Genome Editing for Crop Improvement – Applications in Clonally Propagated Polyploids With a Focus on Potato (Solanum tuberosum L.)

Genome-editing has revolutionized biology. When coupled with a recently streamlined regulatory process by the U.S. Department of Agriculture and the potential to generate transgene-free varieties, genome-editing provides a new avenue for crop improvement. For heterozygous, polyploid and vegetatively propagated crops such as cultivated potato, Solanum tuberosum Group Tuberosum L., genome-editing presents tremendous opportunities for trait improvement. In potato, traits such as improved resistance to cold-induced sweetening, processing efficiency, herbicide tolerance, modified starch quality and self-incompatibility have been targeted utilizing CRISPR/Cas9 and TALEN reagents in diploid and tetraploid clones. However, limited progress has been made in other such crops including sweetpotato, strawberry, grapes, citrus, banana etc., In this review we summarize the developments in genome-editing platforms, delivery mechanisms applicable to plants and then discuss the recent developments in regulation of genome-edited crops in the United States and The European Union. Next, we provide insight into the challenges of genome-editing in clonally propagated polyploid crops, their current status for trait improvement with future prospects focused on potato, a global food security crop

Field Assessment of AtCBF1 Transgenic Potato Lines (Solanum tuberosum) for Drought Tolerance

Abstract Drought prone areas have been increasing around the world and it is expected that these areas will continue to expand and become more severe due to climate change. Increasing the drought stress tolerance of cultivated potato (Solanum tuberosum) could aid in feeding the growing global population. The Arabidopsis CBF1 gene (AtCBF1), which has been shown to increase drought tolerance in other plants, was transformed into a cultivated potato line under the control of the stress inducible promoter COR15a. The expression of the AtCBF1 transgene was verified by RT-PCR and the transformed lines were evaluated in field trials to assess agronomic performance under sub-optimal water management. Despite expression of the AtCBF1 gene, none of the transgenic lines out-performed the control cultivar under drought-stressed conditions. Abiotic stress responsive genes from cultivated potato and wild related species may yield more promising results thus CBF1 genes from S. tuberosum and S. commersonii will be transformed into the potato cultivar Desiree and will be field tested for drought tolerance. Resumen Las áreas con riesgo de sequía se han estado incrementando alrededor del mundo y se espera que estas superficies continuarán en expansión volviéndose más severas debido al cambio climático. El aumento a la tolerancia al agobio hídrico de la papa cultivada (Solanum tuberosum) pudiera ayudar en la alimentación de la población global en crecimiento. El gen de Arabidopsis CBF1 (AtCBF1) que se ha demostrado que aumenta la tolerancia a la sequía en otras plantas, se introdujo en una línea de papa cultivada bajo el control del promotor de inducción de agobio COR15a. La expresión del transgen AtCBF1 se verificó mediante RT-PCR y se evaluaron las líneas transformadas en ensayos de campo para analizar el comportamiento agronómico bajo manejo subóptimo de agua. A pesar de la expresión del gen AtCBF1, ninguna de las líneas transgénicas superó en comportamiento a la variedad testigo bajo condiciones de agobio hídrico. Genes de respuesta de agobio abiótico de papa cultivada y de especies silvestres relacionadas pudieran rendir resultados más promisorios, de manera que los genes CBF1 de S. tuberosum y S. commersonii serán incorporados a la variedad de papa Desiree y serán probados en el campo para tolerancia a sequía

Evaluation of Methods to Assess in vivo Activity of Engineered Genome-Editing Nucleases in Protoplasts

Genome-editing is being implemented in increasing number of plant species using engineered sequence specific nucleases (SSNs) such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems (CRISPR/Cas9), Transcription activator like effector nucleases (TALENs), and more recently CRISPR/Cas12a. As the tissue culture and regeneration procedures to generate gene-edited events are time consuming, large-scale screening methodologies that rapidly facilitate validation of genome-editing reagents are critical. Plant protoplast cells provide a rapid platform to validate genome-editing reagents. Protoplast transfection with plasmids expressing genome-editing reagents represents an efficient and cost-effective method to screen for in vivo activity of genome-editing constructs and resulting targeted mutagenesis. In this study, we compared three existing methods for detection of editing activity, the T7 endonuclease I assay (T7EI), PCR/restriction enzyme (PCR/RE) digestion, and amplicon-sequencing, with an alternative method which involves tagging a double-stranded oligodeoxynucleotide (dsODN) into the SSN-induced double stranded break and detection of on-target activity of gene-editing reagents by PCR and agarose gel electrophoresis. To validate these methods, multiple reagents including TALENs, CRISPR/Cas9 and Cas9 variants, eCas9(1.1) (enhanced specificity) and Cas9-HF1 (high-fidelity1) were engineered for targeted mutagenesis of Acetolactate synthase1 (ALS1), 5-Enolpyruvylshikimate- 3-phosphate synthase1 (EPSPS1) and their paralogs in potato. While all methods detected editing activity, the PCR detection of dsODN integration provided the most straightforward and easiest method to assess on-target activity of the SSN as well as a method for initial qualitative evaluation of the functionality of genome-editing constructs. Quantitative data on mutagenesis frequencies obtained by amplicon-sequencing of ALS1 revealed that the mutagenesis frequency of CRISPR/Cas9 reagents is better than TALENs. Context-based choice of method for evaluation of gene-editing reagents in protoplast systems, along with advantages and limitations associated with each method, are discussed

