[Construction and application of a universal detection system for crop variety identification based on universal...

Universal tailed-sequences (UTS) are the tail sequences of universal primers for amplification. They can achieve fluorescence detection by combining common primers with fluorescent universal primers and have the potential to reduce the cost of primer synthesis. However, a universal identification scheme for multiple crops based on the UTS system has not been established. There is a lack of verification for the system has not yet been practically applied in standardized variety identification scenarios. In addition, the data result docking schemes with conventional fluorescence primer systems remain to be developed, leading to the incompatibility with existing fluorescence databases. To address the difficulty in data exchange between UTS genotyping results and traditional fluorescent fingerprint databases and develop a standardized UTS-based molecular identification procedure applicable to diverse crops, this study leveraging fluorescent capillary electrophoresis, we developed a universal identification scheme based on the UTS system for seven distinct crop species (sorghum, sunflower, Chinese cabbage, melon, maize, upland cotton, and common wheat) to validate the robustness of this approach across diverse genomic backgrounds and ploidies. We introduced two universal tail sequences, designated UTS01 and UTS02, and evaluated their performance against conventional fluorescent primers by simple sequence repeat (SSR) and insertion deletion (InDel) markers. Our results demonstrated that the amplification success rate exceeded 90.0% for all seven crops, reaching 100.0% for melon. We observed a high degree of consistency between the UTS system and conventional fluorescent primers, as evidenced by stable linear fitting relationships and identical genotyping results across replicates. For the sites with poor amplification performance, the UTS can be replaced for optimization. We then established a calibration method by using reference samples to align UTS data with existing fluorescent fingerprint databases, thereby resolving data compatibility barriers. This UTS-based universal detection system offers high throughput, low cost, and superior stability, providing technical support for the rapid establishment of a standardized testing system for the crops without identification standards and a standardized technical solution for variety rights protection, seed quality management, germplasm identification, and molecular marker-assisted breeding.