ISSR Analysis On Genetic Diversity Of Yibeimu And Plants In Fritillaria L. From Xinjiang By Molecular Techniques

Objective:Analysis of genetic diversity of the Yibeimu germplasm resources and plants in Fritillaria L. from Xinjiang by molecular marking technique of ISSR, and it provide the basis for the species protection, comprehensive evaluation of resources and varieties classification. Methods:1) Using 3 different methods to extract medicinal plants total DNA of Fritillaria L., choose the most suitable extraction method of total DNA in medicinal Fritillaria L., to obtain high quality genomic DNA.2) Comprehensive utilization of single factor experiment and L16 (45) orthogonal test, the factors of Taq DNA polymerase, dNTPs, Mg2+, concentration of primers, template DNA content is optimized of the ISSR-PCR system of Fritillaria L..3) The geranium ISSR-PCR system of Fritillaria L. was determined, and it was used to comprehensive analysis the level of genetic diversity of the 128 individuals from 16 populations.4) Inter-simple sequence repeat (ISSR) were used to comprehensive analysis the level of genetic diversity of the 23 individuals. Results:1) Higher yield of genomic DNA can be extracted from the plant Fritillaria L. by 3 kinds of methods, but the kit total DNA extracted highest purity, best quality, all samples were amplified and the rich can clear bands, and simple short-consuming operation.2) Every factors in different levels had the significant effects on the result of PCR, and the most remarkable factor was the concentration of Mg2+. ISSR-PCR optimal system for total DNA of plants in Fritillaria L. From Xinjiang as the 50μL:5μL 10×Buffer,2.5 mmol/L Mg2+,0.30 mmol/L dNTPs,0.8μmol/L primer, 0.75 U Taq DNA polymerase,1.0 ng/μL template DNA,33.35μL ddH2O.3) Yibeimu genetic diversity:15 primers were selected to produce highly reproducible ISSR bands from 74 primers. A total of 15 primers amplified 239 loci, including 227 polymorphic loci (percentage of polymorphic bands was 94.98%). Low levels of genetic diversity were observed in Yibeimu (Na=1.9498, Ne=1.3426,H=0.2190,I=0.3503,Ht=0.2227±0.0266, Hs=0.1046±0.0055, Gst=0.5304, Nm*=0.4427), genetic identity (I) was 0.7344-0.9666, genetic distance (D) was 0.0340～0.3088. The results of cluster analysis showed that Fritillaria walujewii and Fritillaria pallidiflora can be distinguished, the genetic similarity coefficient between 0.69-0.95. To build Yibeimu fingerprints.4) 8 species in Fritillaria L. From Xinjiang genetic diversity:a total of 16 primers were selected from74 primers amplified 199 loci, including 195 polymorphic bands with the percentage of polymorphic bands at 97.99%. Low levels of genetic diversity were observed in all 8 species in Fritillaria L. (Na=1.9799, Ne=1.5230,H=0.3084,I=0.4669, Gst=0.6985, Nm*= 0.2159), genetic identity (I) and genetic distance (D) were 0.7474 and 0.3014, respectively. Clustering analysis showed that the all Fritillaria L could be distinctively classified into 4 groups with genetic similarity coefficient ranged from 0.58～0.99, and to build its indexes of Fritillaria L. based on ISSR marker. Conclusion:1) Kit method is the best way to total DNA’s extracted from medicinal plants of Fritillaria L., and can be used directly in PCR of Fritillaria L. in Xinjiang and Pharmacopoeia of the rest of the medicinal Fritillaria L. and other biological research.2) The established and optimized ISSR reaction system is stable and credible and ISSR-PCR amplification effect is obvious according to the testing results of 23 samples of 8 species of plants in Fritillaria L. from Xinjiang.3) The resources of Yibeimu had high genetic diversity in general, Genetic diversity variation is mostly exists between populations, and in degree of gene flow between populations is limited.4) Using ISSR molecular marker fingerprinting can be fast identification in Yibeimu.5) The resources of 8 species in Fritillaria L. had high genetic diversity in general, Genetic diversity variation is mostly exists between species, the degree of gene flow among species is limited. Using indexes of Fritillaria L. based on ISSR marker can be fast, accurate identification in Fritillaria L. from Xinjiang. To ensure Fritillaria L. source in accurate classification is meaningful.6) Revealed the levels of genetic diversity of the Yibeimu germplasm resources and the plants in Fritillaria L. from Xinjiang, it provide the basis for the germplasm resources, varieties classification, species protection and comprehensive evaluation of resources.