Genetic Diversity Analysis of Isatis indigotica Germplasm Materials by Chloroplast DNA Variation and ISSR Molecular Marker / 中国实验方剂学杂志

ObjectiveTo analyze the sequence variation and genetic diversity of 47 Isatis indigotica germplasm materials， and carry out the study on the genetic differentiation and structure. MethodGenomic DNA of 47 I. indigotica germplasm materials were extracted by kit extraction method. Two chloroplast DNA （cp DNA） sequences and five inter-simple sequence repeat （ISSR） primers were used for amplification and sequencing. Chromas， Mega 7.0， DanSP5， and GenALEx were used to calibrate， splice， and analyze the sequence characteristics. PERMUT and PopGen 1.31 were used to analyze the genetic diversity parameters and genetic structure， and NTSYS was used to obtain the unweighted pair-group method with arithmetic means（UPGMA） clustering tree plot of 47 I. indigotica germplasm materials. ResultA total of 129 samples from 47 I. indigotica germplasm materials were successfully amplified and sequenced. The length of 2 cp DNA sequences after spliced was 1 412 bp， and there were 377 polymorphic variation loci， and 36 haplotypes. Fu and Li's D* test was significant （P<0.01）. The values of Pi， HS， and HT based on cp DNA were 0.119 89， 0.787， and 0.891， respectively. The genetic differentiation coefficients of gene differentiation coefficient（Gst）， nucleotide differentiation coefficient（Nst）， and fixation index（Fst） were 0.117， 0.468， and 0.488， respectively， and the gene flow （Nm） was 0.615. The mean values of PPB， Shannon information diversity index（I）， Nei's genetic diversity index（H）， and Gst based on ISSR were 78.85%， 0.334 8， 0.218 6， and 0.754 4， respectively， and the Nm value was 0.162 8. ConclusionI. indigotica has high genetic diversity and abundant haplotypes at the species level， with abundant haplotypes. Genetic differentiation among different germplasm materials is obvious， and gene exchange is not frequent. Genetic variation mainly exists among populations. The population has accumulated various low-frequency gene mutations recently， suggesting that it has experienced significant regional expansion in the history.

Genetic Diversity Analysis of Isatis indigotica Germplasm Materials by Chloroplast DNA Variation and ISSR Molecular Marker / 中国实验方剂学杂志

ObjectiveTo analyze the sequence variation and genetic diversity of 47 Isatis indigotica germplasm materials， and carry out the study on the genetic differentiation and structure. MethodGenomic DNA of 47 I. indigotica germplasm materials were extracted by kit extraction method. Two chloroplast DNA （cp DNA） sequences and five inter-simple sequence repeat （ISSR） primers were used for amplification and sequencing. Chromas， Mega 7.0， DanSP5， and GenALEx were used to calibrate， splice， and analyze the sequence characteristics. PERMUT and PopGen 1.31 were used to analyze the genetic diversity parameters and genetic structure， and NTSYS was used to obtain the unweighted pair-group method with arithmetic means（UPGMA） clustering tree plot of 47 I. indigotica germplasm materials. ResultA total of 129 samples from 47 I. indigotica germplasm materials were successfully amplified and sequenced. The length of 2 cp DNA sequences after spliced was 1 412 bp， and there were 377 polymorphic variation loci， and 36 haplotypes. Fu and Li's D* test was significant （P<0.01）. The values of Pi， HS， and HT based on cp DNA were 0.119 89， 0.787， and 0.891， respectively. The genetic differentiation coefficients of gene differentiation coefficient（Gst）， nucleotide differentiation coefficient（Nst）， and fixation index（Fst） were 0.117， 0.468， and 0.488， respectively， and the gene flow （Nm） was 0.615. The mean values of PPB， Shannon information diversity index（I）， Nei's genetic diversity index（H）， and Gst based on ISSR were 78.85%， 0.334 8， 0.218 6， and 0.754 4， respectively， and the Nm value was 0.162 8. ConclusionI. indigotica has high genetic diversity and abundant haplotypes at the species level， with abundant haplotypes. Genetic differentiation among different germplasm materials is obvious， and gene exchange is not frequent. Genetic variation mainly exists among populations. The population has accumulated various low-frequency gene mutations recently， suggesting that it has experienced significant regional expansion in the history.

