Genetic diversity analysis and fingerprinting of 175 Chimonanthus praecox germplasm based on SSR molecular marker / 生物工程学报

The elucidation of resources pertaining to the Chimonanthus praecox varieties and the establishment of a fingerprint serve as crucial underpinnings for advancing scientific inquiry and industrial progress in relation to C. praecox. Employing the SSR molecular marker technology, an exploration of the genetic diversity of 175 C. praecox varieties (lines) in the Yanling region was conducted, and an analysis of the genetic diversity among these varieties was carried out using the UPDM clustering method in NTSYSpc 2.1 software. We analyzed the genetic structure of 175 germplasm using Structure v2.3.3 software based on a Bayesian model. General linear model (GLM) association was utilized to analyze traits and markers. The genetic diversity analysis revealed a mean number of alleles (Na) of 6.857, a mean expected heterozygosity (He) of 0.496 3, a mean observed heterozygosity (Ho) of 0.503 7, a mean genetic diversity index of Nei՚s of 0.494 9, and a mean Shannon information index of 0.995 8. These results suggest that the C. praecox population in Yanling exhibits a rich genetic diversity. Additionally, the population structure and the UPDM clustering were examined. In the GLM model, a total of fifteen marker loci exhibited significant (P < 0.05) association with eight phenotypic traits, with the explained phenotypic variation ranging from 14.90% to 36.03%. The construction of fingerprints for C. praecox varieties (lines) was accomplished by utilizing eleven primer pairs with the highest polymorphic information content, resulting in the analysis of 175 SSR markers. The present study offers a thorough examination of the genetic diversity and SSR molecular markers of C. praecox in Yanling, and establishes a fundamental germplasm repository of C. praecox, thereby furnishing theoretical underpinnings for the selection and cultivation of novel and superior C. praecox varieties, varietal identification, and resource preservation and exploitation.

Methods for Identifying Seeds of Chinese Medicinal Materials： A Review / 中国实验方剂学杂志

Seeds are the source for the production of Chinese medicinal materials. The seed authenticity and quality of directly affect the effectiveness and safety of Chinese medicinal materials. The seed quality is faced with the problems such as mixed sources， existence of adulterants and seeds stocked for years， low maturity， and low purity. To ensure the high-quality and sustainable development of the Chinese medicinal material industry， it is urgent to standardize the seed market and identify and evaluate the quality of the seeds circulating in the market. Seed identification methods include visual inspection， microscopic observation， micro-character identification， chemical fingerprinting， molecular identification， electronic nose， X-ray diffraction， electrochemical fingerprinting， spectral imaging， and artificial intelligence. These methods have different application scopes and unique advantages and disadvantages. According to the different species of Chinese herbal medicines and different requirements of testing sites， suitable methods can be selected to achieve rapid and accurate identification with low costs. In the future， the seed identification methods should be developed based on emerging technologies with interdisciplinary knowledge， and intelligent， nondestructive， and single-grain detection methods are needed for the modern Chinese medicinal material industry. This paper introduces the seed identification technologies currently applied in research and production， compares the principles， applicability， advantages， and disadvantages of different technologies， and provides an outlook on the future development of seed identification technologies， aiming to provide a reference for the identification and quality evaluation of seeds of Chinese medicinal material.

Estudio Piloto Comparativo entre la Identificación Dactiloscópica y Poroscópica con Fines de Identificación Humana en el Perú / Comparative Pilot Study Between Dactyloscopy Identification and Poroscopy for Human Identification Purposes in Peru

La dactiloscopía o papiloscopía corresponde al estudio científico de las impresiones digitales, palmares y plantares, que tiene por finalidad la identificación infalible o indubitada del individuo. Existen tres niveles para identificar con mayor certeza nivel 1 (tipo o patrón dactilar), el nivel 2 (minucias o puntos característicos) y el nivel 3 (poroscopia y crestoscopia). Por ello, es necesario analizar las características de las impresiones dactilares directas y las huellas dactilares directas con la finalidad de verificar la presencia de puntos y poros característicos para mejorar el proceso de identificación humana. Se analizaron 80 muestras (54 mujeres y 26 hom- bres). A partir de ellos, se capturaron 800 impresiones y 800 huellas dactilares directas con tampón dactilar y polvo black. En huellas con tampón se identificaron 71.25 % y 1.25 % con 14 y 6 Puntos Característicos respectivamente y en grupos de poros el 84 % y 35 % para un grupo de 1 y grupos de 7 y 8 poros respectivamente. Con polvo black solo se identificaron Puntos Característico y no Poros. La cantidad de poros en hombres fue mayor igual a 10 (LR= 2.08) y en mujeres menor igual a 6 (LR= 1.93). Los grupos de poros fueron para hombres menores o iguales a 12 poros (LR= 1.04) y mayores o iguales a grupos de 13 poros (LR=1.28) para mujeres. Se consiguieron identificar grupos de poros con tampón dactilar pero no con polvos químicos lo que podría emplearse para implementar un protocolo para el uso del nivel 3 de identificación.

