Establishment Of SSR Evaluation System Of Fraxinus Core Germplasm And Fraxinus Velutina Clones

Fraxinus sp. is one of the best tree species of timber, ornamental andafforestation in saline and alkaline land. The constructed of DNA fingerprintsdatabase have great significance to using Fraxinus sp. resources in ourcountry, variety identification and protection of intellectual property rights. Primerswith high polymorphism were screened out. DNA fingerprints database wasconstructed and the genetic diversity was analyzed. In this study, some primers withhigh polymorphism were selected out, and SSR markers were employed to analysisFraxinus sp. species and Fraxinus velutina clones. DNA fingerprints database ofFraxinus sp. species and Fraxinus velutina clones was constructed and the geneticdiversity was analyzed. The main results were summarized as follows:1. This study established and optimized SSR-PCR reaction system of Fraxinus sp.These primer pairs could be used for further research. This experiment establishedthrough L16(45) orthogonal design which selected from four levels of five factors (TaqDNA polymerase, DNA, dNTPs concentration, primers concentration and Mg2+concentration) in SSR-PCR system and optimized through single factor experimentabout the main factors of the PCR reactions. The optimal SSR-PCR reaction systemconsisted of Mg2+(25mmol/L)0.8μL, containing forward-and reversed-primer (10μmol/L)0.2μL respectively, dNTP(10mmol/L)0.3μL, Taq DNA polymerase (5U/μL)0.05μL、DNA(10ng/μL)2.00μL,10×PCR Buffer1.0μLand ddH2O5.45μL.The reaction system was the most conformable one for Fraxinus’SSR-PCR, and wasestablished as the good foundation of SSR-PCR marker technique on Fraxinus sp..2.Using12core germplasms of Fraxinus sp. as experimental materials,10pairs of primers were selected from156pairs of primers for analysis on fingerprintingand genetic diversity based on their reproducible and clear banding patterns. The10primer pairs amplified a total of35polymorphic alleles among the12coregermplasms of Fraxinus sp., with a mean of3.5, and the percentage of polymorphicloci was100%. The polymorphic information content values (PIC) ranged from0.3648to0.6725, with a mean of0.5480, and the maximum PIC was primer of NO. 20, NO.30was the lowest. DNA fingerprinting of12Fraxinus sp. species wasestablished by the molecular fingerprinting combination of4primers which were NO.42,18,26and30. This DNA fingerprint could distinguish all of the12Fraxinus sp.species. UPGMA clustering and genetic diversity analysis could separate differentFraxinus sp. species roughly, and could reflect the genetic relationship betweendifferent kinds of Fraxinus sp.. So, the Fraxinus sp. SSR fingerprint has an importantreference significance in Fraxinus sp. Species identification, germplasm resources,breeding and the protection of intellectual property rights.3. Using46clone in Fraxinus velutina Torr as experimental materials,17pairsof primers were selected from156pairs of primers for analysis on fingerprinting andgenetic diversity based on their reproducible and clear banding patterns. The17primer pairs amplified a total of68polymorphic alleles among the46clone inFraxinus velutina Torr, with a mean of4.0, and the percentage of polymorphic lociwas100%. The polymorphic information content values (PIC) ranged from0.7059to0.1970, with a mean of0.5184, and the maximum PIC was primer of NO.42, NO.16was the lowest. DNA fingerprinting of46clone in Fraxinus velutina Torr wasestablished by the molecular fingerprinting combination of13primers which wereNO.12,16, E3,17,24,5,7, F3,4,42, C3,37and30. This DNA fingerprint coulddistinguish all of the46clone in Fraxinus velutina Torr. By cluster analysis ofUPGMA, Fraxinus velutina Torr clones in different characters and plant can begenerally distinguished.Genetic diversity analysis of data shows that the diversity ofFraxinus velutina Torr clones is rich, and that has the reference value to theestablished evaluation system.