杞柳AFLP反应体系的建立与引物筛选

Establishment of AFLP Reaction System for Salix integra and Primer Selection

以杞柳F1群体为试验材料,采用改良的CTAB法提取基因组DNA,经过酶切、连接、预扩增和选择性扩增,建立杞柳AFLP的反应体系,筛选EcoRI和MseI各16条选择性扩增引物组成256对引物组合。结果表明:320 ng基因组DNA采用5U EcoRI和MseI双酶切6 h,20℃连接12 h,连接产物稀释10倍进行预扩增,预扩增产物稀释15倍进行选择性扩增,选择性扩增产物采用ABI-3130检测,可获得条带清晰的指纹图;从256对引物组合中共筛选出98对多态性较高的引物组合。

Taking Salix integra F1 progeny as experimental material,the genomic DNA was extracted from the leaves by modified CTAB extraction method.Subsequently,the optimized AFLP experimental protocol was established through enzyme incision,conjunction,pre-amplification and selective amplification.256 pairs of primer combinations were composed with 16 EcoRI and 16 MseI selective amplification primers respectively.The results showed that clear and repeatable gel profiles could be obtained under the reaction protocol as: 320 ng genomic DNA was digested at 37℃ for 6 hours by 5U of EcoRI and 5U MseI,then conjugated at 20℃ for 12 hours,the ligation products were 10 times diluted,and used as the template for pre-amplification.After pre-amplification,the products were diluted for 15 times,then used as templates for selective amplification.The selective amplification products were detected by ABI-3130 electrophoresis and the clear and repeatable gel profiles were obtained.98 pairs of primer combinations that yielded high polymorphic bands were selected from the 256 pairs of AFLP primer combinations for AFLP analysis of S.integra.