Studies of verticillium wilt and characterization of candidate verticillium wilt resistance genes in the mint species Mentha longifolia (L) Huds

To investigate the genetic basis of verticillium wilt resistance in mint (Mentha L., Lamiaceae), wild-collected germplasm obtained from the United States Department of Agriculture was employed to develop breeding populations for wilt resistance screening and molecular genetic study, including cloning of candidate verticillium wilt resistance genes. A collection of fourteen Mentha longifolia accessions from Europe, Asia and South Africa was analyzed for morphological traits, oil composition, and verticillium wilt resistance. In addition, a preliminary molecular diversity assessment was conducted utilizing randomly amplified polymorphic DNA (RAPD) markers. The accessions were found to be diverse regarding all observed traits and the South African accessions in particular were shown to possess unique features. Most importantly, highly wilt-resistant and highly wiltsusceptible accessions were observed. A collection of twenty-seven resistance gene analogs (RGAs) was isolated from M. longifolia accessions using a PCR-based approach with primers targeting the conserved nucleotide binding site (NBS) domain found in most plant disease resistance genes. The mint RGAs shared predicted amino acid sequence similarity with disease resistance genes and RGAs from various other plant species, and were grouped into seven distinct families based on DNA and predicted amino acid sequence similarity. In addition to the NBS-related RGAs, a fragment of a verticillium wilt resistance gene candidate was isolated from a verticillium-resistant M. longifolia accession using a combination of PCR-based approaches that exploited known sequences of tomato Ve (Verticillium resistance) genes. Finally, the complete coding region of the mint verticillium wilt resistance candidate gene, mVe1, was cloned and sequenced. Alleles of mVe1 were compared among four M. longifolia accessions used as crossing parents. These seven alleles were highly similar to each other (96.2-99.6% nucleotide identity) and had ∼50% predicted amino acid sequence identity to the tomato Ve genes. F1 and F2 populations were genotyped with respect to mVe1 alleles, and individuals from these populations were screened for wilt resistance. No correlation was found between any mVe allele and resistance or susceptibility to verticillium wilt in plants in the studied populations. However, this result does not discount the possibility that an mVe1 gene product plays a role in mint verticillium wilt resistance

In Vivo and In Vitro Chromosome Doubling of ‘I3’ Hemp

Cannabis sativa L. is a diploid (2x) herbaceous plant that provides a wide variety of products such as essential oils, fiber, and medicine. Hemp was defined in the 2018 Farm Bill as a Cannabis plant with a delta-9 tetrahydrocannabinol concentration of not more than 0.3% on a dry-weight basis. Polyploidy is frequently used in plant breeding to manipulate vigor, reproductive fertility, and biochemistry. By inducing polyploidy/chromosome doubling, we may increase the compounds of interest, principally CBD (cannabidiol), produced by hemp. The purpose of this experiment was to evaluate the efficacy of different treatments of colchicine and oryzalin applied in vivo and in vitro to induce polyploidy in ‘I3’ hemp. After treating vegetative cuttings with colchicine or oryzalin, we had a 31% survival rate. Of the 85 survivors, we recovered two tetraploids: one from the 12-h 0.05% colchicine treatment group and the other from the 12-h 0.2% colchicine treatment group. For the in vitro portion of the experiment, the 12-h 50-µM oryzalin treatment yielded one tetraploid and the 36 h 50 µM oryzalin treatment yielded one cytochimera (mixoploid). The relative efficiency of some treatments showed potential for a simple method to induce tetraploids in clonal hemp for breeding

Dynamic DNA cytosine methylation in the Populus trichocarpa genome: tissue-level variation and relationship to gene expression

This is the publisher’s final pdf. The published article is copyrighted by BioMed Central Ltd and can be found at: http://www.biomedcentral.com/.Background: DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood. \ud \ud Results: We surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem) in the reference tree species black cottonwood (Populus trichocarpa). Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq), we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation") had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation. \ud \ud Conclusions: We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation

Rare but diverse off-target and somatic mutations found in field and greenhouse grown trees expressing CRISPR/Cas9

