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Objective: To make a distinction between Ludisia discolor and its relatives genus in molecular level, SCoT markers were employed to assess the genetic relationship and construct the DNA fingerprint. Methods: Orthogonal design method were carried out to optimize the suitable SCoT-PCR reaction system based on five factors. The optimum annealing temperature and SCoT primers were also screened. The 12 germplasm resources were used as materials, the screened primers were selected to analyze the genetic relationship of 12 materials. POPGENE was used to calculate the genetic diversity, NTSYS was performed to analyze cluster, and DNA map was constructed. Results: The optimized SCoT-PCR reaction system was constructed and a total of 12 rich bands were screened out as the primers of SCoT molecular marker with polymorphism ratio of 98.98%. According to Nei's genetic similarity coefficient, a total of 12 materials were divided into three cluster when coefficient was 0.45. Goodyera schlechtendaliana was in category I with seven L. discolor lines, indicating that these samples had close relationship. In category II, there were three samples came from Anoectochilus roxburghii. Moreover, a green L. discolor sample was alone clustered into category III. The DNA fingerprint map by the SC8 primer could identify the 12 materials. Conclusion: There are rich genetic diversities in 12 samples of L. discolor and its relatives genus, and the construction of DNA fingerprint map provides a theoretical basis for the identification of L. discolor and its relatives genus, which were tested in this study.
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Aim: The present study was undertaken to analyze the extent of genetic variability existing among twenty accessions of Lawsonia inermis, collected from Rajasthan and Gujarat states of India, using gene targeted SCoT, arbitrarily amplified ISSR and nuclear rDNA markers. Methodology: Twenty henna accessions, vegetatively established at the Institute were collected from Rajasthan (7) and Gujarat (13). Twenty-six SCoT and twenty ISSR markers generating distinct, unambiguous and scorable fragments were selected, after preliminary screening for assessment of genetic diversity. Data analysis was performed using NTSYS-pc, GenAlEx 6 and POPGENE version 1.31 programs, and dendrograms were generated using unweighted pair group method for arithmetic mean (UPGMA). The internal transcribed spacer (ITS) region of ribosomal DNA was amplified using universal primers followed by sequencing and dendrogram generation. Results: SCoT markers revealed lower values of similarity coefficients ranging from 0.87 - 0.93 compared to 0.93 - 0.98 for ISSR. SCoT markers delineated the L. inermis cultivars into three distinct clusters while ISSR markers demarcated them into five clusters. Interpretation: The Gujarat population of L. inermis was richer in genetic diversity than that of Rajasthan. SCoT markers proved better than the ISSR markers for genetic diversity analysis. Substantial variation in ITS-1 region due to SNPs, INDELS and ITS length polymorphism the nucleotide sequences signified its phylogenetic utility in assessing genetic diversity in of L. inermis.
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Objective: To study the genetic diversity and genetic structure of Paris genus in Shaanxi Province. Methods: Start Codon Targeted Polymorphism (SCoT) molecular markers were performed on 48 samples from six taxas of Paris genus in Shaanxi. Genetic distance was calculated by POPGENE 3.2 software, and cluster analysis was generated by NTSYS 2.10 software based on unweighted mean distance method (UPGMA) method. Results: A total of 152 bands were produced by 12 primers, among which 135 bands were polymorphic bands, and the percentage of polymorphic bands was 88.82%. The average value of Nei’s genetic diversity index (H) was 0.267 4, Shannon’s information index (I) was 0.404 1, genetic differentiation coefficient (Gst) was 0.517 9, and the gene flow (Nm) was 0.465 4. Six taxas were ranked by genetic diversity: Paris polyphylla > Paris polyphylla var. latifolia > Paris fargesii var. petiolata > Paris polyphylla var. stenophylla > Paris polyphylla var. yunnanensis > Paris verticillata. Cluster analysis distinguished others from Paris verticillata; When the genetic distance was certain, six taxas of the genus were completely separated. Conclusion: SCoT molecular markers can obtain polymorphic and clear band amplification map, which indicates that the technology can be used for molecular identification and genetic relationship of major taxas in Paris genus in Shaanxi Province, providing a scientific basis for screening alternative resource types close to the Pharmacopoeia collection and guiding the rational use of local species.
