Study On Genetic Diversity And Genetic Differentiation Of Tilia Amurensis Natural Population

Tilia amurensis is one of Chinese protected plants, in this paper genetic diversity and population genetic structure of 6 natural populations in longitude & latitude and 6 natural populations in altitude were studied by means of the newly developed inter simple sequence repeats (ISSR) marker. Based on the study of population genetic structure, the strategies to conserve the germplasm resources of Tilia amurensis were also discussed. The results as follows:(1) The content of phenolic compound, polysaccharides and other secondary metabolites is high in Tilia amurensis, which influences frequently the yield and quality of genome DNA when being extracted. This paper deals with extraction of DNA from leaves with 4 different methods, and they were tested with ultraviolet spectrophotometer analysis and agarose gel electrophoresis. The results indicated that the improved CTAB was the most suitable methods for genomic DNA isolation.And then the extraction of DNA from young leaf, mature leaf, phloem and gemma of Tilia amurensis by improved CTAB method was studied respectively. The result show that the genomic DNA can be extracted from young leaf, mature leaf and phloem, all can be used for ISSR analysis. The suitable part of extraction is phloem if young leaf cannot be got, because the quality of DNA, which extracted from phloem, is better than mature leaf.(2) There are two methods to optimize ISSR-PCR amplification system, one is a single factor test, and the other is orthogonal design. In this study, two methods were used to optimize ISSR-PCR amplification system on Tilia amurensis in three levels of four factors (Taq DNA polymerase, Mg²⁺, dNTP, primer) respectively. From the results we found that there has some differences between the two methods in suitable levels of factors. Through the deep analyze, a suitable ISSR-PCR reaction system was established. 14 primers with stable amplification and rich polymorphism for ISSR were screened. The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR. The result provided a standardizing ISSR-PCR program for the analysis of genetic diversity of Tilia amurensis.(3) Inter-simple sequence repeats (ISSR) we used to examine the genetic diversity of Tilia amurensis. in different longitude & latitude. We sampled 135 individuals from natureal populations. 14 ISSR primers were selected for PCR and produced 130 band loci, of which 122 (P=93.85%) were polymorphic;the average number of bands per primer was 9.3. At species level, the allelic gene (A) was 1.9385, effective number of allele (Ae) was 1.3961, the gene diversity (H) was 0.2433 and Shannon Index of diversity (I) is 0.3803. According to the genetic diversity of the 6 populations, higher diversity was found with the populations from the centralrange of the species in contrast to those from peripheral areas.(4) Coefficient of genetic differentiation was 0.1965 on average and the majority of genetic variation occurred within populations. The mean of gene flow (Nm) was 2.0049, which showed relatively steady gene flow among populations, reduce partial genetic variance, and prevent genetic differentiation. Furthermore, genetic diversity was found with the populations from the central range of the species in contrast to those from peripheral areas.(') Inter-simple sequence repeats (ISSR) we used to examine the genetic diversity of Tilia amurensis. in different altitude. We sampled 90 individuals from natureal populations. 14 ISSR primers were selected for PCR and produced 122 band loci, of which 114 (P=80.35%) were polymorphic;the average number of bands per primer was 8.1. At species level, the allelic gene (A) was 1.8769, effective number of allele (Ae) was 1.3740, the gene diversity (H) was 0.2305 and Shannon Index of diversity (/) is 0.3599. The genetic diversity of Tilia amurensis will decrease with the altitude rising.(6) Coefficient of genetic differentiation was 0.3011 on average and the majority of genetic variation occurred within populations. The mean of gene flow (Nm) was 1.1603, which lower than the populations in longitude & latitude. The genetic distance and geographic distance of the species have the clear positive relation, and the genetic continuity is obvious and the altitude factors affect the isolation of Tilia amurensis lightly among the populations in different altitude.(7) According to the facts that the majority of genetic variation occurred within populations and diversity was higher was higher with the populations from the central range, the strategies to conserve the genetic diversity of Tilia amurensis should be laid emphasis on conserving the different kinds of individuals in big populations, especially the populations from the central range. Moreover, efforts should be made to keep the Tilia amurensis population from being further destroyed.