白菜的EST标记及其对油菜的通用性

Development of EST (Expressed Sequence Tags) Marker in Chinese Cabbage and its Transferability to Rapeseed

根据白菜的表达序列标签,设计了28对引物。在对引物、dNTP、MgCl2的浓度及退火温度等参数进行测试后,建立了合适的PCR反应体系。在此反应体系下,以构建EST的白菜自交系A的DNA为模板,对设计的引物进行了筛选,发现有18对引物能对白菜DNA扩增出产物。用筛选出来的引物分别对17个白菜类品种进行PCR扩增,用琼脂糖凝胶电泳分析其产物的多态性,发现10对引物有多态性,占筛选引物的55.6%。为检测白菜EST标记的通用性,进一步利用设计的引物对不同油菜品种的DNA进行PCR扩增。在检测的28对引物中,共有24对引物能扩增出产物,占引物总数的85.7%,显示多态性的引物为18对,占引物总数的64.3%.。在对白菜DNA能扩增出产物的18对引物中,对油菜完全可用,且有13对引物产生多态性。而在那些对白菜未扩增出产物的10对引物中,也有6对能扩增出产物,其中5对显示多态性。结果证明,通过EST建立分子标记是可行的,而且这种标记对近缘物种是可通用的。

pairs of primers were designed according to the expressed sequence tags in Chinese cabbage. After testing on the annealing temperature and the concentration of primer, dNTP and MgCl_2, a suitable PCR system was established. Under the condition of reaction system developed, primers designed specific to ESTs were screened against genomic DNA of inbreed line A from which the cDNA library was constructed. Among them, 18 pairs of primers showed the amplification. Then all the primers available in line A were subjected to PCR for DNAs from 17 cabbage varieties. Polymorphism was detected by electrophoresis with agarose gel, and 10 of 18 primer sets could reveal polymorphisms among cabbage varieties, which accounted for 55.6% of primers selected. To examine the transferability of EST markers developed in cabbage, all primers were further used for PCR-mediated amplification of genomic DNA from (different) varieties of rapeseeds. Of 28 pairs of primers, 24 were able produce amplified product(s) and 18 showed polymorphisms, accounting for 85.7% and 64.3% of total primers respectively. All of 18 primer sets that amplified in cabbage also showed amplified products in rapeseed and 13 of them were polymorphic. Even amongst the 10 primer sets that were unable to amplify in cabbage, 6 pairs produced amplification and 5 could reveal the polymorphism in rapeseeds. Results obtained in the present paper proved that developing polymorphic markers based on EST could be feasible and this kind of marker would be transferable to closed related species.