Objectives        Collaborators

Potential        Accomplishments

George Vandemark, Research Geneticist
Richard Larsen, Research Plant Pathologist

Objectives

Develop molecular tools to increase precision in the selection of alfalfa plants and populations for resistance to multiple soilborne diseases and apply these tools to develop alfalfa germplasm with enhanced levels of disease resistance. These molecular tools will improve efficiency of breeding programs and the experimental design employed to develop these tools will be applicable to breeding programs focused on all other crop species.

Advances in the understanding of the genetic determinants conditioning resistance to soilborne diseases in alfalfa and Medicago truncatula, a forage model species. This understanding will help forage breeding programs incorporate disease resistance into commercial varieties.

Identify sources of resistance to alfalfa mosaic virus (AMV) in bean (Phaseolus vulgaris). The resistant breeding materials identified in this study will be used to incorporate resistance into commercial varieties.

Potential Benefits
top

The development of improved methods of selection based on quantitative PCR, improved understanding of the genetic basis of resistance, and the development of elite germplasm with multiple disease resistance will facilitate the development of improved commercial varieties of alfalfa. This will reduce grower losses due to soilborne disease, and will also increase the use of alfalfa as both a rotation crop in sustainable cultivation systems and as a bridge crop for transition of land to certified organic use. These benefits will reduce dependence on agrochemical applications, diminish deleterious effects of agrochemicals to humans and other non-target organisms, improve environmental quality, and increase profitability. Quantitative PCR assays and increased understanding of the genetic basis of resistance will be useful to scientists studying mechanisms of disease resistance in plants and virulence in pathogens. Strategies used to develop quantitative PCR assays will be applicable for the development of similar assays for other crop species. Identification of breeding lines with resistance to AMV will facilitate the varietal improvement of bean for virus resistance.

Anticipated Products

Realizing the objectives of this project will result in an improved understanding of the genetic basis of disease resistance in alfalfa and M. truncatula. We will produce accurate assays for quantifying resistance in single plants and populations based on PCR detection of pathogen specific DNA sequences. Proprietary protection may be sought for these assays. Alfalfa germplasm will be developed that has high levels of resistance to several diseases caused by soilborne pathogens. We will identify new bean breeding lines with resistance to AMV.

Collaborations
top

Subobjective 1A: (within ARS) Dr. Nichole O'Neill, USDA-ARS, Beltsville, MD. (External to ARS) Dr. Craig R. Grau, University of Wisconsin, Madison.

Subobjective 1B: (within ARS) None. (External to ARS) Dr. Craig R. Grau, University of Wisconsin, Madison; Dr Mark Smith, Pioneer Hi-Bred Intl., Connell, WA.

Subobjective 2A: (within ARS) Dr. Phillip Miklas, USDA-ARS, Prosser, WA. (External to ARS) None.

Subobjective 2B: (within ARS) Dr. Phillip Miklas, USDA-ARS, Prosser, WA. (External to ARS) None.

Customers

Commercial alfalfa and bean seed companies and public and private scientists will benefit from the availability of improved germplasm and accurate assays for both evaluating resistance and detecting plant pathogens. Growers will have increased opportunity for the profitable cultivation of alfalfa and beans. The general public will benefit from reduced consumer costs and reduced deleterious effects associated with the use of agrochemicals for disease control. Government agencies involved in quarantines and plant health will be able to use these assays for detection and quantification of soilborne plant pathogens.

MAJOR RESEARCH ACCOMPLISHMENTS
top

• Alfalfa - Molecular Probe for Disease Identification: Aphanomyces euteiches causes severe root rot disease of alfalfa, peas, and bean, and it is very difficult to detect the presence of the pathogen in infected plants and soil. A method based on the use of molecular probes was successfully developed that can detect the presence of A. euteiches in both soil samples and the roots of field grown alfalfa and peas. This method makes it easier to determine the presence of pathogens in fields, assists growers in their decisions regarding the choice of varieties to be cultivated, and facilitates the disease diagnosis of infected plants.

Isolation and identification of the fungus Phoma sclerotioides, the causal agent of brown root rot of alfalfa, requires up to 60 days using standard methods. A molecular DNA technique was developed in cooperation with University of Wyoming scientists for detecting the pathogen in less than one day.

• Alfalfa Germplasm Enhancement: Our strategy for developing enhanced alfalfa germplasm pools involves allowing two generations of random mating among elite alfalfa varieties prior to selecting for any specific traits. This approach will allow us to produce plants that have new combinations of genes that may work together to improve simultaneously several traits of agronomic importance. We have successfully completed and harvested seed from the first generation of random mating and have the second generation currently in the field for the production of PEAC (Prosser Elite Alfalfa Composite).

• The development of a novel method based on the detection of fluorescent-labeled PCR products that can distinguish between genomes that are homozygous or heterozygous for molecular markers of interest: Many molecular markers that are linked to genes of interest are dominant markers, meaning that genomes that have two copies of the marker (homozygotes) cannot be distinguished from those having only a single copy (heterozygotes). This results in increased costs for plant breeding programs and also delays progress, as an additional generation of seed production is required to produce progeny, and progeny must then be evaluated for the trait of interest in order to distinguish between homozygotes and heterozygotes. We developed conceptual approach and experimental design to realize the objective, and performed all the laboratory work required to develop and validate the method. The application of the method described here will result in more timely population improvement, as plants that are homozygous for dominant markers of interest can be identified as seedlings. The use of this method will reduce the need for breeding programs to dedicate greenhouse facilities for progeny testing. This assay will also eliminate occupational exposure to hazards associated with gel electrophoresis, such as ethidium bromide and ultraviolet light. The method employed for assigning genotype based on quantitative PCR will be broadly applicable to the genotyping of diploid plants or animals for dominant molecular markers. The ability to rapidly assign genotypes to several loci through the use of a multiplex PCR reaction will considerably reduce costs associated with the selection of parental materials and accelerate progress of breeding programs.

• We developed a unique and effective inoculation technique to infect alfalfa by alfalfa mosaic virus (AMV) to facilitate rapid screening and evaluation of varieties for resistance to AMV.

• Alfalfa variety “Winema” was released containing high stable resistance to alfalfa mosaic virus, along with other favorable traits, such as improved plant vigor, longevity of stand, and increased hay yields.

• Studies are in progress to release chickpea and lentil germplasms resistant to pea enation mosaic virus (PEMV). We developed a technique utilizing reverse-transcriptase polymerase chain reaction (RT-PCR) that can detect PEMV and bean leafroll luteovirus in single aphid species.

• We have developed a PCR-based assay using sequence characterized DNA markers for the identification and detection of the soilborne fungus Aphanomyces euteiches. This fungus causes severe root rot disease in alfalfa, peas and beans. The assay can be used for detecting the presence of the fungus in infected plant materials and also in soil samples. This approach allows for the detection of the fungus in soil samples in less than 3 hours, as compared to previous methods of detection that required up to 3 weeks for completion. This assay also is useful as a rapid method for screening breeding materials for resistance to the fungus.

• We have developed a simpler, more cost effective, and more environmentally friendly technique for performing molecular marker analysis using Amplified Fragment Length Polymorphisms (AFLPs). Previous techniques have required the use of very costly equipment, such as DNA sequencing gels, high voltage power sources, and dedicated facilities for either the use of radioactive materials or fluorescent detection systems. Previous techniques have also required the use of toxic reagents such as acrylamide or radioactive nucleotides. Our technique can be performed using relatively inexpensive apparatus for agarose gel electrophoresis and does not require the use of either acrylamide or radioactive compounds. Our technique has been proven effective for AFLP analysis of both plant and fungal populations