SUMMARY: Dactyloscopy or papiloscopy corresponds to the scientific study of digital, palmar and plantar impressions, whose purpose is the infallible or indubitable identification of a subject. There are three levels to identify with greater certainty level 1 (type or fingerprint pattern), level 2 (minutiae or characteristic points) and level 3 (poroscopy and crestoscopy). Therefore, it is necessary to analyze the characteristics of direct fingerprints and direct fingerprints in order to verify the presence of characteristic points and pores to improve the human identification process. 80 samples (54 women and 26 men) were analyzed. Of these, 800 impressions and 800 direct fingerprints with fingerprint buffer and black powder were captured. In footprints with buffer, 71.25 % and 1.25 % were identified with 14 and 6 Characteristic Points respectively and in groups of pores 84 % and 35 % for a group of 1 and groups of 7 and 8 pores respectively. With black powder, only Characteristic Points and no Pores were identified. The number of pores in men was greater than 10 (LR= 2.08) and in women less than 6 (LR= 1.93). The groups of pores were less than or equal to 12 pores (LR= 1.04) for men and greater than or equal to groups of 13 pores (LR=1.28) for women. It was possible to identify groups of pores with a fingerprint buffer but not with chemical powders, which could be used to implement a protocol for the use of level 3 identification.

Prediction of quality markers and medicinal value of sea buckthorn leaves based on network pharmacology, content determination, and activity evaluation / 中国中药杂志

The leaves of sea buckthorn(Hippophae rhamnoides), considered as common food raw materials, have records of medicinal use and diverse pharmacological activities, showing a potential medicinal value. However, the active substances in the sea buckthorn leaves and their mechanisms of action remain unclear. In addition, due to the extensive source and large variety variations, the quality evaluation criteria of sea buckthorn leaves remain to be developed. To solve the problems, this study predicted the main active components, core targets, key pathways, and potential pharmacological effects of sea buckthorn leaves by network pharmacology and molecular docking. Furthermore, ultra-performance liquid chromatography with diode-array detection(UPLC-DAD) was employed to determine the content of active components and establish the chemical fingerprint, on the basis of which the quality markers of sea buckthorn leaves were predicted and then verified by the enzyme activity inhibition method. The results indicated that sea buckthorn leaves had potential therapeutic effects on a variety of digestive tract diseases, metabolic diseases, tumors, and autoimmune diseases, which were consistent with the ancient records and the results of modern pharmacological studies. The core targets of sea buckthorn leaves included PTPN11, AKT1, PIK3R1, ESR1, and SRC, which were mainly involved in the PI3K-AKT, MAPK, and HIF-1 signaling pathways. In conclusion, the active components of sea buckthorn leaves are associated with the rich flavonoids and tannins, among which quercitrin, narcissoside, and ellagic acid can be used as the quality markers of sea buckthorn leaves. The findings provide a reference for the quality control and further development and utilization of sea buckthorn leaves as medicinal materials.

Assessment of genotyping markers in the molecular characterization of a population of clinical isolates of Fusarium in Colombia / Evaluación de marcadores de genotipificación en la caracterización molecular de una población de aislamientos clínicos de Fusarium en Colombia

Introduction: Fusarium is a very heterogeneous group of fungi, difficult to classify, with a wide range of living styles, acting as saprophytes, parasites of plants, or pathogens for humans and animals. Prevalence of clinical fusariosis and lack of effective treatments have increased the interest in the precise diagnosis, which implies a molecular characterization of Fusarium populations. Objective: We compared different genotyping markers in their assessment of the genetic variability and molecular identification of clinical isolates of Fusarium. Materials and methods: We evaluated the performance of the fingerprinting produced by two random primers: M13, which amplifies a minisatellite sequence, and (GACA)4, which corresponds to a simple repetitive DNA sequence. Using the Hunter Gaston Discriminatory Index (HGDI), an analysis of molecular variance (AMOVA), and a Mantel test, the resolution of these markers was compared to the reference sequencing-based and PCR genotyping methods. Results: The highest HGDI value was associated with the M13 marker followed by (GACA)4. AMOVA and the Mantel tests supported a strong correlation between the M13 classification and the reference method given by the partial sequencing of the transcription elongation factor 1-alpha (TEF1-α) and rDNA 28S. Conclusion: The strong correlation between the M13 classification and the sequencing-based reference together with its higher resolution demonstrates its adequacy for the characterization of Fusarium populations.

