Optimized method for dna extraction and PCR amplification in aroeira tree

Molecular markers are important tools in the characterization of plant genetic diversity and can provide support for conservation strategies for endangered populations. The different molecular techniques involve the evaluation of many individuals; therefore, it is crucial to have fast, efficient, and inexpensive methods for DNA extraction. Given the importance of the Aroeira (Myracrodruon urundeuva Fr. All.) it is pertinent to optimize a protocol that allows the obtainment of intact and pure DNA, aiming to assist conservation strategies for this species that is threatened with extinction. Thus, this study aimed to compare five DNA extraction methods: Dellaporta et al. (1983), Doyle and Doyle (1987) modified, Ferreira and Grattapaglia (1995), Romano and Brasileiro (2015), and Khanuja et al. (1999) and optimize the most efficient protocol for M. urundeuva. The modified DNA extraction protocol proposed by Doyle and Doyle (1987), using 100 mg of leaf tissue and 6 µl of ß-mercaptoethanol was the protocol that presented the sharpest bands after DNA electrophoresis and after the reactions of amplification employing Polymerase Chain Reaction (PCR). Therefore, it is suggested to use the protocol described by Doyle and Doyle (1987) modified for the extraction of DNA from young M. urundeuva leaves to carry out techniques involving molecular markers.

Comparación de métodos de extracción de ADN de Giardia spp. medidos por PCR convencional / Comparison of methods of DNA extraction from Giardia spp. measured by conventional PCR

RESUMEN Objetivos . Comparar diferentes métodos de extracción de ADN a partir de quistes y trofozoítos de Giardia spp. mediante la técnica de reacción en cadena de la polimerasa (PCR) convencional. Materiales y métodos. Se aislaron quistes de Giardia spp. a partir de 65 muestras coprológicas procedentes de hospitales de referencia nacional, obteniéndose una carga promedio de 5x104 parásitos. Asimismo, se cultivaron trofozoítos de Giardia intestinalis (ATCC® 30957™) obteniéndose una carga parasitaria de 5x106. Se compararon once métodos de extracción para quistes y seis para trofozoítos. La concentración y pureza del ADN extraído se determinó por espectrofotometría y el rendimiento de la extracción se evaluó mediante la amplificación de los genes beta giardina (bg) y glutamato deshidrogenasa (gdh) por PCR semi-anidada. Resultados. Se observó que el método I mostró la mayor concentración de ADN a partir de quistes (12,24 ng/µL), pureza (1,4) y mejor rendimiento (100% amplificación bg, 60% gdh) en comparación con los otros métodos evaluados. En el caso de los trofozoítos el método que no tuvo pretratamientos presentó la mayor concentración de ADN, pureza y rendimiento (26,56 ng/µL; 1,85; 100% amplificación bg y gdh). Conclusiones. Los pretratamientos mecánicos, de choque térmico y enzimáticos son necesarios para la ruptura de la pared quística de Giardia spp., siendo el marcador molecular bg de mayor rendimiento para detección de ADN de quistes. Los trofozoítos no requieren pretratamientos para lograr resultados satisfactorios. Se cuenta con una metodología reproducible para la extracción de ADN de Giardia spp. a partir de cualquier estadio evolutivo.

ABSTRACT Objectives. To compare different methods of DNA extraction from cysts and trophozoites of Giardia spp. using the conventional polymerase chain reaction (PCR) technique. Materials and Methods. Cysts of Giardia spp. were isolated from 65 coprological samples from national reference hospitals, obtaining an average load of 5x104 parasites. In addition, Giardia intestinalis trophozoites (ATCC® 30957™) were cultured obtaining a 5x106 parasitic load. Eleven extraction methods for cysts and six for trophozoites were compared. The concentration and purity of the extracted DNA were determined by spectrophotometry and the extraction yield was assessed by amplification of the ß-giardin (bg) and glutamate dehydrogenase (gdh) genes with a semi nested PCR assay. Results. It was observed that method 1 showed the highest concentration of DNA from cysts (12.24 ng/µL), purity (1.4) and best performance (bg: 100% amplification; gdh: 60% amplification) compared to the other methods evaluated. In the case of trophozoites, the method without pre treatment showed the highest level of DNA concentration, purity, and yield (26.56 ng/µL; 1.85; 100% amplification of bg and gdh, respectively). Conclusions . Mechanical, thermal shock, and enzymatic pre-treatments are necessary for the rupture of the cystic wall of Giardia spp. making it the highest-yielding bg molecular marker for detecting cyst DNA. Trophozoites do not require pre-treatment to achieve satisfactory results. A reproducible methodology for the extraction of DNA from Giardia spp. from any evolutionary stage is available.

