Fecha/hora: 16/06/2024 05:00:16

Biblioteca(s): | INIA Las Brujas.
Fecha corriente: | 06/05/2021
Fecha de la última actualización: | 10/08/2021
Autoria: | NARANCIO, R.; JOHN, U.; MASON, J.; GIRALDO, P.; SPANGENBERG, G.
Afiliación: | RAFAEL NARANCIO FERES, INIA (Instituto Nacional de Investigación Agropecuaria), Uruguay; School of Applied Systems Biology, La Trobe University, Melbourne, VIC, Australia; Agriculture Victoria Research, AgriBio, Centre for AgriBioscience, Melbourne, VIC, Australia; ULRIK JOHN, Agriculture Victoria Research, AgriBio, Centre for AgriBioscience, Melbourne, VIC, Australia; JOHN MASON, School of Applied Systems Biology, La Trobe University, Melbourne, VIC, Australia; Agriculture Victoria Research, AgriBio, Centre for AgriBioscience, Melbourne, VIC, Australia; PAULA GIRALDO, Agriculture Victoria Research, AgriBio, Centre for AgriBioscience, Melbourne, VIC, Australia; Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Melbourne, VIC, Australia; GERMAN SPANGENBERG, School of Applied Systems Biology, La Trobe University, Melbourne, VIC, Australia; Agriculture Victoria Research, AgriBio, Centre for AgriBioscience, Melbourne, VIC, Australia.
Título: | Digital PCR (dPCR) and qPCR mediated determination of transgene copy number in the forage legume white clover (Trifolium repens).
Año de publicación: | 2021
Fuente/Imprenta: | Molecular Biology Reports, 2021, volume 48, Issue 4, Pages 3069 - 3077. Doi: https://doi.org/10.1007/s11033-021-06354-5
ISSN: | 0301-4851
DOI: | 10.1007/s11033-021-06354-5
Idioma: | Inglés

Notas:

Article history: Received, 03 February 2021; Accepted, 12 April 2021; Published, 16 April 2021.
This project was funded by Agriculture Victoria, and Dairy Australia.

Contenido:

ABSTRACT.
Obtaining data on transgene copy number is an integral step in the generation of transgenic plants. Techniques such as Southern blot, segregation analysis, and quantitative PCR (qPCR) have routinely been used for this task, in a range of species. More recently, use of Digital PCR (dPCR) has become prevalent, with a measurement accuracy higher than qPCR reported. Here, the relative merits of qPCR and dPCR for transgene copy number estimation in white clover were investigated. Furthermore, given that single copy reference genes are desirable for estimating gene copy number by relative quantification, and that no single-copy genes have been reported in this species, a search and evaluation of suitable reference genes in white clover was undertaken. Results demonstrated a higher accuracy of dPCR relative to qPCR for copy number estimation in white clover. Two genes, Pyruvate dehydrogenase (PDH), and an ATP-dependent protease, identified as single-copy genes, were used as references for copy number estimation by relative quantification. Identification of single-copy genes in white clover will enable the application of relative quantification for copy number estimation of other genes or transgenes in the species. The results generated here validate the use of dPCR as a reliable strategy for transgene copy number estimation in white clover, and provide resources for future copy number studies in this species. 
© 2021, The Author(s), under exclusive licence to Springer Nature B.V.

Palabras-Clave: | DdPCR; Reference genes; Relative quantification; Single copy genes; Trifolium repens; Zygosity determination.
Categoría del tema: | F30 Genética vegetal y fitomejoramiento
Registro original: | INIA Las Brujas (LB)
Ejemplares: | LB - (102649-1) - PXI - AP - DD - PP/MOLECULAR BIOLOGY REPORTS/2021 -  -  - DIS