Biology subjects

Dilkes, B. P.

Publications and source records attributed to Dilkes, B. P..

17 recordsLinked to original sources

bioRxiv2026

Hybridization between toxic bloom-forming algae of the Prymnesium parvum sensu lato species complex

The potential for hybridization to act as a driver of genetic diversity and adaptation in harmful algal bloom-forming species has received scarce attention, despite growing recognition of its occurrence in diverse protist and algal lineages. Prymnesium parvum s.l. (Haptophyta), is a cryptic species complex whose members form ecosystem-disruptive toxic algal blooms around the world. A prior genome analysis showed that UTEX2797, a widely used laboratory strain, originated via hybridization between clade A1 and clade A2 of this species complex. To assess the extent of A1xA2 hybridization in P. parvum s.l., we screened the genomes of 28 strains and identified 16 additional A1xA2 hybrid strains isolated from inland Texas or the eastern United States between 2001 and 2020. Chloroplast haplotypes indicated that hybridization between A1 and A2 may have occurred multiple times, and hybrids with different chloroplast haplotypes have been co-isolated from blooms in Texas in 2013 and 2020. Additionally, strain NIES1812 from Okinawa, Japan was sufficiently divergent from A1 and A2 to warrant designation as a separate clade, which we name A3. These results provide evidence for a facultative sexual life cycle in P. parvum s.l. and expand our understanding of the extensive cryptic genetic diversity present in the species complex. The frequent isolation of hybrid strains from North American blooms suggests that hybridization is common and represents a significant source of adaptive potential in these economically and ecologically damaging organisms.

evolutionary biology↗

bioRxiv2026

Life, the universe, and everything for $42: ultra-low pass sequencing of maize for genotyping, mapping, and pedigree analysis

Convenient and economical genotyping methods and simplified bioinformatic workflows are critical for genetic studies and breeding. The declining cost of sequencing library construction, sample multiplexing, and the advent of skim sequencing has reduced costs and enabled large-scale genetic and genomic experiments. Here, we present a simple skim sequencing and bioinformatics pipeline sufficient for various genotyping applications. Our low-depth skim sequencing method costs 21 USD per sample and provides an average of 144k reads. Our approach uses a double-stranded DNA sample to prepare libraries for genome sequencing. We demonstrate various uses for this strategy in maize, a complex and large (2.5 Gbp) genome. DNA from multiple pedigreed populations, including advanced backcrossed progenies, bi-parental populations, near-isogenic lines, and recombinant inbred lines, were used to map loci, detect donor introgressions, and determine introgression haplotypes. Read counts at known polymorphic positions detected donor genotypes even when derived from parents of unknown origin and could localize mutations of phenotypic impact via bulked segregant analysis. Remarkably, the small amount of sequencing data produced were sufficient to identify the haplotypes of introgressions of unknown origin from by comparison to known genotypes. Correct haplotype identification enabled more accurate allele frequencies to be calculated when mapping loci. This is of exceptional value in maize, where a rich collection of mutants from the 20th century are of unknown pedigree. One sentence summaryEconomical and efficient whole genome ultra-low pass sequencing of DNA samples for numerous genetic and genomic applications.

genetics↗

bioRxiv2024

A brassinosteroid receptor kinase is required for sex determination in the homosporous fern Ceratopteris richardii

Most ferns, unlike all seed plants, are homosporous and produce sexually undifferentiated spores. Sex in many homosporous ferns is environmentally regulated by the biosynthesis of antheridiogens. Secreted antheridiogen from female or hermaphrodite gametophytes is perceived by undetermined gametophytes which respond by developing as male. In the fern Ceratopteris richardii (Ceratopteris), several hermaphroditic (her) mutants develop as hermaphrodites even in the presence of antheridiogen. A set of five mutant alleles are tightly linked to her7, a locus of unknown molecular identity. The immense size of the Ceratopteris genome has made efficient identification of the underlying genes a challenge. Here, we used bulked segregant RNA-seq (BSR-Seq) to map two her mutants (her7-14 and her7-19) to a 16 Mbp interval on chromosome 29 of the Ceratopteris genome. A brassinosteroid receptor-like kinase (BRL) within this interval encoded a deletion mutation in her7-14 and a missense mutation in her7-19. Three other linked her mutants (her7-1, her7-11, and her7-15) encoded missense mutations in the same gene, which we name HER7, providing strong support for HER7 being required for antheridiogen-mediated sex determination in Ceratopteris. HER7-GFP fusion protein is localized in the plasma membrane and cytoplasm, consistent with its predicted function as a brassinosteroid receptor kinase. RNA-seq analysis of gene expression shows that numerous transcripts encoding enzymes, including those for brassinosteroid biosynthesis, accumulate in hermaphrodites compared to male gametophytes. The antheridiogen of Ceratopteris is currently unknown and our finding that a BRL mutant is insensitive to antheridiogen suggests that antheridiogen in Ceratopteris may be a brassinosteroid. Additional work is required to fully resolve the sex determination pathway and determine the chemical composition of antheridiogen in this species. Our work demonstrates we can identify the molecular basis of mutants in large genomes, such as those in homosporous ferns, which can be further applied to the extensive remaining collection of mutants in Ceratopteris.

