Analysis of Equivalence Between Traditional and Nontraditional Medicinal Varieties of Citri Reticulatae Semen from Sichuan Province / 中国实验方剂学杂志

Objective:To study the differences in genetic relationship， shape， size， and flavonoid content between traditional and nontraditional medicinal varieties of Citri Reticulatae Semen produced in Sichuan province as well as their equivalence. Method:Six batches of traditional medicinal Citri Reticulatae Semen （<italic>Citrus reticulata</italic> 'Dahongpao'） and 23 batches of nontraditional medicinal varieties were collected， and their genetic relationship was explored using sequence-related amplified polymorphism （SRAP） markers. Following the observation of their shapes and sizes under a stereomicroscope， the contents of naringin， hesperidin， and neohesperidin were measured by high performance liquid chromatography （HPLC）. SIMCA 14.1 software was used for cluster analysis of their shapes， sizes， and flavonoid contents， thus figuring out the similarities between the traditional and nontraditional medicinal varieties in character， size， and chemical components. Result:SRAP markers-based genetic relationship analysis effectively distinguished different Citri Reticulatae Semen varieties from each other. Some samples collected from the same or adjacent places exhibited a close genetic relationship and they shared high similarities in shape， size， and flavonoid content. However， the traditional medicinal Citri Reticulatae Semen was still quite different from most nontraditional medicinal varieties. Conclusion:The analysis of differences in genetic materials， appearance， character， and active ingredient content between the traditional and nontraditional medicinal varieties revealed that the equivalence<italic> </italic>of <italic>C.</italic> <italic>reticulata</italic> 'Ponkan' samples from some regions with the traditional medicinal variety was the largest， enabling them to be considered as the emerging medicinal variety.

Optimization of SRAP-PCR System for Valeriana officinalis var. latifolia and Primer Screening / 中国实验方剂学杂志

Objective:To establish the sequence-related amplified polymorphism （SRAP）-polymerase chain reaction （PCR） system for <italic>Valeriana officinalis</italic> var. <italic>latifolia</italic>，so as to lay the theoretical and technical foundations for the breeding of<italic> V. officinalis </italic>var. <italic>latifolia</italic>. Method:Single factor test was applied to investigate the effects of <italic>Taq</italic> Mix dose，Mg²⁺concentration，template DNA concentration，and <italic>Taq </italic>DNA polymerase content on SRAP-PCR amplification of <italic>V. officinalis </italic>var. <italic>latifolia</italic>，based on which the orthogonal experiments were performed to optimize the SRAP-PCR system for <italic>V. officinalis </italic>var. <italic>latifolia</italic>. The effective primers that could be used for genetic diversity studies of <italic>V. officinalis</italic> var. <italic>latifolia </italic>were selected under the optimal reaction condition. Result:The results of the single factor test showed that <italic>Taq </italic>Mix dose within the range of 8-11 μL resulted in better amplification. The addition of a low concentration of Mg²⁺，the medium to low concentrations of template DNA，or the low concentration of <italic>Taq</italic> DNA polymerase enhanced the amplification efficiency or richness. As demonstrated by the orthogonal experiments，the influencing degrees of related factors on SRAP-PCR amplification of <italic>V. officinalis</italic> var. <italic>latifolia </italic>were sorted in a descending order as follows： <italic>Taq</italic> Mix dose><italic>Taq</italic> DNA polymerase content>Mg²⁺ concentration>template DNA concentration. The optimal reaction system for <italic>V. officinalis</italic> var. <italic>latifolia </italic>was determined to consist of 11 μL of <italic>Taq</italic> Mix，30 ng of template DNA，0.025 mmol·L^-1Mg²⁺，1.5 U<italic> </italic>of<italic> Taq </italic>DNA polymerase，5 μmol·L^-1 forward primer，and 5 μmol·L^-1 reverse primer，which was supplemented to 20 μL with ddH₂O. The optimal annealing temperature was 36.8 ℃. A total of 17 pairs of effective primers with high band resolution and polymorphism were selected from 88 primer pairs for SRAP-PCR of <italic>V. officinalis</italic> var. <italic>latifolia</italic>. Conclusion:The established SRAP-PCR system for <italic>V. officinalis</italic> var. <italic>latifolia</italic> is stable， which can be used for genetic diversity studies of <italic>V. officinalis</italic> var. <italic>latifolia</italic>.

