Key Open Laboratory of Rubber Biology, Ministry of Agriculture/State Key Laboratory Breeding Base of Cultivation & Physiology for Tropical Crops, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou 571737, Hainan, China.
Mol Biotechnol. 2012 Jun;51(2):148-59. doi: 10.1007/s12033-011-9449-8.
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.
为了构建富含基因特异性标记的橡胶树（Hevea brasiliensis Muell. Arg.）连锁图谱的长期目标，我们利用与割胶排干（TPD）相关的橡胶树 EST 开发了内含子侧翼 PCR 标记。在下载和组装与 TPD 相关的橡胶树 EST 后，我们通过将橡胶树转录本与蓖麻（Ricinus communis L.）的基因组序列进行比对来预测外显子/外显子交界处（E/E）。基于预测的 E/E，设计了侧翼内含子和无内含子的引物。与传统方法设计的标记相比，基于预测的 E/E 设计的标记的 PCR 成功率提高了 28-30%，而内含子侧翼 EST-PCR 标记的多态性率约增加了 3.43 倍。因此，内含子侧翼标记比传统方法设计的标记更具有多态性产生效率。此外，分析橡胶树种质资源的多态性信息含量（PIC）表明，野生橡胶树品系的多态性高于栽培橡胶树无性系和橡胶树物种的多态性。本研究丰富了橡胶树分子标记的类别和数量，开发的标记将在橡胶树 DNA 指纹图谱、标记辅助选择和遗传图谱构建中得到广泛应用。本研究还表明，利用蓖麻基因组作为参考序列，有可能开发橡胶树内含子侧翼 EST-PCR 标记，为没有基因组信息的橡胶树或其他植物物种开发内含子侧翼 EST-PCR 标记提供了新的思路。
