Project description:Somatic hybridization has been frequently used to overcome sexual incompatibility between potato and its secondary germplasm. The primary objective of this study was to produce and evaluate somatic hybrids of Solanum tuberosum (Stub) and S. bulbocastanum (Sblb) for breeding purposes. In 2007, 23 somatic hybrids were produced using an electrofusion of mesophyll protoplasts of diploid (2n = 2x = 24) potato line StubDH165 and S. bulbocastanum PI24351 (Sblb66). Phenotype of somatic hybrids in field conditions were evaluated, together with constitution and stability of 30 nuclear (ncSSR) and 27 cytoplasmic (cpSSR) microsatellite markers and content of main glycoalkaloids. All somatic hybrids had very high field resistance against late blight, but the plants were infertile: the viability of pollen grains insignificantly varied between 0.58 and 8.97%. A significant somaclonal variation was observed in terms of the morphology of plants, the date of emergence, the quantity of harvested tubers, the content of glycoalkaloids in foliage, and nuclear microsatellite markers (ncSSR). The analysis of ncSSR identified five distinct genotypes of hybrids partly associated with phenotype variations. The process of somatic hybridization with regeneration of shoots was identified as the most likely source of somaclonal variation because the ncSSR genotypes of hybrids, which were maintained in vitro, remained stable for more than 10 years. The infertile somatic hybrids have no practical breeding potential, but they are considered very suitable for advanced studies of the differential expression of genes in the pathways linked to dormancy of tubers and synthesis of glycoalkaloids.
Project description:Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating potato disease in the world. Control of late blight in the United States and other developed countries relies extensively on fungicide application. We previously demonstrated that the wild diploid potato species Solanum bulbocastanum is highly resistant to all known races of P. infestans. Potato germplasm derived from S. bulbocastanum has shown durable and effective resistance in the field. Here we report the cloning of the major resistance gene RB in S. bulbocastanum by using a map-based approach in combination with a long-range (LR)-PCR strategy. A cluster of four resistance genes of the CC-NBS-LRR (coiled coil-nucleotide binding site-Leu-rich repeat) class was found within the genetically mapped RB region. Transgenic plants containing a LR-PCR product of one of these four genes displayed broad spectrum late blight resistance. The cloned RB gene provides a new resource for developing late blight-resistant potato varieties. Our results also demonstrate that LR-PCR is a valuable approach to isolate genes that cannot be maintained in the bacterial artificial chromosome system.
Project description:BackgroundWild potato Solanum bulbocastanum is a rich source of genetic resistance against a variety of pathogens. It belongs to a taxonomic group of wild potato species sexually isolated from cultivated potato. Consistent with genetic isolation, previous studies suggested that the genome of S. bulbocastanum (B genome) is structurally distinct from that of cultivated potato (A genome). However, the genome architecture of the species remains largely uncharacterized. The current study employed Diversity Arrays Technology (DArT) to generate a linkage map for S. bulbocastanum and compare its genome architecture with those of potato and tomato.ResultsTwo S. bulbocastanum parental linkage maps comprising 458 and 138 DArT markers were constructed. The integrated map comprises 401 non-redundant markers distributed across 12 linkage groups for a total length of 645 cM. Sequencing and alignment of DArT clones to reference physical maps from tomato and cultivated potato allowed direct comparison of marker orders between species. A total of nine genomic segments informative in comparative genomic studies were identified. Seven genome rearrangements correspond to previously-reported structural changes that have occurred since the speciation of tomato and potato. We also identified two S. bulbocastanum genomic regions that differ from cultivated potato, suggesting possible chromosome divergence between Solanum A and B genomes.ConclusionsThe linkage map developed here is the first medium density map of S. bulbocastanum and will assist mapping of agronomical genes and QTLs. The structural comparison with potato and tomato physical maps is the first genome wide comparison between Solanum A and B genomes and establishes a foundation for further investigation of B genome-specific structural chromosome rearrangements.
Project description:Solanum bulbocastanum is a wild diploid tuber-bearing plant. We here demonstrate transgene-free genome editing of S. bulbocastanum protoplasts and regeneration of gene-edited plants. We use ribonucleoproteins, consisting of Cas9 and sgRNA, assembled in vitro, to target a gene belonging to the nitrate and peptide transporter family. Four different sgRNAs were designed and we observed efficiency in gene-editing in the protoplast pool between 8.5% and 12.4%. Twenty-one plants were re-generated from microcalli developed from individual protoplasts. In three of the plants we found that the target gene had been edited. Two of the edited plants had deletion mutations introduced into both alleles, whereas one only had a mutation in one of the alleles. Our work demonstrates that protocols for the transformation of Solanum tuberosum can be optimized to be applied to a wild Solanum species.