Cargando…

An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Głowacka, Katarzyna, Kromdijk, Johannes, Leonelli, Lauriebeth, Niyogi, Krishna K., Clemente, Tom E., Long, Stephen P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5021166/
https://www.ncbi.nlm.nih.gov/pubmed/26670088
http://dx.doi.org/10.1111/pce.12693
_version_ 1782453312426082304
author Głowacka, Katarzyna
Kromdijk, Johannes
Leonelli, Lauriebeth
Niyogi, Krishna K.
Clemente, Tom E.
Long, Stephen P.
author_facet Głowacka, Katarzyna
Kromdijk, Johannes
Leonelli, Lauriebeth
Niyogi, Krishna K.
Clemente, Tom E.
Long, Stephen P.
author_sort Głowacka, Katarzyna
collection PubMed
description 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 T(1) progeny from 26 T(0) 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.
format Online
Article
Text
id pubmed-5021166
institution National Center for Biotechnology Information
language English
publishDate 2016
publisher John Wiley and Sons Inc.
record_format MEDLINE/PubMed
spelling pubmed-50211662016-09-23 An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants. Głowacka, Katarzyna Kromdijk, Johannes Leonelli, Lauriebeth Niyogi, Krishna K. Clemente, Tom E. Long, Stephen P. Plant Cell Environ Original Articles 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 T(1) progeny from 26 T(0) 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. John Wiley and Sons Inc. 2016-01-21 2016-04 /pmc/articles/PMC5021166/ /pubmed/26670088 http://dx.doi.org/10.1111/pce.12693 Text en © 2015 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution (http://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Articles
Głowacka, Katarzyna
Kromdijk, Johannes
Leonelli, Lauriebeth
Niyogi, Krishna K.
Clemente, Tom E.
Long, Stephen P.
An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.
title An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.
title_full An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.
title_fullStr An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.
title_full_unstemmed An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.
title_short An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.
title_sort evaluation of new and established methods to determine t‐dna copy number and homozygosity in transgenic plants.
topic Original Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5021166/
https://www.ncbi.nlm.nih.gov/pubmed/26670088
http://dx.doi.org/10.1111/pce.12693
work_keys_str_mv AT głowackakatarzyna anevaluationofnewandestablishedmethodstodeterminetdnacopynumberandhomozygosityintransgenicplants
AT kromdijkjohannes anevaluationofnewandestablishedmethodstodeterminetdnacopynumberandhomozygosityintransgenicplants
AT leonellilauriebeth anevaluationofnewandestablishedmethodstodeterminetdnacopynumberandhomozygosityintransgenicplants
AT niyogikrishnak anevaluationofnewandestablishedmethodstodeterminetdnacopynumberandhomozygosityintransgenicplants
AT clementetome anevaluationofnewandestablishedmethodstodeterminetdnacopynumberandhomozygosityintransgenicplants
AT longstephenp anevaluationofnewandestablishedmethodstodeterminetdnacopynumberandhomozygosityintransgenicplants
AT głowackakatarzyna evaluationofnewandestablishedmethodstodeterminetdnacopynumberandhomozygosityintransgenicplants
AT kromdijkjohannes evaluationofnewandestablishedmethodstodeterminetdnacopynumberandhomozygosityintransgenicplants
AT leonellilauriebeth evaluationofnewandestablishedmethodstodeterminetdnacopynumberandhomozygosityintransgenicplants
AT niyogikrishnak evaluationofnewandestablishedmethodstodeterminetdnacopynumberandhomozygosityintransgenicplants
AT clementetome evaluationofnewandestablishedmethodstodeterminetdnacopynumberandhomozygosityintransgenicplants
AT longstephenp evaluationofnewandestablishedmethodstodeterminetdnacopynumberandhomozygosityintransgenicplants