MicroRNA-based molecular markers: a novel PCR-based genotyping technique in Brassica species

DNA-based molecular markers play a significant role in gene mapping, genetic diversity analysis, germplasm evaluation and molecular marker-assisted selection. A combination of desirable marker characteristics such as abundant polymorphism, good stability, ease of production and high efficiency is difficult to achieve when utilizing traditional molecular marker systems. MicroRNAs are a type of endogenous non-coding RNAs prevalent in the genomes of many organisms. The high conservation of miRNA and pre-miRNA sequences provides an opportunity to develop a novel molecular marker type. We downloaded 82 miRNA sequences from the Brassica miRBase database and designed 46 single miRNA-based primers which could be randomly combined to generate primer pairs. A proportion of these primer pairs were validated for transferability and polymorphism using DNA from 16 varieties of six Brassica species. The percentage of polymorphic primer pairs were 28.1%, and the average polymorphism information content (PIC) value for the 34 primer pairs was 0.43. Good transferability was verified across species. These results indicate that miRNA-based markers provide a novel genotyping technique with low costs, high efficiency, stability and good transferability.