genetics↗

bioRxiv2024

Natural variation in tetrapyrrole biosynthetic enzymes and their regulation modify the chlorophyll mutant Oy1-N1989

Tetrapyrroles are macrocyclic compounds present in and required for all life on Earth. Mutants with defects in tetrapyrrole pathway enzymes can be used to uncover natural variation in this pathway and study pathway regulation. We report here the effects of the Oy1-N1989/+ mutation, a semi-dominant allele of subunit I in the Mg-chelatase enzyme with reduced chlorophyll biosynthesis, on global gene expression and morphological traits. Coordinate regulation of the tetrapyrrole pathway was observed as transcriptional feedback regulation of genes in the tetrapyrrole pathway in Oy1-N1898/+ mutants. Natural variation in the wild-type allele at oy1 modulated the severity of the impact of Oy1-N1989/+ on gene expression. We also studied the effects of previously identified cis-acting expression variation at oy1 in wild-type plants. The seventy transcripts encoding biosynthetic enzymes in the tetrapyrrole pathway exhibited similar transcriptional co-regulation in response to cis-variation in oy1 transcript accumulation in wild-type plants as observed in the RNAseq of Oy1-N1989/+ mutants. This demonstrated that the coordinate regulation of the pathway also occurs during physiologically-relevant variation in OY1 abundance. Cis variants at seven tetrapyrrole pathway genes were linked to variation in chlorophyll accumulation in Oy1-N1989/+ mutant or wild-type plants. Analysis of trans-acting transcriptional variation by eGWAS detected multiple transcriptional hotspots, which disproportionately affected the expression of a subset of tetrapyrrole pathway genes, indicating that some genes are repeated targets of transcriptional regulation. The trans-regulatory hotspots coordinately regulated this pathway and may work to limit the accumulation of phototoxic intermediates. Author SummaryThe mutant allele and interactions with the genetic background determine the severity of mutant phenotypes. We used Oy1-N1989/+ mutants, affecting Mg-chelatase subunit I, to identify natural variants in the tetrapyrrole pathway affecting transcriptional regulation and variation in mutant traits. By integrating GWAS and transcriptomics, we identified similar feedback regulation of porphyrin and chlorophyll metabolism in mutants as well as in wild-type plants affected by natural variation. This is the first demonstration of natural variation affecting transcriptional feedback in the tetrapyrrole pathway under normal conditions. Using GWAS as a regulatory discovery tool, we identified unexplored regulatory mechanisms in a pathway that is critical for energy capture from the sun and chemical energy utilization in all living things and multiple human diseases.