Introducción. Fusarium es un grupo heterogéneo de hongos, difícil de clasificar y con una amplia gama de estilos de vida, que actúa como saprófito, parásito de plantas o patógeno de humanos y animales. La prevalencia de la fusariosis clínica y la falta de tratamientos han incrementado el interés en su diagnóstico preciso, lo que conlleva la caracterización molecular de las poblaciones. Objetivo. Comparar marcadores de genotipificación en la evaluación de la variabilidad genética e identificación de aislamientos clínicos de Fusarium. Materiales y métodos. Se evaluó la huella genética producida por dos cebadores aleatorios: M13, que amplifica una secuencia minisatélite, y (GACA)4, que corresponde a una secuencia repetitiva de ADN. Utilizando el índice discriminatorio de Hunter Gaston (HGDI), el análisis de varianza molecular (AMOVA) y una prueba de Mantel, se comparó la resolución de estos marcadores con métodos de genotipificación basados en secuenciación y PCR. Resultados. El mayor HGDI se asoció con el marcador M13, seguido de (GACA)4. Las pruebas AMOVA y Mantel mostraron correlación entre las clasificaciones obtenidas con M13 y la referencia basada en la secuenciación parcial del factor de elongación de transcripción 1-alfa (TEF1-α) y el ADNr 28S. Conclusión. La fuerte correlación entre la clasificación obtenida con M13 y el método de referencia, así como su alta resolución, demuestran su idoneidad para la caracterización de poblaciones de Fusarium.

SSR marker-based DNA fingerprinting of Sub1 introgressed lines in the background of traditional rice varieties of Assam India-Supplementary data

Rice varieties are usually characterized by agro-morphological descriptors used for seed certification and seed characterization by following distinctiveness, uniformity, and stability (DUS) test. But in fact, these primary distinguishing morphological descriptors among rice varieties are very limited and hence face problems to distinguish germplasm accessions. Germplasm certification in NBPGR requires a DNA fingerprinting profile to explain germplasm uniqueness compared to existing varieties. Varietal identification has gained a key role worldwide, particularly in plant variety protection. Sixty-two morphological descriptors studies have shown the Sub1 introgressed advanced lines E-6, C-210, C-196, 1189-1 and 1160-1 are distinct from the other varieties for more than 15morphological traits, based on these variations the lines were selected for DNA fingerprinting. About68 SSRs markers were used for DNA fingerprinting in seven genotypes, two of which were parents (Ranjit, Bahadur) and three Sub1 introgressed advanced lines (E6, C210, C196) in Ranjit background, and two Sub1 introgressed advanced lines (1189-1, 1160-1) in Bahadur background. DNA fingerprinting was done on these genotypes of rice using SSR markers. Among the 68 SSR markers, total 65 markers were amplified and three were found not amplified. Out of 65 markersfour of them viz. RM 152, RM 172, RM 251, and RM 346 showed better polymorphism with amplicon size ranges from 155-163 bp, 150-159 bp, 137-147 bp, and 166-175 bp, respectively, and remaining 61 showed monomorphic amplification. Therefore, SSR (Simple-sequence repeats) based DNA fingerprinting helped to differentiate Ranjit, Bahadur, E-6, C-210, C-196, 1189-1, and 1160-1. Hence, the research reveals that newly developed high-yielding Sub1 introgressed advanced lines in the background of traditional Assam rice varieties (Ranjit and Bahadur) are unique in their identity.

SSR marker-based DNA fingerprinting of Sub1 introgressed lines in the background of traditional rice varieties of Assam India-Supplementary data

Rice varieties are usually characterized by agro-morphological descriptors used for seed certification and seed characterization by following distinctiveness, uniformity, and stability (DUS) test. But in fact, these primary distinguishing morphological descriptors among rice varieties are very limited and hence face problems to distinguish germplasm accessions. Germplasm certification in NBPGR requires a DNA fingerprinting profile to explain germplasm uniqueness compared to existing varieties. Varietal identification has gained a key role worldwide, particularly in plant variety protection. Sixty-two morphological descriptors studies have shown the Sub1 introgressed advanced lines E-6, C-210, C-196, 1189-1 and 1160-1 are distinct from the other varieties for more than 15morphological traits, based on these variations the lines were selected for DNA fingerprinting. About68 SSRs markers were used for DNA fingerprinting in seven genotypes, two of which were parents (Ranjit, Bahadur) and three Sub1 introgressed advanced lines (E6, C210, C196) in Ranjit background, and two Sub1 introgressed advanced lines (1189-1, 1160-1) in Bahadur background. DNA fingerprinting was done on these genotypes of rice using SSR markers. Among the 68 SSR markers, total 65 markers were amplified and three were found not amplified. Out of 65 markersfour of them viz. RM 152, RM 172, RM 251, and RM 346 showed better polymorphism with amplicon size ranges from 155-163 bp, 150-159 bp, 137-147 bp, and 166-175 bp, respectively, and remaining 61 showed monomorphic amplification. Therefore, SSR (Simple-sequence repeats) based DNA fingerprinting helped to differentiate Ranjit, Bahadur, E-6, C-210, C-196, 1189-1, and 1160-1. Hence, the research reveals that newly developed high-yielding Sub1 introgressed advanced lines in the background of traditional Assam rice varieties (Ranjit and Bahadur) are unique in their identity.

