Development and Application of InDel Marker Detection Technology for Field Identification of Maize Inbred Lines

Variety identification is vital for the security of agricultural production and integrity of seed markets. Current molecular approaches for identification of crop varieties predominantly require specialized laboratory equipment, resulting in relatively slow turnaround times and high costs. The Maize Point-of-Genuineness (M-POG) identification kit aims to extend variety identification from the laboratory to the field, address the lack of rapid on-site detection methods, and support the establishment of a rapid, efficient, and traceable molecular detection system for the modern seed industry.

Candidate InDel loci from the MaizeIDP50K chip were screened using a stepwise dichotomous partitioning method in combination with a dynamic optimization algorithm. Nine maize elite inbred lines and three closely related maize varieties were used to design dominant and co-dominant primers, which were evaluated by conducting quantitative real-time PCR and fluorescence capillary electrophoresis. An optimal combination algorithm was used to screen and determine the core set of loci. Based on the cycle threshold (Ct) value for the dominant primer for the core markers, a barcode conversion mechanism suitable for field detection was established. The detection thresholds were set using the core loci fingerprints of 2270 maize inbred lines. The consistency and single sample detection efficiency were evaluated by comparison with the traditional method for variety identification using simple sequence repeat (SSR) markers, thereby verifying the accuracy and practicability of the M-POG method.

Eighty InDel candidate loci from the chip were screened for which 76 pairs of dominant primers were designed. Among these loci, 31 high quality loci showed clear qPCR amplification curves, high repeatability, and uniform amplification efficiency. Ultimately, 16 highly discriminative core loci were identified, achieving a variety recognition rate of 95.83% across the 2270 inbred lines. The fingerprint detection threshold for each locus was calculated; loci with Ct<28 were classified as dominant type (coded as 1), and those with Ct>30 were classified as recessive type (coded as 0), enabling generation of a DNA fingerprinting for M-POG detection. Pairwise comparisons among the 2270 inbred lines revealed that 98.29% of the pairs of lines differed at four or more loci. A comparative analysis between the M-POG method and the SSR molecular marker method, conducted using elite inbred lines and closely related varieties, showed that the samples were consistently discriminated by both methods in 90.91% of the comparisons. This result demonstrated the reliability of the M-POG method. Thus, the M-POG approach enables efficient screening of genetically dissimilar inbred lines, thereby substantially reducing the number of samples requiring further laboratory analysis. The complete field detection process required only 42 min, approximately one-quarter of the time required by standard identification procedures.

Sixteen core InDel loci were selected for the M-POG method, achieving a variety recognition rate exceeding 95%. Based on the InDel on-site detection results, the minimum number of differential loci required for variety discrimination was determined. Development of the M-POG kit enables rapid and accurate identification of maize inbred lines in the field.