A simple and cost-effective method for DNA extraction suitable for PCR in "sucupira branca"

"Sucupira branca" is a plant found in the Brazilian Cerrado and is adapted to low fertility soils, and its fruit extract has anti-inflammatory, healing, antiulcerogenic, antimicrobial, cercaricidal, leishmanicidal and antioxidant activities. Furthermore, it provides protection against oxidative stress, is a natural biocontrol agent of Aedes aegypti, has very resistant wood, is a melliferous plant and has been used in reforestation programs. The development of conservation strategies is important for maintaining diversity in natural populations of "sucupira branca" since these populations are in the process of genetic erosion. Molecular biology techniques, which are important for characterizing the genetic diversity of plants to develop conservation strategies, require sufficient high-quality genomic deoxyribonucleic acid (DNA). This study aimed to compare five methods to extract DNA from "sucupira branca". The quality and concentration of DNA were revealed by agarose gel electrophoresis, and only the protocols of Dellaporta, Wood and Hicks et al. (1983) and Khanuja et al. (1999) did not result in satisfactory quantities of DNA. When PCR (Polymerase Chain Reaction) was performed with three inter-simple sequence repeat (ISSR) primers, DNA was successfully amplified from extractions performed with the protocols proposed by Doyle and Doyle (1987), Romano and Brasileiro (1998) and Ferreira and Grattapaglia (1995), which are less expensive than commercial purification kits. These protocols resulted in DNA of sufficient quality and quantity after the amplification reactions were performed.

Study on Sparganium stoloniferum from different regions in China based on appearance characters and ISSR markers / 中国中药杂志

Based on the characteristics and ISSR molecular marker technology, the study is aimed to compare and perform genetic diversity analysis on Sparganium stoloniferum from 7 regions. Molecular identification method was established for S. stoloniferum from Hunan province. Differences among Sparganii Rhizoma samples from seven habitats were analyzed via measuring weight, length, width and thickness of them. Genetic diversity of S. stoloniferum from 7 regions was analyzed by screening out primers amplifying clear band and showing rich polymorphism, then a cultivars dendrogram was built. The target primer was screened out, and the specific band was sequenced. Nine ISSR primers were selected to amplified clear band, rich polymorphism. A total of 73 bands were amplified by nine ISSR primers selected from 27 ISSR primers. On average, each primer produced 8.0 bands. A total of 38 bands were polymorphic, which occupied 52.8% of all bands. The cultivars dendrogram showed the genetic similarity was 0.54-0.94. Genetic similarity coefficient of S. stoloniferum from Jiangsu province, Anhui province and Jiangxi province was big, indicating the differences among them were slight on genetic level. S. stoloniferum from Hunan province is quite different from samples from the other six habitats on appea-rance and genetic level. A specific band(327 bp) in S. stoloniferum from Hunan province was obtained via ISSR-857 primer, and was sequenced. According BLASTn database, there were few sequences similar to the gene fragment and had little correlation with the growth process of plant. ISSR molecular marker technology provides a new idea for the identification of S. stoloniferum. This result confirmed the particularity of S. stoloniferum from ancient Jingzhou.

