The McCoy laboratory is focused on molecular genetic analysis of alfalfa, an autotetraploid species, and related Medicago species. In relation to alfalfa's prominence in United States agriculture, the amount of genetic information available for this species is incredibly limited. As an experimental organism alfalfa has several advantages (McCoy and Bingham 1988), including:
1) ploidy level can be easily manipulated to produce diploid populations containing cultivated germplasm transferred down from the tetraploid level; 2) there is extensive genetic variation within breeding populations, 3) clonal propagation of individuals is easy, and 4) selected genotypes can be routinely transformed and regenerated. However, genetic analysis of alfalfa has been difficult because of tetrasomic inheritance and severe inbreeding depression. We are now able to circumvent these difficulties by carrying out genetic studies with cultivated diploid plants, rather than with tetraploid plants. Segregation and linkage analysis of RFLP and RAPD markers in a diploid population has resulted in the first linkage map for alfalfa (Echt et al.1992; Echt et al. submitted). This map consists of more than 160 markers, and although low density, it enables us to test hypotheses relative to genome organization that were previously not feasible.
For example, Ray and Bingham (1992) have recently succeeded in inbreeding diploid alfalfa to a level of F = 0.97. This represents the most inbred materials ever recovered in alfalfa. Our lab is conducting an extensive inbreeding program, and we have succeeded in producing numerous S5 lines. Molecular markers dispersed throughout the genome will be analyzed to determine whether we are approaching expected levels of homozygosity, or if selection for heterozygous loci is determining progeny survival. Long-term experiments involving analysis of molecular markers in inbred diploids and their tetraploid counterparts, coupled with hybridization experiments using these inbred materials, willenable us to determine the genetic basis of heterosis and fitness in autotetraploid alfalfa.
Additional studies on manipulating Medicago species genomes have important implications for basic understanding of genomes as well as for potential use of diverse Medicago germplasm in improvement of forage resources. Current cultivated germplasm is limited to the biological species M. sativa; however, wild Medicago species contain genes coding for disease and insect resistance as well as tolerance to water and salt stress (McCoy and Bingham 1988). Genetic access to these species was prevented by the inability to cross M. sativa with these wild species. McCoy and Smith (1986) developed an in vitro approach to recovering hybrids, which (with species-specific modifications) has resulted in the recovery of hybrids between M. sativa and all other perennial Medicago species. The potential for successful genetic introgression of useful genes into cultivated germplasm can now be determined by measuring segregation and recombination of molecular markers in interspecific hybrids between alfalfa and other Medicago species, and via classical and molecular cytogenetic analysis. The utility of this approach has recently been demonstrated by analyzing hybrids of M. sativa x M. papillosa (McCoy et al. 1991), whereby cytogenetic analysis coupled with segregation analysis confirmed the lack of genomic affinity. We propose to combine genetic analysis of molecular markers with cytogenetic analysis of genomic affinity to ascertain introgression potential, and subsequently use molecular markers to facilitate introgression of genes from the wild species. Importantly, there are two alternatives, both of potential value. If high levels of genomic affinity are observed, this indicates excellent potential for introgression of genes of interest. Alternatively, if limited genomic affinity is observed, then these species may offer potential for developing novel forage resources with alternative cytogenetic structures e.g., allohexaploids or alloautohexaploids, that have superior fitness.
