sample-preparation-for-in-situ-cryotomography-of-mammalian-cells

Sample Preparation for <em>In Situ</em> Cryotomography of Mammalian Cells

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly ...

University of Minnesota

Availability of readily transformable germplasm, as well as efficient pipelines for gene discovery are notable bottlenecks in the application of genome editing in potato. To study and introduce traits such as resistance against biotic and abiotic factors, tuber quality traits and self-fertility, model germplasm that is amenable to gene editing and regeneration is needed. Cultivated potato is a heterozygous autotetraploid and its genetic redundancy and complexity makes studying gene function challenging. Genome editing is simpler at the diploid level, with fewer allelic variants to consider. A readily transformable diploid potato would be further complemented by genomic resources that could aid in high throughput functional analysis. The heterozygous Solanum tuberosum Group Phureja clone 1S1 has a high regeneration rate, self-fertility, desirable tuber traits and is amenable to Agrobacterium-mediated transformation. We leveraged its amenability to Agrobacterium-mediated transformation to create a Cas9 constitutively expressing line for use in viral vector-based gene editing. To create a contiguous genome assembly, a homozygous doubled monoploid of 1S1 (DM1S1) was sequenced using 44 Gbp of long reads generated from Oxford Nanopore Technologies (ONT), yielding a 736 Mb assembly that encoded 31,145 protein-coding genes. The final assembly for DM1S1 represents a nearly complete genic space, shown by the presence of 99.6% of the genes in the Benchmarking Universal Single Copy Orthologs (BUSCO) set. Variant analysis with Illumina reads from 1S1 was used to deduce its alternate haplotype. These genetic and genomic resources provide a toolkit for applications of genome editing in both basic and applied research of potato.

Genome Report: Genome sequence of 1S1, a transformable and highly regenerable diploid potato for use as a model for gene editing and genetic engineering.

Jacob Jensen

Department of Biochemistry,
Molecular Biology and Biophysics,
Medical School,
Department of Biochemistry, Molecular Biology and Biophysics, Medical School

University of Minnesota

Sample Preparation for In Situ Cryotomography of Mammalian Cells

Jacob Jensen

Department of Biochemistry,Molecular Biology and Biophysics,Medical School,Department of Biochemistry, Molecular Biology and Biophysics, Medical School

University of Minnesota

Sample Preparation for In Situ Cryotomography of Mammalian Cells

Department of Biochemistry,
Molecular Biology and Biophysics,
Medical School,
Department of Biochemistry, Molecular Biology and Biophysics, Medical School