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ABSTRACT: The objective of this study is to research the genetic diversity of the ' Zuijinxiang ' grape and its mutant breeding F1 plants, we screened the excellent mutant plants with potential breeding value. 50 mutated single plants obtained from 137Cs-γ irradiated 'Zuijinxiang' grape seeds were used as research objects, and SCoT molecular marker technology was used for genetic diversity and variation analysis, and clustering research was carried out. The results showed that: (1) 36 SCoT primers produced abundant polymorphisms, and the amplification results showed obvious bright bands, and the amplification efficiency and polymorphism rate were 100%. (2) A total of 221 bands were amplified by 36 primers, of which 175 were rich in polymorphism, the average polymorphic percentage was 80.3%, and the average genetic similarity coefficient was 0.916. (3) The number of observed alleles (Na) ranged from 4 to 8, with an average of 6.1389; the number of effective alleles (Ne) ranged from 1.2772 to 5.6322 with an average of 3.5968; the desired heterozygosity (He) The range is from 0.2192 to 0.8344, the average is 0.6965; the observed heterozygosity (Ho) ranges from 0.1656 to 0.7808 with an average of 0.3035; the Nei's gene diversity index (H) ranges from 0.2170 to 0.8224 with an average of 0.6863; Shannon-Wiener The index (I) ranges from 0.5186 to 1.8597 with an average of 1.4517. (4) UPGMA clustering of 51 materials showed that the test materials could be divided into three groups when the genetic distance was 0.856. The experiment shows that the genetic diversity of the 'Zuijinxiang' radiation variation germplasm resources is rich. In addition, SCoT molecular marker technology can distinguish the materials with close genetic distance, and can be used for early identification techniques of grape mutant materials. This study provides a theoretical basis for the development of excellent mutant germplasm of 'Zuijinxiang' grapes.
RESUMO: O objetivo deste estudo é investigar a diversidade genética da uva 'Zuijinxiang' e de suas plantas F1 reprodutoras mutantes. Foram selecionadas as melhores plantas mutantes com potencial e valor genético. Utilizaram-se como objeto de pesquisa 50 plantas individuais mutantes obtidas de sementes de uva irradiadas com 137Cs-γ 'Zuijinxiang', e a tecnologia de marcadores moleculares SCoT para análise de diversidade genética e variação, e foi realizada uma pesquisa de agrupamento. Os resultados mostraram que: (1) 36 iniciadores de SCoT produziram polimorfismos abundantes, e os resultados de amplificação mostraram bandas claras óbvias, e a eficiência de amplificação e taxa de polimorfismo foram de 100%. (2) Um total de 221 bandas foi amplificado por 36 iniciadores, dos quais 175 eram ricos em polimorfismo, a porcentagem polimórfica média foi de 80,3% e o coeficiente médio de similaridade genética foi de 0,916. (3) O número de alelos observados (Na) variou de 4 a 8, com uma média de 6,1389; o número de alelos efetivos (Ne) variou de 1,2772 a 5,6322 com uma média de 3,5968; a heterozigosidade desejada (He), o intervalo é de 0,2192 a 0,8344, a média é de 0,6965; a heterozigosidade observada (Ho) varia de 0,1656 a 0,7808 com uma média de 0,3035; o índice de diversidade genética (H) de Nei varia de 0,2170 a 0,8224 com uma média de 0,6863; Shannon-Wiener o índice (I) varia de 0,5186 a 1,8597 com uma média de 1,4517. (4) O agrupamento de 51 materiais da UPGMA mostrou que os materiais de teste poderiam ser divididos em três grupos quando a distância genética era de 0,856. O experimento mostra que a diversidade genética dos recursos de germoplasma de variação de radiação "Zuijinxiang" é rica. Além disso, a tecnologia de marcadores moleculares da SCoT pode distinguir os materiais com uma distância genética próxima, e pode ser usada para técnicas de identificação precoce de materiais mutantes da uva. Este estudo fornece uma base teórica para o desenvolvimento de germoplasma mutante excelente de uvas "Zuijinxiang".