The Montana State University barley genetics program (Blake lab)is a member of the North American Barley Genome Mapping Project (NABGMP). A medium density map (Botstein et al. 1980) consisting of 295 markers was produced with an average spacing of 4.6 cM (Kleinhofs et al. in press). Five of seven centromeres and six of fourteen telomeres have been mapped to date. Our lab has specialized in turning RFLP markers into PCR markers (Innis et al. 1988; Mullis and Faloona 1987; Beckman 1988; Weining et al. 1991). Since RAPD markers (Williams et al. 1990) proved unreliable (of 150 polymorphisms identified, 14 wound up on the map) we focused on sequences amplified under the direction of primers which flank well characterized sequences. The PCR polymorphisms derived from well-characterized sequences originated from two sources: published sequences and sequences of clones which had been mapped in the NABGMP mapping effort. This approach is analogous to the Sequence-Tagged-Site approach (Olson et al. 1989; Cole et al. 1991) now widely used in human genome mapping experiments. Quantitative Trait Locus analysis (Lander and Botstein 1989) permitted the location of genes with profound impact on agronomic and malthouse performance and markers for these genes are currently being utilized in selection experiments to test our system. Known sequence PCR primers have provided reliable tools for genotypic selection in segregating populations using minute leaf tissue samples. One problem found with many of the RFLP markers tested in barley is that they hybridize to multiple loci. Because of the specificity of PCR, complex and problematic groups of polymorphisms can be dealt with one at a time. Since PCR provides products which can be easily characterized and sequenced, it has been possible to determine the level of homology among related sequences. We recently completed comparative sequence analysis of 5srRNA spacer sequences across the Hordeum genus, and provided the first evidence suggesting the likely diploid sources of genomes within the species H. arizonicum, H. procerum and H. lechlerii. We have been utilizing PCR based on primer sequences derived from mapped clones to map genes in barley (Tragoonrung et al. 1992), and have been collaborating with the Talbert laboratory in transferring these markers to wheat (D'Ovidio et al. 1990). In addition to permitting comparisons of sequence homology across species and genera, this will permit our laboratories to determine whether QTL loci, like their cognate RFLP markers, tend to map to homoeologous locations across
The Talbert lab has focused its efforts on molecular genetic analysis of wheat. Cultivated wheat and its relatives have evolved through divergence at the diploid level, followed by evolutionary convergence via polyploidy. This type of reticulate evolution has led to immense variability in the wheat tribe, but also to difficulties in evolutionary analysis. Most evolutionary inferences in the wheat tribe have come from studies of chromosome pairing in hybrids. This work has provided very useful insights; however, a limitation of chromosome pairing analysis is that relatively few individuals may be analyzed due to the labor-intensiveness of the technique. Thus, a concern regards whether or not germplasm groups can be adequately sampled. Molecular approaches have the advantage of allowing a larger number of individuals to be assayed.
The hypothesis of pivotal-differential evolution (Feldman 1965; Kimber and Yen 1988) is central to our perception of the evolution of wheat and its relatives. According to this hypothesis, one genome of an allopolyploid such as wheat remains stable during evolution, and serves as the "pivotal" genome. The other genomes become modified (i.e., differential), partially due to crossing with other sympatric polyploid species which have a common pivotal genome. Acceptance of this hypothesis has not only academic implications, but also influences the way plant breeders design and implement their crossing programs (Kimber 1984). We are developing molecular markers to test the hypothesis that crossing among polyploid wheats with a common genome is an important evolutionary process. Previous studies utilizing genome-specific repetitive DNA families have provided equivocable results in regard to this question (Talbert et al. 1991; Talbert et al. 1993). Thus, our focus is on developing multiple PCR markers for low copy sequences on each of the chromosome arms of wheat and using these to compare allopatric and sympatric accessions of polyploid wild wheats. Our basic hypothesis is that sympatric accessions will share more sequences than allopatric accessions if introgression is a common event. Preliminary results suggest that introgression among the polyploid wild wheats may be less common than previously supposed.
The Lavin lab is integrating plant molecular evolution with organismal evolution (Lavin 1993). Research is focused at the species level where genetic structure of populations is used to infer origins and migration routes of cultivated legume trees (Lavin et al. 1991), and at higher taxonomic levels where the origins and relationships of the North American tropical biotas are addressed (Lavin and Luckow 1993). This latter aspect includes evaluating alternative hypotheses that invoke over-water dispersal from the paleotropics to the neotropics, migration from South America since the formation of the Panamanian Isthmus, and migration from Eurasia prior to the dissolution of the North Atlantic land bridge. As with the introduction of a crop into a new environment, the establishment of lineages into new geographical areas over evolutionary time needs to be assessed in terms of drift, founder effect, and rates of evolution. Lavin has identified a number of odel plant groups from the family Leguminosae that are appropriate for testing such hypotheses. Predictions drawn from such hypotheses are tested by phylogenetic analysis of morphological, chemical, and DNA data that bear on relatedness of species within these model plant groups. Because an emphasis of these studies pertains to the evolutionary analysis of DNA sequence data, questions pertinent to molecular evolution are addressed.
To summarize, the plant genetics and evolution group utilizes a common set of technologies to test hypotheses concerning interactions between levels of available genetic diversity, degree of reproductive isolation and rate of evolutionary change using several model plant systems. Three crop species, alfalfa, barley and wheat, and their wild relatives are contrasted with a broad array of non-cultivated tropical legumes. Through appropriate use of well-characterized molecular marker systems and appropriate analysis using the tools of modern systematics, we will be able to determine how recently (in relative terms) related species diverged, and within species how much residual genetic variance is extant. We hope that in time the fundamental knowledge acquired from these investigations will lead to improvements in the way in which scientists deal with plant germplasm resources.