Qinba Mountain Salvia Habitat Group Of Molecular Genetic And Epigenetic Diversity And Genetic Structure

Salvia miltiorrhiza is a traditional Chinese herb plant, its root has been used for centuries to treat diseases such as stems medicine, coronary heart disease, cardiovascular disease, cirrhosis and other diseases. Because the majority of Salvia miltiorrhiza has distributed by artificial harvesting and processing in a long time, and different areas have different ecological environments, the use of seeds, seedlings, cultivation techniques, harvesting period and seasonal, so lead to a great difference of medicinal ingredients in different areas of Salvia miltiorrhizae, and seriously affect the quality of herbs stability of Salvia miltiorrhiza.Research about genetic diversity, genetic structure and epigenetics of different Salvia miltiorrhiza geographical populations would benefit to scientific evaluation and rational utilization of germplasm resources of Salvia miltiorrhiza, guiding elite germplasm breeding and promoting the modernization of Chinese medicine. In this paper, the population genetic structure, genetic diversity, gene flow and Nei's Genetic Distance of 5 populations of shaanxi were studied based on AFLP. epigenetics diversity were studied based on MSAP, and detected the relativity of AFLP and MSAP. Main results obtained were as follows:1. The high quality genomic DNA of Salvia miltiorrhiza. which is suitable for AFLP and MSAP analysis, was extracted by an improved method of CTAB.2.144 loci were amplified from 99 individuals of Salvia miltiorrhiza with five primer combinations of AFLP, the order of the percentage of polymorphic loci(P) was NS(72.22%)> NQ(68.75%)>SZ(68.06%)> DF(55.56%)>SN(50.00%).3. About 69.90% of the total genetic variation occurred within populations,30.10% occurred among populations in Salvia miltiorrhiza. There was a small gene flow (Nm) among the five wild populations of Salvia miltiorrhiza (Nm=1.1612), the bigest gene flow was between SN and DF(Nm=3.3882), the smallest gene flow was between SN and NQ(Nm=1.1500).4. Based on Nei's unbiased measures of genetic distance and UPGMA cluster analysis measures of AFLP, the genetic distance was obtained. The smallest genetic distance was between SN and DF. and the biggest was between SN and NQ. and the individuals of every popolation were clustered into one group. Mantel test indicated that there was a high relativity between genetic distance and geographical distance among the five populations(r= 0.9214).5. The optimized reaction system of MSAP was obtained:Restriction digestion of genomic DNA was performed by using two restriction enzymes, around 500 ng DNA digested with 10 units of EcoR I and Msp I enzymes respectively in a reaction volume of 25μL,and incubated at 37℃for 8h. The 25μL preamplification reaction mixture contained the follow factors, such as Mg2+ (25 mmol/L) 1.0μL, dNTPs (2.5mmol/L) 2.0μL, DNA template 2.5μL, Taq DNA polymerase(5U/μL) 0.4μL, Primers E00/HM00(50ng/μL) 1.0μL respectively. The preamplification products were diluted 100 times for the selective amplification. The 25μL selective amplification reaction mixture contained Mg2+(25mmol/L) 2.0μL. dNTPs(2.5mmol/L) 2.0μL. DNA template 1.5μL, Taq DNA polymerase(5U/μL) 0.1μL, Primers E-ACG/HM-CAA(50ng/μL) 0.8μL respectively.6. DNA methylation pattern of wild population of Salvia miltiorrhiza. which may be related to different habitat, was characteristic of each population. AMOVA analysis generated from MSAP indicated that about 66.87% of the total DNA methylation variation occurred within populations. Mantel test indicated that there was a relativity between epigenetic distance and geographical distance among the populations(r= 0.7032).7. The relativity between AFLP, MSAP, MSP and MISP. The highest relativity was between MSAP and MISP(r=0.99984), which indicated that MISP data had a dominant position in original MSAP data. The coefficient of relativity between AFLP and MSAP was 0.65120. The lowest relativity was between AFLP and MSP(r=0.50499).8. AMOVA analysis generated from MSAP indicated that DNA methylation variation occurred within populations was higher than corresponding genetic variation occurred within populations, and the relativity between epigenetic distance and geographical distance was lower than relativity between genetic distance and geographical distance, and UPGMA cluster analysis measures of AFLP and MSAP had a difference, and the Mantel test indicated that the coefficient of relativity between AFLP and MSAP was only 0.65120, all of which indicated that the methylation pattern change was not only related to DNA sequence variation, but also maybe regulated by other controlling systems.