Generation and characterization of SCARs by cloning and sequencing of RAPD products: A strategy for species-specific marker development in bamboo
Generation and characterization of SCARs by cloning and sequencing of RAPD products: A strategy for species-specific marker development in bamboo
复制标题
DOI：
10.1093/aob/mci088
复制
发表时间：
2005-04-01
期刊：
影响因子：
4.2
通讯作者：
Pal, A
中科院分区：
文献类型：
--
作者：
Das, M;Bhattacharya, S;Pal, A
中心点背景和目的本研究的目的是建立毛竹和毛竹的物种特异性分子标记，以便于正确识别它们，以避免无意的掺假而影响纸浆生产的质量和数量。通过Southern杂交证实了这两个标记的种属特异性，将快速凝胶印迹并与放射性标记克隆快速标记杂交。Southern杂交分析还验证了从16个不同种群分别扩增出的BB(836)和Bt(609)市场带的同源性。从Bb(836)和Bt(609)序列中设计了20聚体寡核苷酸引物，构建了Bb(836)和Bt(609)的序列特征扩增区(SCAR)标记。中心点关键结果表明，BB(836)和Bt(609)的扩增带与Bb(836)和Bt(609)的扩增产物杂交，这两个标记与所研究的其他14个竹种的扩增产物均不杂交。这两对SCAR引物仅在各自的物种中扩增出目标序列。这些物种特有的疤痕片段分别命名为Balco(936)和Tuldo(609)，分别代表B.balcoa和B.tulda。从16个赤眼蜂种群的80个个体的基因组DNA中扩增出种特异性的‘Balco(836)’。同样，在所评估的代表16个种群的所有80个个体中都注意到了‘Tuldo(609)’的存在。中心点结论这两个分子标记可能有助于监管机构确立巴尔库亚和图尔达的种质主权。此外，这是竹子物种特异性SCAR标记开发的首次报道。
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.