plant biology↗

bioRxiv2023

A large effect genetic trade-off is caused by a single mutation in CBF2

Identifying the genetic basis of local adaptation and fitness trade-offs across environments is a central goal of evolutionary biology. Cold acclimation is an adaptive plastic response for surviving seasonal freezing, and costs of acclimation may be a general mechanism for fitness trade-offs across environments in temperate zone species. Starting with locally adapted ecotypes of Arabidopsis thaliana from Italy and Sweden, we examined the fitness consequences of a naturally occurring functional polymorphism in CBF2. This gene encodes a transcription factor that is a major regulator of cold-acclimated freezing tolerance, and resides within a locus responsible for a genetic trade-off for long-term mean fitness. We estimated the consequences of alternate genotypes of CBF2 on 5-year mean fitness and fitness components at the native field sites by comparing near isogenic lines with alternate genotypes of CBF2 to their genetic background ecotypes. The effects of CBF2 were validated at the nucleotide level using gene edited lines in the native genetic backgrounds grown in simulated parental environments. The foreign CBF2 genotype in the local genetic background reduced long-term mean fitness in Sweden by more than 10%, primarily via effects on survival. In Italy, fitness was reduced by more than 20%, primarily via effects on fecundity. At both sites, the effects were temporally variable and much stronger in some years. The gene edited lines confirmed that CBF2 encodes the causal variant underlying this genetic trade-off. Additionally, we demonstrated a substantial fitness cost of cold acclimation, which has broad implications for potential maladaptive responses to climate change.

evolutionary biology↗

bioRxiv2023

The Auxin Response Factor ARF27 is required for maize root morphogenesis

Crop root systems are central to nutrition acquisition and water usage. Root hairs and lateral roots contribute to fine-scale patterning of root systems and can offer many advantages for improving root function without drastically impacting overall system architecture. Key genetic regulators underpinning root hair morphogenesis have been well characterized in the model plant Arabidopsis but are less understood in maize. Here, we identify a novel determinant of root hair morphogenesis and auxin responses in maize, AUXIN RESPONSE FACTOR27 (ARF27), using both reverse and quantitative genetic approaches. ARF27 is enriched in maize primary root tissues at both the transcript and protein level. Loss of ARF27 leads to short primary roots and reduced root hair formation, while lateral root density is unaltered. In arf27 roots, auxin-responsive gene expression is dysregulated, which is consistent with the predicted function of this transcription factor. Moreover, a genome wide association study (GWAS) to uncover genetic determinants of auxin-dependent root traits identified ARF27 as a candidate gene. Furthermore, auxin hypersensitive maize genotypes exhibit altered crown root length and surface area in field-grown plants. A gene regulatory network (GRN) was reconstructed and an ARF27 subnetwork was integrated with DAP-seq and GWAS data to identify ARF27 target genes. The ARF27 GRN includes known maize root development genes, such as ROOTLESS CONCERNING CROWN ROOTS (RTCS), ROOTHAIRLESS 3 (RTH3) and RTH6. Altogether this work identifies a novel genetic driver of auxin-mediated root morphogenesis in maize that can inform agricultural strategies for improved crop performance.

plant biology↗

bioRxiv2023

Two teosintes made modern maize

Despite its global importance as a crop with broad economic, dietary, and cultural importance, the origins of maize and its closest wild relatives remained the topic of vigorous debate for nearly a century. Molecular analyses ultimately concluded that maize was domesticated once from a common ancestor with its closest extant relative, the lowland wild grass Zea mays ssp. parviglumis. But neither the current genetic model nor earlier models based on archaeological data account for the totality of available data, and recent work has highlighted the potential contribution of a second wild relative, the highland Zea mays ssp. mexicana. Here we present a detailed population genetic analysis of the contributions of both wild taxa to modern maize diversity using the largest sample of traditional maize varieties sequenced to date. We show that all modern maize can trace its origin to an ancient admixture event between domesticated ancient maize and Zea mays ssp. mexicana in the highlands of Mexico ca 5300 cal BP, some 4,000 years after domestication began. We show that variation in admixture is a key component of modern maize genetic and phenotypic diversity, both at the level of individual loci and as a factor driving a substantial component of additive genetic variation across a number of agronomic traits. Our results clarify the long-debated origin of modern maize, highlight the potential contributions of crop wild relatives to agronomic improvement, and raise new questions about the anthropogenic mechanisms underlying multiple waves of dispersal throughout the Americas. One-Sentence SummaryOur results clarify the long-debated origin of modern maize and highlight the contributions of crop wild relatives to the agronomic improvement of modern varieties.

evolutionary biology↗

bioRxiv2022

Genome-wide association identifies impacts of chlorophyll levels on reproductive maturity and architecture in maize

