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The traditional method for detecting the copy numbers of exogenous gene in transgenic plants is Southern hybridization, which is costly and time-consuming, and is difficult to meet the breeding needs of high-throughput detection of the copy numbers of exogenous gene. Therefore, this study aims to establish a fast and high-throughput method for detecting the copy numbers of exogenous gene in transgenic papayas. In this study, two single copy genes, Cpa03g018830 and Cpa03g018770, were selected from the papaya genome. Using the known exogenous gene as a single copy integrated transgenic papaya as a reference, the copy numbers were identified by digital PCR. Further, using them as reference genes, and the commonly used screening marker gene NPTII in papaya transgenic breeding as an exogenous target gene, methods for detecting the copy numbers of exogenous gene in transgenic papaya using digital PCR and fluorescence quantitative PCR were established. The results showed that Cpa03g018830 and Cpa03g018770 were both single copy genes; the established digital PCR method was accurate and reliable in detecting the copy numbers of exogenous gene in transgenic papayas, while fluorescence quantitative PCR had a higher accuracy in detecting single or low copy integration of exogenous genes in transgenic papayas, and a lower accuracy in detecting multiple copy integration of exogenous genes. Therefore, fluorescence quantitative PCR is suitable for initially screening out single or low copy integration plants from a large number of transgenic plants. The single copy Cpa03g018830 and Cpa03g018770 identified in this study can be used as reference genes for detecting the copy numbers of exogenous gene in transgenic papayas. The established methods for detecting the copy numbers of exogenous gene in transgenic papayas using digital PCR and fluorescence quantitative PCR techniques are simple, fast and suitable for batch detection, providing new methods for selecting single or low copy lines in transgenic resistance breeding of papayas.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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