High polymorphism and resolution in targeted fingerprinting with combined β-tubulin introns

Tubulin-Based-Polymorphism (TBP) was originally introduced as a novel method for assaying genetic diversity in plants. TBP is based on polymorphism resulting from the PCR-mediated amplification of the first intron in the coding region of the β-tubulin gene family. Although, the method was successful in genetic assessment of some plant species and varieties, it suffered from low number of molecular markers due to limited variation in the first intron of β-tubulin gene family. We have now rectified this limitation by introducing the second intron of the β-tubulin genes as a valuable source of molecular markers. We show that the combined use of the two introns substantially increases the number of molecular markers and results in a reliable assessment of species/varieties relationships. After a preliminary validation on Brassica, this new combinatorial method was tested on species of Eleusine and Arachis. For both, reliable assessment of species relationships were obtained that were consistent with recently published studies resulting from more elaborated methods including DNA sequencing. Combinatorial TBP is a reliable, reproducible, simple, fast, and easy to score method that is very useful for breeding programs and species and variety assessments.