The semi-dominant mutant allele Oy1-N1989 encodes a dominant-negative subunit I of Mg-Chelatase that catalyzes the first committed step of chlorophyll biosynthesis. Analyses of mutant F1 hybrids from crosses of Oy1-N1989 to diverse maize lines identified a natural variant at oil yellow1 as major modifier of chlorophyll content. Chlorophyll reduction increased the time to reproductive maturity, decreased stalk width, and had complex effects on plant height. We explored these effects by genome wide association studies of F1 hybrids crosses of Oy1-N1989 to a maize diversity panel. The delay in reproductive maturity in Oy1-N1989/+ mutants neither altered the vegetative-to-adult phase change nor total leaf number but did slow the rate of organ emergence from the whorl. In addition to the allele at oy1, analysis of tetrapyrrole, heme, and chlorophyll biosynthesis pathway genes identified multiple modifiers of mutant traits. Consistent with organ emergence, rather than a developmental delay, affecting the reproductive delay of Oy1-N1989/+ mutants, known flowering time regulators did not affect variation in the reproductive delay affected by chlorophyll loss. Plant height showed a complex relationship with chlorophyll. The Oy1-N1989/+ mutant F1 plants with a modest reduction in chlorophyll were taller than their wildtype siblings, but F1 plants with dramatically decreased chlorophyll were shorter than their wildtype siblings. Perturbations in the biosynthesis of the phytochrome chromophore increased plant height in wild-type plants but dramatically reduced plant height and chlorophyll contents of Oy1-N1989/+ mutants. The mechanism by which the interaction between chlorophyll and bilin affects height and chlorophyll remains to be explored. One sentence summaryChlorophyll reduction in maize delays reproductive maturity by altering growth and not development, and depending on the reduction can either increase or decrease plant height.

plant biology↗

bioRxiv2022

Disruption of Zea mays isochorismate synthase1 decreases PHENYLALANINE AMMONIA LYASE activity and suppresses hypersensitive response-induced metabolism

ISOCHORISMATE SYNTHASE (ICS) catalyzes the isomerization of chorismate to isochorismate, an essential precursor in the biosynthesis of the Photosystem I electron carrier phylloquinone and of one of two pathways for the biosynthesis of the defense response hormone salicylic acid (SA). We characterized a Zea mays ics1 mutant for impacts on metabolism, photosynthesis, and immune signaling. Phylloquinone was reduced in the mutant resulting in low electron transfer rates and high electron backflow rates. SA accumulation induced by autoactive alleles of the nucleotide-binding leucine-rich repeat (NLR) gene Resistance to Puccinia sorgi1 (Rp1) required ics1. Induced accumulation of SA was not required for lesion formation by the autoactive Rp1-D21#4 allele. Metabolomic analyses and SA supplementation of Rp1-D21#4 mutants, ics1-1 mutants and Rp1-D21#4; ics1-1 double mutants demonstrated that most hypersensitive response-induced metabolism required ics1 but this was independent of SA accumulation. Both the PAL and ICS pathways contributed to SA biosynthesis in maize as labeled phenylalanine was incorporated into SA glucoside. Maize ics1-1 mutants had low PHENYLALANINE AMMONIA LYASE activity, accumulated phenylalanine, and decreased abundance of phenylalanine derived metabolites. This demonstrates that the ICS and PAL pathways interact by a yet unknown mechanism complicating the interpretation of SA biosynthesis in plants from genetics alone.

plant biology↗

bioRxiv2021

slim shady is a novel allele of PHYTOCHROMEB present in the T-DNA line SALK_015201

Auxin is a hormone that is required for hypocotyl elongation during seedling development. In response to auxin rapid changes in transcript and protein abundance occur in hypocotyls and some auxin responsive gene expression is linked to hypocotyl growth. To functionally validate proteomic studies, a reverse genetics screen was performed on mutants in auxin-regulated proteins to identify novel regulators of plant growth. This uncovered a long hypocotyl mutant, which we called slim shady, in an annotated insertion line in IMMUNOREGULATORY RNA-BINDING PROTEIN (IRR). Overexpression of the IRR gene failed to rescue the slim shady phenotype and characterization of a second T-DNA allele of IRR found that it had a wild-type hypocotyl length. The slim shady mutant has an elevated expression of numerous genes associated with the brassinosteroid-auxin-phytochrome (BAP) regulatory module compared to wild-type, including transcription factors that regulate brassinosteroid, auxin and phytochrome pathways. Additionally, slim shady seedlings fail to exhibit a strong transcriptional response to auxin. Using whole genome sequence and transcriptomics data for SALK_015201C we determined that a novel single nucleotide polymorphism in PHYTOCHROME B was responsible for the slim shady phenotype. This is predicted to convert induce a frameshift and premature stop codon at leucine 1125, within the histidine kinase-related domain of the carboxy terminus of PHYB, which is required for phytochrome signaling and function. Genetic complementation analyses with phyb-9 confirmed that slim shady is a mutant allele of PHYB. This study advances our understanding of the molecular mechanisms in seedling development, by furthering our understanding of how light signaling is linked to auxin dependent cell elongation. Furthermore, this study highlights the importance of confirming the genetic identity of research material before attributing phenotypes to known mutations sourced from T-DNA stocks.