Genetic diversity of Indo-China rice varieties using ISSR, SRAP and InDel markers

Rice is believed to have originated from Indo-China, area between China and India, and then spread throughout the world. The Indochina region mainly includes countries like Thailand, Laos and Vietnam, which are the world’s major rice exporters. Rice varieties grown in this area are highly diverse due to their different environment, ecosystem and climatic conditions. The objective of this study was to evaluate the genetic relationship of Indochina rice varieties using intersimple sequence repeat (ISSR), sequence-related amplified polymorphism (SRAP) and insertion–deletion (InDel) markers. Forty-six rice varieties, including 16, 4,11 and 15 from Thailand, China, Laos and Vietnam, respectively were used in this study. Seventeen of the 20 ISSR primers showed 82.96% polymorphism. At the same time, 17 of the 30 primer pairs of SRAP marker showed clearDNA amplification, which resulted in 84.79% polymorphism. Ninety-seven of 133 InDel markers have about 99.47% polymorphism. Three markers showed average PIC score ranging from 0.20 to 0.26. When the analysis was conducted using UPGMA clustering method, it was found that the combined data from three markers gave a better result than each marker separately. The results from clustering analysis showed that all accessions can be grouped based on their location and can be categorized into two major groups. Useful results from this study could bring substantial benefits and ultimately help the rice breeders to develop elite rice varieties in future.

Development of a Molecular Marker for Fruiting Body Pattern in Auricularia auricula-judae

The fruiting body pattern is an important agronomic trait of the edible fungus Auricularia auricula-judae, and an important breeding target. There are two types of fruiting body pattern: the cluster type and the chrysanthemum type. We identified the fruiting body pattern of 26 test strains, and then constructed two different near-isogenic pools. Then, we developed sequence characterized amplified region (SCAR) molecular markers associated with the fruiting body pattern based on sequence-related amplified polymorphism (SRAP) markers. Ten different bands (189–522 bp) were amplified using 153 pairs of SRAP primers. The SCAR marker “SCL-18” consisted of a single 522-bp band amplified from the cluster-type strains, but not the chrysanthemum strains. This SCAR marker was closely associated with the cluster-type fruiting body trait of A. auricula-judae. These results lay the foundation for further research to locate and clone genes controlling the fruiting body pattern of A. auricula-judae.

SRAP analysis on genetic diversity for germplasm resources of Dipsacus asper / 中草药

Objective: To study the genetic diversity of Dipsacus asper from different populations and provide a reference for the rational utilization of its germplasm. Methods: The genetic diversity of the 14 populations of D. asper was analyzed by SRAP molecular markers. Results: Ten pairs of primers produced 124 sites, among which 102 were polymorphic sites. The percentage of polymorphic loci (PPL) was 82.26%. The Nei's genetic diversity index (H) and the Shannon's information index (I) were 0.2800 and 0.4353, respectively. At the population level, PPL was 53.92%, H was 0.1212-0.2440, and I was 0.1796-0.3611. The genetic diversity values of the five populations were relatively high, and the populations had the characteristics of high altitude and microhabitat. Genetic differentiation coefficient (Gst) was 0.2930, gene flow (Nm) was 1.2064. Cluster analysis based on genetic similarity indicated that the 14 populations could be divided into three groups. Conclusion: The genetic diversity among the populations of D. asper was at relatively high level. The genetic variance of D. asper mainly existed within the populations. The high genetic diversity could be attributed to the geographical position (altitude) and climate, while geographic isolation (microhabitat) was another important factor for the genetic variance within the populations.

