Issr Analysis Of Genetic Diversity In Ligustrum Lucidum Ait. Germplasm

Ligustrum lucidum Ait., evergreen shrubs or small trees, which belongs to the genus of Ligustrum Linn. in Oleaceae, is a precious Kudingcha beverage plant and medicinal plants, with a variety of medicinal value and health care functions. Currently, due to environmental changes and the impact of human activities, there is some danger of progressive loss to the genetic diversity. The genetic diversity and relationships of different populations materials of Ligustrum lucidum Ait. were investigated with ISSR (inter-simple sequence repeat) to provide the theoretical base for the breeding and variety protection of Ligustrum lucidum Ait.The modified CTAB method has been developed to extract the high quality genomic DNA from the leaf of Ligustrum lucidum Ait. which contains abundant polysaccharide, polyphenols, organic acid and some other secondary metabolites. The results of ratio of A260 and A280, electrophoresis and amplification suggested that the modified CTAB method could obtain higher quality genomic DNA with better purity and integrity.A suitable ISSR-PCR reaction system (25μl) for Ligustrum lucidum Ait. was established by Single-factor test, i. e.2.5μl 10×buffer,2.0-3.0 mmol·L-1 MgCl2, 150-300μmol·L-1 dNTPs,0.3-0.4μmol·L-1 primer,5-320 ng template DNA and 1.0-1.5 unit of DNA polymerase. The parameters were:4 min at 94℃for initial activation step, followed by 35 cycles of 40 s at 94℃,45 s at annealing temperature 52-62℃and a 2 min extension at 72℃. At the end, amplified products were elongated for 8 min at 72℃and preserved at 4℃.The genetic diversity of Ligustrum lucidum Ait. were studied by ISSR.12 primers were screened in 66 typical Ligustrum lucidum Ait. materials, which could produce polymorphic bands. A total of 237 DNA fragments ranging from 0.15-2.5kb were amplified, among which 221 were polymorphic, using these 12 primers. The average number of DNA band produced by each primer was 19.75. Total percentage of polymorphic loci (P)%was 93.2%, while Shannon's information index was 0.3151 and Nei's gene diversity (I) was 0.2005, the observed number of alleles (Na) was 1.7222 and the effective number of alleles (Ne) was 1.3217 respectively.According to the result of PCR, the genetic similarity analysis for 66 cultivars was calculated by the software of NTSYS pc-2.1. The result of genetic similarity analysis showed that the Jaccard's coefficient ranged from 0.4-0.5. The 66 Ligustrum lucidum Ait. cultivars were divided into 4 groups based on UPGMA cluster analysis. The study indicated that ISSR produced high polymorphism on Ligustrum lucidum Ait.. ISSR analysis can be used for the molecular evaluation and studies of genetic diversity of Ligustrum lucidum Ait. germplasm.