Generation and characterization of SCARs by cloning and sequencing of RAPD products: A strategy for species-specific marker development in bamboo

center dot Background and Aims The aim of this study was to develop species-specific molecular markers for Bambusa balcooa and B. tulda to allow for their proper identification, in order to avoid unintentional adulteration that affects the quality and quantity of paper pulp production. center dot Methods Two putative, species-specific RAPD markers, Bb(836) for B. balcooa and Bt(609) for B. tulda were generated using a PCR-based RAPID technique. Species-specificity of these two markers was confirmed through Southern hybridization in which RAPID gels were blotted and hybridized with radiolabelled cloned RAPID markers. Southern hybridization analyses were also performed to validate homology of the co-migrating Bb(836) and Bt(609) market bands amplified from 16 different populations of B. balcooa and B. tulda, respectively. Sequence-characterized amplified region (SCAR) markers were developed from Bb(836) and Bt(609) sequences, using 20-mer oligonucleotide primers designed from both the flanking ends of the respective RAPID primers. center dot Key Results As anticipated, Bb(836) hybridized with an amplified band from B. balcooa and Bt(609) hybridized only with an amplified product from B. tulda; the two markers did not hybridize with the amplified products of any of the other 14 bamboo species studied. The two pairs of SCAR primers amplified the target sequences only in the respective species. The species-specific SCAR fragments were named as 'Balco(936)' for B. balcooa and 'Tuldo(609)' for B. tulda. The species-specific 'Balco(836)' was amplified from the genomic DNA of 80 individuals of 16 populations of B. balcooa studied. Similarly, the presence of 'Tuldo(609)' was noted in all the 80 individuals representing 16 populations of B. tulda assessed. These SCAR fragments contained no obvious repetitive sequence beyond the primers. center dot Conclusion These two molecular markers are potentially useful for regulatory agencies to establish sovereign rights of the germplasms of B. balcooa and B. tulda. In addition, this is the first report of species-specific SCAR marker development in bamboo.