title: Accurate measurement of transgene copy number in crop plants using droplet digital PCR.
abstract: Genetic transformation is a powerful means for the improvement of crop plants, but requires labor- and resource-intensive methods. An efficient method for identifying single-copy transgene insertion events from a population of independent transgenic lines is desirable. Currently, transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization is a convincing and reliable method, but it also is expensive, time-consuming and often requires a large amount of genomic DNA and radioactively labeled probes. Alternatively, qPCR requires less DNA and is potentially simpler to perform, but its results can lack the accuracy and precision needed to confidently distinguish between one- and two-copy events in transgenic plants with large genomes. To address this need, we developed a droplet digital PCR-based method for transgene copy number measurement in an array of crops: rice, citrus, potato, maize, tomato and wheat. The method utilizes specific primers to amplify target transgenes, and endogenous reference genes in a single duplexed reaction containing thousands of droplets. Endpoint amplicon production in the droplets is detected and quantified using sequence-specific fluorescently labeled probes. The results demonstrate that this approach can generate confident copy number measurements in independent transgenic lines in these crop species. This method and the compendium of probes and primers will be a useful resource for the plant research community, enabling the simple and accurate determination of transgene copy number in these six important crop species.
authors.affiliation: Western Regional Research Center, Crop Improvement and Genetics Research Unit, USDA-ARS, 800 Buchanan Street, Albany, CA, 94710, USA.
authors.forename: Ray
authors.initials: R
authors.lastname: Collier
authors.affiliation: Department of Plant Sciences, University of California, One Shields Avenue, Davis, CA, 95616, USA.
authors.forename: Kasturi
authors.initials: K
authors.lastname: Dasgupta
authors.affiliation: Western Regional Research Center, Crop Improvement and Genetics Research Unit, USDA-ARS, 800 Buchanan Street, Albany, CA, 94710, USA.
authors.forename: Yan-Ping
authors.initials: YP
authors.lastname: Xing
authors.affiliation: Department of Plant Sciences, University of California, One Shields Avenue, Davis, CA, 95616, USA.
authors.forename: Bryan Tarape
authors.initials: BT
authors.lastname: Hernandez
authors.affiliation: Department of Plant Sciences, University of California, One Shields Avenue, Davis, CA, 95616, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Shao
authors.affiliation: Plant Gene Expression Center, USDA-ARS, 800 Buchanan Street, Albany, CA, 94710, USA.
authors.forename: Dominica
authors.initials: D
authors.lastname: Rohozinski
authors.affiliation: Plant Gene Expression Center, USDA-ARS, 800 Buchanan Street, Albany, CA, 94710, USA.|Department of Plant & Microbial Biology, University of California, Berkeley, CA, 94720, USA.
authors.forename: Emma
authors.initials: E
authors.lastname: Kovak
authors.affiliation: Western Regional Research Center, Crop Improvement and Genetics Research Unit, USDA-ARS, 800 Buchanan Street, Albany, CA, 94710, USA.
authors.forename: Jeanie
authors.initials: J
authors.lastname: Lin
authors.affiliation: USDA-ARS Subtropical Insects and Horticulture Research Unit, Fort Pierce, FL, 34945, USA.
authors.forename: Maria Luiza P
authors.initials: MLP
authors.lastname: de Oliveira
authors.affiliation: USDA-ARS Subtropical Insects and Horticulture Research Unit, Fort Pierce, FL, 34945, USA.
authors.forename: Ed
authors.initials: E
authors.lastname: Stover
authors.affiliation: Western Regional Research Center, Crop Improvement and Genetics Research Unit, USDA-ARS, 800 Buchanan Street, Albany, CA, 94710, USA.
authors.forename: Kent F
authors.initials: KF
authors.lastname: McCue
authors.affiliation: Plant Gene Expression Center, USDA-ARS, 800 Buchanan Street, Albany, CA, 94710, USA.|Department of Plant & Microbial Biology, University of California, Berkeley, CA, 94720, USA.
authors.forename: Frank G
authors.initials: FG
authors.lastname: Harmon
authors.affiliation: Western Regional Research Center, Crop Improvement and Genetics Research Unit, USDA-ARS, 800 Buchanan Street, Albany, CA, 94710, USA.
authors.forename: Ann
authors.initials: A
authors.lastname: Blechl
authors.affiliation: Western Regional Research Center, Crop Improvement and Genetics Research Unit, USDA-ARS, 800 Buchanan Street, Albany, CA, 94710, USA.
authors.forename: James G
authors.initials: JG
authors.lastname: Thomson
authors.affiliation: Western Regional Research Center, Crop Improvement and Genetics Research Unit, USDA-ARS, 800 Buchanan Street, Albany, CA, 94710, USA.
authors.forename: Roger
authors.identifier: http://orcid.org/0000-0002-6591-6699
authors.initials: R
authors.lastname: Thilmony
mesh_terms: D018556:Crops, Agricultural / Q000235:genetics*; D018551:Solanum lycopersicum / Q000235:genetics; D012275:Oryza / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D011198:Solanum tuberosum / Q000235:genetics; D019076:Transgenes / Q000235:genetics*; D014908:Triticum / Q000235:genetics; D003313:Zea mays / Q000235:genetics
keywords: 
  Citrus sinensis
  ; 
  Oryza sativa
  ; 
  Solanum lycopersicum
  ; 
  Solanum tuberosum
  ; 
  Triticum aestivum
  ; 
  Zea mays
  ; droplet digital PCR; transgene copy number