plant biology↗

bioRxiv2020

Mutation of the nuclear pore complex component, aladin1, disrupts asymmetric cell division in Zea mays (maize)

The nuclear pore complex (NPC) regulates the movement of macromolecules between the nucleus and cytoplasm. Dysfunction of many components of the NPC results in human genetic diseases, including triple A syndrome (AAAS) as a result of mutations in ALADIN. Here we report a nonsense mutation in the maize ortholog, aladin1 (ali1-1), at the orthologous amino acid residue of an AAAS allele from humans, alters plant stature, tassel architecture, and asymmetric divisions of subsidiary mother cells (SMCs). Crosses with the stronger nonsense allele ali1-2 identified complex allele interactions for plant height and aberrant SMC division. RNA-seq analysis of the ali1-1 mutant identified compensatory transcript accumulation for other NPC components as well as gene expression consequences consistent with conservation of ALADIN1 functions between humans and maize. These findings demonstrate that ALADIN1 is necessary for normal plant development, shoot architecture, and asymmetric cell division in maize.

genetics↗

bioRxiv2020

Maize brace roots provide stalk anchorage

Mechanical failure, known as lodging, negatively impacts yield and grain quality in crops. Limiting crop loss from lodging requires an understanding of the plant traits that contribute to lodging-resistance. In maize, specialized aerial brace roots are reported to reduce root lodging. However, their direct contribution to plant biomechanics has not been measured. In this manuscript, we find that brace roots establish a rigid base (i.e. stalk anchorage) to limit plant deflection in maize. The more brace root whorls that contact the soil, the greater the contribution of brace roots to anchorage. Previous studies have linked the number of brace root whorls to flowering time in maize. To determine if flowering time selection alters the brace root contribution to anchorage, a subset of the Hallauers Tuson tropical population was analyzed. Despite a significant change in flowering time, selection neither altered the number of brace root whorls in the soil nor the overall contribution of brace roots to anchorage. These results demonstrate that brace roots provide a rigid base in maize, but the contribution to anchorage is not linearly related to flowering time.

plant biology↗

bioRxiv2020

Endosidin20 targets cellulose synthase catalytic domain to inhibit cellulose biosynthesis

Cellulose is synthesized by rosette structured cellulose synthase (CESA) complexes (CSCs), each of which is composed of multiple units of CESAs in three different isoforms. CSCs rely on vesicle trafficking for delivery to the plasma membrane where they catalyze cellulose synthesis. Although the rosette structured CSCs were observed decades ago, it remains unclear what amino acids in plant CESA that directly participate in cellulose catalytic synthesis. It is also not clear how the catalytic activity of CSCs influences their efficient transport at the subcellular level. Here we report characterization of the small molecule Endosidin20 (ES20) and present evidence that it represents a new CESA inhibitor. We show data from chemical genetic analyses, biochemical assays, structural modeling, and molecular docking to support our conclusion that ES20 targets the catalytic site of Arabidopsis CESA6. Further, chemical genetic analysis reveals important amino acids that potentially form the catalytic site of plant CESA6. Using high spatiotemporal resolution live-cell imaging, we found that inhibition of CSC catalytic activity by inhibitor treatment, or by creating missense mutation at amino acids in the predicted catalytic site, causes reduced efficiency in CSC transport to the plasma membrane. Our results show that the catalytic activity of plant CSCs is integrated with subcellular trafficking dynamics. One sentence summaryEndosidin20 targets cellulose synthase at the catalytic site to inhibit cellulose synthesis and the inhibition of catalytic activity reduces cellulose synthase complex delivery to the plasma membrane.

