Progress 05/01/12 to 09/30/14
Outputs
Target Audience: The target audience for PLRV, PVY, and nematode resistance research is primarily potato breeders, however this work will benefit in the long term potato growers and the larger potato industry by facilitating the incorporation of virus resistance into new potato varieties. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Attend and participate in the American Society of Plant Biologists meeting in Portland, Oregon, July 2014. At this conference a poster was presented and numerous seminars attended on the latest research in plant science. Graduate student Mike Wall and I attended and participated in the annual meeting of the Potato Association of America in Spokane, WA, July 2014. How have the results been disseminated to communities of interest? A poster was presented at the Project Director Meeting for Plant Biology in Washington, D.C. May 2014, titled Characterization of potato leafroll virus resistance derived from Solanum etuberosum. The poster reported progress in developing markers to track resistance to potato leafroll virus in potato. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 3. Evaluate priority crop core subsets and other selected germplasm with morphological descriptors, and key agronomic or horticultural traits, such as general adaptation, phenology, and growth potential. Identify accessions with desirable economical traits for multiple location tests and potential release to broaden the genetic base of breeding gene pools. A diverse set of 32 potato lines were evaluated for resistance to Globodera pallida, pale cyst nematode. These lines included Europe and US cultivars as well as breeding lines developed in the US with the goal of incorporating nematode resistance. Two replicated trials were recently completed and the data will soon be analyzed. This data will be combined with results evaluating the same lines for resistance to G. ellingtonae and G. rostochiensis. Objective 4. Apply molecular marker techniques to assess diversity, detect duplicated accessions, identify taxa that were difficult to classify with morphological characteristics and associated DNA polymorphism with variations of important economical traits in selected crops. Molecular markers flanking Rlretb were identified at 9.3 and 2.1 cM. Molecular markers 1367-8 and DMB32-11 are currently being used for marker assisted selection. A novel series of DNA-based screening protocols are being developed using pyrosequencing to detect resistance gene copy number in tetraploid potato. A protocol has been designed for Ryadg that appears to accurately detect dosage in a limited number of individuals. A new strategy using nested PCR has been initiated for Rysto and Rx1/Gpa2.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Ali, M.C., J.S. Rowley, J.C. Kuhl, S.M Gray, and A.V. Karasev (2014) Evidence of a monogenic nature of the Nz gene conferring resistance against Potato virus Y strain Z (PVYZ) in potato. American Journal of Potato Research.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: The target audience for the PLRV resistance research reported is primarily potato breeders, however this work indirectly will benefit potato growers and the larger potato industry. Target audiences for potato marker dosage research are potato breeders and other potato researchers. Target audiences for development of a new IMI diagnostic assay are wheat breeders and the wheat industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Attend and participate in the American Society of Plant Biologists meeting in Providence Rhode Island, July 2013. At this conference a poster was presented and numerous seminars attended on the latest research in plant science. Graduate student Ian Fullmer attended and participated in the annual meeting of the Potato Association of America in Quebec, Canada. How have the results been disseminated to communities of interest? A talk was presented at the Potato Association of America conference in Quebec, Canada titled Molecular Characterization of Vacuolar Acid Invertase in a Population of Potatoes which shows Phenotypic Segregation for Fry Color in July 2013. This talk reported on the application of a pyrosequencing assay to quantify different alleles of vacuolar acid invertase in potato. A poster was presented at the Project Director Meeting for Plant Biology in Washington, D.C. May 2013. This reported progress in developing markers to track resistance to potato leafroll virus in potato. What do you plan to do during the next reporting period to accomplish the goals? Once BAC sequence data is available, alignment analysis will help identify the fewest possible contigs for the sequenced BACs. This will form a physical map on which the associated molecular markers can be placed and surrounding genes identified. Depending of the extent of sequence coverage, additional BACs may be identified for sequencing. Sequence from S. etuberosum will be evaluated for evolutionary change when compared to potato and tomato sequence from homologous regions on chromosome 4. Additional markers can be developed and evaluated for linking to Rlretb. Sequence data from linked CAPS markers will be used to develop assays for quantifying alleles dosage in potato. Specifically sequence data from markers linked to potato genes Rysto and Rx1/Gpa2. Following assays design reaction conditions will be optimized and the assay tested against segregating progeny in a number of different potato populations.
Impacts
What was accomplished under these goals?