Integration of Two Diploid Potato Linkage Maps with the Potato Genome Sequence

To facilitate genome-guided breeding in potato, we developed an 8303 Single Nucleotide Polymorphism (SNP) marker array using potato genome and transcriptome resources. To validate the Infinium 8303 Potato Array, we developed linkage maps from two diploid populations (DRH and D84) and compared these maps with the assembled potato genome sequence. Both populations used the doubled monoploid reference genotype DM1-3 516 R44 as the female parent but had different heterozygous diploid male parents (RH89-039-16 and 84SD22). Over 4,400 markers were mapped (1,960 in DRH and 2,454 in D84, 787 in common) resulting in map sizes of 965 (DRH) and 792 (D84) cM, covering 87% (DRH) and 88% (D84) of genome sequence length. Of the mapped markers, 33.5% were in candidate genes selected for the array, 4.5% were markers from existing genetic maps, and 61% were selected based on distribution across the genome. Markers with distorted segregation ratios occurred in blocks in both linkage maps, accounting for 4% (DRH) and 9% (D84) of mapped markers. Markers with distorted segregation ratios were unique to each population with blocks on chromosomes 9 and 12 in DRH and 3, 4, 6 and 8 in D84. Chromosome assignment of markers based on linkage mapping differed from sequence alignment with the Potato Genome Sequencing Consortium (PGSC) pseudomolecules for 1% of the mapped markers with some disconcordant markers attributable to paralogs. In total, 126 (DRH) and 226 (D84) mapped markers were not anchored to the pseudomolecules and provide new scaffold anchoring data to improve the potato genome assembly. The high degree of concordance between the linkage maps and the pseudomolecules demonstrates both the quality of the potato genome sequence and the functionality of the Infinium 8303 Potato Array. The broad genome coverage of the Infinium 8303 Potato Array compared to other marker sets will enable numerous downstream applications

Construction of reference chromosome-scale pseudomolecules for potato: integrating the potato genome with genetic and physical maps

The genome of potato, a major global food crop, was recently sequenced. The work presented here details the integration of the potato reference genome (DM) with a new STS marker based linkage map and other physical and genetic maps of potato and the closely related species tomato. Primary anchoring of the DM genome assembly was accomplished using a diploid segregating population, which was genotyped with several types of molecular genetic markers to construct a new ~936 cM linkage map comprising 2,469 marker loci. In silico anchoring approaches employed genetic and physical maps from the diploid potato genotype RH and tomato. This combined approach has allowed 951 superscaffolds to be ordered into pseudomolecules corresponding to the 12 potato chromosomes. These pseudomolecules represent 674 Mb (~93%) of the 723 Mb genome assembly and 37,482 (~96%) of the 39,031 predicted genes. The superscaffold order and orientation within the pseudomolecules is closely collinear with independently constructed high density linkage maps. Comparisons between marker distribution and physical location reveal regions of greater and lesser recombination, as well as regions exhibiting significant segregation distortion. The work presented here has led to a greatly improved ordering of the potato reference genome superscaffolds into chromosomal 'pseudomolecules'.Fil: Carboni, Martín Federico. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires. Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: D'ambrosio, Juan Martín. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional San Cristobal de Huamanga. Laboratorio de Genética y Biotecnología Vegetal; PerúFil: Sharma, Sanjeev Kumar. The James Hutton Institute; Reino UnidoFil: Bolser, Daniel. University of Dundee; Reino UnidoFil: de Boer, Jan. Wageningen University & Researc; Países BajosFil: Sønderkær, Mads . Aalborg University; DinamarcaFil: Amoros, Walter. International Potato Center; PerúFil: de la Cruz, Germán. Universidad Nacional San Cristobal de Huamanga; PerúFil: Di Genova, Alex. Universidad de Chile; ChileFil: Douches, David S.. Michigan State University; Estados UnidosFil: Eguiluz, Maria. Universidad Peruana Cayetano Heredia; PerúFil: Guo, Xiao. Shandong Academy of Agricultural Sciences; ChinaFil: Guzman, Frank. Universidad Peruana Cayetano Heredia; PerúFil: Hackett, Christine A.. Biomathematics and Statistics Scotland; Reino UnidoFil: Hamilton, John P.. Crops Environment and Land Use Programme; IrlandaFil: Li, Guangcun. Shandong Academy of Agricultural Sciences; ChinaFil: Li, Ying. The New Zealand Institute for Plant & Food Research; Nueva ZelandaFil: Lozano, Roberto. Universidad Peruana Cayetano Heredia; PerúFil: Maass, Alejandro. Universidad de Chile; ChileFil: Marshall, David. The James Hutton Institute; Reino UnidoFil: Martinez, Diana. Universidad Peruana Cayetano Heredia; PerúFil: McLean, Karen. The James Hutton Institute; Reino UnidoFil: Mejía, Nilo. Instituto de Investigaciones Agropecuarias. Centro Regional de Investigación La Platina; ChileFil: Milne, Linda. The James Hutton Institute; Reino UnidoFil: Munive, Susan. International Potato Center; PerúFil: Nagy, Istvan. Crops Environment and Land Use Programme; IrlandaFil: Ponce, Olga. Universidad Peruana Cayetano Heredia; PerúFil: Ramirez, Manuel. Universidad Peruana Cayetano Heredia; PerúFil: Simon, Reinhard. International Potato Center; PerúFil: Thomson, Susan J.. Chinese Academy of Agricultural Sciences; Chin

