广藿香AFLP反应体系的优化及引物筛选

Optimization of AFLP Reaction System and Screening of Primers for Pogostemon cablin

以广藿香叶子为试材,采用AFLP分子标记方法,研究了广藿香AFLP反应体系,包括酶切连接体系、连接产物稀释倍数、预扩增产物稀释倍数等关键因素,并对AFLP引物组合(E/M组合)进行筛选,以期为后续的广藿香优良种质资源筛选奠定基础。结果表明:酶切体系为20μL,含300 ng模板DNA,在37℃下反应3 h;连接反应在16℃下反应12 h;连接产物的最适稀释倍数为3倍;预扩增产物的最适稀释倍数为20倍。采用优化好的反应体系筛选出了12对条带清晰、多态性丰富的引物组合。优化好的反应体系适用于广藿香AFLP分子标记研究,筛选出的引物也具有较好的多态性,可为后续的广藿香优良种质筛选提供技术支持。

The Pogostemon cablin leaves were used as the test material and the AFLP molecular labeling method was used to study the AFLP reaction system of Pogostemon cablin,including key factors such as reaction system of digestion and ligation dilution multiple of ligation product and pre-amplification product,and then screened the AFLP primer combinations(E/M combinations)in order to lay the foundation for the subsequent screening of superior germplasm resources of P.cablin.The results showed that the digestion system was 20μL,containing 300 ng of template DNA,and reacted at 37℃for 3 hours;the ligation reaction was carried out at 16℃for 12 hours;the optimal dilution multiple of the ligation product was 3 times;the optimal dilution multiple of the pre-amplified product was 20 times.12 pairs of primer combinations with clear and polymorphic bands were screened by optimized reaction system.The optimized reaction system was suitable for the AFLP analysis of P.cablin,and the selected primers also had good polymorphism,which can provide technical support for the subsequent screening of excellent germplasm of P.cablin.