Molecular characterization of phosphate solubilizing fungi Aspergillus niger and its correlation to sustainable agriculture

Aim: The present study aimed to investigate the phosphate solubilization potential of agriculturally important fungi, i.e., Aspergillus sp. isolated from the rhizosphere of healthy plants in Abha city, Saudi Arabia.Methodology: Sixteen Aspergillus sp. isolated and tested for phosphate solubilization potential were identified by 5.8S-ITS region sequencing and characterized by 11 ISSR-PCR markers. Finally, the highest phosphate solubilization potential isolates were used in field experiments on cucumber and tomato plants. Results: All Aspergillus niger isolates showed 96–100% similarity to A. niger strains available at GenBank database, Isolate ASAB-5 was most efficient at solubilizing phosphate on Pikovskaya’s medium, with a solubilization index of 2.67, and 235.22 mg l-1 of solubilized phosphate. ISSR-PCR markers revealed is total 142 bands in all isolates, with about 32.3% showing monomorphism and 67.6% polymorphism. Based on genetic similarity and intraspecies variability, the Aspergillus isolates were grouped into two different clusters with about 67.9% genetic similarity. The results of field experiments showed no significant difference between seeds treated with culture filtrate or conidial suspension of ASAB-5; however, both differed remarkably from untreated seeds. Interpretation: The current study confirms the existence of several useful phosphate solubilizing fungi in plants, which may serve as potential biological fertilizers. They are safer than chemical fertilizers and increase the bioavailability of soil phosphates for plants

SSR and ISSR markers in assessing genetic diversity in Gallus gallus domesticus: a quantitative analysis of scientific production / Marcadores SSR e ISSR na avaliação da diversidade genética em Gallus gallus domesticus: uma análise quantitativa da produção científica

ABSTRACT: Poultry meat is a major source of animal protein in the world. Research indicates a high inbreeding rate derived from a relative absence of heterozygous subpopulations of chicken from different suppliers. Molecular markers can provide information for the genetic basis of chicken consumed in rural areas and help establishing a chicken database for product quality and warranty. The bibliometric research, comprises between 1994 and 2018, from five previously selected databases: Google Scholar, PubMed, ScienceDirect, Scopus and Web of Science, using the following descriptors: 'microsatellites', 'SSR', 'ISSR', 'genetic variability' and 'genetic diversity', all of them coupled to 'chicken' and/or 'birds' results in 66 scientific publications. The publications were then categorized according to their titles to the use of ISSR or SSR markers. They were also addressed by countries according first author cited. The publications data appointed that countries with the height production of poultry meat and hens are the most interested in the genetic diversity study of these species. The SSR markers, due to its more specific characteristic, are more frequently applied to genetic diversity assignment, compared to ISSR.

RESUMO: A carne de frango é uma das principais fontes de proteína animal do mundo. Pesquisas indicam uma alta taxa de endogamia derivada de uma relativa ausência de subpopulações heterozigotas de frango de diferentes fornecedores. Marcadores moleculares podem fornecer informações para a base genética de frango consumido em áreas rurais, e ajudar a estabelecer um banco de dados de frango para qualidade e garantia do produto. A pesquisa bibliométrica compreende entre 1994 e 2018, a partir de cinco bancos de dados selecionados anteriormente: Google Scholar, PubMed, ScienceDirect, Scopus e Web of Science, usando os seguintes descritores: 'microssatélites', 'SSR', 'ISSR', 'variabilidade genética' e 'diversidade genética', todos eles associados a resultados de 'galinha' e / ou 'aves' o que resultou em 66 publicações científicas. As publicações foram então categorizadas de acordo com seus títulos para o uso de marcadores ISSR ou SSR. Eles também foram abordados pelos países, segundo o primeiro autor citado. Os dados das publicações obtidas apontam que os países com grande produção de carnes de frangos são os mais interessados no estudo da diversidade genética dessas espécies. Os marcadores SSR, devido à sua característica mais específica, são frequentemente aplicados à atribuição de diversidade genética, em comparação com o ISSR.

