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Primer design and real-time PCR for mRNA expression in velvetleaf and common sunflower
Abstract
The acceptance of glyphosate-resistant crops and the subsequent increase in glyphosate use has dramatically changed weed management programs within agricultural crop production. It is possible that the high market share of glyphosate-resistant soybean and cotton, together with an increased use of glyphosate-resistant corn, could lead to glyphosate applications on more than 100 million acres of cropland per year in the U.S. A sensitive and powerful molecular biology technique for the analysis of mRNA expression is real-time PCR. This technique could be useful in weed science research and a step to better understanding the basic biology of our weed populations. However, one problem is that real-time PCR requires specific sequence information and there is little sequence information publicly available for weed species. This research focused on using publicly available sequence from other plant species to design real-time PCR primers for use in two common weed species, velvetleaf and common sunflower. Since glyphosate is such a widely used herbicide, this research also focused on developing these primers for the enzyme that glyphosate inhibits, EPSPS, and two other enzymes that belong to the same pathway. Computer software alignments were used to design primers for real-time PCR amplification of three genes of the shikimic acid pathway, DAHP synthase, EPSPS, and chorismate synthase. Multiple pairs of primers were designed and tested for real-time PCR suitability. The three primer pairs chosen for this work all showed little primer-dimer formation, gene-specific product amplification, and very high primer efficiency. These primers were used to study mRNA expression with leaf number, time of day, and glyphosate application in velvetleaf and common sunflower leaf samples. The most significant result of the expression analysis is the agreement between previous gene-expression work and that reported here. This is a validation of the technique described herein to design real-time PCR primers without species-specific sequence information and collect reliable mRNA expression data.
Subject Area
Agronomy|Molecular biology
Recommended Citation
Waltz, Aaron L, "Primer design and real-time PCR for mRNA expression in velvetleaf and common sunflower" (2005). ETD collection for University of Nebraska-Lincoln. AAI3176806.
https://digitalcommons.unl.edu/dissertations/AAI3176806