SSR marker-based DNA fingerprinting of Sub1 introgressed lines in the background of traditional rice varieties of Assam India

Rice varieties are usually characterized by agro-morphological descriptors used for seed certification and seed characterization by following distinctiveness, uniformity, and stability (DUS) test. But in fact, these primary distinguishing morphological descriptors among rice varieties are very limited and hence face problems to distinguish germplasm accessions. Germplasm certification in NBPGR requires a DNA fingerprinting profile to explain germplasm uniqueness compared to existing varieties. Varietal identification has gained a key role worldwide, particularly in plant variety protection. Sixty-two morphological descriptors studies have shown the Sub1 introgressed advanced lines E-6, C-210, C-196, 1189-1 and 1160-1 are distinct from the other varieties for more than 15morphological traits, based on these variations the lines were selected for DNA fingerprinting. About68 SSRs markers were used for DNA fingerprinting in seven genotypes, two of which were parents (Ranjit, Bahadur) and three Sub1 introgressed advanced lines (E6, C210, C196) in Ranjit background, and two Sub1 introgressed advanced lines (1189-1, 1160-1) in Bahadur background. DNA fingerprinting was done on these genotypes of rice using SSR markers. Among the 68 SSR markers, total 65 markers were amplified and three were found not amplified. Out of 65 markersfour of them viz. RM 152, RM 172, RM 251, and RM 346 showed better polymorphism with amplicon size ranges from 155-163 bp, 150-159 bp, 137-147 bp, and 166-175 bp, respectively, and remaining 61 showed monomorphic amplification. Therefore, SSR (Simple-sequence repeats) based DNA fingerprinting helped to differentiate Ranjit, Bahadur, E-6, C-210, C-196, 1189-1, and 1160-1. Hence, the research reveals that newly developed high-yielding Sub1 introgressed advanced lines in the background of traditional Assam rice varieties (Ranjit and Bahadur) are unique in their identity.

Diagnosing lung cancer through metabolic fingerprint based on machine learning / 中华检验医学杂志

Objective:To screen out the differentially regulated metabolites by the analysis of serum metabolic fingerprints, and to provide potential biomarkers for diagnosis of lung cancer.Methods:A total of 228 subjects were enrolled in Changhai Hospital from January 27, 2021 to June 4, 2021, including 97 newly diagnosed lung cancer patients and 131 healthy individuals. Serum samples were collected from the enrolled cohort according to a standard procedure, and the enrolled cohort was divided into a training set and a completely independent validation set by stratified random sampling. The metabolic fingerprints of serum samples were collected by previously developed nano-assisted laser desorption/ionization mass spectrometry (nano-LDI MS). After age and gender matching of the training set, a diagnostic model based on serum metabolic fingerprints was established by machine learning algorithm, and the classification performance of the model was evaluated by receiver operating characteristic (ROC) curve.Results:Serum metabolic fingerprint for each sample was obtained in 1 minute using a novel nano-LDI MS, with consumption of only 1 μl original serum sample. For the training set, the area under ROC curve (AUC) of the constructed classifier for diagnosis of lung cancer was 0.92 (95% CI 0.87-0.97), with a sensitivity of 89% and specificity of 89%. For the independent validation set, the AUC reached 0.96 (95% CI 0.90-1.00) with a sensitivity of 91% and specificity of 94%, which showed no significant decrease compared to training set. We also identified a biomarker panel of 5 metabolites, demonstrating a unique metabolic fingerprint of lung cancer patients. Conclusion:Serum metabolic fingerprints and machine learning were combined to establish a diagnostic model, which can be used to distinguish between lung cancer patients and healthy controls. This work sheds lights on the rapid metabolic analysis for clinical application towards in vitro diagnosis.

Expression, purification, and functional identification of immunoglobulin degrading enzyme IdeS in Escherichia coli / 药学学报

In the process of evolution, pathogenic Streptococcus pyogenes secretes an immunoglobulin G-degrading enzyme IdeS which can specifically cleave the hinge region of immunoglobulin G in order to escape the immune response against the host. On the one hand, IdeS can be used for IgG fingerprinting as a tool enzyme combined with mass spectrometry technology. On the other hand, IdeS can be used to treat the antibody-responsive diseases produced by autoimmunity as a therapeutic protein. In this study, the backbone of plasmid pCold was used to construct two expression vectors of recombinant protein IdeS, which were heterologously expressed in Escherichia coli Shuffle T7. After purification by affinity chromatography, the recombinant IdeS activity was detected and their activity differences between the two were compared. Among them, the yield of the recombinant IdeS containing the His6-tag at the N-terminus was 4 mg·L-1, and the cleavage reaction with antibody IgG1 at 1∶200 (m/m) at 37 ℃ for 30 min could complete. However, the yield of the recombinant IdeS containing both the N-terminal His6 tag and the C-terminal silica affinity tag (silica bing peptide, SiBP) is 1.5 mg·L-1, and the degradation reaction with antibody IgG1 at 1∶20 (m/m) at 37 ℃ for 30 min could reach the end. The C-terminal fusion peptide has a great influence on the yield and activity of IdeS, which is not conducive to subsequent application in drug development. Above all, the recombinant IdeS containing the His6-tag at the N-terminus expressed by this system has high activity and can fully meet the needs of antibody drug development and mapping analysis of IgG.