Application of Touch DNA in Investigation Practice / 法医学杂志

With the continuous development of DNA extraction and testing technology, the DNA left at a crime scene plays a decisive role in the determination of criminal suspects in criminal investigation. But in the meanwhile, the anti-reconnaissance awareness of suspect is growing, which leads to a decrease of evidence left at scene during and after a crime. Therefore, in the process of evidence collection at scene, the finding and extraction of touch biological evidence, and the DNA detection are more and more important. At present, the proportion of touch evidence at the crime scene increases, which plays an increasingly important role in the detection of cases. However, with the characteristics of minute quantities, small size and secrecy, these touch evidence is difficult to be observed. What's more, various forms of pollution at the scene greatly accelerate the degradation rate of trace material, thus, the test and analysis of such material has become the emphasis and difficulty of the forensic evidence identification. This article reviews different kinds, collection and extraction methods of touch DNA, the factors that affect the detection and the problems may meet in the detection for providing an application prospect to the forensic practice.

The use of Proteinase K to access genitalia morphology, vouchering and DNA extraction in minute wasps

ABSTRACT Genitalia are rich source of characters in insect taxonomy. Usually, they are examined after dissection and cleaning with potassium hydroxide (KOH), procedure that may damage both genital morphological structures and intracellular molecular contents. Enzymatic procedure with Proteinase K has been used to clean muscle off the genitalia while extract DNA, but its damage to the genital structures has not been evaluated. Herein, we qualitatively compare the use of KOH and Proteinase K to prepare genital structures in minute insects (Hymenoptera: Bethylidae). We show that Proteinase K is better to preserve the genital structure and provides quality DNA.

Development of a real-time PCR assay for the detection of the golden mussel ( Limnoperna fortunei, Mytilidae) in environmental samples

ABSTRACT The golden mussel, Limnoperna fortunei, is among the most devastating invasive species in freshwater habitats worldwide, leading to severe environmental disturbances and economic losses. Therefore, management efforts would be greatly improved by methods that efficiently detect and quantify the abundance of the golden mussel in freshwater habitats, particularly in early stages of colonization. In this study, we describe a highly-sensitive real-time PCR assay targeting a 100-bp region of the COI mitochondrial gene of the golden mussel. The method was able to detect as little as 0.225 pg of target DNA. This assay represents an important contribution to surveillance methods, as well as to optimize field measures to contain and manage populations of the golden mussel in its introduced range.

Human DNA extraction from whole saliva that was fresh or stored for 3, 6 or 12 months using five different protocols

Abstract Saliva when compared to blood collection has the following advantages: it requires no specialized personnel for collection, allows for remote collection by the patient, is painless, well accepted by participants, has decreased risks of disease transmission, does not clot, can be frozen before DNA extraction and possibly has a longer storage time. Objective and Material and Methods This study aimed to compare the quantity and quality of human DNA extracted from saliva that was fresh or frozen for three, six and twelve months using five different DNA extraction protocols: protocol 1 – Oragene™ commercial kit, protocol 2 – QIAamp DNA mini kit, protocol 3 – DNA extraction using ammonium acetate, protocol 4 – Instagene™ Matrix and protocol 5 – Instagene™ Matrix diluted 1:1 using proteinase K and 1% SDS. Briefly, DNA was analyzed using spectrophotometry, electrophoresis and PCR. Results Results indicated that time spent in storage typically decreased the DNA quantity with the exception of protocol 1. The purity of DNA was generally not affected by storage times for the commercial based protocols, while the purity of the DNA samples extracted by the noncommercial protocols typically decreased when the saliva was stored longer. Only protocol 1 consistently extracted unfragmented DNA samples. In general, DNA samples extracted through protocols 1, 2, 3 and 4, regardless of storage time, were amplified by human specific primers whereas protocol 5 produced almost no samples that were able to be amplified by human specific primers. Depending on the protocol used, it was possible to extract DNA in high quantities and of good quality using whole saliva, and furthermore, for the purposes of DNA extraction, saliva can be reliably stored for relatively long time periods. Conclusions In summary, a complicated picture emerges when taking into account the extracted DNA’s quantity, purity and quality; depending on a given researchers needs, one protocol’s particular strengths and costs might be the deciding factor for its employment.

