The Study On Genetic Diversity Of Curculigo Gaertn Germplasm Resources

Curculigo plant including Sect. Curculigo and Sect. Molineria, belongs to perennial herbs. It has high medicinal value. Nowadays, the number of natural population of Curculigo plant has declined, and most populations only have several trees, facing the risk of population extinction. Therefore, studying of the genetic diversity and protecting the genetic resource of Curculigo plant is particularly important and urgent. This paper took 3 wild populations with 134 samples about the Curculigo plant as example, to carry on the analysis of genetic diversity. The main results are as follows:(1) The reagent kit method and improved CTAB method had extracted high-quality genome DNA from these leaves of C. orchioides Gaetn. The purity and yields of DNA extracted from the leaves preserved in silica gel were not significantly different from those of-80℃ leaves. However, in the extraction process, the leaves preserved in silica gel were easy to make the colour become browning and DNA partial degradation, so we can take the method to preserve the leaves when the wild environment is not allow. The reagent kit method had obtained high-quality DNA from-80℃leaves, and they could meet the requirement of PCR amplification.The DNA extraction of roots could use 2% CTAB, adding PVP to 0.2g and NaCl 4 mol/L, which could got purity DNA to meet the molecular experiment.(2) The important factors of the inter-simple sequence repeat PCR(ISSR-PCR) amplification system, such as DNA template, primers, dNTPs, Mg2+ concentration, dose of Taq DNA polymerase and annealing temperature for Curculigo were optimized using the single factor experiment and orthogonal design experiments. The results show that the optimal reacting system with the volume of 25μL was established as follows:50ng DNA template,2.5mmol/L Mg2+,0.2 mmol/L dNTPs,0.4μmol/L primers, and 1.5U Taq DNA polymerase.(3) The genetic diversity of 134 individuals from 13 natural populations of this plant was assessed using ISSR-PCR amplification system. The results showed that 256 total ISSR discernible bands with 15 ISSR primers. The percentage of polymorphic bands was 91.79%. The highest polymorphic bands of populations was YiBin of SiChuan province, and the lowest was Yun-nan nine which adapting ability were range, threat to the survival of the species. At the population level with the percent of polymorphic bands ranged from 4.69% to 29.30%. At the population level with the Shannon’s information index (I) from0.0283 to 0.1605. The highest genetic diversity of populations was YiBin of SiChuan province (P=29.30%, I=0.1605), and the lowest genetic diversity of populations was Yun-nan nine (P=4.69%,I=0.0283).(4)According to the genetic identity and Nei’s genetic distance,13 species of Curculigo were analyzed by UPGMA cluster analysis, clustering results showed that the genetic relationship of Curculigo was more complex.The GuangXi population、Yunnan and Sichuan population were being in the same group; the other populations were the other group. The result indicates the relationship between YiBin and WuGong mountain is the closest, YaChang and YanBian population is the closest in the other group.