Biology subjects

Parrott, W. A.

Publications and source records attributed to Parrott, W. A..

9 recordsLinked to original sources

bioRxiv2026

A fast neutron mutant resource for Ipomoea purpurea reveals dose-dependent genomic and phenotypic variation

A central goal in evolutionary biology is to understand the genomic basis of phenotypic traits, yet this pursuit is often constrained by a lack of accessible phenotypic and genetic variation, particularly in non-model organisms. Fast neutron irradiation offers a powerful approach for generating such variation, as it does not require prior genomic resources, established transformation systems, or reference mutant collections. Here, we describe the development and characterization of the first fast neutron mutant library in Ipomoea purpurea (common morning glory), a noxious agricultural weed and established model system in evolutionary ecology. We exposed an I. purpurea reference genotype to three levels of irradiation and developed 1,043 lines third generation lines and characterized the phenotypic and genetic variation in a subset of 200. Phenotypic screens identified seven mutant lines exhibiting eleven distinct phenotypes, and revealed higher phenotypic variance for anther-stigma distance, flower number, and pollen count relative to a non-irradiated control line. We further identified genetic variation across eight phenotypic traits among the mutant lines. Together, these results show that fast neutron mutagenesis generates novel phenotypic and genetic variation in ecologically relevant traits, offering the research community a new genomic resource for an established model in evolutionary ecology.

plant biology↗

bioRxiv2025

Loss of flavonol 3-O-glucosyltransferase activity confers soybean resistance to leaf-chewing insects

Caterpillars and beetles are among the most economically damaging defoliating insects, and their economic damage is predicted to increase in the coming decades. Hence the use of genetically derived resistance to supplement other pest control strategies is warranted. In soybean (Glycine max (L.) Merr.), a major determinant for resistance is the quantitative trait locus, QTL-M. Glyma07g14530, the gene underlying QTL-M, encodes a feeding-inducible flavonol 3-O-glycosyltransferase (F3GlcT or UGT78D2) that glucosylates kaempferol, as well as quercetin, myricetin, and isorhamnetin. The resistant allele has a premature stop codon in it, thus preventing the glucosylation and sequestration of flavonols in the vacuole, leading to a concomitant accumulation of proanthocyanidins and manifestation of resistance. Expressing the dominant (susceptible) allele in resistant plants restores susceptibility and silencing the susceptible allele results in resistance. The discovery and characterization of GmF3GlcT helps clarify the role of flavonoids in resistance to leaf-chewing insects and facilitates the development of insect-resistant cultivars that ultimately can lower production costs and reduce insecticide applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/679769v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@10e2e77org.highwire.dtl.DTLVardef@ca905org.highwire.dtl.DTLVardef@12cb46corg.highwire.dtl.DTLVardef@1f9328b_HPS_FORMAT_FIGEXP M_FIG C_FIG The loss of flavonol 3-O-glucosyltransferase in soybean reduces feeding damage from defoliating insects and is accompanied by a concomitant increase in proanthocyanidins (red arrowhead). SignificanceUnraveling the biochemical and genetic basis of soybean resistance to leaf-chewing insects facilitates the development of new, naturally insect resistant varieties. Such varieties contribute to on-farm profitability and reduced concerns over pesticide residues in the field.

plant biology↗

bioRxiv2024

Single component CRISPR-mediated base-editors for Agrobacterium and their use to develop an improved suite of strains

Agrobacterium mediated plant transformation largely depends on two distinct strain lineages - C58 and Ach5. To better serve the plant transformation community, we have created a suite of auxotrophic and auxotrophic recombinant deficient mutants of C58 derivatives EHA105, GV3101::pMP90, and Ach5 derivative LBA4404. While these derivatives are useful, having additional strain backgrounds available would help expand the repertoire for plant transformation even further. Toward that end, two underutilized hypervirulent strains are K599 (NCPPB 2659), and Chry5--but disarmed variants are not easily accessible. To improve availability, we produced disarmed versions of A. rhizogenes strain K599 and A. tumefaciens strain Chry5 and introduced the same desirable mutations as with the other lineages. Each thymidine auxotrophy and recombination deficiency were introduced to existing and newly disarmed Agrobacterium strains via loss of function mutations conferred to thyA and recA, respectively, through CRISPR-mediated base-editing of codons amenable to nonsense mutation. To streamline the editing process, we created a series of visually marked single component base-editor vectors and a corresponding guide-filtering Geneious Prime wrapper plugin for expedited guide filtering. These new strains, the simplified CRISPR-mediated base-editor plasmids, and streamlined workflow will improve the ease with which future Agrobacterium strain derivatives are created while also supporting plant transformation at large.

plant biology↗

bioRxiv2024

Identification and characterization of a temperature sensitive chlorotic soybean mutant

Screening a transposon-mutagenized soybean population led to the discovery of a recessively inherited chlorotic phenotype. This "vir1" phenotype results in smaller stature, weaker stems, and a smaller root system with smaller nodules. Genome sequencing identified 15 candidate genes with mutations likely to result in a loss of function. Amplicon sequencing of a segregating population was then used to narrow the list to a single candidate mutation, a single-base change in Glyma.07G102300 that disrupts splicing of the second intron. Single cell transcriptomic profiling indicates that this gene is expressed primarily in mesophyll cells and RNA sequencing data indicates it is upregulated in germinating seedlings by cold stress. Previous studies have shown that mutations to Os05g34040, the rice homolog of Glyma.07G102300, produced a chlorotic phenotype that was more pronounced in cool temperatures. Growing soybean vir1 mutants at lower temperatures also resulted in a more severe phenotype. In addition, transgenic expression of wild type Glyma.07G102300 in the knockout mutant of the Arabidopsis homolog At4930720 rescues the chlorotic phenotype, further supporting the hypothesis that the mutation in Glyma.07G102300 is causal of the vir1 phenotype.

