Submission Type
Oral Presentation
Abstract
Diet analysis is a valuable tool to understand animal feeding behavior. However, validation of workflows for DNA-based diet analysis often lack experimental evaluation with positive controls. To establish a methodology for diet analysis in organic, pasture-raised chickens (as part of ongoing research), we conducted a feeding trial experiment with a mock diet community to determine the efficacy of commonly used DNA extraction kits and primer sets. Six backyard hens were hand fed 22 known food items including insects and plants and monitored for their defecation for eight hours. We evaluated the efficacy of three DNA extraction kits, three primer sets for arthropod identification (targeting COI), and three primer sets for plant identification (targeting rbcL, trnL, and ITS2 ). Overall, average successful detection of our positive control food items ranged from ~15-46% for all kit and primer combinations. Linear modeling suggests that the extraction kits using bead-based homogenization had positive significant effects on recovery rate of insect and plant DNA in excreta samples. Two COI primer sets significantly outperformed the third for amplification success, and both recovery success rate and taxonomic resolution from ITS2 was significant higher than those from rbcL and trnL. No significant effect from variation among birds was found. Overall, this protocol was able to detect a wide range of poultry food items, although multiple markers will be required for detection of diverse diets. This validated protocol will be an invaluable tool for future diet analysis studies in poultry systems, and can potentially be applied in other Avian species.
Included in
Assessing the performance of DNA extraction kits and loci for DNA metabarcoding diet analysis of pastured chickens
Diet analysis is a valuable tool to understand animal feeding behavior. However, validation of workflows for DNA-based diet analysis often lack experimental evaluation with positive controls. To establish a methodology for diet analysis in organic, pasture-raised chickens (as part of ongoing research), we conducted a feeding trial experiment with a mock diet community to determine the efficacy of commonly used DNA extraction kits and primer sets. Six backyard hens were hand fed 22 known food items including insects and plants and monitored for their defecation for eight hours. We evaluated the efficacy of three DNA extraction kits, three primer sets for arthropod identification (targeting COI), and three primer sets for plant identification (targeting rbcL, trnL, and ITS2 ). Overall, average successful detection of our positive control food items ranged from ~15-46% for all kit and primer combinations. Linear modeling suggests that the extraction kits using bead-based homogenization had positive significant effects on recovery rate of insect and plant DNA in excreta samples. Two COI primer sets significantly outperformed the third for amplification success, and both recovery success rate and taxonomic resolution from ITS2 was significant higher than those from rbcL and trnL. No significant effect from variation among birds was found. Overall, this protocol was able to detect a wide range of poultry food items, although multiple markers will be required for detection of diverse diets. This validated protocol will be an invaluable tool for future diet analysis studies in poultry systems, and can potentially be applied in other Avian species.
Comments
Delia Scott Hicks2, David Gonthier1, and Julian Dupuis1
1 Department of Entomology, University of Kentucky, Agricultural Science Center, S-225, Lexington, KY 40508, USA
2 Department of Horticulture, University of Kentucky, Agricultural Science Center, N-322, Lexington, KY 40546, USA