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  1. Free, publicly-accessible full text available June 1, 2024
  2. Abstract Extrachromosomal circular DNAs (eccDNAs) are found in many eukaryotic organisms. EccDNA-powered copy number variation plays diverse roles, from oncogenesis in humans to herbicide resistance in crop weeds. Here, we report interspecific eccDNA flow and its dynamic behavior in soma cells of natural populations and F1 hybrids of Amaranthus sp. The glyphosate-resistance (GR) trait is controlled by eccDNA-based amplification harboring the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (eccDNA replicon), the molecular target of glyphosate. We documented pollen-mediated transfer of eccDNA in experimental hybrids between glyphosate-susceptible Amaranthus tuberculatus and GR Amaranthus palmeri. Experimental hybridization and fluorescence in situ hybridization (FISH) analysis revealed that the eccDNA replicon in Amaranthus spinosus derived from GR A. palmeri by natural hybridization. FISH analysis also revealed random chromosome anchoring and massive eccDNA replicon copy number variation in soma cells of weedy hybrids. The results suggest that eccDNAs are inheritable across compatible species, contributing to genome plasticity and rapid adaptive evolution. 
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  3. Abstract The introgression of chromosome segments from wild relatives is an established strategy to enrich crop germplasm with disease-resistance genes 1 . Here we use mutagenesis and transcriptome sequencing to clone the leaf rust resistance gene Lr9 , which was introduced into bread wheat from the wild grass species Aegilops umbellulata 2 . We established that Lr9 encodes an unusual tandem kinase fusion protein. Long-read sequencing of a wheat Lr9 introgression line and the putative Ae. umbellulata Lr9 donor enabled us to assemble the ~28.4-Mb Lr9 translocation and to identify the translocation breakpoint. We likewise cloned Lr58 , which was reportedly introgressed from Aegilops triuncialis 3 , but has an identical coding sequence compared to Lr9 . Cytogenetic and haplotype analyses corroborate that the two genes originate from the same translocation event. Our work sheds light on the emerging role of kinase fusion proteins in wheat disease resistance, expanding the repertoire of disease-resistance genes for breeding. 
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    Free, publicly-accessible full text available June 1, 2024
  4. In agriculture, various chemicals are used to control the weeds. Out of which, glyphosate is an important herbicide invariably used in the cultivation of glyphosate-resistant crops to control weeds. Overuse of glyphosate results in the evolution of glyphosate-resistant weeds. Evolution of glyphosate resistance (GR) in Amaranthus palmeri (AP) is a serious concern in the USA. Investigation of the mechanism of GR in AP identified different resistance mechanisms of which 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification is predominant. Molecular analysis of GR AP identified the presence of a 5- to >160-fold increase in copies of the EPSPS gene than in a glyphosate-susceptible (GS) population. This increased copy number of the EPSPS gene increased the genome size ranging from 3.5 to 11.8%, depending on the copy number compared to the genome size of GS AP. FISH analysis using a 399-kb EPSPS cassette derived from bacterial artificial chromosomes (BACs) as probes identified that amplified EPSPS copies in GR AP exist in extrachromosomal circular DNA (eccDNA) in addition to the native copy in the chromosome. The EPSPS gene-containing eccDNA having a size of ∼400 kb is termed EPSPS-eccDNA and showed somatic mosacism in size and copy number. EPSPS-eccDNA has a genetic mechanism to tether randomly to mitotic or meiotic chromosomes during cell division or gamete formation and is inherited to daughter cells or progeny generating copy number variation. These eccDNAs are stable genetic elements that can replicate and exist independently. The genomic characterization of the EPSPS locus, along with the flanking regions, identified the presence of a complex array of repeats and mobile genetic elements. The cytogenomics approach in understanding the biology of EPSPS-eccDNA sheds light on various characteristics of EPSPS-eccDNA that favor GR in AP. 