DNA barcoding of Gentiana crassicaulis in Lijiang and evaluation of root processing methods / 药学学报

The key factors for producing the best quality Chinese herbal medicines are high-quality germplasm, suitable cultivation area and the proper processing methods for herbal raw materials. Gentiana crassicaulis in Gentiana (Sect. Cruciata), Gentianaceae is one of the original plants of the Chinese herb Qinjiao (Gentianae Macrophyllae Radix), and its type specimen was collected in Lijiang, Yunnan. There is a long planting history of the herb in this area. In this study a sampling plot was designated in these traditional planting areas. G. crassicaulis was planted and herbal raw materials were harvested from the plot. The raw materials were prepared locally and at a pharmaceutical factory in Shanghai using processing methods such as "sweating" or "no sweating", "slicing" or "no slicing" (whole root), and "stoving" or "no stoving" (air drying). The quality of all processed samples was evaluated. In addition, molecular markers were determined for identifying cultivated and wild samples from Lijiang, Yunnan. The results are as follows: ① Samples from the sampling plot and the field are taxonomically identified as Gentiana crassicaulis. ② A total of 270 sequences of trnC-GCA-petN, atpB-rbcL, psbN, ndhB-rps7 and ycf1 were obtained, and three genotypes were determined from the cultivated samples; the type III was shared by both cultivated and wild plants. Based on the molecular markers, a DNA barcoding method to identify cultivated and wild samples of G. crassicaulis from Lijiang, Yunnan was established. ③ Total content of loganic acid and gentiopicroside in all samples was ≥ 2.5%, and above the Chinese Pharmacopoeia (2020) limit. ④ In HPLC fingerprinting, 9 common peaks were assigned and similarity between all samples was > 0.999; and ⑤ In a PCA score plot all slice samples were clustered, while whole root samples were scattered. Therefore, our studies could provide basic data for optimizing the processing method, producing best quality Gentianae Macrophyllae Radix, and evaluating the quality of different ecotype varieties and the multiple origin of herbal medicines.

An ultra-robust fingerprinting method for quality assessment of traditional Chinese medicine using multiple reaction monitoring mass spectrometry / 药物分析学报

Chromatographic fingerprinting has been perceived as an essential tool for assessing quality and chemical equivalence of traditional Chinese medicine.However,this pattern-oriented approach still has some weak points in terms of chemical coverage and robustness.In this work,we proposed a multiple reaction monitoring(MRM)-based fingerprinting method in which approximately 100 constituents were simultaneously detected for quality assessment.The derivative MRM approach was employed to rapidly design MRM transitions independent of chemical standards,based on which the large-scale finger-printing method was efficiently established.This approach was exemplified on QiShenYiQi Pill(QSYQ),a traditional Chinese medicine-derived drug product,and its robustness was systematically evaluated by four indices:clustering analysis by principal component analysis,similarity analysis by the congruence coefficient,the number of separated peaks,and the peak area proportion of separated peaks.Compared with conventional ultraviolet-based fingerprints,the MRM fingerprints provided not only better discriminatory capacity for the tested normal/abnormal QSYQ samples,but also higher robustness under different chromatographic conditions(i.e.,flow rate,apparent pH,column temperature,and column).The result also showed for such large-scale fingerprints including a large number of peaks,the angle cosine measure after min-max normalization was more suitable for setting a decision criterion than the unnormalized algorithm.This proof-of-concept application gives evidence that combining MRM tech-nique with proper similarity analysis metrices can provide a highly sensitive,robust and comprehensive analytical approach for quality assessment of traditional Chinese medicine.

Evaluation of the production of antifungal metabolites against Colletotrichum gloeosporioides in Streptomyces 5.1 by random mutagenesis