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OBJECTIVE: To identify Belamcandae Rhizoma, Iridis Tectori Rhizoma and their adulterants by ISSR and SCoT markers. METHODS: The genome of Belamcandae Rhizoma, Iridis Tectori Rhizoma and its adulterants were amplified by the optimized ISSR and SCoT PCR conditions and screened primers. Genome polymorphism analysis and the dendrogram construction were calculated by the POPGENE 1.32 and the NTSYS-pc version 2.10 respectively. RESULTS: The 130 and 143 bands were amplified by the screened ISSR and SCoT primers respectively. The percentage of polymorphic bands were 96.2% and 97.9% respectively. Shannon diversity index(I) were 0.540 2 and 0.500 3, Nei's gene diversity index (H) were 0.363 0 and 0.327 3 respectively. The genetic distance calculated based on SCoT marker was higher than that of ISSR marker, which suggested that Elamcandae Rhizoma, Iridis Tectori Rhizoma and their adulterants have great genetic diversity. Cluster analysis based on SCoT genetic similarity indicated that Belamcandae Rhizoma, Iridis Tectori Rhizoma formed independent group with far relationship among their adulterants. CONCLUSION: Belamcandae Rhizoma, Iridis Tectori Rhizoma and their adulterants could be distinguished stablely, fastly and clearly by SCoT marker. The ISSR marker could only be used for identification of Belamcandae Rhizoma and its adulterants. The SCoT marker is better suitable for genetic diversity research on the low taxonomic category in genus Iris.
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OBJECTIVE:To establish PCR reaction system of DNA of Marchantia convoluta in Guangxi marked with SCoT polymorphism marker technique by screening primer and optimizing reaction condition. METHODS:Modified CTAB method was used to extract DNA of M. convolute from Guangxi;gel electrophoresis and UV spectrophotometry were used to investigate purity and concentration of DNA. Using sample DNA as template,PCR amplification of 36 SCoT primers was conducted,and suitable primers were screened after electrophoresis,staining and imaging of products. The orthogonal experiment of 5 factors and 4 levels was conducted by 5 main factors as DNA concentration,Mg2 + concentration,dNTP concentration,primer concentration,Taq DNA polymerase concentration. The condition of SCoT-PCR reaction system was optimized. RESULTS:Extracted sample DNA bands were neat without RNA contamination,degradation or dispersion of fluorescence;sample well was clear. UV absorbance ratio ranged 1.7-2.0 at 260 nm and 280 nm;purity and concentration of DNA were both suitable for follow-up test. PCR results of 36 primers showed that product band of No. 4 primer was neat without diffuse fluorescence but with best luminance,so No. 4 primer was used for PCR reaction. The optimal SCoT-PCR reaction system contained 30.00 μg/mL DNA,2.00 mmol/L Mg2+,0.20 mmol/L dNTP, 0.40 μmol/L primer,0.50 U/mL Taq DNA polymerase(total reaction volume of 20 μL). CONCLUSIONS:Suitable SCoT-PCR primer of DNA is screened,and reaction system is optimized. It provides technologic basis for variety identification and genetic relationship analysis of M. convoluta in Guangxi.
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Objective To detect the genetic relationship among nine medicinal species of Dipsacus in China. Methods The polymorphic bands were detected by three labeling methods (ISSR, SCoT, and SRAP). The genetic distances were calculated by TREECONW software and the systematic diagram of genetic relationship was clustered by UPGMA method. Results The percentage of polymorphism by ISSR, SCoT and SRAP markers showed little difference with value at 90.4%, 88.5%, and 88.2%, respectively, which indicated that the genetic polymorphism of the tested materials was very rich. The genetic distance between Dipsacus chinensis and D. japonicus was the largest, which indicated their genetic relationship was the most distant. Nine medicinal species of Dipsacus were all divided into three groups by three markers, that D. chinensi and D. lijiangensis, D. asperoides and D. japonicas were respectively clustered into one group, D. asperoides var. omiensis was alone clustered, the other four species were clustered into one group. The clustering results labeled by ISSR and SRAP were basically the same with a slight difference between D. daliensis and D. asperoides. Conclusion The clustering results by there markers was between well consistent with the classical classification, which confirmed that molecular markers can be used as one of the effective methods to reveal the genetic relationship among medicinal species of Dipsacus.
