Genetic diversity analysis of germplasm of authentic Astragalus membranaceus in Inner Mongolia / 中草药

Objective: To establish an optimum reaction system suitable for ISSR analysis of Astragalus membranaceus and to analyze the genetic diversity of wild populations in Inner Mongolia. Methods: A stable and reliable ISSR reaction system was set up combining the concentration gradient of the single factor test and orthogonal test. The genetic diversity of 30 A. membranaceus populations in nine zones of Inner Mongolia was analyzed using NTSYS2.1 software. Results: The optimal ISSR reaction system (20 μL) contained 10 × PCR buffer 2.0 μL, 1.5 mmol/L MgCl2, 0.4 mmol/L deoxyribonucleotide triphosphate (dNTP), 1.5 U Taq DNA polymerase, 0.5 μmol/L primer, and 40 ng template DNA. A total of 169 amplified loci were detected by 15 ISSR primers, in which 157 loci were polymorphic loci with the percentage of 75%~100%. The genetic distance amplitude ranged between 0.242 7-0.730 8. The clustering analysis showed that 30 A. membranaceus populations could be divided into two categories, and most of them corresponded to the geographical distribution. Conclusion: ISSR-PCR reaction system for A. membranaceus is stable and reliable. Wild resources of A. membranaceus in Inner Mongolia have higher genetic diversity. The genetic relationship of the populations is correlated with its geographic location.

Optimization for ISSR-PCR reaction system of Illicium difengpi by orthogonal design / 中草药

Objective: To establish a stable, reproducible, and suitable reaction system for ISSR analysis of genetic differences in Illicium difengpi. Methods: The ISSR-PCR amplification system on I. difengpi in five factors (Mg2+, dNTPs, primers, Taq DNA polymerase, and DNA template) was optimized by orthogonal design, and the PCR result was analyzed by SPSS. Then based on the optimal ISSR-PCR amplification system, the annealing temperature and cycle times in PCR were proposed by gradient determenation. Results: Most of the factors in different levels had the significant effects on the result of PCR, and the most remarkable factor was the quantity of Taq DNA polymerase. The optimized ISSR-PCR reaction system (20 μL) for I. difengpi was constructed of Mg2+ (1.60 mmol/L), dNTP (0.22 mmol/L), primer (0.90 μmol/L), Taq polymerase (0.50 U), and DNA template (70.00 ng). The optimized annealing temperature and cycle times were 51.8 °C and 40 cycles, respectively. Thirteen ISSR primers with stable amplification and abundant polymorphism were selected from 62 ISSR primers. Conclusion: The established and optimized ISSR reaction system is stable and credible according to the testing results of 16 samples of I. difengpi, and provides the basis for the genetic analysis of I. difengpi.

Population genetic structure of Sisyrinchium micranthum Cav. (Iridaceae) in Itapuã State Park, Southern Brazil

Sisyrinchium micranthum Cav. is a member of the family Iridaceae, which is distributed over the American continent. In Brazil, this species is found, not only in disturbed areas and coastal regions, but is also very common in urban centers, such as public parks, during the spring. Chromosome counts for North American specimens are 2n = 32 and 2n = 48, whereas in southern Brazil, there is a polyploidy series with three chromosome numbers, 2n = 16, 2n = 32, and 2n = 48. Population analyses using DNA molecular markers are inexistent for this species, in spite of its wide distribution and morphological variation. To study the genetic population structure of S. micranthum, five natural populations were accessed in a conservation park within the Atlantic Rain Forest Biome in southern Brazil. Here, the chromosome numbers 2n = 16 and 2n = 48 had already been described. Molecular analysis showed that the populations are highly structured with low gene flow among them. The population with 2n = 48 was genetically less variable than and distinct from the other populations. Population genetics in relation to cytogenetic data provided new insights regarding the genetic diversification and mating system of S. micranthum.