Study on genetic diversity and genetic variation law of Polygonatum cyrtonema / 中草药

Objective: To study the genetic diversity and geographical distribution of Polygonatum cyrtonema resources. Methods: ISSR technique was applied to analyze 118 individuals from 20 P. cyrtonema provenances in six provinces, including Anhui, Jiangxi, Fujian, Hunan, Hubei, and Zhejiang. Results: The results showed that 130 clear bands were amplified by 16 primers with 123 polymorphic bands and the average percentage of polymorphic loci (PPL) was 94.62%, PPL within provenances was 33.85%-60.00%. Nei's genetic diversity index (He) was 0.183 8, Shannon's information index (I) was 0.267 4 and gene differentiation index (Gst) was 0.529 3. There were abundant genetic diversities existing in wild resources of P. cyrtonema. The UPGMA clustering analysis revealed that individuals from the same provenance were almost clustered together firstly, explaining that the genetic differentiation among different provenances was higher than those within provenances. When genetic similarity (GS) was 0.61, 118 germplasms can be divided into four categories, including Wuyi Mountains, Wuling and Luoxiao Mountains, Dabie Mountains, Donggong, and Tianmu Mountains. Conclusion: P. cyrtonema has high genetic diversity, genetic variation was closely related to mountains, and the isolation of plains and water areas between mountains was one of the main causes of genetic differentiation among groups. This study had essentially theoretical value and practical significance for the protection of the germplasm resources and the breeding of the species.

Genetic diversity analysis on cultivar and wild population of Rehmannia glutinosa based on ISSR markers / 中草药

Objective: To study the genetic diversity and genetic relationship of cultivar and wild population of Rehmannia glutinosa and its relative species by ISSR molecular marker technique, and provide the reference for R. glutinosa germplasm protection and breeding. Methods: 106 samples including cultivar and wild population of R. glutinosa and its relative species were studied by ISSR-PCR markers. Nei's genetic diversity index (H) and other parameters of genetic information were calculated by POPGEN 32, and a cluster dendrogram of different samples was established based on the unweighted pair-group method with arithmetic mean (UPGMA) by NTSYS-pc software. Results: Seven ISSR primers generated 85 loci of which 83 loci were polymorphic. The percentage of polymorphie bands (PPB) of all samples was 97.65%. Nei's genetic diversity index (H) and Shannon's information index (I) were 0.2659 and 0.4125. The percentage of polymorphie bands (PPB) of cultivar of R. glutinosa was 30.59%. The percentage of polymorphie bands (PPB) of wild population of R.glutinosa in Henan province was 83.53%. In the cluster dendrogram, all samples were clustered into seven groups at the level of Genetic similarity coefficient (GS) 0.67. Conclusion: The results of ISSR analysis revealed that the level of genetic diversity between wild populations of R. glutinosa was higher than that within cultivar populations of R. glutinosa. The genetic diversity among wild populations of R. glutinosa in Henan province was higher than other region, which was consistent with authentic producing areas of R. glutinosa in this area. The relationships of wild population of R. glutinosa had no obvious correlation with their geographical distribution pattern.

ISSR Analysis of Genetic Relationship and Population Structure of Turpinia arguta Fresh Leaves in Jiangxi Province / 中国实验方剂学杂志

Objective:To explore genetic relationship and population structure of Turpinia arguta in six locations of Jiangxi province by inter-simple sequence repeat （ISSR） molecular marker technique， and to provide theoretical basis for the protection and utilization of this medicinal material resource. Method:A total of 22 samples from six locations in four counties in Jiangxi province were collected， and genomic DNA was extracted by kit method. Polymerase chain reaction （PCR） amplification was performed using sixty-four universal ISSR molecular marker primers， and the products were detected with polyacrylamide gel electrophoresis （PAGE）. NTsys 2.10e software was selected to calculate the genetic similarity coefficient by unweighted pair group method with arithmetic mean （UPGMA） and cluster analysis. Population genetic structure was analyzed by Structure 2.1 software. Result:A total of forty-eight ISSR primers were amplified to obtain the product， the percent of polymorphic bands ranged from 45.45% to 100%. UPGMA cluster analysis showed that these plant individuals could not be clustered according to their respective executive locations. Analysis of population genetic structure showed that 22 samples of T. arguta could be divided into three populations. Conclusion:There is gene exchange among the populations of T. arguta in Jiangxi province， and it can affect the genetic structure of germplasm resources from different geographical sources.

