An efficient and accurate droplet digital PCR method for rapid transgene copy number detection and homozygous identification in cotton (Gossypium hirsutum)

Abstract

Transgene copy number characterization and homozygous identification are important steps for both the scientific research and transgenic breeding in plant. Traditionally, Southern blotting, quantitative PCR, genome sequencing and genetic analysis are the major methods to achieve these objectives. These methods are laborious, time-consuming and cost-expensive. Digital PCR is a breakthrough technology that is able to provide sensitive and absolute nucleic acid quantification. However, its application is rarely studied in transgene copy number detection and homozygous identification, especially in cotton, an important industrial crop worldwide. Here, we developed a droplet digital PCR (ddPCR) system to rapidly and efficiently detect the transgene copy number in cotton. The GhTPS, a single gene in per subgenome of Gossypium hirsutum, and NPT II, a marker gene in the transgenic T-DNA, were selected as the endogenous reference gene and exogenous insertion sequences, respectively. Results showed that probe concentration at 150 nM and DNA concentration at 5.0 ng/µL were the best conditions to perform ddPCR in tetraploid cotton. The detected T-DNA copy numbers obtained from the developed ddPCR system were in consistent with the results from Southern blotting analysis and genome resequencing results, respectively, demonstrating that the developed ddPCR system is accurate and efficient in identifying the T-DNA copy number. Moreover, the homozygous and heterozygous T1 offspring plants from a T0 plant were efficiently distinguished using this approach, greatly shortening the selection cycles of homozygous plants. The ddPCR system was further applied to detect the transgenic Bt copy numbers in commercialized cotton varieties, indicating the suitability and convenience in identifying transgenic cotton with different origins. This study provides a novel, rapid and efficient method for copy number and homozygous determination in transgenic cotton.