RAPD And ISSR Analysis On Genetic Diversity Of The Ancient Ginkgo Trees In Suizhou

Ginkgo biloba L., a "living fossil" belonging to Ginkgoaceae and Ginkgo, is one of the oldest remained tree species. China is the native land of Ginkgo biloba and there are lots of ancient Ginkgo trees in many places in the country. Suizhou of Hubei Province is one of the main concentrated distribution regions of ancient Ginkgo trees. In this paper, we collected leaf samples from 121 ancient Ginkgo trees and seed samples from 30 ancient Ginkgo trees in Suizhou as research samples, analyzed the genetic diversity of ancient Ginkgo trees in Suizhou at population level and individual level by applying RAPD and ISSR molecular markers. Major results are as follows:(1) A total of 104 bands (including 103 polymorphic bands) were amplified using 9 RAPD primers and a total of 107 bands (including 105 polymorphic bands) were amplified using 9 ISSR primers, which were based on the DNA of those 121 Ginkgo individuals of 7 populations. The percentage of polymorphic bands (PPB) were 99.04% for RAPD and 98.13% for ISSR respectively. The genetic diversity of Ginkgo at species level (H_E = 0.3241, H_O= 0.4887) and population level (H_E = 0.2302, H_O = 0.3440) revealed by RAPD were both similar to those by ISSR (species level: H_E=0.2516, H_O= 0.3913; population level: H_E=0.1775, H_O=0.2697). Correlation coefficient between the genetic identity based on RAPD and that based on ISSR was 0.6932 (P=99.65%) and was significant.(2) Genetic differentiation among the ancient Ginkgo populations in Suizhou was revealed by Nei's genetic diversity analysis and AMOVA (Analysis of Molecular Variance) based on RAPD and ISSR experiment data. The RAPD results revealed that for the 7 polulations of ancient Ginkgo trees in the Dahong Mountain region, G_ST=0.2896,φ_ST= 24.22%; while ISSR results revealed that G_ST=0.2944, φ_ST = 23.12%. The genetic differentiation value was even higher when the Caodian population was concerned: G_ST=0.3380, φ_ST=32.05%.(3) According to the data of RAPD and ISSR molecular markers of the leaf samples, UPGMA cluster was made at population level and individual level. The clustering analysis of the 129 ancient Ginkgo individuals of 8 populations at individual level showed that most individuals from a same population could be clustered together in thedendrogram of genetic relationship. It showed that there was great genetic differentiation among these populations. In particular, the genetic differentiation between Caodian population and other populations was the highest and the regional difference was distinct. But this difference was not so obvious in the dendrogram at population level. The genetic differentiation among populations might be caused by human production activities and barriers to gene flow.(4) Six ISSR primers which could amplify more bands were screened out secondly from those 9 polymorphic ISSR primers. These effective primers were used to analyze the genetic diversity of 150 seeds samples of 30 ancient Ginkgo trees at individual level. As a result, 91 bands were generated, of which polymorphic bands was 90 based on haploid data and 64 based on diploid data, the percentage of polymorphism bands were 98.90% and 70.33% respectively. Heterozygosity test showed that the average number of heterozygous loci was 39 and the average heterozygosity was 43.53%. The highest heterozygosity was 54.95%, and the lowest was 34.07%. These results indicated that high heterozygosity existed in the ancient Ginkgo trees in Suizhou. Genetic diversity at individual level was: He=0.1617, #0=0.2393. GSt and ?&St among the 30 Ginkgo individuals were 0.4111 and 25.65% respectively. Among the 3 seed nucleus types, Gst was 0.0674, and ?&st was 4.82%.(5) According to the amplification results with the 6 ISSR primers to the 30 ancient Ginkgo individuals, similar but not exactly the same dendrograms were generated through molecular genetic clustering at haploid level and diploid level. The former based on haploid level was more accurate than the latter based on diploid level, so the former was more credible than the latter. The result of genetic dendrogram was different from conventional classification based on seed nucleus morphological characteristics. As the genetic relationship among the Ginkgo individuals based on DNA was more accurate, it is necessary to use both molecular markers and morphological markers to classify or identify the Ginkgo types or varieties.