Genetics characterization of fresh water clam Spathopsis rubens using ISSR-PCR markers

Spathopsis rubens is freshwater bivalves distributed in the Nile River and its main canals all over Egypt.Four populations of S. rubens on Al-Mahmoudia irrigation canal at Damnhour, Egypt were collected andinvestigated for morphometric characters and genetic diversity by using inter simple sequence repeats (ISSR).The results declared that both the length and height measurements of the collected samples from the differentlocations showed slight significant differences; however, no significant differences between the total weightand the width of the samples have found. High degree of correlation between temperature and total weight,height and length was reported in samples collected from the third location. Genomic DNA from the selectedsamples of each population was extracted and amplified using 10 ISSR primers. The primers (M2, M3, M8,M12, M17, F2, F4, and F9) showed 100% polymorphism. Unweighted pair group method with arithmetic meandendrogram characterized the samples of S. rubens into two main definite clusters. The cluster of genotypes2 and 3 recorded the highest similarity and distance indices at a distance of 0.60859, while genotypes 1 and 3recorded the lowest similarity and distance indices at a distance of 0.1716.

Genetic diversity in henna based on gene targeted SCoT, arbitrarily amplified ISSR and nuclear rDNA multiplicity

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.

Pharmacognostical study of Ochrosia elliptica Labill. (Apocynaceae)

Ochrosia elliptica Labill. is a small shrub belonging to family Apocynaceae and well-known as a promising anticancer agent. Botanical study of the plant was done for the young and old stems, stem bark, and leaves. Laticiferoustubes with yellowish brown content, isodiametric stone cells (sclereids), sclerenchyma (rod-like), and calcium oxalateclusters are the main diagnostic elements observed in this plant. Furthermore, DNA fingerprinting was done usingrapid amplified (RAPD) and inter simple sequence repeat (ISSR) techniques with the identification of a total of 30RAPD markers and 17 ISSR markers. Carbohydrates, sterols, catechol tannins, flavonoids, and alkaloids were presentin all the organs under investigation. This study could be valuable for quality control of the plant.

Genetic Diversity and Association Analysis between Medicinal Components and ISSR Markers in Astragali Radix / 中国药学杂志

OBJECTIVE: To study and exploit Chinese medicine Astragali Radix, the molecular markers that relates to the phenotypic traits on medicinal components of Astragali Radix and would be detected. METHODS: The genetic diversity of 43 Astragali Radix samples was analyzed with ISSR molecular marker technique and then the population genetic structure was studied through 13 selected markers. The association analysis between ISSR markers and 4 phenotypic traits of medicinal components were performed with GLM (general linear model) programs in Tassel 2.1. Certain genetic diversity was discovered among the 43 Astragali Radix samples. RESULTS: The genetic distance varied between 0.050 6 and 0.743 8, with an average of 0.274 1. Moreover, the cultivated Astragali Radix from Ningxia and Gansu province closely related to the wild Astragali Radix collected from Liupanshan town in Ningxia. On the other hand, No. 340 had the farthest relationship with other Astragali Radix. The content of polysaccharide, total saponins, total flavonoids, and Astragaloside IV ranged between 7.693-27.840 mg•g-1, 7.167-17.579 mg•g-1, 2.212-6.164 mg•g-1 and 6.070-107.920 μg•g-1, respectively. Meanwhile, linear regression analysis indicated that there was a significant positive correlation between the content of the total saponins and that of flavonoids (r=0.650 5,P=2.3×10-6<0.01), while the content of astragaloside had no significant correlation with that of polysaccharide, total saponins and total flavonoids. The population genetic structural analysis showed that the 43 samples were divided into 4 subgroups. There were total of 34 locus in 13 ISSR markers significantly associated (P<0.01) with the content of polysaccharide,total saponins, flavonoids and astragaloside , and the rate of explanation on the phenotype of related marker ranged from 8.14% to 51.39%. Among the locus, 15 were related with astragaloside content at interpretation rates above 30%, 1 with polysaccharide content an interpretation rate reached as high as 51.39% with high threshold (P<1×10-5). CONCLUSION: These results would provide supporting evidence for identification and protection of germplasm resources as well as molecular marker-assisted breeding.

