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Title: HIGH THROUGHPUT DNA EXTRACTION AND ALLELE SPECIFIC PCR PRIMERS ENABLES EFFICIENT SCREENING FOR MUTANT (GSF) AND WILD-TYPE (GSF) ALLELES IN EASTERN GAMAGRASS

Author
item Goldman, Jason

Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/29/2005
Publication Date: 1/24/2006
Citation: Goldman, J.J. 2006. High throughput DNA extraction and allele specific pcr primers enables efficient screening for mutant (gsf) and wild-type (GSF) alleles in eastern gamagrass. Crop Science. 46:362-364.

Interpretive Summary: Eastern gamagrass is a perennial warm-season bunchgrass with high forage potential. Low seed production is one factor that has limited wide spread use of this native grass. A mutant form of gamagrass was found that produces entirely female flowers, rather than the predominantly male wild-type form. This mutation has the potential to increase seed production. The mutation, caused by a deletion at the DNA level is recessive, meaning plants can carry the mutation and not express it. With traditional breeding methods, it normally requires an additional breeding generation to determine which plants are carriers of the mutation but do not express it. Polymerase chain reaction (PCR) primers were designed that specifically amplify a portion of the mutant or wild-type DNA when present in a plant. By combing the PCR screen with a high-throughput DNA extraction protocol, large scale screening of plants was possible, enabling practical use of this technology in a gamagrass breeding program.

Technical Abstract: Eastern gamagrass is a perennial warm-season bunchgrass with high forage potential. Low seed production is one factor that has limited wide spread use of this native grass. A recessive gynomonoecious sex form (GSF) mutation previously found in a wild population has the potential to increase seed production, although it may also be linked to other deleterious traits. Allele specific primers were designed to detect the wild-type (GSF) and mutant (gsf) allele. Primers were successful at confirming the genotype of 15 plants tested using template DNA from a high-throughput extraction protocol. A tissue-pooling strategy enabled detection of the mutant allele when present in a predominantly wild-type background. The low cost, lack of toxic waste, and speed of the DNA extraction protocol combined with the ability to efficiently genotype a large number of individuals makes this a practical tool in a breeding program working with the GSF trait.