Streptomyces 5.1 is a bacterium isolated from rice soils in the south of the Tolima department (Colombia). This microorganism is characterized by its antagonistic activity against rubber tree phytopathogens like Colletotrichum gloeosporioides, the causal agent of leaf anthracnose. The antifungal activity of this Streptomyces isolate has been associated with secondary metabolites production. However, the identity of those metabolites is unknown because its purification and identification have not been possible through classic chemical studies. Therefore, aiming to contribute in the study of the secondary metabolites produced by 5.1 from a molecular approach, this research seeks to identify -preliminarily- the genomic fingerprint changes associated with the production of antifungal secondary metabolites produced by Streptomyces 5.1 through the evaluation of a mutant library of 5.1 obtained by random mutagenesis using controlled ultraviolet light exposure. The antifungal activity of obtained mutants was evaluated using Colletotrichum gloeosporioides (C1) fungus as a biosensor, isolated by the Biotechnology Institute of Universidad Nacional de Colombia. In this way, the library of mutants of 5.1, initially formed by 300 isolations, was classified into two phenotypic groups of interest: enhanced mutants (1 isolate) and null mutants (11 isolates) of secondary metabolites. The genomic changes in both groups were analyzed by obtaining the genomic profile of the isolates using Repetitive Extragenic Palindromic (Rep-PCR). The obtained profiles evidenced the presence of one additional band in the enhanced mutant, and the absence of a specific band in the non-producing mutants, both in comparison with the original strain. These bands are proposed for a future sequencing study which will define their role in the production process of metabolites with antifungal activity in Streptomyces 5.1.

Genetic traceability of Staphylococcus aureus strains isolated from primiparous dairy cows mastitis, humans and environment in the Northeast region of Brazil / Rastreabilidade genética de cepas de Staphylococcus aureus isoladas de mastite de vacas primíparas, ordenhadores e ambiente de ordenha na região Nordeste do Brasil

ABSTRACT: This research aimed to investigate the genotypic relatedness of 18 Staphylococcus aureus strains isolated from intramammary infections in primiparous cows and extramammary sites on five dairy herds by rep-PCR using RW3A primers, and by PFGE using the endonuclease SmaI. The isolates were also evaluated in vitro for the susceptibility against beta-lactam antimicrobials drugs (penicillin and oxacillin), considering that beta-lactams are frequently used for treating staphylococcal intrammamary infections. The rep-PCR typing was highly discriminatory (D value= 0.9804) and a total of 15 patterns were detected. The PFGE method was also highly discriminatory (D value= 0.9667) and a total of 13 patterns were observed. A total of 15 out of 18 (83%) isolates were resistant to penicillin and one out of 18 (6%) to oxacillin. In conclusion, these findings confirmed the occurrence of a high genetic diversity of S. aureus strains at the herds and the presence of clonally-related strains only at the same herd, emphasizing a variety of genotypic profiles among the isolates.

RESUMO: Objetivou-se com este estudo investigar a correlação genética de 18 cepas de Staphylococcus aureus isoladas de infecções intramamárias em vacas primíparas e de locais extramamários em cinco propriedades leiteiras através das técnicas de PCR por sequências palindrômicas extragênicas repetitivas (rep-PCR), usando iniciadores RW3A, e de eletroforese em gel de campo pulsado (PFGE), usando a endonuclease SmaI. Os isolados também foram avaliados in vitro quanto à suscetibilidade aos antimicrobianos beta-lactâmicos (penicilina e oxacilina). A tipagem por rep-PCR foi altamente discriminatória (valor D = 0,9804) e um total de 15 padrões foram detectados. Os isolados de S. aureus foram agrupados em três grupos diferentes (A a C), com 80% de similaridade. A técnica de PFGE também foi altamente discriminatória (valor D = 0,9667) e um total de 13 padrões foi observado. A análise do dendrograma com um coeficiente de similaridade de 80% gerou dois grupos diferentes (A e B). Além disso, cepas clonais isoladas do leite foram identificadas na mesma propriedade pelos dois métodos de tipificação e, apesar da presença de cepas dominantes, nossos resultados sugerem uma alta diversidade genética dentre as cepas de S. aureus analisadas. Um total de 15, dos 18 (83%) isolados, eram resistentes à penicilina e um dos 18 (6%) à oxacilina. Assim, esses achados confirmam a ocorrência de uma alta diversidade genética de cepas de S. aureus nas propriedades e a presença de cepas clonalmente relacionadas apenas na mesma propriedade, enfatizando uma variedade de perfis genotípicos entre os isolados.

First Genetically Differentiation between Upupa epops major and Upupa epops epops (Family: Upupidae)

Abstract Hoopoe has been traditionally treated as a single species within the order Coraciiformes. Presently, however, various authors have suggested separating the hoopoe into two or more species and even its order, Bucerotiformes. So, this work aimed to use the RAPD PCR and DNA sequences of the COI gene barcodes to confirm and to assess whether the Egyptian hoopoe is a different species named Upupa epops major from the European hoopoe called Upupa epops epops, and to determine the relationships among them. Five primers were used in this technique. Two hoopoes were taken in this work as studying birds, migratory and resident one. The results showed the highest genetic distance between them using different random primers while genetic identity was in general low, overall primers. DNA fingerprinting detected greater genetic distance between Upupa epops major and Upupa epops epops and low genetic identity, this may indicate that both hoopoes fall into two separate species. Furthermore, using mitochondrial cytochrome oxidase subunit I (COI) sequences in this study suggests the separation of Upupa epops major into a new species.