Investigation of genetic diversity of yam cultivars by SRAP markers / 中国药学杂志

OBJECTIVE: To investigate the genetic diversity of yam resources in China and provide reliable molecular evidences for cultivar identification and genetic relationship. METHODS: Sequence-related amplified polymorphism (SRAP) was applied to detect the genetic diversity of 21 yam cultivars from eight cultivated populations, Popgene software (version 1.32) was used to calculate genetic parameters, UPGMA clustering was carried out using Mega software (version 4.1), and principal component analysis was performed with the help of Ntsys-pc (Version 2.1) program. RESULTS: Three hundreds and nine bands were amplified by 20 SRAP primer combinations, of which 289 bands were polymorphic. The Nei's gene diversity index (H=0.2881), Shannon's information index (I=0.4416) and total genetic diversity (Ht=0.2891) revealed a relatively high genetic diversity level among yam populations. Within populations, HNWX population showed the highest genetic diversity (PPB= 35.92%), whereas the lowest diversity (PPB=0) was observed in FJSM and ZJRG populations. The genetic differentiation coefficient (Gst=0.8658) and genetic flow (Nm=0.0075) indicated that the genetic diversity among populations was higher than that of within populations. A well-separated groups coupling with cultivated species was formed according to the genetic distance (GD) ranging from 0.0498-0.4879 of the investigated populations. Twenty-one investigated cultivars could be distinguished and be effectively grouped into four origins, i.e., Dioscorea opposita Thunb., Dioscorea alata Linn., Dioscorea persimilis Prain et Burkill. and Dioscorea fordii Prain et Burkill.. CONCLUSION: The genetic diversity level of yam resources is high, and relatively high genetic variation exists among four yam species. SRAP marker is an effective method to identify and classify numerous yam cultivars.

Genetic diversity of Lonicera macranthoides geographical populations revealed by SRAP markers / 中草药

Objective: To research genetic diversity of different Lonicera macranthoides geographical populations. Methods: Seventeen materials were estimated by the approach of sequence-related amplified polymorphism (SRAP). The data of amplified bands were analyzed by Popgene 1.31 and Treeconw software. The system diagram of genetic relationship was built by UPGMA. Results: The 24 pairs of SRAP primers combination employed produced a total of 239 discernable and reproducible amplified fragments. Among them there were 210 polymorphic bands. The percentage of polymorphic bands within different populations was 87.8%; Genetic diversity analysis showed that Nei's gene diversity (He) was 0.239 9 and Shannon's genetic diversity index (I) was 0.372 4. Basis on the DNA molecular level, the results indicated that there was abundant genetic diversity among the tested materials. Genetic similarity coefficient ranges changed from 0.442 3 to 0.940 2. The dendrogram including all samples was obtained by UPGMA. In the dendrogram, there were two cluster groups. Conclusion: The genetic diversity of L. macranthoides geopraphical population in China is plentiful. SRAP markers can be effectively applied to genetic analysis in L. macranthoides populations.

Genetic diversity of Liriope muscari using SRAP markers / 中草药

Objective: To study the genetic diversity of Liriope muscari. Methods: The genetic diversity of 47 populations was analyzed by SRAP marker technique. Results: Fifteen primers amplified 323 polymorphic bands, and the average percentage of polymorphic bands reached to 88.47%. The average of polymorphism information content (PIC) was 0.90. Nei's gene diversity index (H) was 0.197 7 and Shannon's information index (I) was 0.319 0. Gene differentiation index (Gst) was 0.252 8. Cluster analysis using UPGMA method showed that genetic similarity coefficient ranged in 0.59-0.99. Conclusion: L. muscari shows higher genetic diversity and the majority of genetic variation occurs in populations.

Optimization of sequence-related amplified polymorphism system in Dendrobium nobile based on orthogonal design / 中草药

primer.Conclusion The present reaction system could provide clear bands,abundant polymorphisms,and reliable reaction.It is proved to be suitable for molecular biology research of D.nobile.

