松杨栅锈菌小种特异性DNA片段的SCAR标记转换

SCAR markers transfer for race-specific DNA fragments of Melampsora larici-populina

用SCAR标记技术对松杨栅锈菌生理小种特异性DNA片段进行了转化和检测研究。通过对RAPD随机引物的扩增筛选,发现随机引物BAO118扩增产生的约700bp的DNA片段与松杨栅锈菌小种特异性相关联。将该片段进行回收、TA克隆、双向测序和序列整合后,序列全长685bp,3’端有BAO118随机引物序列。根据整合后序列,用Primerse-lect软件设计了一对包含随机引物序列的简并引物对。用该引物对对供试菌样进行PCR扩增,成功获得了松杨栅锈菌小种685bp的SCAR标记。用该标记进行田间混合孢子菌样、单孢子接种菌样DNA检测,均得到了685bp的DNA片段,不受寄主DNA和孢子菌系类型的影响,具有较好的稳定性和特异性。

Sequence characterized amplified region （SCAR） markers were used to transfer and verify the DNA fragment related to physiology race of Melampsora larici-populina. A DNA fragment with the length of 700 bp, which was produced by using random amplification polymorphic DNA （RAPD） primer BA0118,was found to be closely related to the race C3 and race C4 in the electrophoresis map. The DNA fragment was recycled, then cloned, sequenced from its two ends, and finally combined. The combined DNA sequence has a length of 685bp. Based on the sequence discovered, one pair of primers with the primer BA0118 sequence at 3＇ end was designed by using Primerselect software package. The designed primers were used to identify the race-specific SCAR markers successfully from 11 mono-urediniospore isolates by running polymerase chain reaction （PCR）. The advanced application with the designed primers showed that the fragment with the length of 685bp can be obtained from the DNA of urediniospores mixture collected from the field and mono-urediniospores cultivated in the laboratory, which indicated the SCAR markers were stable and unique, and were not influenced by the DNA of the host and the types of urediniospore.