Twins from different fathers: A heteropaternal superfecundation case report in Colombia / Gemelos de diferentes padres: un caso de superfecundación heteropaternal en Colombia

Abstract: Heteropaternal superfecundation is an extremely rare phenomenon that occurs when a second ova released during the same menstrual cycle is additionally fertilized by the sperm cells of a different man in separate sexual intercourse. In August, 2018, the Grupo de Genética de Poblaciones e Identificación at Universidad Nacional de Colombia received a request to establish the paternity of a pair of male twins with genetic markers. The following analyses were performed: amelogenin gene, autosomal short tandem repeat (STR), and Y-STR analyses by means of human identification commercial kits, paternity index, and the probability of paternity calculation and interpretation. A paternity index of 2.5134E+7 and a probability of paternity of 99.9999% for twin 2 were obtained while 14 out of 17 Y-chromosome markers and 14 out of 21 autosomal short tandem repeats were excluded for twin 1. The results indicated that the twins have different biological fathers. Although heteropaternal superfecundation is rarely observed among humans given its low frequency, in paternity disputes for dizygotic twins it is mandatory to demand the presence of the two twins in the testing to avoid wrong conclusions.

Resumen: La superfecundación heteropaternal es un fenómeno extremadamente raro que se produce cuando un segundo óvulo, liberado durante el mismo ciclo menstrual, es fertilizado por un espermatozoide de un hombre diferente en relaciones sexuales separadas. En agosto de 2018, el Grupo de Genética de Poblaciones e Identificación de la Universidad Nacional de Colombia recibió una solicitud para establecer la paternidad mediante marcadores genéticos de un par de mellizos varones, en quienes se hizo el análisis del gen de amelogenina, el análisis de repeticiones cortas en tándem (Short Tandem Repeats, STR) autosómicas y del cromosoma Y (Y-STR) mediante kits comerciales de identificación humana y cálculos e interpretación del índice de paternidad y probabilidad de paternidad. Se obtuvo un índice de paternidad de 2,5134E+7 y una probabilidad de paternidad de 99,9999 % para el gemelo 2, en tanto que en el gemelo 1 se excluyeron 14 de los 17 marcadores del cromosoma Y y 14 de los 21 sistemas STR autosómicos evaluados. Los resultados indicaron que los gemelos tienen diferentes padres biológicos. A pesar de que la superfecundación heteropaternal rara vez se observa en humanos debido a su baja frecuencia, en las disputas de paternidad para los gemelos dicigóticos, es obligatorio exigir en la prueba la presencia de los dos gemelos para evitar conclusiones incorrectas.

Use of molecular markers SSR and SCAR for identification of olive accessions / Uso de marcadores moleculares SSR e SCAR para identificação de acessos de oliveira

Morphological and agronomical describers are traditionally used in plant characterization. However, the usage of these describers have some limitations such as susceptibility to abiotic and biotic stress and environmental factors. Furthermore, the describers are not stable over time and many can only be evaluated during the adult phase of the plants, which requires time and physical space. Molecular markers offer numerous advantages compared to the conventional alternatives based on phenotype: they are stable and detectable in all vegetable tissues, and are independent of the environment and development phase. One of the main advantages of the use of molecular markers is the time reduction in the identification of genetic diversity among the studied subjects, as the genotypes may even be described for the seed or seedling phase. Many countries have already adopted molecular markers to identify olive cultivars more accurately. The aim of this study was to evaluate the genetic identity of eight olive accessions supposedly belonging to cultivar Arbequina by using microsatellite (SSR) and Sequence Characterized Amplified Region (SCAR) markers. One accession corresponding to the cultivar was also incorporated into the analysis as a reference genotype. The molecular marker data were analyzed on the software GENALEX6.The markers generated an accumulated PI and PE of 1.26 x 10-6 and 0.949, respectively.The results supported the hypothesis that all accessions belong to the cultivar Arbequina, and the markers can therefore be applied to other varieties of olive species.

Descritores morfológicos e agronômicos são tradicionalmente utilizados na caracterização de plantas. Apesar de recomendado, o emprego destes descritores apresenta algumas limitações como a influência a estresses abióticos e bióticos e aos efeitos do ambiente. Além disso, não são estáveis ao longo do tempo e muitos só podem ser avaliados durante a fase adulta das plantas, o que requer tempo e espaço físico para as avaliações. Os marcadores moleculares oferecem numerosas vantagens relativamente às alternativas convencionais baseadas no fenótipo, pois são estáveis e detectáveis em todos os tecidos vegetais, independente do ambiente e fase de desenvolvimento e uma das principais vantagens da utilização destes é proporcionar a redução do tempo na identificação da diversidade genética entre os indivíduos trabalhados, podendo ser avaliadas genótipos ainda na fase de semente ou de plântula. O objetivo deste estudo foi avaliar a identidade genética de oito acessos de oliveira supostamente pertencentes a cultivar Arbequina usando microssatélites (SSR) e Sequence Characterized Amplified Region (SCAR) marcadores. Um acesso correspondente a cultivar também foi incorporado na análise como o genótipo de referência. Os dados de marcadores moleculares foram analisados com o software GENALEX 6. Como resultado, os marcadores SSR geraram um PI acumulada e PE de 1,26 x 10- 6 e 0,949, respectivamente. Os resultados suportam a hipótese de que todos os acessos pertencem a cultivar Arbequina, e, por conseguinte, esses marcadores podem ser aplicados em situações semelhantes em outras variedades de espécies de oliveira.