Comparison of MPure-12 Automatic Nucleic Acid Purification and Chelex-100 Method / 法医学杂志

<p><b>OBJECTIVES</b>To explore the forensic application value of MPure-12 automatic nucleic acid purification （MPure-12 Method） for DNA extraction by extracting and typing DNA from bloodstains and various kinds of biological samples with different DNA contents.</p><p><b>METHODS</b>Nine types of biological samples, such as bloodstains, semen stains, and saliva were collected. DNA were extracted using MPure-12 method and Chelex-100 method, followed by PCR amplification and electrophoresis for obtaining STR-profiles.</p><p><b>RESULTS</b>The samples such as hair root, chutty, butt, muscular tissue, saliva stain, bloodstain and semen stain were typed successfully by MPure-12 method. Partial alleles were lacked in the samples of saliva, and the genotyping of contact swabs was unsatisfactory. Additional, all of the bloodstains （20 μL, 15 μL, 10 μL, 5 μL, 1 μL） showed good typing results using Chelex-100 method. But the loss of alleles occurred in 1 μL blood volume by MPure-12 method.</p><p><b>CONCLUSIONS</b>MPure-12 method is suitable for DNA extraction of a certain concentration blood samples．Chelex-100 method may be better for the extraction of trace blood samples．This instrument used in nucleic acid extraction has the advantages of simplicity of operator, rapidity, high extraction efficiency, high rate of reportable STR-profiles and lower man-made pollution.</p>

Coexistence of resistance to thyroid hormone and ectopic thyroid: ten-year follow-up

SUMMARY Resistance to thyroid hormone (RTH) coexisting with ectopic thyroid is rare. Here we report a case of RTH with ectopic thyroid. A ten-year-old girl had been misdiagnosed as congenital hypothyroidism and treated with levothyroxine since she was born. Ten-year follow-up showed that the elevated thyrotropin was never suppressed by levothyroxine and no signs indicating hyperthyroidism or hypothyroidism despite elevated FT3 and FT4 levels. Therefore the girl developed no defects in physical and cognitive development. Pituitary adenoma was excluded by magnetic resonance imaging. Ultrasonography did not find the thyroid gland in the normal place, while the thyroid scan found a large lingual thyroid gland. The octreotide inhibition test showed a reduction in thyrotropin by 41.98%. No mutation was detected in the thyroid hormone receptor (THR) β, THRα, thyrotropin receptor (TSHR), and GNAS1 genes. To our knowledge, it is an interesting RTH case coexisting with lingual thyroid.

Polimorfismo (+3954c>T) del gen IL-1B y su asociación con la resorción radicular apical externa post-tratamiento ortodóntico / IL-1B (+3954c>T) gene polymorphism and its association with external root resorption after orthodontic treatment

El objetivo fue determinar la presencia del polimorfismo rs1143634 (+3954C>T) del gen Interleuquina 1 Beta (IL-1B) y su asociación con la resorción radicular apical externa (RRE) post-tratamiento ortodóntico. Se realizó un estudio piloto de individuos tratados con aparatología ortodontica, 13 (casos) presentaron RRE posterior al tratamiento ortodóntico y 22 (controles) estaban clínicamente sanos. A partir de muestras de células epiteliales de mucosa bucal se extrajo ADN y se genotipificó el polimorfismo rs1143634 (+3954C>T) del gen IL-1B mediante la reacción en cadena de la polimerasa y digestión del producto con la enzima de restricción TaqI. Se estimaron las frecuencias alélicas y genotípicas del rs1143634; además, se evaluó la desviación del equilibrio de Hardy-Weinberg. Las frecuencias alélicas y genotípicas se compararon mediante la prueba de c2 con razón deverosimilitud (p <0,05). El promedio de edad de los participantes fue 28,1 (DE=11,5) años y el 68,6 % era mujeres. Al comparar la distribución de los genotipos del polimorfismo IL-1B (+3954C>T) entre grupos no se encontró una diferencia estadísticamente significativa (p=0,0926). Sin embargo, se observó una diferencia significativa en la distribución de alelos (p= 0,035), siendo el alelo T (alelo 2) más prevalente en el grupo control. El polimorfismo IL-1B (+3954C>T) se encontró presente en la población de estudio. Aunque no existieron diferencias en la distribución de los genotipos que apoyara una asociación entre este polimorfismo y la RRE, si hubo una diferencia en la distribución de los alelos, sugiriendo que el alelo T posiblemente actúa como factor protector contra el desarrollo de la RRE.