Introduction: CRISPR gene editing, while highly efficient in creating desired mutations, also has the potential to cause off-target mutations. This risk is especially high in clonally propagated plants, where editing reagents may remain in the genome for long periods of time or in perpetuity. We studied a diverse population of Populus and Eucalyptus trees that had CRISPR/Cas9-containing transgenes that targeted one or two types of floral development genes, homologs of LEAFY and AGAMOUS.Methods: Using a targeted sequence approach, we studied approximately 20,000 genomic sites with degenerate sequence homology of up to five base pairs relative to guide RNA (gRNA) target sites. We analyzed those sites in 96 individual tree samples that represented 37 independent insertion events containing one or multiples of six unique gRNAs.Results: We found low rates of off-target mutations, with rates of 1.2 × 10−9 in poplar and 3.1 × 10−10 in eucalypts, respectively, comparable to that expected due to sexual reproduction. The rates of mutation were highly idiosyncratic among sites and not predicted by sequence similarity to the target sites; a subset of two gRNAs showed off-target editing of four unique genomic sites with up to five mismatches relative to the true target sites, reaching fixation in some gene insertion events and clonal ramets. The location of off-target mutations relative to the PAM site were essentially identical to that seen with on-target CRISPR mutations.Discussion: The low rates observed support many other studies in plants that suggest that the rates of off-target mutagenesis from CRISPR/Cas9 transgenes are negligible; our study extends this conclusion to trees and other long-lived plants where CRISPR/Cas9 transgenes were present in the genome for approximately four years

Crop wild relatives as germplasm resource for cultivar improvement in mint (Mentha L.)

Mentha is a strongly scented herb of the Lamiaceae (formerly Labiatae) and includes about 30 species and hybrid species that are distributed or introduced throughout the globe. These fragrant plants have been selected throughout millennia for use by humans as herbs, spices, and pharmaceutical needs. The distilling of essential oils from mint began in Japan and England but has become a significant industrial product for the US, China, India, and other countries. The US Department of Agriculture (USDA), Agricultural Research Service, National Clonal Germplasm Repository (NCGR) maintains a mint genebank in Corvallis, Oregon. This facility preserves and distributes about 450 clones representing 34 taxa, hybrid species, advanced breeder selections, and F1 hybrids. Mint crop wild relatives are included in this unique resource. The majority of mint accessions and hybrids in this collection were initially donated in the 1970s by the A.M. Todd Company, located in Kalamazoo, Michigan. Other representatives of diverse mint taxa and crop wild relatives have since been obtained from collaborators in Australia, New Zealand, Europe, and Vietnam. These mints have been evaluated for cytology, oil components, verticillium wilt resistance, and key morphological characters. Pressed voucher specimens have been prepared for morphological identity verification. An initial set of microsatellite markers has been developed to determine clonal identity and assess genetic diversity. Plant breeders at private and public institutions are using molecular analysis to determine identity and diversity of the USDA mint collection. Evaluation and characterization includes essential oil content, disease resistance, male sterility, and other traits for potential breeding use. These accessions can be a source for parental genes for enhancement efforts to produce hybrids, or for breeding new cultivars for agricultural production. Propagules of Mentha are available for distribution to international researchers as stem cuttings, rhizome cuttings, or seed, which can be requested through the GRIN-Global database of the US National Plant Germplasm System, subject to international treaty and quarantine regulations

Genome resequencing reveals multiscale geographic structure and extensive linkage disequilibrium in the forest tree Populus trichocarpa

