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Title: Utilizing Single Primers as Molecular Markers

item Kindiger, Bryan

Submitted to: Grassland Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/9/2009
Publication Date: 7/30/2009
Citation: Kindiger, B.K., Conley, T. 2009. Utilizing single primers as molecular markers. Grassland Science. 55:206-215.

Interpretive Summary: The development and implementation of molecular marker based systems for marker assisted improvement in grass forages has lagged behind the rapid development of molecular markers for cereal grass species such as wheat, corn, rice, sorghum, etc. This is due primarily to the high cost and the time-consuming nature of marker development and the lack of emphasis placed on non-food grasses utilized as a livestock forage. If such molecular markers were available, they could have a direct and immediate application in grass forage studies similar to those applications used in advancing breeding improvements in human-food based cereal grass species. The development of a rapid and inexpensive molecular marker approach that builds upon prior marker development in these cereal grass species would also reduce the cost and the time consuming nature of marker development for forage grass species. In addition, an approach that would take advantage of unique DNA regions found in the genomes of many grass forages could have wide application. This research presents a simple and effective approach, utilizing presently available molecular markers generated for other grass species, to identify inverted repetitive DNA regions in bluegrass and bluegrass hybrids. Results of the study suggest that inverted DNA sequence regions in bluegrass can be highly specific within and across species. The availability of these markers and their high specificity makes these markers useful for DNA fingerprinting, cultivar identification and marker-assisted-selection studies. The results of this study will be of value to researchers working with turf and forage grass species.

Technical Abstract: A genotyping approach is described whereby single SSR primers are utilized at high annealing temperature to identify palindrome or near-palindrome regions. Palindromes or quasi-palindromes are unique genome sequences considered to be potentially abundant in polyploid plant genomes and the development of a marker approach that could identify such sequences could have desirable application in the study of complex polyploid plant species. Utilization of the method focused on Poa arachnifera, a highly polyploid, genetically diverse, sexually reproducing Poa sp. that is native to the southern plains of the USA. The procedure was effective in identifying markers that coexpress in Poa arachnifera and generated interspecific Poa hybrids. Sequence analysis of the resulting PCR products identified an array of homeologous loci within and across the Poa samples. From the study, 157 single primers were identified that provided informative and identify polymorphisms across P. arachnifera. Polymorphic index coefficients were also calculated and provided for each PCR product. This approach requires no prior genome information and is analogous to RAPD, AP-PCR or LSSP-PCR methods. The approach appears promising for genotyping turf and forage grass species.