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

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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.

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Availability of data and materials

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

References

  1. Zhong GY (2001) Genetic issues and pitfalls in transgenic plant breeding. Euphytica 118(2):137–144. https://doi.org/10.1023/A:1004048019670

    Article  CAS  Google Scholar 

  2. Spangenberg G, Kalla R, Lidgett A, Sawbridge T, Ong EK, John U (2001) Transgenesis and genomics in molecular breeding of forage plants. Molecular breeding of forage crops. Kluwer Academic Publishers, Dordrecht, pp 1–28

    Chapter  Google Scholar 

  3. Woodfield DR, White DWR (1996) Breeding strategies for developing transgenic white clover cultivars. Agronomy Society of New Zealand Special Publication 11/Grassland Research and Practice Series No. 6(11):125–130

  4. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98(3):503–517. https://doi.org/10.1016/S0022-2836(75)80083-0

    Article  CAS  PubMed  Google Scholar 

  5. Głowacka K, Kromdijk J, Leonelli L, Niyogi KK, Clemente TE, Long SP (2016) An evaluation of new and established methods to determine T-DNA copy number and homozygosity in transgenic plants. Plant Cell Environ 39(4):908–917. https://doi.org/10.1111/pce.12693

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wang X, Jiang D, Yang D (2014) Fast-tracking determination of homozygous transgenic lines and transgene stacking using a reliable quantitative real-time PCR Assay. Appl Biochem Biotechnol 175(2):996–1006. https://doi.org/10.1007/s12010-014-1322-3

    Article  CAS  PubMed  Google Scholar 

  7. Ingham DJ, Beer S, Money S, Hansen G (2001) Quantitative real-time PCR assay for determining transgene copy number in transformed plants. Biotechniques 31(1):132–140

    Article  CAS  Google Scholar 

  8. Bubner B, Baldwin IT (2004) Use of real-time PCR for determining copy number and zygosity in transgenic plants. Plant Cell Rep 23(5):263–271. https://doi.org/10.1007/s00299-004-0859-y

    Article  CAS  PubMed  Google Scholar 

  9. Bubner B, Gase K, Baldwin IT (2004) Two-fold differences are the detection limit for determining transgene copy numbers in plants by real-time PCR. BMC Biotechnol 4:1–11. https://doi.org/10.1186/1472-6750-4-14

    Article  CAS  Google Scholar 

  10. Baker M (2012) Digital PCR hits its stride. Nat Methods 9(6):541–544. https://doi.org/10.1038/nmeth.2027

    Article  CAS  Google Scholar 

  11. Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Bright IJ, Lucero MY, Hiddessen AL, Legler TC, Kitano TK, Colston BW (2011) High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83(22):8604–8610. https://doi.org/10.1021/ac202028g

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Bharuthram A, Paximadis M, Picton ACP, Tiemessen CT (2014) Comparison of a quantitative real-time PCR assay and droplet digital PCR for copy number analysis of CCL4L genes. Infect Genet Evol 25:28–35. https://doi.org/10.1016/j.meegid.2014.03.028

    Article  CAS  PubMed  Google Scholar 

  13. Collier R, Dasgupta K, Xing Y-P, Hernandez BT, Shao M, Rohozinski D, Kovak E, Lin J, de Oliveira ML, Stover E, McCue KF, Thilmony R (2017) Accurate measurement of transgene copy number in crop plants using droplet digital PCR. Plant J 90(5):1014–1025. https://doi.org/10.1111/tpj.13517

    Article  CAS  PubMed  Google Scholar 

  14. Sun Y, Aiyar P (2017) Application of droplet digital PCR to determine copy number of endogenous genes and transgenes in sugarcane. Plant Cell Rep 36(11):1775–1783. https://doi.org/10.1007/s00299-017-2193-1

    Article  CAS  PubMed  Google Scholar 

  15. Narancio R, Ding YL, Lin YH, Sahab S, Panter S, Hayes M, John U, Anderson H, Mason J, Spangenberg G (2020) Application of linked and unlinked co-transformation to generate triple stack, marker-free, transgenic white clover (Trifolium repens L.). Plant Cell Tissue Organ Cult 142(3):635–646. https://doi.org/10.1007/s11240-020-01891-6

    Article  CAS  Google Scholar 

  16. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3—new capabilities and interfaces. Nucleic Acids Res 40(15):1–12. https://doi.org/10.1093/nar/gks596

    Article  CAS  Google Scholar 

  17. Baeumler S, Wulff D, Tagliani L, Song P (2006) A real-time quantitative PCR detection method specific to widestrike transgenic cotton (Event 281–24-236/3006-210-23). J Agric Food Chem 54(18):6527–6534. https://doi.org/10.1021/jf0610357

    Article  CAS  PubMed  Google Scholar 

  18. Hand ML, Cogan NOI, Sawbridge TI, Spangenberg GC, Forster JW (2010) Comparison of homoeolocus organisation in paired BAC clones from white clover (Trifolium repens L.) and microcolinearity with model legume species. BMC Plant Biol. https://doi.org/10.1186/1471-2229-10-94

    Article  PubMed  PubMed Central  Google Scholar 

  19. De Smet R, Adams KL, Vandepoele K, Van Montagu MCE, Maere S, Van de Peer Y (2013) Convergent gene loss following gene and genome duplications creates single-copy families in flowering plants. Proc Natl Acad Sci USA 110(8):2898–2903. https://doi.org/10.1073/pnas.1300127110