This is the publisher’s final pdf. The article is copyrighted by the New Phytologist Trust and published by John Wiley & Sons, Inc. It can be found at: http://onlinelibrary.wiley.com/journal/10.1111/%28ISSN%291469-8137. To the best of our knowledge, one or more authors of this paper were federal employees when contributing to this work.•Plant population genomics informs evolutionary biology, breeding, conservation and bioenergy feedstock development. For example, the detection of reliable phenotype–genotype associations and molecular signatures of selection requires a detailed knowledge about genome-wide patterns of allele frequency variation, linkage disequilibrium and recombination.\ud •We resequenced 16 genomes of the model tree Populus trichocarpa and genotyped 120 trees from 10 subpopulations using 29 213 single-nucleotide polymorphisms.\ud •Significant geographic differentiation was present at multiple spatial scales, and range-wide latitudinal allele frequency gradients were strikingly common across the genome. The decay of linkage disequilibrium with physical distance was slower than expected from previous studies in Populus, with r² dropping below 0.2 within 3–6 kb. Consistent with this, estimates of recent effective population size from linkage disequilibrium (N[subscript e] ≈ 4000–6000) were remarkably low relative to the large census sizes of P. trichocarpa stands. Fine-scale rates of recombination varied widely across the genome, but were largely predictable on the basis of DNA sequence and methylation features.\ud •Our results suggest that genetic drift has played a significant role in the recent evolutionary history of P. trichocarpa. Most importantly, the extensive linkage disequilibrium detected suggests that genome-wide association studies and genomic selection in undomesticated populations may be more feasible in Populus than previously assumed

Epigenetic regulation of adaptive responses of forest tree species to the environment

Epigenetic variation is likely to contribute to the phenotypic plasticity and adaptative capacity of plant species, and may be especially important for long-lived organisms with complex life cycles, including forest trees. Diverse environmental stresses and hybridization/polyploidization events can create reversible heritable epigenetic marks that can be transmitted to subsequent generations as a form of molecular “memory”. Epigenetic changes might also contribute to the ability of plants to colonize or persist in variable environments. In this review, we provide an overview of recent data on epigenetic mechanisms involved in developmental processes and responses to environmental cues in plant, with a focus on forest tree species. We consider the possible role of forest tree epigenetics as a new source of adaptive traits in plant breeding, biotechnology, and ecosystem conservation under rapid climate chang

Methylome reorganization during in vitro dedifferentiation and regeneration of Populus trichocarpa

Background: Cytosine DNA methylation (5mC) is an epigenetic modification that is important to genome stability and regulation of gene expression. Perturbations of 5mC have been implicated as a cause of phenotypic variation among plants regenerated through in vitro culture systems. However, the pattern of change in 5mC and its functional role with respect to gene expression, are poorly understood at the genome scale. A fuller understanding of how 5mC changes during in vitro manipulation may aid the development of methods for reducing or amplifying the mutagenic and epigenetic effects of in vitro culture and plant transformation. Results: We investigated the in vitro methylome of the model tree species Populus trichocarpa in a system that mimics routine methods for regeneration and plant transformation in the genus Populus (poplar). Using methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq), we compared the methylomes of internode stem segments from micropropagated explants, dedifferentiated calli, and internodes from regenerated plants. We found that more than half (56%) of the methylated portion of the genome appeared to be differentially methylated among the three tissue types. Surprisingly, gene promoter methylation varied little among tissues, however, the percentage of body-methylated genes increased from 9% to 14% between explants and callus tissue, then decreased to 8% in regenerated internodes. Forty-five percent of differentially-methylated genes underwent transient methylation, becoming methylated in calli, and demethylated in regenerants. These genes were more frequent in chromosomal regions with higher gene density. Comparisons with an expression microarray dataset showed that genes methylated at both promoters and gene bodies had lower expression than genes that were unmethylated or only promoter-methylated in all three tissues. Four types of abundant transposable elements showed their highest levels of 5mC in regenerated internodes. Conclusions: DNA methylation varies in a highly gene-and chromosome-differential manner during in vitro differentiation and regeneration. 5mC in redifferentiated tissues was not reset to that in original explants during the study period. Hypermethylation of gene bodies in dedifferentiated cells did not interfere with transcription, and may serve a protective role against activation of abundant transposable elements.Keywords: Jacq, Palm Elaeis guineensis, Expression analysis, Genome, Cytosine methylation, Arabidopsis cells, Somaclonal variation, Plants, Tissue culture, Gene

Dynamic DNA cytosine methylation in the Populus trichocarpa genome: tissue-level variation and relationship to gene expression

Background: DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood. Results: We surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem) in the reference tree species black cottonwood (Populus trichocarpa). Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq), we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation") had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation. Conclusions: We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation.Keywords: Populus, DNA methylation, 5-methylcytosine, Epigenetics, Epigenomic