Objective 3. Evaluate priority crop core subsets and other selected germplasm with morphological descriptors, and key agronomic or horticultural traits, such as general adaptation, phenology, and growth potential. Identify accessions with desirable economical traits for multiple location tests and potential release to broaden the genetic base of breeding gene pools. Thirty-five BC4 potato lines (9 immune, 9 resistant, 8 moderately resistant and 9 susceptible) have been identified for grafting and aphid inoculation experiments and all lines entered into virus testing and clean-up. Lines have tested virus free and initial experiments started to determine virus movement across graft unions. Objective 4. Apply molecular marker techniques to assess diversity, detect duplicated accessions, identify taxa that were difficult to classify with morphological characteristics and associated DNA polymorphism with variations of important economical traits in selected crops. Approximately 72 new primer sets were designed and screened, 34% were polymorphic, of which 72% could be place on the chromosome 4 linkage map. Molecular markers flanking Rlretb were identified at 8.8 and 1.5 cM. Molecular markers 1367-8 and DMB32-11 are currently being used for marker assisted selection. A novel DNA-based screening protocol was developed using pyrosequencing to screen for a imidazolinone tolerant mutation in hexaploid wheat. One assay is shown to successfully detect zero to four copies of the mutation, while additional assays were shown to detect the presence of the mutation in individual wheat genomes. This work was published in Crop Science, Ellison et al. 2013. Another pyrosequencing assay was developed and applied to tetraploid potato to detect different alleles of vacuolar acid invertase. This work was reported at the annual meeting of the Potato Association of America in Quebec, Canada.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Ellison, M.T., Fischinger, F., Zemetra, R.S., and Kuhl, J.C. (2013). Use of pyrosequencing technology to genotype imidazolinone-tolerant wheat, Crop Science, 53: 2021-2027.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Research related to PLRV resistance has focused on Rlretb, a resistance locus from Solanum etuberosum mapped to potato chromosome 4. Previously indentified molecular markers were applied to the 109 genotypes from two backcross 4 populations and scored. A linkage map was generated using Map Manager. A BAC library generated from ~109 selfed S. etuberosum seedlings was screened with molecular markers tightly linked to Rlretb. This screen identified 33 BACs which were subsequently submitted for sequencing. All identified BACs were screened with available molecular markers, thereby generating a series of overlapping BACs based on shared molecular markers. Additional efforts are being made to develop and characterize new molecular markers closer to Rlretb for use in marker assisted breeding. Research was initiated to develop and evaluate diagnostic assays, using PCR and pyrosequencing, to replace existing PCR protocols for four potato resistance genes including Ryadg, Rysto, Rx1 and Gpa2. The goal of this work is to develop assays that can quantify resistance allele copy number in tetraploid potato cultivars. Initial work included reproducing previous reports, focusing on CAPS markers closely linked to the resistance genes. PCR fragments from resistant and susceptible lines were cloned and sequenced to determine sequence variation at the CAPS loci. A consensus sequence was used to design PCR primers and pyrosequencing primers that could be used to determine resistance allele dosage. Currently the assay for Ryadg is being optimized prior to conducting pyrosequencing. Sequences are being analyzed to design assays for Rx1 and Gpa2 prior to assay development. PARTICIPANTS: The principal investigator for this project was Joseph Kuhl. He directed this research providing necessary technical and scientific support. He organized the research, developed the procedures that would be applied and oversaw application and recovery of results. He analyzed and interpreted results from the project mentioned above. The principal support scientist was Margaret Dibble. She applied protocols and collected results. Data was prepared and presented to the principal investigator. She applied the necessary protocols for the molecular markers used in the PLRV resistance research. She also applied the PCR assay used in the cloning of the vacuolar acid invertase project and the preparation of DNA for sequencing. Additional work was provided by undergraduate and graduate students working on various components of the above research. TARGET AUDIENCES: The target audience for the PLRV resistance research reported above is primarily potato breeders, however this work indirectly will benefit potato growers and the larger potato industry. Target audiences for development of a new IMI diagnostic assay are wheat breeders and the wheat industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
New molecular markers were used to generate a linkage map of the distall end of the long arm of potato chromosome 4. These markers flank the PLRV resistance locus, Rlretb, with the closest markers 1-5 centiMorgans away. These markers and ones nearby can be used for marker assisted selection to help potato breeders track resistance in breeding lines. They will also provide additional information to obtain even closer markers, screen diploid populations, screen the newly created BAC library, provide new data for grant applications, and facilitate structural characterization of the chromosome region containing the resistance. A publication reporting this work will be submitted for publication in 2013 in collaboration with Rich Novy and Jonathan Whitworth. A new assay has been established using pyrosequencing to determine copy number of the IMI herbicide resistance allele in wheat. This assay provides wheat breeders with a fast and reliable assay for tracking resistance and will be particularly useful when breeders combine resistance from different genomes, the manuscript will soon be submitted.
Publications
- No publications reported this period
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