    Article  PubMed  Google Scholar 

  20. Silver N, Best S, Jiang J, Thein SL (2006) Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol 7(1):33. https://doi.org/10.1186/1471-2199-7-33

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lievens A, Jacchia S, Kagkli D, Savini C, Querci M (2016) Measuring Digital PCR quality: performance parameters and their optimization. PLoS ONE 11(5):e0153317. https://doi.org/10.1371/journal.pone.0153317

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res 29(9):16–21

    Article  Google Scholar 

  23. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3(6):1101–1108. https://doi.org/10.1038/nprot.2008.73

    Article  CAS  PubMed  Google Scholar 

  24. Rossello FJ (2011) Production and characterization of transgenic white clover for Alfalfa Mosaic Virus resistance and aluminium tolerance. La Trobe University, Bundoora

    Google Scholar 

  25. Sallaud C, Meynard D, van Boxtel J, Gay C, Bès M, Brizard JP, Larmande P, Ortega D, Raynal M, Portefaix M, Ouwerkerk PB, Guiderdoni E (2003) Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. Theor Appl Genet 106(8):1396–1408. https://doi.org/10.1007/s00122-002-1184-x

    Article  CAS  PubMed  Google Scholar 

  26. Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14(6):745–750. https://doi.org/10.1038/nbt0696-745

    Article  CAS  PubMed  Google Scholar 

  27. Heredia NJ, Belgrader P, Wang S, Koehler R, Regan J, Cosman AM, Saxonov S, Hindson B, Tanner SC, Brown AS, Karlin-Neumann G (2013) Droplet DigitalTM PCR quantitation of HER2 expression in FFPE breast cancer samples. Methods 59(1):183–186. https://doi.org/10.1016/j.ymeth.2012.09.012

    Article  CAS  Google Scholar 

  28. Xu X, Peng C, Wang X, Chen X, Wang Q, Xu J (2016) Comparison of droplet digital PCR with quantitative real-time PCR for determination of zygosity in transgenic maize. Transgenic Res 25(6):855–864. https://doi.org/10.1007/s11248-016-9982-0

    Article  CAS  PubMed  Google Scholar 

  29. Verhaegen B, Reu KD, Zutter LD, Verstraete K, Heyndrickx M, Coillie EV (2016) Comparison of droplet digital PCR and qPCR for the quantification of thiga Toxin-producing Escherichia coli in bovine feces. Toxins 8:1–11. https://doi.org/10.3390/toxins8050157

    Article  CAS  Google Scholar 

  30. Huggett JF, Foy CA, Benes V, Emslie K, Garson JA, Haynes R, Hellemans J, Kubista M, Mueller RD, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT, Bustin SA (2013) The digital MIQE guidelines: minimum information for publication of quantitative digital PCR experiments. Clin Chem 59(6):892–902. https://doi.org/10.1373/clinchem.2013.206375

    Article  CAS  PubMed  Google Scholar 

  31. Mieog JC, Howitt CA, Ral JP (2013) Fast-tracking development of homozygous transgenic cereal lines using a simple and highly flexible real-time PCR assay. BMC Plant Biol. https://doi.org/10.1186/1471-2229-13-71

    Article  PubMed  PubMed Central  Google Scholar 

  32. Forster JW, Cogan NOI, Abberton MT (2014) White clover. In: Cai H, Yamada T, Kole C (eds) Genetics, genomics and breeding of forage crops. Taylor & Francis, Abingdon, pp 250–286

    Google Scholar 

  33. Caradus J, Woodfield DR, Stewart AV (1996) Overview and vision for white clover. Agronomy Society of New Zealand Special Publication No. 11. Grassland Research and Practice Series No. 6(11):1–6

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Funding

This project was funded by Agriculture Victoria, and Dairy Australia.

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Authors and Affiliations

  1. Instituto de Investigación Agropecuaria (INIA), Unidad de Biotecnología, Estación Experimental INIA Las Brujas, Ruta 48 km 10, Canelones, Uruguay

    Rafael Narancio

  2. School of Applied Systems Biology, La Trobe University, Melbourne, VIC, 3086, Australia

    Rafael Narancio, John Mason & German Spangenberg

  3. Agriculture Victoria Research, AgriBio, Centre for AgriBioscience, Melbourne, VIC, 3086, Australia

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

  4. Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Melbourne, VIC, 3052, Australia

    Paula Giraldo

Authors
  1. Rafael Narancio
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  2. Ulrik John
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  3. John Mason
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  4. Paula Giraldo
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  5. German Spangenberg
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Contributions

Material preparation, data collection and analysis were performed by Rafael Narancio. Paula Giraldo contributed with data collection and analysis. The first draft of the manuscript was written by Rafael Narancio and all authors contributed on the edition of previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Ulrik John.

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Narancio, R., John, U., Mason, J. et al. Digital PCR (dPCR) and qPCR mediated determination of transgene copy number in the forage legume white clover (Trifolium repens). Mol Biol Rep 48, 3069–3077 (2021). https://doi.org/10.1007/s11033-021-06354-5

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  • DOI: https://doi.org/10.1007/s11033-021-06354-5

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