Development and characterization of intron-flanking EST-PCR markers in rubber tree (Hevea brasiliensis Muell. Arg.)

With a long-term goal of constructing a linkage map enriched with gene-specific markers in rubber tree (Hevea brasiliensis Muell. Arg.), we utilized rubber tree ESTs associated with tapping panel dryness (TPD) to develop intron-flanking PCR markers. After downloading and assembling the rubber tree ESTs associated with TPD, we predicted the exon/exon junction sites (E/E) by aligning rubber tree transcripts with the genomic sequences of castor bean (Ricinus communis L.). Based on the predicted E/E, the primers flanking intron(s) and no intron were designed. Compared with the markers designed by conventional method, the PCR success rate of the markers designed with the predicted E/E increased 28-30%, whereas the polymorphism rate of intron-flanking EST-PCR markers was approximately 3.43-fold increase. Therefore, the intron-flanking marker was more polymorphism-generating efficient than the markers designed by conventional methods. In addition, analyzing the polymorphic information content (PIC) among Hevea germplasm showed that the polymorphism of wild rubber tree accessions was higher than one of cultivated rubber tree clones and Hevea species. This study enriches the categories and numbers of molecular markers in rubber tree, and the markers developed in this research will have a wide application in DNA fingerprinting, marker-assisted selection and genetic mapping in rubber tree. This research also indicates that it is possible to develop intron-flanking EST-PCR markers of rubber tree with castor bean genome as reference sequences, which provides new insights into developing intron-flanking EST-PCR markers for rubber tree or other plant species without genomic information.