High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes

BACKGROUND: Genetic engineering of crop plants has been successful in transferring traits into elite lines beyond what can be achieved with breeding techniques. Introduction of transgenes originating from other species has conferred resistance to biotic and abiotic stresses, increased efficiency, an...

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Autores principales: Edwards, Brianne, Hornstein, Eli D., Wilson, Nathan J., Sederoff, Heike
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9533571/
https://www.ncbi.nlm.nih.gov/pubmed/36195834
http://dx.doi.org/10.1186/s12864-022-08918-6
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author Edwards, Brianne
Hornstein, Eli D.
Wilson, Nathan J.
Sederoff, Heike
author_facet Edwards, Brianne
Hornstein, Eli D.
Wilson, Nathan J.
Sederoff, Heike
author_sort Edwards, Brianne
collection PubMed
description BACKGROUND: Genetic engineering of crop plants has been successful in transferring traits into elite lines beyond what can be achieved with breeding techniques. Introduction of transgenes originating from other species has conferred resistance to biotic and abiotic stresses, increased efficiency, and modified developmental programs. The next challenge is now to combine multiple transgenes into elite varieties via gene stacking to combine traits. Generating stable homozygous lines with multiple transgenes requires selection of segregating generations which is time consuming and labor intensive, especially if the crop is polyploid. Insertion site effects and transgene copy number are important metrics for commercialization and trait efficiency. RESULTS: We have developed a simple method to identify the sites of transgene insertions using T-DNA-specific primers and high-throughput sequencing that enables identification of multiple insertion sites in the T(1) generation of any crop transformed via Agrobacterium. We present an example using the allohexaploid oil-seed plant Camelina sativa to determine insertion site location of two transgenes. CONCLUSION: This new methodology enables the early selection of desirable transgene location and copy number to generate homozygous lines within two generations. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-022-08918-6.
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spelling pubmed-95335712022-10-06 High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes Edwards, Brianne Hornstein, Eli D. Wilson, Nathan J. Sederoff, Heike BMC Genomics Research BACKGROUND: Genetic engineering of crop plants has been successful in transferring traits into elite lines beyond what can be achieved with breeding techniques. Introduction of transgenes originating from other species has conferred resistance to biotic and abiotic stresses, increased efficiency, and modified developmental programs. The next challenge is now to combine multiple transgenes into elite varieties via gene stacking to combine traits. Generating stable homozygous lines with multiple transgenes requires selection of segregating generations which is time consuming and labor intensive, especially if the crop is polyploid. Insertion site effects and transgene copy number are important metrics for commercialization and trait efficiency. RESULTS: We have developed a simple method to identify the sites of transgene insertions using T-DNA-specific primers and high-throughput sequencing that enables identification of multiple insertion sites in the T(1) generation of any crop transformed via Agrobacterium. We present an example using the allohexaploid oil-seed plant Camelina sativa to determine insertion site location of two transgenes. CONCLUSION: This new methodology enables the early selection of desirable transgene location and copy number to generate homozygous lines within two generations. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-022-08918-6. BioMed Central 2022-10-05 /pmc/articles/PMC9533571/ /pubmed/36195834 http://dx.doi.org/10.1186/s12864-022-08918-6 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Edwards, Brianne
Hornstein, Eli D.
Wilson, Nathan J.
Sederoff, Heike
High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes
title High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes
title_full High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes
title_fullStr High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes
title_full_unstemmed High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes
title_short High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes
title_sort high-throughput detection of t-dna insertion sites for multiple transgenes in complex genomes
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9533571/
https://www.ncbi.nlm.nih.gov/pubmed/36195834
http://dx.doi.org/10.1186/s12864-022-08918-6
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