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item Kindiger, Bryan

Submitted to: United States Japan Natural Resources Forage Seed Panel
Publication Type: Proceedings
Publication Acceptance Date: 11/20/2004
Publication Date: 3/15/2005
Citation: Kindiger, B.K. 2005. Genotyping polyploid grass genomes with single primers. United States Japan Natural Resources Forage Seed Panel. 3:6-12.

Interpretive Summary: Polyploid grass species which provide multiple copies of their genomes provide few molecular based molecular marker techniques for cultivar identification and marker assisted breeding. Polyploid species which reproduce by sexual means are especially difficult. Texas bluegrass, an indigenous, sexually reproducing, polyploid grass species was submitted to a new molecular marker technique which would utilize the occurrence of chromosome rearrangements or inverted repeats in its genome to develop a new set of molecular markers. Utilizing a series of molecular markers developed in other species, modifications in the usage of those markers resulted in a robust and efficient method for identifying the proposed chromosome rearrangements which reside in Texas bluegrass. This new approach resulted in rapid development of over 100 new molecular markers associated with the chromosome rearrangements in Texas bluegrass. Application of this technique to other polyploid species such as orchardgrass, bromegrass, intermediate wheatgrass, perennial ryegrass and creeping bentgrass suggest the approach will have wide application. This research will be of value to geneticists and plant breeders working on grass species having complex polyploid genomes. This technique can be applied towards cultivar identification and marker-to-trait selective breeding programs.

Technical Abstract: We describe an approach utilizing single SSR primers at high annealing temperature to identify and utilize inverted repeat regions in polyploid genomes to provide locus specific genotyping for polyploid grass species. Genome regions associated with the inverted repeats were sequenced and found to be naturally occurring or associated with transposable element transposition events. The procedure has been effective in identifying both dominant and co-dominant markers in Poa ssp. which supports both a complex polyploid genome and a sexual or apomictic form of reproduction. DNA sequence analysis of PCR amplification products identified orthologous and non-orthologous regions at the genus and species level and identified the occurrence of multiple alleles at a single locus. Similar to RAPD-PCR, this approach requires no prior genome information, utilizes agarose gels and can be visualized with ethidium bromide. Application of this approach appears widespread among grasses and has particular benefits for utilization in polyploid species. Preliminary evaluations of additional Poa ssp., Bromus inermis, Dactylis glomerata, Thinopyrum ponticum, Lolium perenne, Aragostis ssp. and Arabidopsis thaliana suggest wide utility of this approach in genotyping both polyploid and diploid germplasm.