Application of droplet digital PCR to determine copy number of endogenous genes and transgenes in sugarcane
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Droplet digital PCR combined with the low copy ACT allele as endogenous reference gene, makes accurate and rapid estimation of gene copy number in Q208 A and Q240 A attainable.
Sugarcane is an important cultivated crop with both high polyploidy and aneuploidy in its 10 Gb genome. Without a known copy number reference gene, it is difficult to accurately estimate the copy number of any gene of interest by PCR-based methods in sugarcane. Recently, a new technology, known as droplet digital PCR (ddPCR) has been developed which can measure the absolute amount of the target DNA in a given sample. In this study, we deduced the true copy number of three endogenous genes, actin depolymerizing factor (ADF), adenine phosphoribosyltransferase (APRT) and actin (ACT) in three Australian sugarcane varieties, using ddPCR by comparing the absolute amounts of the above genes with a transgene of known copy number. A single copy of the ACT allele was detected in Q208 A , two copies in Q240 A , but was absent in Q117. Copy number variation was also observed for both APRT and ADF, and ranged from 9 to 11 in the three tested varieties. Using this newly developed ddPCR method, transgene copy number was successfully determined in 19 transgenic Q208 A and Q240 A events using ACT as the reference endogenous gene. Our study demonstrates that ddPCR can be used for high-throughput genetic analysis and is a quick, accurate and reliable alternative method for gene copy number determination in sugarcane. This discovered ACT allele would be a suitable endogenous reference gene for future gene copy number variation and dosage studies of functional genes in Q208 A and Q240 A .
KeywordsDroplet digital PCR (ddPCR) Sugarcane Copy number Brown rust resistance locus (Bru1)
We thank Dr Bert Collard and Dr Frikkie Botha for providing critical suggestion to improve this paper and Kate Wathen-Dunn for performing the Southern blots. We gratefully acknowledge Sugar Research Australia for funding the research. We also thank DuPont for supplying the gat4621 transgene.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Abdel-Ghany SE, Golovkin M, Reddy ASN (2015) Engineering of plants for the production of commercially important products: approaches and accomplishments. In: Bahadur B, Venkat Rajam M, Sahijram L, Krishnamurthy KV (eds) Plant biology and biotechnology: volume II: plant genomics and biotechnology. Springer India, New Delhi, pp 551–577Google Scholar
- Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Bright IJ, Lucero MY, Hiddessen AL, Legler TC, Kitano TK, Hodel MR, Petersen JF, Wyatt PW, Steenblock ER, Shah PH, Bousse LJ, Troup CB, Mellen JC, Wittmann DK, Erndt NG, Cauley TH, Koehler RT, So AP, Dube S, Rose KA, Montesclaros L, Wang SL, Stumbo DP, Hodges SP, Romine S, Milanovich FP, White HE, Regan JF, Karlin-Neumann GA, Hindson CM, Saxonov S, Colston BW (2011) High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83:8604–8610CrossRefPubMedPubMedCentralGoogle Scholar
- Huggett JF, Foy CA, Benes V, Emslie K, Garson JA, Haynes R, Hellemans J, Kubista M, Nolan R, 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 59:892–902CrossRefPubMedGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis Y (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Express, Cold Spring HarborGoogle Scholar
- Tohidfar M, Khosravi S (2015) Transgenic crops with an improved resistance to biotic stresses. A review. Biotechnol Agron Soc 19:62–70Google Scholar
- Uchiyama Y, Nakashima M, Watanabe S, Miyajima M, Taguri M, Miyatake S, Miyake N, Saitsu H, Mishima H, Kinoshita A, Arai H, Yoshiura K, Matsumoto N (2016) Ultra-sensitive droplet digital PCR for detecting a low-prevalence somatic GNAQ mutation in Sturge-Weber syndrome. Sci Rep 6:22985CrossRefPubMedPubMedCentralGoogle Scholar
- Wu H, Awan FS, Vilarinho A, Zeng QC, Kannan B, Phipps T, McCuiston J, Wang WL, Caffall K, Altpeter F (2015) Transgene integration complexity and expression stability following biolistic or Agrobacterium-mediated transformation of sugarcane. In Vitro Cell Dev Biol Plant 51:603–611CrossRefGoogle Scholar