1a. Objectives (from AD-416):
Develop reliable high throughput methods of phenotyping common scab resistance. Accelerate selection of breeding lines combining chip processing, late blight and scab resistance.
1b. Approach (from AD-416):
Common scab initiates and progresses on developing potato tubers underground, where it is difficult to visualize. In cooperation with Sklarczyk Minitubers, Johannesburg, MI, a a soil-less hydroponic potato tuberization system (nutrient film technology or NFT) has been set up at Michigan State. Experiments are being conducted to examine how nutrient solution pH, inoculum concentration and timing of Streptomyces inoculation influence scab lesion development on a set of four lines that differ in their scab reaction under field conditions. The validated NFT system will be further used to evaluate commonly grown varieties and breeding material, as well as for phenotyping scab tolerance in populations developed during other research projects. Evaluation of 117 crosses comprising about 500 selections segregating for common scab resistance, late blight resistance and chip-processing will allow selection of optimal lines with good agronomic traits that also combine these three characteristics for further advancement in the ARS breeding program. In addition, 53 lines that may combine scab resistance, late blight resistance and chip-processing will be evaluated in replicated trials for agronomic performance in 2009 and also screened in replicated trials for late blight and scab resistance.
3. Progress Report:
We are developing a scab screening protocol using minitubers produced with a soil-less hydroponic potato tuberization system (nutrient film technology or NFT). The NFT system we implemented in the past two years to initiate an RNA-seq experiment to identify potential candidate genes involved in scab resistance. We are further optimizing the NFT system so that we can evaluate multiple different cultivars/breeding lines at the same time. NFT optimization: The biggest challenge for utilizing the NFT system for scab trials is getting multiple different potato clones to tuberize at the same time and/or determining when tuberization is occurring. To address this challenge we have made three changes to our protocol. First, the plants are put into the system without rockwool so that tuber development can be monitored. Second, the nutrient solution has been reduced to a 0.5X concentration. We have found that some clones will continue to produce long stolons but never tuberize under full concentration. At half-strength nutrient concentration clones still thrive and are more likely to tuberize. Third, after an initial 16/8 hr light-dark cycle the ratio is reduced to a 10/14 hr light/dark cycle to promote tuber formation. While tuber formation is still variable this new protocol narrows the range between early tuberizing clones and later tuberizing clones. RNA-seq: Using the new protocol described above, we initiated a trial to conduct an RNA-seq study. Two cultivars with different scab responses were planted in the NFT system: Kalkaska (R) and Tundra (S). Tubers approximately 5mm in diameter were placed in filter paper lined Petri dishes (to avoid contaminating the NFT nutrient solution) and inoculated with filter paper discs soaked in either 50 µM Thaxtomin or an equivalent control solution (no thaxtomin). At various time-points after inoculation, tubers were harvested, quickly diced and frozen in liquid nitrogen for later RNA extraction. One replication has been completed and a second replication is in progress. RNA-seq data collected from this experiment will be used to compare gene expression in controls vs. treated tubers at each time-point. In 2011 we had two locations to evaluate scab resistance: a commercial field with a history of severe scab infection and a highly infected site at the Montcalm Research Farm in the commercial production area. The commercial site and the new site at the Montcalm Research Farm both gave us the high infection levels. A summary of the lines that were evaluated is below. The control varieties, Snowden and Atlantic, were classified as susceptible and Pike as resistant. Based upon the data collected, the level of scab resistance is increasing in the breeding program. Resistant Susceptible Fifth year selections: 21 13 Fourth year selections: 49 22 Third year selections: 73 107 Advanced selections: 39 93 In 2011, we collected replicated (4-5 hill plots) scab infection data from our Montcalm Research Farm scab field on 200 progeny from a cross between resistant and susceptible varieties Of the 200 progeny, 27 were highly resistant and 48 were moderately resistant. The next step is to conduct QTL analysis with the SNP data. We will retest the 200 progeny in 2012. Only the early generation selections with a late blight resistance pedigree were evaluated for late blight resistance in inoculated trials at the Clarksville Research Center. Of the 170 lines tested 67 were classified as having resistant to moderately resistant foliage. Ten different resistant sources can be found in the pedigrees of these lines. Of the 155 advanced selections tested, 41 were classified as having resistant to moderately resistant foliage.