QTL mapping of foliar glycoalkaloid aglycones in Solanum tuberosum×S. berthaultii potato progenies: quantitative variation and plant secondary metabolism

 Glycoalkaloids are quantitatively inherited in Solanum, and in high concentrations they can be toxic to humans. The increased use of wild potato germplasm to improve the pest resistance, yield, and quality characteristics of cultivated potato may elevate or introduce new, more toxic glycoalkaloids into the cultivated gene pool. Therefore, it is important to increase our understanding of their inheritance, accumulation, and biosynthesis. Glycoalkaloids have two basic constituents – a glycosidic grouping and a steroid alkaloid skeleton. Steroid alkaloids are classified as solanidanes and spirosolanes, of which solanidine and solasodine are, respectively, representatives. RFLP-mapped, diploid, reciprocal backcross potato progenies involving the parents S. tuberosum and S. berthaultii, which produce solanidine and solasodine, respectively, were analyzed for segregation of the glycoalkaloids solanine, chaconine, solasodine and solamargine to identify quantitative trait loci (QTLs) for the production of the aglycones solanidine and solasodine. The F₁ clone M200-30 exhibited low to nondetectable levels of solasodine and solanidine, suggesting that expression was controlled by recessive genes. In a backcross to berthaultii (BCB) and backcross to tuberosum (BCT), several QTLs for the accumulation of solasodine and solanidine were identified. Three QTLs explaining approximately 20% of the variation in solasodine were identified in BCB on chromosomes 4, 6, and 12. Similarly, three QTLs were identified in BCT on chromosomes 4, 8 and 11, but these accounted for only 10% of the variation observed in solasodine accumulation. Two QTLs for solanidine were identified in BCT on chromosomes 1 and 4. The QTL located on chromosome 1 was highly significant, accounting for 17% and 22% of the variation in solanidine accumulation in 1994 and 1995, respectively. This same QTL was also detected in BCB. The QTLs detected in this study probably represent structural and/or regulatory genes controlling the accumulation of solasodine and solanidine. Results are discussed in the context of steroid alkaloid accumulation and biosynthesis. Received: 27 August 1997 / Accepted: 16 March 1998     

Isolation of Functional RNA from Periderm Tissue of Potato Tubers and Sweet Potato Storage Roots

A reliable and efficient protocol is given for the isolation of mRNA from the periderm of potato tubers and sweet potato storage roots. The method relies on a urea-based lysis buffer and lithium chloride to concentrate total RNA away from most of the cytoplasmic components and to prevent oxidation of phenolic complexes. To enhance the physical separation of the RNA from other macromolecular components, the RNA fraction was incubated in the presence of the cationic surfactant Catrimox-14. Poly(A)⁺ mRNA was separated from total RNA and other contaminants by using Promega's MagneSphere technology. The mRNA was suitable for cDNA library construction and RNA fingerprinting.     

Rapid Assessment of Primer Combinations and Recovery of AFLP™ Products using Ethidium Bromide Staining

AFLP is a novel high-resolution fingerprinting method that can be used to delineate intraspecific relationships among a large variety of fungi and plants. We demonstrate that with the appropriate technical modifications, ethidium bromide staining and non-denaturing polyacryalmide minigels can be an inexpensive and time saving alternative for screening DNA samples for suitable AFLP primer pairs. Furthermore, the recovery of ethidium bromide stained polymorphic DNA fragments is not as tedious as the recovery of isotopic DNA fragments.Abbreviations: EDTA, ethylene diamine tetraacetic acid; BSA, bovine serum albumin.     

Involvement of acetosyringone in plant-pathogen recognition

In this study, acetosyringone was identified as one of the major extracellular phenolics in tobacco suspension cells and was shown to have bioactive properties that influence early events in plant-bacterial pathogenesis. In our model system, tobacco cell suspensions treated with bacterial isolate Pseudomonas syringae WT (HR+) undergo a resistant interaction characterized by a burst in oxygen uptake several hours after inoculation. When the extracellular concentration of acetosyringone in tobacco cell suspensions was supplemented with exogenous acetosyringone, the burst in oxygen uptake occurred as much as 1.5h earlier. The exogenous acetosyringone had no effect on tobacco suspensions undergoing susceptible interactions with Pseudomonas tabaci or a non-resistant interaction with a near-isogenic mutant derivative of isolate P. syringae WT (HR+). Resistant interactions with isolate P. syringae WT (HR+) also produce an oxidative burst which oxidizes the extracellular acetosyringone. This study demonstrates that acetosyringone, and likely other extracellular phenolics, may have bioactive characteristics that can influence plant-bacterial pathogenesis.     

