of maize mutants

In pursuing our long-term goals of identifying causal genes for mutant phenotypes in maize, we have developed a new, phenotype-to-genotype approach for transposon-based resources, and used this to identify candidate genes that co-segregate with visible kernel mutants. The strategy incorporates a redesigned Mu-seq protocol (sequence-based, transposon mapping) for high-throughput identification of individual plants carrying Mu insertions. Forward-genetic Mu-seq also involves a genetic pipeline for generating families that segregate for mutants of interest, and grid designs for concurrent analysis of genotypes in multiple families. Critically, this approach not only eliminates gene-specific PCR genotyping, but also profiles all Mu-insertions in hundreds of individuals simultaneously. Here, we employ this scalable approach to study 12 families that showed Mendelian segregation of visible seed mutants. These families were analyzed in parallel, and 7 showed clear co-segregation between the selected phenotype and a Mu insertion in a specific gene. Results were confirmed by PCR. Mutant genes that associated with kernel phenotypes include those encoding: a new allele of Whirly1 (a transcription factor with high affinity for organellar and single-stranded DNA), a predicted splicing factor with a KH domain, a small protein with unknown function, a putative mitochondrial transcription-termination factor, and three proteins with pentatricopeptide repeat domains (predicted mitochondrial). Identification of such associations allows mutants to be prioritized for subsequent research based on their functional annotations. Forward-genetic Mu-seq also allows a systematic dissection of mutant classes with similar phenotypes. In the present work, a high proportion of kernel phenotypes were associated with mutations affecting organellar gene transcription and processing, highlighting the importance and non-redundance of genes controlling these aspects of seed development.

Genetic and molecular analyses of UniformMu transposon insertion lines.

The UniformMu transposon population is a large public resource for reverse genetics and functional genomics of maize. Users access the collection of UniformMu genetic stocks that are freely distributed by the Maize Cooperation Stock Center using online tools maintained at MaizeGDB.org. Genetic and molecular analyses of UniformMu stocks (UFMu insertion lines) typically require development of genotyping assays that use a gene-specific polymerase chain reaction (PCR) to follow segregation of transposon insertions in genes of interest. Here we describe methods for accessing the resource and recommended protocols for genotyping of transposon insertion alleles.

Contrasting insect attraction and herbivore-induced plant volatile production in maize

Main conclusionThe maize inbred line W22 has lower herbivore-induced volatile production than B73 but both fall armyworm larvae and the wasps that parasitize them prefer W22 over B73.AbstractMaize inbred line W22 is an important resource for genetic studies due to the availability of the UniformMu mutant population and a complete genome sequence. In this study, we assessed the suitability of W22 as a model for tritrophic interactions between maize, Spodoptera frugiperda (fall armyworm) and the parasitoid wasp Cotesia marginiventris. W22 was found to be a good model for studying the interaction as S. frugiperda prefers W22 over B73 and a higher parasitism rate by C. marginiventris was observed on W22 compared to the inbred line B73. W22 also produced lower amounts of many herbivore-induced volatile terpenes and indole emission upon treatment with S. frugiperda oral secretions. We propose that some of the major herbivore-induced terpene volatiles are perhaps impeding S. frugiperda and C. marginiventris preference and that as yet unidentified compounds are produced at low abundance may be positively impacting these interactions.

The maize W22 genome provides a foundation for functional genomics and transposon biology

The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using short-read sequencing technologies. We show that significant structural heterogeneity exists in comparison to the B73 reference genome at multiple scales, from transposon composition and copy number variation to single-nucleotide polymorphisms. The generation of this reference genome enables accurate placement of thousands of Mutator (Mu) and Dissociation (Ds) transposable element insertions for reverse and forward genetics studies. Annotation of the genome has been achieved using RNA-seq analysis, differential nuclease sensitivity profiling and bisulfite sequencing to map open reading frames, open chromatin sites and DNA methylation profiles, respectively. Collectively, the resources developed here integrate W22 as a community reference genome for functional genomics and provide a foundation for the maize pan-genome.Sequencing and de novo assembly of the maize W22 reference genome enable accurate placement of Mutator (Mu) and Dissociation (Ds) transposable element insertions, providing a foundation for maize functional genomics and transposon biology.