Abstract
qPCR (quantitative polymerase chain reaction) and random amplified polymorphic DNA (RAPD) were utilized to investigate genetic stability of Palmer amaranth cloned plants over 10 generations. DNA from original parent Palmer amaranth plants (grown from seeds) was re-analyzed using qPCR, and confidence levels for determining ΔΔCt (threshold crossing) values were established. ANOVA was used to determine variation (margin of error) of these ΔΔCt values. This margin of error was applied to qPCR analysis of DNA from eight individual parent plants and their descendants (10th generation) so that possible differences in EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene copy number could be ascertained. This method (and the associated error) indicated a lack of agreement in ΔΔCt values of DNA from plants of these two generations. qPCR analysis showed that in five out of eight clones, EPSPS gene copy number varied more than the calculated error (P = 0.05). A second technique to monitor genetic stability, RAPD was used to determine possible changes in genomic DNA due to extended cloning of these regenerated plants. RAPD analysis showed that four out of the eight clones differed when the profiles of the two generations were compared. Results show that qPCR and RAPD analysis point to the fact that several Palmer amaranth clones experienced changes in genome structure over 10 generations. Although the glyphosate resistance trait was retained, results suggest that during cloning studies, the genetic stability of macro-vegetatively propagated lines should be monitored.
Highlights
Weeds have an estimated potential to cause nearly a 35% loss in crop yield when six crops were examined on a worldwide basis [1]
In our investigations of glyphosate resistance in Palmer amaranth plants of resistant and susceptible populations, plants grown from seed were tested using whole plant spraying and leaf disc bioassay techniques [27] [33]
The mechanism of glyphosate resistance we are primarily investigating is increased EPSPS gene copy number [22] and an important technique to estimate increased gene copy number is by Quantitative polymerase chain reaction (qPCR)
Summary
Weeds have an estimated potential to cause nearly a 35% loss in crop yield when six crops were examined on a worldwide basis [1]. There are other obvious additional economic factors associated with weeds including, herbicide application costs and crop yield losses due to competition and contamination. Annual costs (damage and control costs) associated with alien plant species introduced into the US has been estimated at $34 billion [3]. An additional facet of weed control or weed management is the development of herbicide resistance. 238 species of weeds have become resistant to various herbicides in 65 countries, with 30 weeds documented as resistant to glyphosate [N-(phosphonomethyl)glycine] [4]. Glyphosate use in glyphosate-resistant (GR) crops to control weeds has exacerbated the development of weed resistance to this herbicide [5]
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