ISSR Molecular Markers And Genetic Diversity Analvsis Of Juglans Hopeiensis Hu

Abstract ISSR (inter-simple sequence repeat), with high efficiency, simplicity and speed, is a new type of DNA molecular markers with micro-satellite repeat sequences as primers to detect the polymorphic of species. Nuts of Juglans hopeiensis Hu, with big and thick shell and diverse wrinkled texture, are suitable for engraving, watching and playing, etc, so folk called them "playing walnut". With the improvement of living standards, the interest of playing with Juglans hopeiensis Hu gets increasingly strong, and more people have focused on collection of the Juglans hopeiensis Hu. Although the germplasm of walnut was rich and widely distributed in China, research on the genetic diversity of the Juglans hopeiensis Hu was still a blank. The phenomenon of same name for different cultivars and the same cultivar for different names was serious. To provide the basis for the identification, germplasm conservation and breeding of Juglans hopeiensis Hu varieties, this study established the fingerprinting and analyzed the genetic diversity for the 138 walnut materials by using ISSR. The main results were as follows:1. The optimum of DNA extraction was 3% PVP40 high-salt. The extract components were: 0.1mol/L NaAC, 0.5mol/L NaCl, 0.05mol/L EDTA, 1.4% SDS, 0.1mol/Lβ-Mercaptoethanal.2. A suitable system for the ISSR analysis of Juglans hopeiensis Hu was: 10×PCR Buffer (Mg2+ Plus) 2μL, dNTPs (Each 2.5mM) 1.6μL, TaqDNA Polymerase 1.0U, Primer 25 mΜ, Template DNA 50ng, added ddH2O to 20μL.3. Seven primers were selected for ISSR analysis from twenty primers according to high polymorphism, clear bands and uniform distribution. 53 bands were scored and 32 bands were polymorphic(The percentage of polymorphic bands was 60.38%). The dendrogram of 138 tested materials(119 Juglans hopeiensis Hu, 15 J.regia L., 2 J.mandshurica Max. and 2 J.nigra L.) was established in the light of these bands.4. 138 walnut materials in this study were separated into 3 groups. Their genetic diversity was discussed by NTSYS-PC software.