Genetic diversity analysis of Gardenia jasminoides based on ISSR and SRAP molecular markers / 中草药

Objective To analyze of genetic diversity of Gardenia jasminoides and provide the information for the conservation and new variety breeding of G. jasminoides. Methods 12 ISSR primers and 9 primer combinations of SRAP were used to assess the polymorphisms, genetic diversity, and cluster analysis within 21 G. jasminoides materials from three populations. Results The results showed that 100 (80.00%) of 125 and 74 (92.50%) of 80 bands were polymorphic by ISSR and SRAP primers amplification, respectively. In ISSR results, the populations of species level of observed number of alleles (Na), effective number of alleles (Ne), Nei’s gene diversity (H), Shannon’s information index (I), total genetic diversity for species (Ht), and the mean heterozygosity with populations (Hs) were 1.461 3, 1.307 7, 0.173 1, 0.254 5, 0.239 1, and 0.173 1, respectively. Comparatively, for SRAP primers, the Na, Ne, H, I, Ht, and Hs value was 1.579 2, 1.342 1, 0.197 4, 0.295 9, 0.289 9, and 0.197 4, respectively. The coefficient of gene differentiation (Gst) for population was 0.276 2 and 0.318 9, which indicated that the within-population component accounted for 73.38% and 68.11%, respectively. The average mean of gene flow (Nm > 1) suggested that there certainly gene flow among the populations. UPGMA analysis showed that 21 samples were clustered into 2 branches, and a hierarchical dendrogram based on SRAP was more consistent with actual populations. Conclusion The gene diversity of G. jasminoides populations was high. The characteristics of genetic structure included genetic differentiation that occurs mainly within populations, which provided a reference for conservation and breeding of G. jasminoides germplasm.

Genetic diversity of Forsythia suspensa based on ISSR / 中草药

Objective: To investigate the genetic diversity of Forsythia suspensa from different populations. Methods: ISSR markers were used to analyze the genetic diversity of 25 populations of F. suspensa. Percentage of species level polymorphic bands (P), Nei’s genetic diversity index (H), and Shannon’s information index (I) of genetic information were calculated by POPGEN 32. UPGMA relationship dendrogram was clustered by NTSYS. Results: Thirteen primers produced 353 bands and P was 100%. H and I were 0.252 3 and 0.394 0, and genetic differentiation coefficient (Gst) and gene flow (Nm) were 0.331 8 and 1.007 0 within the population levels. The genetic distance varied from 0.031 0 to 0.155 5. NTSYSpc software was used to cluster the system. A total of 25 clusters were divided into two categories and seven groups. Cluster analysis was conducted among Forsythia individuals. A total of 195 samples were divided into two categories and five groups. Conclusion: The genetic diversity among the populations of F. suspensa is at higher level. However, the genetic distance among the Forsythia population is not related to the geographical distance, which is mainly dependent on the ecological factors and the growth environment.