The objective of this study was to determine the presence of Interleukin 1 beta (IL-1B) rs1143634 (+3954C>T) gene polymorphism and its association with external apical root resorption (ERR) after orthodontic treatment. We conducted a pilot study of individuals treated with orthodontic treatment, 13 (cases) had ERR after orthodontic treatment and 22 (controls) were clinically healthy. DNA was extracted from samples of epithelial cells from the oral cavity and IL-1B rs1143634 (+3954C>T) gene polymorphism was genotyped by polymerase chain reaction and digestion product through the TaqI restriction enzyme. Genotype and allele frequencies of rs1143634 were estimated; in addition, the deviation from Hardy-Weinberg equilibrium was assessed. Allele and genotype frequencies were compared using the c2 test with likelihood ratio (p <0.05). The mean age of participants was 28.6 (SD= 11.5) years and 68.6 % were females. No statistically significant association was found between the genotypes distribution of IL-1B (+3954C>T) polymorphism with ERR (p= 0.0926). However, a significant difference in the alleles distribution (p= 0.035) was observed, where the allele T (allele 2) was more prevalent in the control group. IL-1B (+3954C>T) polymorphism was present in the study population. Although there were no differences in the genotypes distribution to support an association between this polymorphism with ERR, there was a difference in the alleles distribution, suggesting that the allele T possibly acts as a protective factor against the development of ERR.

A rare case of tick infestation of the eyelid: case report and literature review / Um caso raro de infestação de carrapatos da pálpebra: relato de caso e revisão da literatura

ABSTRACT This article presents a case of tick infestation of the lower eyelid by a previously unreported species. A 71-year-old male presented with a tick attached to the lower eyelid. The tick was identified morphologically, and then molecularly via polymerase chain reaction (PCR) and sequencing of its DNA. In addition, a review of the literature relevant to the genera of ticks associated with infestation of the human eye is provided. The tick, which was in the nymphal developmental stage, was first identified according to taxonomic keys as Dermacentor sp. For complete species identification, 16s rDNA gene PCR and sequencing were performed, which showed that the tick was D. marginatus. Systematizing tick species could assist physicians in determining the potential for transmission of tick-borne human diseases.

RESUMO Este artigo apresenta um caso de infestação por carrapatos da pálpebra inferior por uma espécie previamente não declarada. Um homem de 71 anos de idade apresentou-se com um carrapato grudado na pálpebra inferior. O carrapato foi identificado morfologicamente, e, em seguida, uma estrutura molecular através de reacção em cadeia da polimerase (PCR) e a sequenciação do seu DNA. Além disso, uma análise da literatura pertinente aos gêneros de carrapatos associados à infestação do olho humano é fornecido. O carrapato, que estava em fase de desenvolvimento das ninfas, foi identificado pela primeira vez de acordo com chaves taxonômicas com o Dermacentor sp. Para identificação de espécies completa, gene 16S rDNA PCR e sequenciamento foram realizadas, que mostrou que o carrapato foi D. marginatus. Sistematizando espécie de carrapato poderia ajudar os médicos a determinar o potencial de transmissão de doenças humanas transmitidas por carrapatos.

Comparison of Different Pretreatment Methods for DNA Extraction from Teeth / 法医学杂志

OBJECTIVE@#To compare the concentration of teeth DNA extracted by three different pretreatment methods and to explore a simple, economical and practical pretreatment method with high concentration of extracted DNA from teeth.@*METHODS@#A total number of 21 molars were collected from 7 corpses. The pretreatment of 3 molars from each individual was randomly performed by tooth crumb method, ball-milling method and liquid nitrogen milling method and 50 mg tooth crumb was weight and DNA was extracted by AutoMate Express forensic DNA extraction system. Subsequently, the concentration of DNA and corresponding STR genotyping of three methods were compared.@*RESULTS@#The DNA concentration extracted by tooth crumb method, ball-milling method and liquid nitrogen milling method was 0.055 6-1.989 1 ng/μL, 0.036 6-1.175 6 ng/μL and 0.037 8-1.249 0 ng/μL, respectively. The DNA concentration obtained by tooth crumb method was higher (P < 0.05) and the success rate of STR genotyping was high.@*CONCLUSION@#Combined with AutoMate Express forensic DNA extraction system, tooth crumb method is an efficient and feasible method to extract DNA from teeth, which can be applied in forensic practice.