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Objective To carry out genetic polymorphism analysis of Citrus reticulata Blanco cv. chachiensis Tanaka and its relatives by using SCoT molecular marker method. Methods Five factors of Mg2+, dNTPs, TaqDNA polymerase, primer, and template DNA concentration were used to screen the suitable SCoT-PCR reaction system for C. reticulata and its relatives by the method of orthogonal design. The optimum annealing temperature was screened by gradient temperature, and its polymorphic primers were verified. Results A total of 12 clear and rich bands were finally screened out as the primers of SCoT molecular marker for C. reticulata and its relatives in the optimized PCR reaction system, and the genetic distance and the UPGMA clustering tree of C. reticulata and its relatives were got by NTSYS software analysis. Conclusion The optimized SCoT-PCR reaction system was validated by using three different places of citrus genomic DNA to obtain the polymorphism and the clear amplified bands, which showed that the SCoT molecular marker system of Citrus reticulata is stable and reliable. The results of cluster analysis were able to make a preliminary separation of 13 kinds of materials scientifically and intuitively in molecule level.
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Objective: The genetic variation and relationship of 47 pieces of Gardenia jasminoides germplasms were analyzed by SCoT molecular markers. Methods: The genetic similarity coefficient was calculated by NTSYS version 2.1 software and the dendrogram was constructed by UPGMA method. Results: More than 20 polymorphic primers were screened from 36 primers and PCR amplificated to test materials. The primers generated totally 154 bands among which 120 bands were found to be polymorphic, with an average of 78.14%. The genetic similarity coefficient among the germplasms ranged from 0.655 8 to 0.980 5, and the average content was 0.784 1. The clustering results showed that the genetic diversity of G. jasminoides was rich and the genetic relationship was complex. Conclusion: SCoT markers were feasible and effective to analyze the genetic diversity of G. jasminoides germplasm resources. The results provide a reliable theoretical basis for breeding, classification, preservation and utilization of G. jasminoides.
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Rubia cordifolia L. has been used as a traditional Chinese medicine for several centuries. In recent years, the resources of wild Rubia cordifolia have been declined sharply due to increased utilization and rising price. Therefore, it is of great urgency to evaluate and protect resources of wild plant of Rubia cordifolia. In our study, sixty-four individuals that represent eight wild populations of Rubia cordifolia L. were analyzed by Start Codon Targeted Polymorphism (SCoT) molecular markers. Genetic distance was calculated by POPGENE 3.2 software, cluster analysis was generated by NTSYS 2.10 software based on UPGMA method and Mantel Test was used to analysis the relationship between the genetic distances and geographical distance among the wild populations. The results showed a high genetic diversity of wild populations of Rubia cordifolia L. in Shaanxi province. A total of 182 bands were produced by 14 primers, among which 163 bands were polymorphic bands, and the percentage of polymorphic bands was 89.56%. The average value of Nei's genetic diversity index (H) was 0.293 6, Shannon's information index (I) was 0.444 6, genetic differentiation coefficient (Gst) was 0.555 3, and the gene flow (Nm) was 0.440 8, the wild populations were ranked by genetic diversity:AK > YL > SL > BJ > TC > YA > WN > XY. Mantel Test analysis demonstrated that the significant correlation was found between the genetic distances and geographical distances (r=0.776 4, PRubia cordifolia L. germplasms.
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The genetic diversity and genetic relationship among four medicinal species of Coptis were detected by the approach of sequence-related amplified polymorphism (SCoT). The associated genetic parameters were calculated by POPGENE1.31. The systematic diagram of genetic relationship were clustered by TREECONW. The results showed that a total of 434 bands were produced by using 28 primers, of which 430 were polymorphic loci. There was a high level of genetic diversity among species (PPB=99.1%,Na=1.990 6,Ne=1.329 3,H=0.212 2,I=0.337 8). However, genetic diversity was lower within species, the average of genetic parameters wasPPB=16.8%,Na=1.168 2, Ne=1.073 0,H=0.043 7,I=0.067 7. The Nei's genetic differentiation coefficient was 0.794 0, that indicated that most of the genetic variation existed among species. By clustering analysis, different individuals gathered in the same group. The results confirmed that SCoT marker can be used as one of the effective methods to reveal the genetic diversity and relationship among medicinal species of Coptis.