The research approach originally proposed in the Illinois-Missouri Biotechnology Trust Fund Agreement No. 58-6216-8-022 entitled "Mapping and Cloning of Apomixis Genes in Tripsacum By Positional Cloning" was modified when brought under new guidance by Dr. T.L. Kamps at the USDA-ARS Southern Plains Range Research Station. Included in the revised objectives was constructing a Tripsacum dactyloides genetic linkage map based on simple sequence repeat (SSR) molecular markers. Importantly, these markers are more desirable than the previously employed RAPDs because of their better applicability to genetic research. Moreover, the markers to be tested were derived from Sorghum bicolor (Moench) L. and maize. This factor provides the added benefit to perform and utilize direct comparative mapping as a means to more quickly define genetic regions of interest. SSR marker transfer across plant genomes is only beginning to be embraced. This is due in part to the limited success reported by earlier investigations exploring this idea. Consequently, my research program first conducted a pilot project examining the basic feasibility of the technology by testing polymerase chain reaction (PCR) amplification of Tripsacum DNA with primers designed to amplify sorghum and maize DNA. The results indicated that the approach was feasible but substantial improvement might be realized with modifications to the laboratory technology. The subsequent research was devised to determine the effects of changing the PCR conditions on improving the frequency for achieving cross-taxa amplification, and the protocol that would optimize the potential for identifying informative SSR markers derived from other genomes. Inherent to this goal is determining the expectation for marker transferability. This is a function of the genetic diversity of the loci. The sum of this research is in my manuscript currently undergoing peer review in the publication process. The highlights are that my modifications resulted in an important improvement to the technology. This is supported by the interest in this work expressed by other researchers and their now positive consideration for including cross-taxa SSR technology in their mapping programs. Within my program, the collection of sorghum SSR primers have undergone a preliminary screening. It is now poised to screen the proposed maize SSR primer collection. The DNA has been prepared from the mapping population plants. These are ready to be assayed subsequent to identifying the polymorphically informative loci necessary to construct the linkage map.

The proposal to employ Genomic In Situ Hybridization (GISH) technology to monitor inheritance and DNA introgressions of the Tripsacum and maize in hybrid plants has progressed through a pilot investigation. This study successfully demonstrated a capacity to differentially "paint" the DNA of these two species. Further refinement, however, is necessary prior to full application to investigations involving hybrids between maize and Tripsacum.

The data pertaining to fertility and characterization of the resulting progeny of the maize-Tripsacum F1 hybrids is currently being summarized. If desired, an addendum report on this work can be supplied in the future to the IMBA.

Should further detail on the SSR research be desired by the IMBA, the manuscript entitled "A Method to Increase Efficiency in Obtaining SSR Markers From Cross-taxa PCR" by T.L. Kamps can be made available. Two research abstracts on this work are currently published and can also be produced upon request.

Lastly, no new patents were developed from the research conducted.

Apomixis is a form of reproduction by which plants make identical copies of themselves through seeds. Harnessing and applying apomixis could greatly impact agricultural production practices as well as increase basic scientific knowledge about the different ways, and why, plants reproduce as they do. Much of the immediate interest in apomixis is aimed toward identifying the gene(s) responsible for this trait, learning how to manipulate them, and ultimately transfer this technology into other agronomically important crops. Achieving this goal would, for example, allow a grower to perpetually maintain a desirable genetic hybrid simply by planting the clonally produced descendent seed season after season. This true breeding feature also offers the potential to augment novel breeding and transgenic based programs.

The USDA-ARS research program at Woodward, OK is taking basic science approaches to identify and exploit the gene(s) responsible for apomictic reproduction in Tripsacum dactyloides (eastern gamagrass). Eastern gamagrass is the closest wild, apomictically reproducing relative to maize and fertile progeny can be obtained from cross pollinations. Our main investigations on apomixis currently include visually characterizing the chromosomes of selected offspring plants obtained from crossing maize with gamagrass and, making the map of the gamagrass DNA.

Genetic maps provide vital information of where genes are found within the DNA of a species and allows for them to be tracked from generation to generation. These locations are discovered by identifying other genes or markers that are linked together when they are passed from parents to offspring. The basic need for a gamagrass genetic map for furthering our investigations has progressed the 2001 research program mainly toward developing the laboratory tools necessary for rapid molecular marker identification. This research has also been successful at identifying a number of useful markers for mapping purposes, finding markers that distinguish between gamagrass and maize DNA, and providing an estimate of how much useful genetic variation is available in the gamagrass mapping population. The type of molecular markers that we are pursuing are known as simple sequence repeat (SSR) containing loci. The laboratory method used to see these markers involves mixing together all the components needed to replicate DNA and providing the appropriate regiment of temperature conditions for the reaction to occur. The technique is a chain reaction event that in a matter of hours results in amplifying a few copies of a specific DNA sequence to over a million copies of the fragment. DNA fragments in this quantity are clearly visible as distinctive bands once they have been slowly pushed through a gel slab and stained with a dye that attaches to the DNA. To amplify a specific fragment of DNA requires controlling where the chain reaction for replication starts and ends on the DNA. This is achieved by the ingredient in the reaction mix known as "primers". Primers are short, unique sequences of DNA that must locate and bond with the compatible sequence of the DNA to be amplified. When the DNA does not contain these sequences the reaction fails to occur. Identifying and developing these primers for a particular species is a time consuming and labor intensive process. Consequently we are first attempting to use the primer collections available from the sorghum and maize genome mapping programs. We have systematically tested modifications to the reaction conditions to determine the feasibility of efficiently increasing the number of these primers capable of amplifying gamagrass DNA. Positive results from these adjustments were obtained for both sorghum and maize derived primers. It is notable that the influence of these changes appears to be greater for the primers developed from sorghum, the more distant relative to gamagrass. Approximately 8% of the tested primers from each species showed evidence of being useful for constructing our gamagrass map. We were also able to find primers that distinguish between the DNA contributed by each parent of the maize-gamagrass hybrids. It is anticipated that the markers these primers detected will be useful to the apomixis project through an ability to track the maintenance and loss of the parental DNA in the descendants of these hybrids.