Construction of DNA molecular identity card of proso millet in Ningxia based on fluorescent SSR

Objective proso millet (Panicum miliaceum L.) possesses a rich in germplasm and demonstrates advantages in arid environments..Developing a DNA identification system for proso millet using fluorescent SSR markers can establish a theoretical foundation and provide a molecular detection tool for digital resource management. In this study, 274 core germplasm samples of Ningxia proso millet were selected as an experimental materials, Proso millet specific SSR markers developed by Shanxi Agricultural University were subjected to multiple rounds of PCR screening and optimization.The core markers were then mapped using BLAST sequence comparison based on the reference genome information of proso millet. The SSR primers were labeled with FAM/HEX at the 5' end, and the genotypes of the materials were obtained through capillary electrophoresis..The presence of amplified bands was recorded using “0,1” binary coding, while the differentiation degree of the materials was assessed using IDAnalysis4.0. The size of the amplified fragments was represented in decimal form (0-9) to generate a unique molecular ID for each material. Genetic diversity, genetic clustering and principal component analysis. were conducted using software such as Popgene, Powermarker, MEGA, and NTSYS. The molecular IDs of the materials were obtained by generating QR codes using an online QR code software ( https://cli.im/ ). PCR amplification results showed that a combination of 10 fluorescent SSR markers (RYW6, RYW125, RYW43, RYW3, RYW40, RYW37, RYW42, RYW8, RYW28, and RYW124) could distinguish all 274 materials. BLAST analysis indicated that RYW124 was located at 7.8 cM on chromosome 12, RYW40 was located at 42.64 cM on chromosome 4, RYW42 was positioned at 34.63 cM on chromosome 13, RYW28 was at 2.34 cM on chromosome 16, and RYW8 was found at 9.90 cM on chromosome 3. Across the 10 loci, a total of 125 alleles were detected at 10 loci in the 274 samples, with an average of 12.5 alleles per locus and a range of variation from 5.0000 (RYW3) to 25.0000 (RYW6). The Shannon diversity index (I) ranged from 1.2458 (RYW3) to 2.6568 (RYW6), with an average value of 1.8532. The observed heterozygosity (Ho) ranged from 0.5185 (RYW40) to 0.9964 (RYW124), averaging at 0.8674. The expected heterozygosity (He) varied from 0.5724 (RYW40) to 0.9108 (RYW42), with an average of 0.7784. Nei’s gene diversity index (Nei) ranged from 0.5711 (RYW40) to 0.9091 (RYW42), averaging at 0.7767. The polymorphism information content (PIC) ranged from 0.6563 (RYW3) to 0.9602 (RYW42), with an average of 0.8399. Cluster analysis and principal component analysis classified the materials into four groups. The electrophoretic strip was digitally encoded, and the combination of the 10 markers was used to construct both the string and two-dimensional code DNA molecular IDs for all materials.