Identification of groundnut (Arachis hypogaea) SSR markers suitable for multiple resistance traits QTL mapping in African germplasm

Background This study aimed to identify and select informative Simple Sequence Repeat (SSR) markers that may be linked to resistance to important groundnut diseases such as Early Leaf Spot, Groundnut Rosette Disease, rust and aflatoxin contamination. To this end, 799 markers were screened across 16 farmer preferred and other cultivated African groundnut varieties that are routinely used in groundnut improvement, some with known resistance traits. Results The SSR markers amplified 817 loci and were graded on a scale of 1 to 4 according to successful amplification and ease of scoring of amplified alleles. Of these, 376 markers exhibited Polymorphic Information Content (PIC) values ranging from 0.06 to 0.86, with 1476 alleles detected at an average of 3.7 alleles per locus. The remaining 423 markers were either monomorphic or did not work well. The best performing polymorphic markers were subsequently used to construct a dissimilarity matrix that indicated the relatedness of the varieties in order to aid selection of appropriately diverse parents for groundnut improvement. The closest related varieties were MGV5 and ICGV-SM 90704 and most distant were Chalimbana and 47-10. The mean dissimilarity value was 0.51, ranging from 0.34 to 0.66. Discussion Of the 376 informative markers identified in this study, 139 (37%) have previously been mapped to the Arachis genome and can now be employed in Quantitative Trait Loci (QTL) mapping and the additional 237 markers identified can be used to improve the efficiency of introgression of resistance to multiple important biotic constraints into farmer-preferred varieties of Sub-Saharan Africa.

Compilation of an informative microsatellite set for genetic characterisation of East African finger millet (Eleusine coracana)

Background Genetic diversity of finger millet (Eleusine coracana), a nutritious neglected staple cereal in Africa and South Asia is largely uncharacterized. This study analysed 82 published SSR markers for finger millet across 10 diverse accessions to compile an informative set for genetic characterisation. Extensive optimization compared single samples with bulked leaf or bulked DNA samples for capturing within accession genetic diversity. The markers were evaluated to determine (1) how efficiently they amplified target loci during high-throughput genotyping with a generic PCR protocol, (2) ease of scoring PCR products and (3) polymorphism and ability to discern genetic diversity within the tested finger millet germplasm. Results Across 88 samples, the 52 markers that worked well amplified 274 alleles, ranging from 2 to 14 per locus with a mean of 4.89. Major allele frequency ranged from 0.18 to 0.93 with a mean of 0.57. Polymorphic Information Content (PIC) ranged from 0.13 to 0.88 with a mean of 0.5 and availability varied between 64 and 100% with a mean of 92.8%. Heterozygosity ranged from 0 to 1.0, with a mean of 0.26. Discussion Five individual samples from an accession captured the largest number of alleles per locus compared to the four different bulked sampling strategies but this difference was not significant. The identified set comprised 20 markers: UGEP24, UGEP53, UGEP84, UGEP27, UGEP98, UGEP95, UGEP64, UGEP33, UGEP67, UGEP106, UGEP110, UGEP57, UGEP96, UGEP66, UGEP46, UGEP79, UGEP20, UGEP12, UGEP73 and UGEP5 and was since used to assess East African finger millet genetic diversity in two separate studies.

DNA from tissues of young mice is optimal for genotyping

Background Genotyping of mice is a common procedure in animal facilities. The aim of this study was to compare the quantity and quality of DNA extracted from samples obtained from young mice (YM; 10 d old) and adult mice (AM; 12 weeks old). We collected samples from the tail and ear of YM and AM. We also sampled blood, check cells (via buccal swabs), hair and fecal pellets of AM, and biopsied distal phalanx of YM. We isolated DNA using commercial kits and determined concentrations and purity by spectrophotometry. The integrity of DNA was evaluated by agarose-gel electrophoresis and polymerase chain reaction (PCR). Results DNA in all samples was amplified successfully but the intensities of bands after electrophoresis was heterogeneous. In general, tissues from YM yielded more DNA than those from AM, with differences being statistically significant for ear samples (38 ± 12 ng/μL for YM; 7 ± 3 ng/μL for AM; P = 0.006). In YM, the most DNA was obtained from ear and tail samples, with differences from the amounts obtained from phalanx samples being statistically significant (P = 0.02 and P = 0.005, respectively). In AM, the most DNA was obtained from tail and blood samples. Samples obtained by non-invasive sampling methods in adults resulted in a deficient DNA extraction. Conclusions The results of the present study do not support the previous recommendations for using non-invasive methods to genotype adult animals. The use of newborn tissue samples showed the highest efficiency for DNA extraction.