plant biology↗

bioRxiv2022

An Agrobacterium strain auxotrophic for methionine is useful for switchgrass transformation

Auxotrophic strains of Agrobacterium tumefaciens can contribute to the development of more efficient transformation systems, especially for crops historically considered recalcitrant. Homologous recombination was used to derive methionine auxotrophs of two common A. tumefaciens strains, LBA4404 and EHA105. The EHA105 strains were more efficient for switchgrass transformation, while both the EHA105 and LBA4404 strains worked equally well for the rice control. Event quality, as measured by transgene copy number, was not affected by auxotrophy, but was higher for the LBA4404 strains than the EHA105 strains. Ultimately, the use of auxotrophs reduced bacterial overgrowth during co-cultivation, with a concomitant decrease in the need for antibiotics. In turn, reduced overgrowth allowed longer co-cultivation periods, with a trend towards higher transformation frequencies for switchgrass, but not for rice.

plant biology↗

bioRxiv2021

Plant-derived insulator-like sequences for control of transgene expression

Stable and consistent transgene expression is necessary to advance plant biotechnology. Stable expression can be achieved by incorporating enhancer-blocking insulators, which are cis-regulatory elements that reduce enhancer interference in gene expression, into transgene constructs. Sufficient insulators for plant use are not available, and their discovery has remained elusive. In this work, we computationally mined the compact genome of Utricularia gibba for insulator sequences and identified short (<1 kb) sequences with potential insulator activity. Based on in vivo tests, three of these effectively mitigate the ectopic transgene expression caused by the Cauliflower Mosaic Virus 35S promoter and do so better than previously reported plant insulators. However, all sequences with apparent insulator activity also decrease the effectiveness of the CaMV 35S promoter, and thus may be more accurately classified as silencers. However, since the insulator effect is proportionately much higher than the silencing effect, these sequences are still useful for plant transformation.

plant biology↗

bioRxiv2020

Fast neutron mutagenesis in soybean creates frameshift mutations

The mutagenic effects of ionizing radiation have been used for decades to create novel variants in experimental populations. Fast neutron (FN) bombardment as a mutagen has been especially widespread in plants, with extensive reports describing the induction of large structural variants, i.e., deletions, insertions, inversions, and translocations. However, the full spectrum of FN-induced mutations is poorly understood. We contrast small insertions and deletions (indels) observed in 27 soybean lines subject to FN irradiation with the standing indels identified in 107 diverse soybean lines. We use the same populations to contrast the nature and context (bases flanking a nucleotide change) of single nucleotide variants. The accumulation of new single nucleotide changes in FN lines is marginally higher than expected based on spontaneous mutation. In FN treated lines and in standing variation, C[->]T transitions and the corresponding reverse complement G[->]A transitions are the most abundant and occur most frequently in a CpG local context. These data indicate that most SNPs identified in FN lines are likely derived from spontaneous de novo processes in generations following mutagenesis, rather than from the FN irradiation mutagen. However, small indels in FN lines differ from standing variants. Short insertions, from 1 - 6 base pairs, are less abundant than in standing variation. Short deletions are more abundant and prone to induce frameshift mutations that should disrupt the structure and function of encoded proteins. These findings indicate that FN irradiation generates numerous small indels, increasing the abundance of loss of function mutations that will impact single genes. Significance StatementIrradiation mutagenesis is commonly viewed as a method to induce large structural variants in genomes. We also find enrichment in small insertion and deletion (indel) variants. The radiation-mutagenized lines averaged 32 indels per line, far exceeding the number estimated to occur by spontaneous processes, indicating that these arose from the irradiation treatment. Nevertheless, naturally-occurring standing variation among soybean accessions is still four orders of magnitude higher than the level of diversity introduced by mutagenesis. Induced mutations from any source are likely to constitute a relatively small portion of the genetic variation present in crop species. However, irradiation mutagenesis is useful for altering genomes by introducing small indels into single genes or disrupting gene clusters by creating structural variants.

genetics↗

bioRxiv2020

Development of mPing-based Activation Tags for Crop Insertional Mutagenesis

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing-based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.

plant biology↗

bioRxiv2018

Genome-scale sequence disruption following biolistic transformation in rice and maize

We biolistically transformed linear 48 kb phage lambda and two different circular plasmids into rice and maize and analyzed the results by whole genome sequencing and optical mapping. While some transgenic events showed simple insertions, others showed extreme genome damage in the form of chromosome truncations, large deletions, partial trisomy, and evidence of chromothripsis and breakage-fusion bridge cycling. Several transgenic events contained megabase-scale arrays of introduced DNA mixed with genomic fragments assembled by non-homologous or microhomology-mediated joining. Damaged regions of the genome, assayed by the presence of small fragments displaced elsewhere, were often repaired without a trace, presumably by homology-dependent repair (HDR). The results suggest a model whereby successful biolistic transformation relies on a combination of end joining to insert foreign DNA and HDR to repair collateral damage caused by the microprojectiles. The differing levels of genome damage observed among transgenic events may reflect the stage of the cell cycle and the availability of templates for HDR.

plant biology↗