Discriminant analysis of principal components and pedigree assessment of genetic diversity and population structure in a tetraploid potato panel using SNPs

The reported narrow genetic base of cultivated potato (Solanum tuberosum) can be expanded by the introgression of many related species with large genetic diversity. The analysis of the genetic structure of a potato population is important to broaden the genetic base of breeding programs by the identification of different genetic pools. A panel composed by 231 diverse genotypes was characterized using single nucleotide polymorphism (SNP) markers of the Illumina Infinium Potato SNP Array V2 to identify population structure and assess genetic diversity using discriminant analysis of principal components (DAPC) and pedigree analysis. Results revealed the presence of five clusters within the populations differentiated principally by ploidy, taxonomy, origin and breeding program. The information obtained in this work could be readily used as a guide for parental introduction in new breeding programs that want to maximize variability by combination of contrasting variability sources such as those presented here.Fil: Deperi, Sofía Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Tagliotti, Martin Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Bedogni, María Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Manrique Carpintero, Norma C.. Michigan State University; Estados UnidosFil: Coombs, Joseph. Michigan State University; Estados UnidosFil: Zhang, Ruofang. Inner Mongolia University; ChinaFil: Douches, David. Michigan State University; Estados UnidosFil: Huarte, Marcelo Atilio. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentin

Use of easy measurable phenotypic traits as a complementary approach to evaluate the population structure and diversity in a high heterozygous panel of tetraploid clones and cultivars

Diversity in crops is fundamental for plant breeding efforts. An accurate assessment of genetic diversity, using molecular markers, such as single nucleotide polymorphism (SNP), must be able to reveal the structure of the population under study. A characterization of population structure using easy measurable phenotypic traits could be a preliminary and low-cost approach to elucidate the genetic structure of a population. A potato population of 183 genotypes was evaluated using 4859 high-quality SNPs and 19 phenotypic traits commonly recorded in potato breeding programs. A Bayesian approach, Minimum Spanning Tree (MST) and diversity estimator, as well as multivariate analysis based on phenotypic traits, were adopted to assess the population structure. Results: Analysis based on molecular markers showed groups linked to the phylogenetic relationship among the germplasm as well as the link with the breeding program that provided the material. Diversity estimators consistently structured the population according to a priori group estimation. The phenotypic traits only discriminated main groups with contrasting characteristics, as different subspecies, ploidy level or membership in a breeding program, but were not able to discriminate within groups. A joint molecular and phenotypic characterization analysis discriminated groups based on phenotypic classification, taxonomic category, provenance source of genotypes and genetic background. Conclusions: This paper shows the significant level of diversity existing in a parental population of potato as well as the putative phylogenetic relationships among the genotypes. The use of easily measurable phenotypic traits among highly contrasting genotypes could be a reasonable approach to estimate population structure in the initial phases of a potato breeding program.Fil: Tagliotti, Martin Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Deperi, Sofía Irene. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Bedogni, María Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Zhang, Ruofang. Inner Mongolia University; ChileFil: Manrique Carpintero, Norma C.. Michigan State University; Estados UnidosFil: Coombs, Joseph. Michigan State University; Estados UnidosFil: Douches, David. Michigan State University; Estados UnidosFil: Huarte, Marcelo Atilio. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentin

Overcoming Self-Incompatibility in Diploid Potato Using CRISPR-Cas9

Potato breeding can be redirected to a diploid inbred/F1 hybrid variety breeding strategy if self-compatibility can be introduced into diploid germplasm. However, the majority of diploid potato clones (Solanum spp.) possess gametophytic self-incompatibility that is primarily controlled by a single multiallelic locus called the S-locus which is composed of tightly linked genes, S-RNase (S-locus RNase) and multiple SLFs (S-locus F-box proteins), which are expressed in the style and pollen, respectively. Using S-RNase genes known to function in the Solanaceae gametophytic SI mechanism, we identified S-RNase alleles with flower-specific expression in two diploid self-incompatible potato lines using genome resequencing data. Consistent with the location of the S-locus in potato, we genetically mapped the S-RNase gene using a segregating population to a region of low recombination within the pericentromere of chromosome 1. To generate self-compatible diploid potato lines, a dual single-guide RNA (sgRNA) strategy was used to target conserved exonic regions of the S-RNase gene and generate targeted knockouts (KOs) using a Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (Cas9) approach. Self-compatibility was achieved in nine S-RNase KO T0 lines which contained bi-allelic and homozygous deletions/insertions in both genotypes, transmitting self compatibility to T1 progeny. This study demonstrates an efficient approach to achieve stable, consistent self-compatibility through S-RNase KO for use in diploid potato breeding approaches

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