Establishment and optimization of ISSR-PCR reaction system in Asparagus cochinchinensis / 中草药

Objective: To establish and optimize ISSR-PCR reaction system for Asparagus cochinchinensis and lay foundation for its genetic diversity research. Methods: The single-factor test and orthogonal design were applied for optimizing seven factors in the ISSR-PCR reaction system including Mg2+, dNTP, primers, Taq DNA polymerase, the template DNA, extension time, and cycle times. Results: The suitable PCR reaction system contained 1.25 mmol/L Mg2+, 320 μmol/L dN TP, 1.2 μmol/L primer, 1.5 U Taq DNA polymerase, and 40 ng template DNA in total 25 μL reaction solution. On this basis, 13 primers were screened with stable amplification and rich polymorphism from 50 ISSR primers. The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR. Conclusion: It is proved that the established and optimized ISSR reaction system would be stable and credible by the germplasm testing result of 17 A. cochinchinensis populations. This would provide the basis for the genetic analysis of A. cochinchinensis.

Optimization for ISSR-PCR reaction system on Schisandra henryi by orthogonal design / 中草药

Objective: To optimize the each factor affecting on ISSR-PCR reaction system of Schisandra henryi and establish the stable ISSR-PCR system. Methods: Based on the analysis of orthogonal design test, an orthogonal design was used to optimize the ISSR-PCR amplification system on S. henryi by five factors (Taq polymerase, Mg2+, DNA template, dNTP, and primer) at four concentration levels, respectively. Results: A suitable ISSR-PCR reaction system was constructed with the 20 μL reaction system containing 1.00 U Taq polymerase, Mg2+1.50 mmol/L, DNA template 40.00 ng, dNTP 0.25 mmol/L, and primer 0.50 μmol/L. Twelve effective ISSR primers were selected and the optional annealing temperature of every one primers was fixed. Conclusion: ISSR-PCR is significantly influenced by the concentration of S. henryi. This ISSR-PCR system could provide clear bands, reliable reaction system, and abundant polymorphisms. It proves a reference for molecular research of S. henryi.

Establishment of ISSR marker technology and optimization of its system in Prunella vulgaris / 中草药

Objective To establish and optimize the ISSR-PCR reaction system for Prunella vulgaris and lay foundation for its genetic diversity research.Methods The single-factor and orthogonal design were applied for optimizing seven factors in the ISSR-PCR reaction system including Mg2+,dNTP,primers,Taq DNA polymerase,the template DNA,annealing temperature,and cycles.Results The suitable PCR reaction system contained 2.2 mmol/L Mg2+,175 ?mol/L dNTP,0.75 ?mol/L primer,1.0 U Taq DNA polymerase,and 30 ng template DNA in total 20 ?L reaction solution.On this basis,18 primers were screened with stable amplification and rich polymorphism from 92 ISSR primers.The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR.Conclusion It is a way to establisb the ISSR-PCR system for orthogonal design combining with single-factor test.And it is proved to be stable and credible for the result of 24 P.vulgaris populations.This optimized ISSR reaction system would provide the basis for the genetic analysis of P.vulgaris.

Application of ISSR-PCR to identification of different Dendrobium Sw. species / 中草药

Objective To identify nine species of Dendrobium Sw. by inter-simple sequence repeats-polymerase chain reaction (ISSR-PCR) method and to investigate the difference among the species of Dendrobium Sw. at DNA molecular level. Methods Ten primers constituted by simple sequence repeats (SSR) were tested for PCR and sepharose electrophoresis of the nine species. Results Seven of the ten primers amplified polymorphic bands. The number of polymorphic band positions of each primer ranged from 7 to 14, and the size of fragments ranged from 220 to 1 260 bp. The amplification patterns of two primers, namely UBC-807 and UBC-864, were higher in terms of polymorphic and amplified band ratio. Each of them was able to distinguish all the examined species. Conclusion ISSR-PCR provides a quick, reliable molecular marker technique for identification of different species of Dendrobium Sw.