The Colorado potato beetle (Leptinotarsa decemlineata) is the most widespread and destructive insect defoliator pest of potato and its control has historically been achieved through the use of insecticide. The diploid potato species Solanum chacoense has been utilized for over four decades in an attempt to introgress glycoalkaloid-based insect resistance into cultivated tetraploid potato. Despite these efforts, insect resistant cultivars have not been achieved, due in part to the complex genetics underlying the trait. The creation of inbred diploid lines would allow more efficient examination and deployment of this economically important trait. We introduced self-compatibility into diploid insect resistant S. chacoense germplasm and developed the first potato recombinant inbred line (RIL) population to study, understand and deploy this mechanism of host-plant insect resistance in cultivated, diploid breeding lines.We first examined the genetic features underlying leptine glycoalkaloid mediated Colorado potato beetle host plant resistance in the F2 generation derived from a cross between S. chacoense lines USDA8380-1 (80-1) and M6. Using biparental linkage mapping, a major overlapping QTL region with dominant effects was identified on chromosome 2 explaining 49.3% and 34.1% of the variance in Colorado potato beetle field resistance and leptine accumulation, respectively. Bulk segregant whole genome sequencing of the same F2 population detected QTL associated with Colorado potato beetle resistance on chromosomes 2, 4, 6, 7, and 12. Candidate genes within these QTL regions were identified by weighted gene co-expression network analysis of parental lines and resistant and susceptible F2 individuals. Second, we exploited M6-mediated self-compatibility and established vigorous, F5 inbred diploid lines to further examine loci associated with Colorado potato beetle resistance and explore the practicality of inbreeding in diploid potato. F5 inbred lines carrying Colorado potato beetle resistance equivalent to the resistant donor parent were created without field selection during the inbreeding process. We report that the ratio of acetylated to non-acetylated glycoalkaloids measured under greenhouse conditions is a powerful metabolite marker to predict field performance without incurring the costs of conducting a Colorado potato beetle field trial. Leptine production was successfully introduced into diploid breeding germplasm. Single nucleotide polymorphism (SNP) genotyping coupled with stylar analysis of pollen tube growth and self-fertility phenotyping of the F4 and F5 generations revealed that multiple factors mediate the self-compatible response in this RIL population. Third, we assessed the initial transcriptional and metabolite response to Colorado potato beetle herbivory in beetle resistant and beetle susceptible S. chacoense lines over a 48-hour time course. To facilitate genome editing modification of the leptine biosynthesis pathway, we characterized the allelic variation between S. chacoense 80-1 and M6 in a candidate leptine biosynthesis gene identified by transcriptional profiling. This work highlights the challenges of establishing inbred germplasm, reinforces the complexity of selecting for self-fertility in diploid potato, and lays the foundation for optimization of potato RIL development. The availability of highly homozygous Colorado potato beetle resistant lines will enable further genomic inquiry of the loci contributing to this trait and will facilitate rapid deployment of beetle resistant diploid potato varieties.
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