ISSR Analysis Of Germplasm Resources Of Bongainvillea Brasiliensis Raeusch.

Bougainvillea (Bongainvillea Brasiliensis Raeusch.), belonging to Bougainvillea genus of Nyctaginaceae, is an evergreen climbing shrub widely cultivated in subtropical and tropic areas. There are over one hundred individuals. The genetic background of the most individuals is complex, and so far, the researches on genetic relationship of bougainvillea are limited. To provide scientific evidences and assist on quality chosen, cross breeding parents selection and fine variety breeding of bougainvillea on molecular level, ISSR molecular marker technology was used to investigate the genetic diversity and genetic relationship of 68 bougainvillea individuals in this study. The main results were indicated as follows:1. To resolve the influence of pigment, polyphenol and polysaccharide on total DNA extraction, 4 different methods were employed and compared in this study. The results showed that the improved CTAB mini method was the fittest method for bougainvillea DNA extraction, and the concentration and purification of DNA were all suitable for ISSR analysis.2. Firstly, the ISSR-PCR reaction system was optimized, and then ISSR-PCR amplification reaction protocol for bougainvillea was built in this study. The amplification reaction was done in a 20μL volume consisting of 1.0 U Taq enzyme, 0.3μmol/L primers, 250μmol/L dNTPs, 20 ng template DNA and 2μL10×Buffer. The PCR protocol consisted of a single denaturation step of 5 min at 94℃, followed by 34 cycles of 60 s denaturation at 94℃, 60 s annealing at optimized temperature, 2 min extension at 72℃, with a final 7 min incubation at 72℃and conservation at 4℃. The annealing temperature was optimized by T-gradient PCR. The agarose gel electrophoresis results indicated that the bands were distinct. It was concluded that the PCR amplification reaction protocol was suitable for ISSR analysis.3. Fourteen ISSR primers were used in the ISSR analysis of 68 bougainvillea individuals. A total of 195 ISSR bands were scored, corresponding to the polymorphic bands were 182, and the percentage of polymorphic was 93.3%. The data showed that ISSR-PCR can produces rich bands and high polymorphic rate. The study also found that there were 11 dinucleotide repeat sequence in 14 primers. It was indicated that dinucleotide repeat sequences were rich in bougainvillea genome. According to ISSR-PCR results, a cluster ananlysis (UPGMA) was used to generate a dendrogram among all 68 individuals. The results showed that genetic similarity among individuals was between 0.50 and 0.97, the average was 0.67. It was indicated that the relationship among all individuals was close and the genetic diversity was poor. According to dendrogram, 68 individuals can be divided into A group and B group at genetic similarity 0.64. The A group can be divided into 2 subgroups and B group can be divided into 6 subgroups. This study revealed the genetic relationship and confirmed the genetic distance among 68 bougainvillea individuals using molecular marker technology.