Identification of Solanum Immune Receptors by Bulked Segregant RNA-Seq and High-Throughput Recombinant Screening

The identification, understanding, and deployment of immune receptors are crucial to achieve high-level and durable resistance for crops against pathogens. In potato, many R genes have been identified using map-based cloning strategies. However, this is a challenging and laborious task that involves the development of a high number of molecular markers for the initial mapping, and the screening of thousands of plants for fine mapping. Bulked segregant RNA-Seq (BSR-Seq) has proven to be an efficient technique for the mapping of resistance genes. The RNA from two bulks of plants with contrasting phenotypes is sequenced and analyzed to identify single-nucleotide polymorphism (SNPs) markers linked to the target gene. Subsequently, the SNP markers that are identified can be used to delimit the mapping interval. Additionally, we designed an in vitro recombinant screening strategy that is advantageous for analyzing a large number of plants, in terms of time, space, and cost. Tips and detailed protocols, including BSR-Seq, bioinformatic analysis, and recombinant screening, are provided in this chapter.