plant biology↗

bioRxiv2018

Adult Plant Resistance in Maize to Northern Leaf Spot Is a Feature of Partial Loss-of-function Alleles of Hm1

Adult plant resistance (APR) is an enigmatic phenomenon in which resistance genes are ineffective in protecting seedlings from disease but confer robust resistance at maturity. Maize has multiple cases in which genes confer APR to northern leaf spot, a lethal disease caused by Cochliobolus carbonum race 1 (CCR1). The first identified case of APR in maize is encoded by a hypomorphic allele, Hm1A, at the hm1 locus. In contrast, wild type alleles of hm1 provide complete protection at all developmental stages and in every part of the maize plant. Hm1 encodes an NADPH-dependent reductase, which inactivates HC-toxin, a key virulence effector of CCR1. Cloning and characterization of Hm1A ruled out differential transcription or translation for its APR phenotype and identified an amino acid substitution that reduced HC-toxin reductase (HCTR) activity. The possibility of a causal relationship between the weak nature of Hm1A and its APR phenotype was confirmed by the generation of two new APR alleles of Hm1 by mutagenesis. The HCTRs encoded by these new APR alleles had undergone relatively conservative missense changes that partially reduced their enzymatic activity similar to HM1A. No difference in accumulation of HCTR was observed between adult and juvenile plants, suggesting that the susceptibility of seedlings derives from a greater need for HCTR activity, not reduced accumulation of the gene product. Conditions and treatments that altered the photosynthetic output of the host had a dramatic effect on resistance imparted by the APR alleles, demonstrating a link between the energetic or metabolic status of the host and disease resistance affected by HC-toxin catabolism by the APR alleles of HCTR.\n\nAUTHOR SUMMARYAdult plant resistance (APR) is a phenomenon in which disease resistance genes are able to confer resistance at the adult stages of the plant but somehow fail to do so at the seedling stages. Despite the widespread occurrence of APR in various plant diseases, the mechanism underlying this trait remains obscure. It is not due to the differential transcription of these genes, and here we show that it is also not due to the differential translation or activity of the APR alleles of the maize hm1 gene at different stages of development. Using a combination of molecular genetics, biochemistry and physiology, we present multiple lines of evidence that demonstrate that APR is a feature or symptom of weak forms of resistance. While the mature parts of the plant are metabolically robust enough to manifest resistance, seedling tissues are not, leaving them vulnerable to disease. Growth conditions that compromise the photosynthetic output of the plant further deteriorate the ability of the seedlings to protect themselves from pathogens.\n\nOne sentence summaryCharacterization of adult plant resistance in the maize-CCR1 pathosystem reveals a causal link between weak resistance and APR.

pathology↗

bioRxiv2017

A Very Oil Yellow1 modifier of the Oil Yellow1-N1989 allele uncovers a cryptic phenotypic impact of cis-regulatory variation in maize

Forward genetics determines the function of genes underlying trait variation by identifying the change in DNA responsible for changes in phenotype. Detecting phenotypically-relevant variation outside protein coding sequences and distinguishing this from neutral variants is not trivial; partly because the mechanisms by which DNA polymorphisms in the intergenic regions affect gene regulation are poorly understood. Here we utilized a dominant genetic marker with a convenient phenotype to investigate the effect of cis and trans-acting regulatory variation. We performed a forward genetic screen for natural variation that suppress or enhance the semi-dominant mutant allele Oy1-N1989, encoding the magnesium chelatase subunit I of maize. This mutant permits rapid phenotyping of leaf color as a reporter for chlorophyll accumulation, and mapping of natural variation in maize affecting chlorophyll metabolism. We identified a single modifier locus segregating between B73 and Mo17 that was linked to the reporter gene itself, which we call very oil yellow1. Based on the variation in OY1 transcript abundance and genome-wide association data, vey1 is predicted to consist of multiple cis-acting regulatory sequence polymorphisms encoded at the wild-type oy1 alleles. The vey1 allele appears to be a common polymorphism in the maize germplasm that alters the expression level of a key gene in chlorophyll biosynthesis. These vey1 alleles have no discernable impact on leaf chlorophyll in the absence of the Oy1-N1989 reporter. Thus, use of a mutant as a simple and efficient reporter for magnesium chelatase activity resulted in the detection of expression-level polymorphisms not readily visible in the laboratory.