Genetic diversity of endophytic fungi strains Fusarium proliferatum isolated from Belamcanda chinensis using ISSR and SRAP markers / 中草药

Objective: In order to understand the genetic diversity of endophytic fungi strain Fusarium proliferatum isolated from Belamcanda chinensis. Methods: A total of 52 ISSR primers and 90 SRAP primers were used to detect the genetic diversity among 17 F. proliferatum strains. Results: The results indicated that 27 ISSR primers and 38 SRAP primers were screened out for the genetic diversity analysis. 178 bands were amplified from 27 ISSR primers, among which 131 (63%) allelic variations were detected. However, 357 bands were amplified by 38 SRAP primers, among which 323 (91%) allelic variations were detected. The value of allelic polymorphism information content (PIC) of ISSR primers ranged from 0.19 to 0.91, with the average of 0.70 per primer. The value of PIC of SRAP primers ranged from 0.00 to 0.93, with the average of 0.72 per primer. The value of Nei's genetic similarity (GS) indexes of 17 strains based on ISSR, SRAP and ISSR + SRAP genetic locus varied from 0.73-0.99, 0.72-0.95 and 0.73-0.95, and with the average of 0.84, 0.85 and 0.85, separately. Cluster analysis showed that the 17 strains in this study could be clustered into three groups, three strains from the roots were clustered together, and F. proliferatum strains isolated from stems and leaves were gathered in other two groups. Cluster analysis revealed that genetic similarity of 17 strains were high, this suggested that the 17 strains had a near relationship, in accordance with the traditional morphology identification. Conclusion: The results show that the ISSR and SRAP technology is more efficient than traditional morphology identification. It is also found that ISSR and SRAP markers could more really reflect the genetic diversity of endophytic fungi strain F. proliferatum from B. chinensis, which can provide the basis for the application of molecular biotechnology in endophytic fungi of F. proliferatum from B. chinensis.

Analysis of the Phylogenetic Relationships of 3 Basic Plants of Tibetan Medicine “Dida”Based on ISSR Technology / 中国药房

OBJECTIVE： To study the phylogenetic relationships of 3 basic plants of Tibetan medicine “Dida”， such as Swertia puricea， Wertia mileensis， Halenia elliptica. METHODS： ISSR technology was used for PCR amplification of 9 samples of S. puricea （ZT-1 to ZT-5 from Gantongsi in Dali Cangshan， ZC-1 to ZC-4 from Binchuan county of Dali）， 2 samples of W. mileensis （QYD-1 to QYD-2） and 2 samples of H. elliptica （HM-1 to HM-2）. Using DNA genome of S. puricea as template， 8 primers were screened and used for PCR reaction. The PCR amplification products were read by hand， the original data matrix was established， and the polymorphic band ratio was calculated. At the same time， genetic similarity coefficient was calculated by using NTSYS 2.1 software， and UPGMA method was used to draw cluster diagram. RESULTS： A total of 113 clear and identifiable amplification product bands were obtained by 8 ISSR primers. The rate of polymorphic site was 100％. The genetic similarity coefficients for totally 13 samples of S. puricea， W. mileensis and H. elliptica ranged 0.301-0.500. Intraspecific genetic similarity coefficients for 9 samples of S. puricea ranged from 0.752 to 0.929. The cluster analysis showed， when the range line was 0.410， 13 samples could be divided into three groups， i.e. S. puricea， W. mileensis， H. elliptica； when the range line was 0.780， 9 samples of S. purpurea could be divided into 2 subgroups， one of which was only sample ZT-1 collected from Gantongsi in Cangshan， and the other contained the remaining 8 samples. CONCLUSIONS： ISSR technology can be used to identify S. punicea， S. glabra and H. elliptica at the molecular level. S. punicea has some genetic relationship with S. glabra and H. elliptica， but the genetic relationship is relatively distant and the genetic difference is large. S. punicea from two different locations in Dali area has little genetic difference and close relationship， but it shows abundant genetic diversity.

