| The formation of traditional Chinese medicine geoherbalism is closely related to the environmental factors, phenotypic traits and genetic diversities. Phenotypic variation of plants is the result of gene-environment interaction. Epigenetic acts as a bridge between the environment and genes. Among its many mechanisms, DNA methylation plays an important role in epigenetic phenomenon, and is of great importance in exploring the formation and evolution of geoherbalism. Cyathula officinalis Kuan is a specie of Cyathula of Amaranthaceae which is widely used in traditional chinese medicine.Researches using Methylation Sensitive Amplified Polymorphism(MSAP), on the statistical features of different Cyathula officinalis Kuan genotypes and DNA methylation patterns under the influence of biotic and abiotic factors along with genes of polymorphic methylation sites are of great importance to explore the mechanism of Cyathula geoherbalism formation, control the stability of Cyathula officinalis Kuan in the genuine producing areas and to predict suitable areas for migrating. Research results are as follows:(1) A MSAP protocol is established and optimized for Cyathula officinalis Kuan and CTAB is found to be the best method to extract DNA, helping solving the key problems about Cyathula officinalis Kuan's digestion, preamplification and selective amplification. Six sets of suitable primers are screened out, serving as technical support for the follow-up experiments.(2) Research on disease resistance difference of Cyathula officinalis Kuan from different areas under the stress from the white rust disease. Strong disease resistance is found in Cyathula officinalis Kuan from Xiaogoucun in comparison to the others. Analyze the methylation patterns, methylation levels, genetic diversity and epigenetic diversity of Cyathula officinalis Kuan. The results show that all four methylation patterns existed in Cyathula officinalis Kuan from Xiaogoucun with the strongest disease resistance. High overall level of methylation (47.5-52.3%), genetic diversity (H= 0.2054, I= 0.2930) and apparent genetic diversity (H= 0.1859, I= 0.2660) are found. Compare the contribution of genetic and epigenetic diversity to resistance difference finding that the genetic diversity of Cyathula is of greater influence on disease resistance than epigenetic diversity.(3) Reseach on genetic background and epigenetic variation of Cyathula officinalis Kuan cultivated in the same environment from different areas and analysis about ecological factors of their origin shows that:the ecological factors in Chongqing vary most, followed by areas in Sichuan, Hubei and Hunan areas. Coefficients of variation were 1.95,1.61,0.87 respectively. Analysis of clustering thier genetic background turns out that the population in Chong Lake showed the highest epigenetic diversity (H=0.1949,I=0.2799)while the population in Leshan shows the least epigenetic diversity(H=0.1541,1=0.2249), and there is the biggest epigenetic distance between the populations in Chong Lake and Leshan(D'=0.1280), and the lowest consistency of phenotype difference(J'=0.8798). The comparison of the two classification results suggest that the genetic backgrounds of the samples from the two lakes and Chongqing are similar to each other, but pertain to two different climatic types. Besides, the genetic backgrounds of the samples from two producing areas in Sichuan with the same climatic type show bigger difference relatively. It is deduced that the difference between the samples in the producing areas of two lakes might be greatly affected by the climatic types. However, the samples from the production areas in Yaan and Leshan of Sichuan are greatly affected by the genetic background.(4) Reseach on genetic background and epigenetic variation of Cyathula officinalis Kuan from different altitude. The former shows:samples at different altitudes are of higher similarity in the genetic background with genetic identity (J) greater than 0.9500. The latter shows:the genetic and epigenetic distance of Cyathula officinalis Kuan from Maogangzi and Xiaogoucun are smaller, resulting in a similar phenotype. So Maogangzi can be used as an alternative to the introduction Cyathula.Comprehensive analysis shows:altitude has a weak influence on Cyathula officinalis Kuan genetic background but a strong influence on epigenetic variation.(5) Research on genetic background and epigenetic stability of Cyathula officinalis Kuan of different growth years from the genuine producing areas. Genetic background analysis shows that the genetic identity (J) of all samples with different growth years is greater than 0.9400, showing higher similarity in the genetic background; epigenetic variation analysis shows that annual and triennial samples are of larger epigenetic distance (D-0.1846) and smaller epigenetic identity (J'= 0.8315); epigenetic stability analysis shows:variation coefficients of Cyathula officinalis Kuan with one, two, three growth years are 28.3,9.9,49.5 respectively, indicating that biennial Cyathula officinalis Kuan is of highest epigenetic stability. Comprehensive analysis shows:growth years have a weak influence on Cyathula officinalis Kuan genetic background but a strong influence on epigenetic variation.These studies explore the contribution of different factors on the formation of Cyathula officinalis Kuan geoherbalism, providing a basis for Cyathula officinalis Kuan cultivation and a guide for suitable choice of producing area. These researches are of great important to explore the correlation between environmental, phenotypic and genetic influences on the formation of geoherbalism, helping to reveal the molecular mechanisms of Chinese medicine geoherbalism. |
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