genetics↗

bioRxiv2017

Phytohormone Inhibitor Treatments Phenocopy Brassinosteroid And Gibberellin Dwarf Mutant Interactions In Maize

Phytohormone biosynthesis produces metabolites with profound effects on plant growth and development. Modulation of hormone levels during developmental events, in response to the environment, by genetic polymorphism, or by chemical application can reveal the plant processes most responsive to a phytohormone. In many cases, chemical inhibitors are applied and the levels of specific phytohormones are measured to determine if, and which, phytohormone is affected by a molecule. In many cases, the sensitivity of biochemical testing has determined multiple pathways affected by a single inhibitor. Genetic studies are not subject to this problem, and a wealth of data about the morphological impacts of hormone biosynthetic inhibition has accumulated through the study of enzyme mutants. We previously identified a complex interplay between brassinosteroid (BR) and gibberellin (GA) in maize, where the interdependence of the two differs dependent on the developmental context. We found that: GA is required for loss of BR to induce retained pistils in the tassel florets (POPIT); BR is required for the loss of GA to induce tiller outgrowth; BR and GA are additive for plant height; BR has no effect on the induction of anther retention in ear florets of GA mutants. In this work, we sought to assess the specificity of three triazole inhibitors of cytochrome P450s by determining their abilities to recapitulate the phenotype of double mutants. The GA biosynthetic inhibitors uniconazole (UCZ) and paclobutrazol (PAC) were applied to the BR biosynthetic mutant na2 and all double mutant phenotypes were recovered in the UCZ treatment. PAC was unable to suppress the retention of pistils in the tassels of nana plant2 (na2) mutant plants. The BR biosynthetic inhibitor propiconazole (PCZ) suppressed tiller outgrowth in the GA biosynthetic mutant dwarf5 (d5). All treatments were additive with genetic mutants for effects on plant height. Due to additional measurements done here but not in previous studies of the double mutants, we detected new interactions between GA and BR biosynthesis affecting plastochron index and tassel branching. These experiments, a refinement of our previous model, and a discussion of the extension of this type of work are presented.

plant biology↗

bioRxiv2016

Whole genome sequence accuracy is improved by replication in a population of mutagenized sorghum.

The accurate detection of induced mutations is critical for both forward and reverse genetics studies. Experimental chemical mutagenesis induces relatively few single base changes per individual. In a complex eukaryotic genome, false positive detection of mutations can occur at or above this mutagenesis rate. We demonstrate here, using a population of ethyl methanesulfonate (EMS) treated Sorghum bicolor BTx623 individuals, that using replication to detect false positive induced variants in next-generation sequencing data permits higher throughput variant detection with greater accuracy. We used a lower sequence coverage depth (average of 7X) from 586 independently mutagenized individuals and detected 5,399,493 homozygous SNPs. Of these, 76% originated from only 57,872 genomic positions prone to false positive variant calling. These positions are characterized by high copy number paralogs where the error-prone SNP positions are at copies containing a variant at the SNP position. The ability of short stretches of homology to generate these error prone positions suggests that incompletely assembled or poorly mapped repeated sequences are one driver of these error prone positions. Removal of these false positives left 1,275,872 homozygous and 477,531 heterozygous EMS-induced SNPs which, congruent with the mutagenic mechanism of EMS, were greater than 98% G:C to A:T transitions. Through this analysis we generated a database of sequence indexed mutants of Sorghum. This collection contains 4,035 high impact homozygous mutations in 3,637 genes and 56,514 homozygous missense mutations in 23,227 genes. Each line contains, on average, 2,177 annotated homozygous SNPs per genome, including seven likely gene knockouts and 96 missense mutations. The number of mutations in a transcript was linearly correlated with the transcript length and also the G+C count, but not with the GC/AT ratio. Analysis of the detected mutagenized positions identified CG-rich patches, and flanking sequences strongly influenced EMS-induced mutation rates. Our method for detecting false-positive induced mutations is generally applicable to any organism, is independent of the choice of in silico variant-calling algorithm, and is most valuable when the true mutation rate is likely to be low, such as in laboratory induced mutations or somatic mutation detection in medicine.

genetics↗