Sequence variation of intergenic mitochondrial DNA spacers (mtDNA‐IGS) of Phytophthora infestans (Oomycetes) and related species

The potato late‐blight disease is caused by the pseudofungus Phytophthora infestans (Oomycetes). This pathogen was of historical importance as it caused the Irish Potato Famine. There is currently a worldwide resurgence of the disease. Following worldwide migrations as well as being able to discriminate P. infestans from related species are key issues. We present sequence variation of five inter‐genic mitochondrial DNA spacers (mtDNA‐IGS) for P. infestans and four related taxa. Intra and inter‐taxon variation was observed showing potential for both molecular ecology and molecular systematic.     

Monitoring the rate of oxygen consumption in plant cell suspensions

A method is described that allows the rate of oxygen consumption to be monitored in plant cell suspensions. The method utilized oxygen electrodes placed in beakers of plant cells subjected to various treatments. The voltage readings from calibrated electrodes were converted to % oxygen (100% equals air equilibration) and the rate of oxygen consumption was estimated by calibration graphs made with no cells present. This system simultaneously monitors one to sixteen or more samples, allowing comparison of treatments on identically treated cells. We have used this method to study the respiratory burst of plant cells produced in response to viable or heat-killed bacteria. Because the system was computer-monitored and open to the atmosphere, data could be collected over several hours. Various factors that affected the measurement of dissolved oxygen concentration with this technique were explored and considered. This revised version was published online in June 2006 with corrections to the Cover Date.     

Partial preparative purification of leptine I from foliage of the wild potato, Solanum chacoense (Bitt.)

Leptine I, a glycoalkaloid only known to occur in the foliage of the wild potato species Solanum chacoense (Bitt.), is a potent feeding deterrent to the economically serious insect pest, the Colorado potato beetle (Leptinotarsa decemlineata Say). In order to demonstrate, systematically, the effectiveness of leptine I, incorporation into synthetic beetle diet trials is necessary. We describe a preparative procedure for the partial purification of leptine I by a series of steps, starting with a solid-phase C18 extraction, followed by sequential silica gel chromatography, and finally reversed-phase preparative HPLC. This preparation yields a white powder, containing leptine I as the sole glycoalkaloid, with an overall purity of greater than 65%, and is entirely suitable for incorporation into synthetic diets.     

Genome Analyses of an Aggressive and Invasive Lineage of the Irish Potato Famine Pathogen

Pest and pathogen losses jeopardise global food security and ever since the 19^th century Irish famine, potato late blight has exemplified this threat. The causal oomycete pathogen, Phytophthora infestans, undergoes major population shifts in agricultural systems via the successive emergence and migration of asexual lineages. The phenotypic and genotypic bases of these selective sweeps are largely unknown but management strategies need to adapt to reflect the changing pathogen population. Here, we used molecular markers to document the emergence of a lineage, termed 13_A2, in the European P. infestans population, and its rapid displacement of other lineages to exceed 75% of the pathogen population across Great Britain in less than three years. We show that isolates of the 13_A2 lineage are among the most aggressive on cultivated potatoes, outcompete other aggressive lineages in the field, and overcome previously effective forms of plant host resistance. Genome analyses of a 13_A2 isolate revealed extensive genetic and expression polymorphisms particularly in effector genes. Copy number variations, gene gains and losses, amino-acid replacements and changes in expression patterns of disease effector genes within the 13_A2 isolate likely contribute to enhanced virulence and aggressiveness to drive this population displacement. Importantly, 13_A2 isolates carry intact and in planta induced Avrblb1, Avrblb2 and Avrvnt1 effector genes that trigger resistance in potato lines carrying the corresponding R immune receptor genes Rpi-blb1, Rpi-blb2, and Rpi-vnt1.1. These findings point towards a strategy for deploying genetic resistance to mitigate the impact of the 13_A2 lineage and illustrate how pathogen population monitoring, combined with genome analysis, informs the management of devastating disease epidemics.