Single nucleotide polymorphism discovery in elite north american potato germplasm

BACKGROUND: Current breeding approaches in potato rely almost entirely on phenotypic evaluations; molecular markers, with the exception of a few linked to disease resistance traits, are not widely used. Large-scale sequence datasets generated primarily through Sanger Expressed Sequence Tag projects are available from a limited number of potato cultivars and access to next generation sequencing technologies permits rapid generation of sequence data for additional cultivars. When coupled with the advent of high throughput genotyping methods, an opportunity now exists for potato breeders to incorporate considerably more genotypic data into their decision-making. RESULTS: To identify a large number of Single Nucleotide Polymorphisms (SNPs) in elite potato germplasm, we sequenced normalized cDNA prepared from three commercial potato cultivars: 'Atlantic', 'Premier Russet' and 'Snowden'. For each cultivar, we generated 2 Gb of sequence which was assembled into a representative transcriptome of (~)28-29 Mb for each cultivar. Using the Maq SNP filter that filters read depth, density, and quality, 575,340 SNPs were identified within these three cultivars. In parallel, 2,358 SNPs were identified within existing Sanger sequences for three additional cultivars, 'Bintje', 'Kennebec', and 'Shepody'. Using a stringent set of filters in conjunction with the potato reference genome, we identified 69,011 high confidence SNPs from these six cultivars for use in genotyping with the Infinium platform. Ninety-six of these SNPs were used with a BeadXpress assay to assess allelic diversity in a germplasm panel of 248 lines; 82 of the SNPs proved sufficiently informative for subsequent analyses. Within diverse North American germplasm, the chip processing market class was most distinct, clearly separated from all other market classes. The round white and russet market classes both include fresh market and processing cultivars. Nevertheless, the russet and round white market classes are more distant from each other than processing are from fresh market types within these two groups. CONCLUSIONS: The genotype data generated in this study, albeit limited in number, has revealed distinct relationships among the market classes of potato. The SNPs identified in this study will enable high-throughput genotyping of germplasm and populations, which in turn will enable more efficient marker-assisted breeding efforts in potato

High-Density SNP Genotyping of Tomato (Solanum lycopersicum L.) Reveals Patterns of Genetic Variation Due to Breeding

The effects of selection on genome variation were investigated and visualized in tomato using a high-density single nucleotide polymorphism (SNP) array. 7,720 SNPs were genotyped on a collection of 426 tomato accessions (410 inbreds and 16 hybrids) and over 97% of the markers were polymorphic in the entire collection. Principal component analysis (PCA) and pairwise estimates of F-st supported that the inbred accessions represented seven sub-populations including processing, large-fruited fresh market, large-fruited vintage, cultivated cherry, landrace, wild cherry, and S. pimpinellifolium. Further divisions were found within both the contemporary processing and fresh market sub-populations. These sub-populations showed higher levels of genetic diversity relative to the vintage sub-population. The array provided a large number of polymorphic SNP markers across each sub-population, ranging from 3,159 in the vintage accessions to 6,234 in the cultivated cherry accessions. Visualization of minor allele frequency revealed regions of the genome that distinguished three representative sub-populations of cultivated tomato (processing, fresh market, and vintage), particularly on chromosomes 2, 4, 5, 6, and 11. The PCA loadings and F-st outlier analysis between these three sub-populations identified a large number of candidate loci under positive selection on chromosomes 4, 5, and 11. The extent of linkage disequilibrium (LD) was examined within each chromosome for these sub-populations. LD decay varied between chromosomes and sub-populations, with large differences reflective of breeding history. For example, on chromosome 11, decay occurred over 0.8 cM for processing accessions and over 19.7 cM for fresh market accessions. The observed SNP variation and LD decay suggest that different patterns of genetic variation in cultivated tomato are due to introgression from wild species and selection for market specialization