ISSR Analysis On The Diversity Of Oil-tea Camellia Germplasms

Using optimized ISSR-PCR reaction system and22primers screening from a set of100ISSR primers to amplify by PCR the genome DNA of98oil-tea Camellia germplasms (51clonal lines and47breeding lines from sexual hybridization), the PCR products were electrophoresized and the ISSR fingerprinting of these oil-tea camellia germplasms was established. The results provided theoretical foundation for the identification of oil-tea Camellia germplasms. The main results were as follows:1. The suitable ISSR reaction system for oil-tea camellia germplasms was optimized. A total volume of25μl reaction system is comprised of12.5μl Mix (DNA polymerase, dNTPs and the optimum buffer,2×concentration),2μl50ng/μl template DNA,2μl0.6μmol/L primer,8.5μl ddH2O. The PCR program is shown as:pre-denaturation at94℃for5min;39cycles of denaturation at94℃for45s, annealing at56℃for45s, and extending at72℃for1min30s; and finally extending at72℃for7min, the product was stored at4℃.2. Using the optimized ISSR-PCR reaction system and the screened22primers to amplify by PCR the genome DNA of51clonal oil-tea camellia germplasms derived from different regions of China, the products were electrophoresized and493discermible loci with distinct electrophoretic bands were obtained and478loci (96.78%) of them were polymorphic, which demonstrated that oil-tea germplasms possess a higher level of genetic diversity. Based on the clustering analysis performed using software NTSYS2.10and Winboot,51clonal oil-tea germplasms were divided into two groups, named Group I (which contained48Camellia oleifera Abel lines) and Group II (which possessed three Camellia oleifera Abel related species). The similarity coefficient of two groups was0.620. Moreover, Group I germplasms were divided into two sub-groups named Group Ⅰ-1and Group Ⅰ-2, and their similarity coefficient (Gs) was0.634. In Group Ⅰ-1, all members originated from Hunan Province, but Group Ⅰ-2possessed partial Hunan lines and all Camellia oleifera Abel ones originated from other regions. Analysed by software POPGENE version1.32, the Shannon’s information index (I*) of genetic polymorphism of51oil-tea germplasms was0.3852, the genetic diversity among different region populations(Ht) was0.2537, the genetic diversity within populations (Hs) was0.1555, the differentiation coefficient of genetic diversity among populations (Gst) was0.3967, the gene flow among populations (Nm*) was0.8262. The Nei’s genetic distances between the Hunan population and those from other regions were shown in order as:Yunnan’s> Fujian’s> Guizhou’s> Guangxi’s> Jiangxi’s> Hubei’s, which implied that geographic isolation strongly influences genetic differentiation among populations. Based on the ISSR fingerprinting,51camellia germplasms in the different areas were distinctly identified.3. The above22ISSR primers were also used to amplify by PCR the genome DNA of13breeding lines of ZB-8(HNDZ-1×XL-F-183),13breeding lines of ZB-7(XL-183-M1×XL-1) and21breeding lines of ZB-9(XL-183-M2×XL-1) respectively. As a result,359.387and382discernible loci with distinct electrophoretic bands were obtained and the polymorphic loci was293.335and328for ZB-8, ZB-7and ZB-9populations respectively. The polymorphic loci percentage was81.62%.86.56%and85.86%respectively, all of them were less than the polymorphic level (96.78%) of51clonal oil-tea germplasms, which means that obvious genetic diversity was shown among different breeding lines derived from the same hybridization combination, but the level of genetic difference is less than that of the clonal germplasms of different regions. The statistical analysis results showed that, the observed number of alleles(Na*) of ZB-8, ZB-7and ZB-9were1.8162.1.8656.1.8586respectively; the effective number of alleles (Ne*) were1.4835.1.4999.1.4502respectively; the genetic heterozygosity(h*)were0.2834.0.2952.0.2717respectively; the Shannon’s information index (I*) were0.4256.0.4449.0.4150respectively.The molecular properties among the breeding lines from each of three hybridization combinations showed obvious diversities and were all distinctly different from their parents, so hybridization is an important way to obtain a plenty of oil-tea camellia germplasms with genetic diversity.Meanwhile, both of seedling root stock grafting and high grafting for tree crown change resulted in gene variation of the breeding offsprings.The above results of ISSR analysis on the genetic diversities of oil-tea camellia germplasms in China provided the theoretic data and method for the identification of oil-tea camellia germplasms, is of great significance for the preservation and utilization of oil-tea camellia.