Genetic Characterisation of Honey Bees (Apis mellifera) Populations from Kurdistan Region of Iraq via ISSR Markers

In the present study, to investigate genetic diversity and phylogenetic relationships among honey bee populations of Iraq ISSR markers were used. Sampling was carried out during summer 2017 from 5 cities of Iraq (Dahuk, Arbil, Sulaymaniyah, Kirkuk, and Kafri). Total DNA was extracted from the head and thorax sections of each worker honey bee, using salting out method with minor modifications. PCR amplification of genomic DNA was performed using 10 ISSR marker primers (A1, A2, A3, A4, A5, A6, A7, A8, A9 and A10). The primers yielded 50 polymorphic bands and number of bands were variable from 8-12 (average 9.62), and percentage of polymorphic loci was 73.6. The estimated genetic diversity for the populations ranged from 0.39 in Kafri population to 0.47 in Arbil population, and total genetic diversity among loci was calculated as 0.47 while average within population genetic diversity was 0.44. GST value was 0.085. The Phylogenetic tree showed two main clusters; the first one comprised of three populations (Dahuk, Arbil, and Sulaymaniyah), and the second one included two communities (Kirkuk and Kafri). Heterozygosity values, Shannon index and the number of alleles of honey bee populations were minimal that could be caused by low definite geographic structure of honey bee populations. This research provided new information regarding genetic diversity in selected local honeybee in Kurdistan region of Iraq and will be useful for selection, future local biodiversity conservation and controlled breeding programs.

Comparison of the phenolic contents and epigenetic and genetic variability of wild and cultivated watercress (Rorippa nasturtium var. aquaticum L.)

Background: Epigenetic modifications are key factors modulating the expression of genes involved in the synthesis of phytochemicals. The knowledge of plant epigenetic and genetic variations can contribute to enhance the production of bioactive compounds. These issues have been little explored thus far in Rorippa nasturtium var. aquaticum L. (watercress), an edible and medicinal plant. The aim of the current study was to determine and compare the phenolic composition and epigenetic and genetic variations between wild and cultivated watercress. Results: Significant differences were found in the quantitative phenolic composition between wild and cultivated watercress. The eight primer combinations used in the methylation-sensitive amplification polymorphism (MSAP) method revealed different epigenetic status for each watercress type, the cultivated one being the most epigenetically variable. The genetic variability revealed by the EcoRI/MspI amplification profile and also by eight inter-simple sequence repeat (ISSR) primers was different between the two types of watercress. The results of the Mantel test showed that the correlation between genetic and epigenetic variations has diminished in the cultivated type. Cluster analyses showed that the epigenetic and genetic characterizations clearly discriminated between wild and cultivated watercress. Conclusions: Relevant chemical, epigenetic, and genetic differences have emerged between wild and cultivated watercress. These differences can contribute to fingerprint and develop quality control tools for the integral and safety use and the commercialization of watercress. The richness of epialleles could support the development of tools to manipulate the watercress epigenome to develop high bioproduct­producing cultivars

Morphometric and molecular differences among Calvertius tuberosus (Coleoptera: Curculionidae) populations associated with Andean and coastal populations of Araucaria araucana in the La Araucanía Region, Chile

Abstract Calvertius tuberosus (Curculionidae) lives exclusively on Araucaria araucana trees (commonly known as pehuen) in southern Chile. In this study, morphometric and molecular genetic analyses of Andean and coastal populations of C. tuberosus were performed to evaluate evolutionary divergence associated with the discontinuity of the Araucaria forest between the coastal and Andean regions. Specimens of C. tuberosus were collected in Nahuelbuta National Park, Villa Las Araucarias, and Malalcahuello National Reserve and were classified and stored at the Animal Biotechnology Researching Laboratory (LINBA), University of La Frontera, Temuco, Chile. Thirteen morphometric parameters and the expression patterns of ISSR (inter-simple sequence repeat) markers were analyzed. Morphometric data revealed high phenotypic similarity between coastal populations. The genetic analysis revealed a high similarity between coastal populations (genetic identity, 93%), which were differentiated from the Andean population (genetic identity, 84%). This study contributes new genotypic and phenotypic data for the C. tuberosus populations in forest ecosystems of A. araucana, and clarifies the associations between these characteristics and the geographic distributions of populations.