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  5. Abstract

    The development of next-generation sequencing (NGS) enabled a shift from array-based genotyping to directly sequencing genomic libraries for high-throughput genotyping. Even though whole-genome sequencing was initially too costly for routine analysis in large populations such as breeding or genetic studies, continued advancements in genome sequencing and bioinformatics have provided the opportunity to capitalize on whole-genome information. As new sequencing platforms can routinely provide high-quality sequencing data for sufficient genome coverage to genotype various breeding populations, a limitation comes in the time and cost of library construction when multiplexing a large number of samples. Here we describe a high-throughput whole-genome skim-sequencing (skim-seq) approach that can be utilized for a broad range of genotyping and genomic characterization. Using optimized low-volume Illumina Nextera chemistry, we developed a skim-seq method and combined up to 960 samples in one multiplex library using dual index barcoding. With the dual-index barcoding, the number of samples for multiplexing can be adjusted depending on the amount of data required, and could be extended to 3,072 samples or more. Panels of doubled haploid wheat lines (Triticum aestivum, CDC Stanley x CDC Landmark), wheat-barley (T.aestivumxHordeum vulgare) and wheat-wheatgrass (Triticum durum x Thinopyrum intermedium) introgression lines as well as known monosomic wheat stocks were genotyped using the skim-seq approach. Bioinformatics pipelines were developed for various applications where sequencing coverage ranged from 1 × down to 0.01 × per sample. Using reference genomes, we detected chromosome dosage, identified aneuploidy, and karyotyped introgression lines from the skim-seq data. Leveraging the recent advancements in genome sequencing, skim-seq provides an effective and low-cost tool for routine genotyping and genetic analysis, which can track and identify introgressions and genomic regions of interest in genetics research and applied breeding programs.

     
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  6. Abstract A-genome diploid wheats represent the earliest domesticated and cultivated wheat species in the Fertile Crescent and include the donor of the wheat A sub-genome. The A-genome species encompass the cultivated einkorn (Triticum monococcum L. subsp. monococcum), wild einkorn (T. monococcum L. subsp. aegilopoides (Link) Thell.), and Triticum urartu. We evaluated the collection of 930 accessions in the Wheat Genetics Resource Center (WGRC) using genotyping by sequencing and identified 13,860 curated single-nucleotide polymorphisms. Genomic analysis detected misclassified and genetically identical (>99%) accessions, with most of the identical accessions originating from the same or nearby locations. About 56% (n = 520) of the WGRC A-genome species collections were genetically identical, supporting the need for genomic characterization for effective curation and maintenance of these collections. Population structure analysis confirmed the morphology-based classifications of the accessions and reflected the species geographic distributions. We also showed that T. urartu is the closest A-genome diploid to the A-subgenome in common wheat (Triticum aestivum L.) through phylogenetic analysis. Population analysis within the wild einkorn group showed three genetically distinct clusters, which corresponded with wild einkorn races α, β, and γ described previously. The T. monococcum genome-wide FST scan identified candidate genomic regions harboring a domestication selection signature at the Non-brittle rachis 1 (Btr1) locus on the short arm of chromosome 3Am at ∼70 Mb. We established an A-genome core set (79 accessions) based on allelic diversity, geographical distribution, and available phenotypic data. The individual species core set maintained at least 79% of allelic variants in the A-genome collection and constituted a valuable genetic resource to improve wheat and domesticated einkorn in breeding programs. 
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  7. null (Ed.)
    Gene transfer from wild wheat relatives to bread wheat is restricted to homologous recombination. The presence of the Pairing homoeologous 1 (Ph1) gene in the long arm of wheat chromosome 5B allows only homologous chromosomes to pair and recombine, resulting in diploid inheritance of polyploid wheat. Previously, we identified a potent homoeologous pairing promotor gene(s) (Hpp-5Mg); its carrier chromosome 5Mg derived from Aegilops geniculata and its wheat homoeologous chromosome 5D freely recombined in the presence of the Ph1 gene. In this study, we investigated the effect of Hpp-5Mg on homoeologous recombination in the absence of Ph1. In Hpp-5Mg/ph1bph1b plants, we observed a vast genome-wide increase in homoeologous recombination and multiple crossovers (CO), including CO breakpoints in proximal regions of the chromosomes where recombination is known to be suppressed. We tested the efficacy of Hpp-5Mg/ph1bph1b-induced homoeologous recombination by producing new recombinants for the wheat streak mosaic virus resistance gene, Wsm3, present in the wheat-Thinopyrum intermedium Robertsonian translocation (RobT T7BS.7S#3L). A recombination frequency of 6.5% was detected by screening the progenies double monosomic for T7BS.7S#3L and 7B by genomic in situ hybridization. This recombination frequency was about 100-fold higher compared with the recombinant frequency of 0.06% observed by using ph1b-induced homoeologous recombination alone. Our results indicate that chromosome 5Mg promotes homoeologous recombination between wheat and wild wheat relative chromosomes, which helps in the generation of pre-breeding materials thereby accelerating wheat crop improvement. 
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