SRAP Analysis of genetic diversity about germplasm in Salvia miltiorrhiza from different sources / 中草药

Objective To solve the key problem on classification and identification of Salvia miltiorrhiza,which was urgently needed to be solved in breeding of S. miltiorrhiza. Methods Sequence-related amplified polymorphism (SRAP) was carried out to analyze the genetic variation of 48 germplasm in S. miltiorrhiza from different sources. Results The data showd that 120 alleles had been found using 15 pairs of primer which had been selected from 100 pairs. Unweighted pair-group method with arithmetical averages (UPGMA) cluster analysis was used to classify the germplasm of S. miltiorrhiza. Two groups could be divided and three subgroups were contained in every group. Conclusion A complex relationship exists between the genetic distance and space distance of differernt S. miltiorrhiza. The genetic diversity in different geographical populations of S. miltiorrhiza in China is rich. The results show that the genetic distance of different germplasm will increase with the stage of domestication.

Genetic relationship among populations of cultivated Coptis chinensis revealed by SRAP / 中草药

Objective To reveal the genetic relationship among populations of cultivated Coptis chinensis.Methods Twenty four populations of cultivated C.chinensis from different habitats were employed to be analyzed by the approach of sequence-related amplified polymorphism(SRAP).Systematic relationship was constructed based on the UPGMA method by Treeconw software.Results A total of 276 bands were scored,among which 120 were polymorphic bands.The average percentage of polymorphic bands was 43.48%,indicating that the materials in the test have low genetic diversity.Genetic similarity coefficients were changed from 0.877 0 to 0.951 9.By cluster analysis,the geographical distribution was not very obvious,but it was also showed some of the cultivated C.chinensis from the same region were in the same group.Conclusion Different germplasms diversity of cultivated C.chinensis population is lower and genetic background is more single.

Establishment and optimization of SRAP reaction system in Tinospora sagittata / 中草药

Objective To develop a new method for Tinospora sagittata species identification and genetic map construction. Methods Sequence-related amplified polymorphism (SRAP) was applied for T. sagittata to carry on PCR amplification of its DNA and optimize the reaction parameter grade by grade. Results The stable and reproducible SRAP reaction system of T. sagittata has been developed. Conclusion SRAP is an effective method for T. sagittata identification in molecular degree and it has set up a foundation for the further species identification and genetic map construction of T. sagittata.

Establishment and optimization of sequence-related amplified polymorphism amplification system for Carthamus tinctorius L / 第二军医大学学报

Objective:To study the factors influencing the amplification of sequence-related amplified polymorphism(SRAP) system in Carthamus tinctorius L.and to establish a stable SRAP reaction system,laying a foundation for molecular marker assistant breeding of Carthamus tinctorius L.Methods: Cetyltrimethylammonium bromide method was used to extract the genomic DNA of Carthamus tinctorius L..Twenty-seven tests with 3 factors at 3 levels were designed,the conditions including Taq polymerase concentrations(0.02,0.04,0.06 U/?l),dNTP concentrations(0.15,0.25,0.30 mmol/L)and Primer concentrations(0.15,0.30,0.45 ?mol/L);another 8 tests were designed based on the single factor of Mg~(2+) concentrations(0.5,1.0,1.5,2.0,2.5,3.0,3.5 and 4.0 mmol/L).Twenty ng DNA template was added into 25 ?l SRAP reaction system;the system was optimized and agarose electrophoresis was used for determination.Results: A SRAP reaction system for Carthamus tinctorius L.was established.In a 25 ?l reaction system,Taq polymerase was 0.02 U/?l,dNTP was 0.25 mmol/L,Primer was 0.30 ?mol/L,and Mg~(2+) was 3.0 mmol/L.Target bands increased after optimization,with good reproducibility,and the amplification results were satisfactory.Conclusion: The SRAP reaction system in this experiment is suitable for analysis of Carthamus tinctorius L.