DNA fingerprinting identification of bile power(bile)medicines / 中国中药杂志

The pig bile powder, bovine bile powder, snake bile, sheep bile, goose bile powder, and bear bile powder were contained by the Chinese Pharmacopoeia. The bile power medicine has a long history in traditional Chinese medicine and definite effect. However, the medicine of bile powder(bile) are similar in morphology. Besides, many medicine lack specific microscopic identification characteristics and chemical characteristics. There is a risk of adulteration, especially when the fake medicine were mixed in authentic medicine, it is difficult to detection. The key to control the quality and ensures the clinical efficacy is the good or bad, true or false of the bile power medicine. The STR typing technology is a method that according to differential typing of PCR amplified lengths to compare and identify individual organisms. Based on the principle of STR typing, the easily, rapid DNA fingerprinting method to identify the bile power and adulteration was established.The original animal or bile powder of pigs, cattle, sheep, chickens, ducks, geese, snakes, bears, fish were collected, the 12 S-L1091/12 S-H1478 and 16 S-L3428/16 S-H3667 was obtained by sifted, the DNA fingerprinting of the bile power and adulteration was obtained by STR typing. Every species has different STR fingerprints, so different species can be identified. Besides, the fingerprints have both the authentic and fake's information, the adulteration of authentic and fake can be identified. Therefore, the method to identify the bile power and adulteration was achieved through the combination of two primers. The DNA fingerprinting method established in this study can also be used for other animal medicine.

Study on quality control of Miao medicine Laportea bulbifera based on fingerprint analysis and quantitative analysis of multi-components / 中草药

Objective: To establish a rational method for Laportea bulbifera quality control. Methods: The fingerprint technique and multi-component quantitation were used to study the quality control of L. bulbifera by UHPLC. The 12 batches of L. bulbifera UHPLC fingerprint were evaluated by the evaluation system on similitude degree of chromatogram fingerprint of traditional Chinese medicine. Results: The quality control methods of Miao medicine L. bulbifera for simultaneous determination of 10 components (including neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, rutin, isoquercitrin, luteoloside, kaempferol-3-O-rutinoside, quercitrin, epigallocatechin and epicatechin) were established. The linear, precision, repeatability and stability are good. The standard recoveries were 95.89%-98.62%, with RSD less than 3%. The common mode of fingerprint was established after determination fingerprints of 12 batches of samples of L. bulbifera by UHPLC. There were 20 common peaks in these samples. The similarity of the 12 batches fingerprints were in the range from 0.805 to 0.931. Conclusion: The fingerprinting and multi-index content determination methods for quantitative control of Miao medicine L. bulbifera have high sensitivity, good accuracy, stability and reliability, which can provide a theoretical and experimental foundation for quantitative control of Miao medicine L. bulbifera.

DNA fingerprinting and genetic diversity in a threatened alpine medicinal plant, Gentiana crassicaulis (Gentianaceae) using AFLP / 药学学报

Jieji Nabao is a common Tibetan herb. According to our ethnobotanical studies, one of its original plants is identified as Gentiana crassicaulis Duthie ex Burk. (Gentianaceae). Endemic to the Qinghai-Tibet Plateau, this medicinal alpine plant is a threatened species. In this study, 163 individuals from 20 populations of G. crassicaulis were collected throughout its geographical range and amplified fragment length polymorphism (AFLP) was used to investigate genetic variation in this species. A cluster analysis was performed on the AFLP data with Halenia elliptica and Gentiana straminea as the outgroups. From 64 pairs of AFLP primer combinations, 12 pairs were selected for amplification and a total of 315 bands were amplified, of which 254 bands were polymorphic, accounting for 80.63%. High genetic differentiation was detected between populations (87%), and low within populations (13%). The UPGMA (unweighted pair-group method with arithmetic means) tree was topologically consistent with the traditional taxonomic treatments at the species level, and the populations of G. crassicaulis were divided into two branches: one from Yunnan and Guizhou, the other from Tibet, Qinghai, Sichuan and Gansu. PCA analysis and the Mantel test showed that there was a positive correlation between genetic distance and geographical distance. In addition, combined with SSR and SNP markers within cpDNA, the genetic differentiation within the Sichuan population S1 was validated.