title: An evaluation of new and established methods to determine T-DNA copy number and homozygosity in transgenic plants.
abstract: Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL-)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T-DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL-PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T1 progeny from 26 T0 plants showed that at least 19% of the lines carried multiple T-DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T-DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided.
authors.affiliation: Carl R. Woese Institute for Genomic Biology, University of Illinois, 1206 W Gregory Drive, Urbana, IL, 61801, USA.|Institute of Plant Genetics, Polish Academy of Sciences, ul. Strzeszyńska 34, 60-479, Poznań, Poland.
authors.forename: Katarzyna
authors.identifier: http://orcid.org/0000-0003-1883-6925
authors.initials: K
authors.lastname: Głowacka
authors.affiliation: Carl R. Woese Institute for Genomic Biology, University of Illinois, 1206 W Gregory Drive, Urbana, IL, 61801, USA.
authors.forename: Johannes
authors.initials: J
authors.lastname: Kromdijk
authors.affiliation: Howard Hughes Medical Institute, Department of Plant and Microbial Biology, 111 Koshland Hall, University of California Berkeley, Berkeley, CA, 94720-3102, USA.
authors.forename: Lauriebeth
authors.initials: L
authors.lastname: Leonelli
authors.affiliation: Howard Hughes Medical Institute, Department of Plant and Microbial Biology, 111 Koshland Hall, University of California Berkeley, Berkeley, CA, 94720-3102, USA.|Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.
authors.forename: Krishna K
authors.initials: KK
authors.lastname: Niyogi
authors.affiliation: Center for Plant Science Innovation, E324 Beadle Center, 1901 Vine Street, Lincoln, NE, 68588, USA.
authors.forename: Tom E
authors.initials: TE
authors.lastname: Clemente
authors.affiliation: Carl R. Woese Institute for Genomic Biology, University of Illinois, 1206 W Gregory Drive, Urbana, IL, 61801, USA.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Long
mesh_terms: D015139:Blotting, Southern / Q000379:methods*; D020090:Chromosome Segregation / Q000235:genetics; D003433:Crosses, Genetic; D004269:DNA, Bacterial / Q000235:genetics*; D018628:Gene Dosage; D056426:Genetic Loci; D006720:Homozygote; D030821:Plants, Genetically Modified; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D014026:Nicotiana / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; C036483:T-DNA
keywords: Southern blot; TAIL-PCR; ddPCR; digital droplet PCR; qPCR; segregation analysis; selectable marker; transformation