Digital PCR (dPCR) and qPCR mediated determination of transgene copy number in the forage legume white clover (Trifolium repens).

Rafael Narancio,Paula Giraldo,John Mason,German Spangenberg,Ulrik John

doi:10.1007/s11033-021-06354-5

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.

Highlights

The first downstream steps in production of genetically modified (GM) crop lines, involve identification of transformed events and determination of transgene copy number
List of candidate single copy reference genes, and the results of BLASTN searches of the NCBI white clover EST database with sequences of single copy genes reported in Arabidopsis
Identification of a reference gene is integral to the conduct of copy number estimations by relative quantification

Summary

Introduction

The first downstream steps in production of genetically modified (GM) crop lines, involve identification of transformed events and determination of transgene copy number. Identification of plants homozygous for the transgene(s) is required as part of generation of stable transgenic lines This task was performed by test crosses, which entails crossing the transgenic plants with non-transgenic plants in order to analyse segregation ratios [2, 3]. This method is time consuming, given that it involves an extra generation for evaluating progenies. QPCR has gained favour [6, 7] This technique enables high throughput copy number estimation in a shorter timeframe, with lower DNA quantities required than Southern blots [6,7,8]. Difficulties differentiating homozygous and hemizygous transgenic plants using qPCR have been reported [9]

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Results

Conclusion