Polymorphism of three milk protein genes in Mexican Jersey cattle

The objective was to estimate the allelic and genotypic frequencies, genetic diversity and polymorphic information content for the β-casein, κ-casein and β-lactoglobulin genes. Blood and frozen semen samples were collected from 453 Jersey individuals registered by the Mexican Jersey Cattle Association. Twenty eight breed specific SNP primers for whole genes were used. The B allele of κ-casein had higher frequency (0.69) than the A (0.26) and E (0.05). For β-lactoglobulin, the highest frequency was for B (0.72), followed by A and C alleles (0.26 and 0.02, respectively). The β-casein allele with the highest frequency was A² (0.71), followed by A¹ (0.19), A³ (0.05), B (0.04) and C (0.01). The average genetic diversity (He) was 0.53. The average locus effective allele number was 1.79. These results indicate a high allelic diversity for κ-caseín, β-casein and β-lactoglobulin that could be included in breeding programs in the population studied, aimed to improve the milk quality traits of economic importance.

Development and significance of RAPD-SCAR markers for the identification of Litchi chinensis Sonn: by improved RAPD amplification and molecular cloning

Background Analysis of genetic diversity is important for the authentication of a species. Litchi (Litchi chinensis Sonn.) is a subtropical evergreen tree. Recently, L. chinensis has been characterized by an improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis. The goal of this study was to develop sequence-characterized amplified region (SCAR) markers from the improved RAPD fragments for the genetic analysis of L. chinensis. Results The improved RAPD fragments from L. chinensis were cloned, sequenced and converted into stable SCAR markers. Sequencing of three cloned RAPD fragments revealed that the clone L7-16 consisted of 222 nucleotides (GenBank accession number KM235222), clone L9-6 consisted of 648 nucleotides (GenBank accession number KM235223), and clone L11-26 consisted of 369 nucleotides (GenBank accession number KM235224). Then, specific primers for SCAR markers L7-16, L9-6, and L11-26 were designed and synthesized. PCR amplification was performed using DNA templates from 24 different samples, including 6 samples of L. chinensis and other plants. The SCAR marker L9-6 was specific for all of the L. chinensis samples, the SCAR marker L11-26 specific for five L. chinensis samples, and the SCAR marker L7-16 only specific for the samples from Luzhou. Conclusions This study developed stable SCAR markers for the identification of L. chinensis by the cloning of the improved RAPD fragments. Combining RAPD and SCAR markers provides a simple and reliable tool for the genetic characterization of plant species.

Genetic diversity in two Italian almond collections

Background Sweet-seeded domesticated almonds were brought to the Mediterranean Basin from central Asia about 4000 years ago. In Italy, most of the almonds produced are cultivated in the southern part of the country. Local populations of the tree in Sardinia are largely seed-derived and mostly self-incompatible, so have developed extensive genetic diversity. The need to protect biodiversity has prompted a revived interest in local genetic materials in almond. Two Italian collections have been established, one in Sardinia and the other in Apulia. These collections were the focus of the present evaluation of genetic diversity. Results Eleven SSRs (microsatellites) were used for fingerprinting. The Sardinian germplasm was highly polymorphic, revealing a mean of 14.5 alleles per locus and a mean heterozygosity of 0.71. Using a model-based clustering approach, two genetic clusters were distinguished: one included all the commercial varieties and most of the Sardinian accessions, and the other most of the Apulian accessions. A similar structure was produced using a distance-based cluster analysis. The Sardinian accessions could still be distinguished from the commercial germplasm with few exceptions. Conclusion The extensive genetic variability present in the Sardinian and Apulian almond germplasm indicates that these materials represent an important source of genes for the improvement of the crop.

Effect of nickel chloride on Arabidopsis genomic DNA and methylation of 18S rDNA

Background In recent years, nickel (Ni) has been widely applied in industrial and agricultural production and has become a kind of environmental pollution. In this study, the effect of nickel chloride (NiCl2) with different concentrations on Arabidopsis genomic stability and DNA methylation has been demonstrated. The nucleolus variation and 18S rDNA methylation after NiCl2 treatment have been analyzed. Results The results are as follows: (1) The NiCl2 could result in heritable genomic methylation variations. The genomic DNA methylation variations have been detected by methylation-sensitive amplified polymorphism (MSAP) molecular markers, and the result showed that after NiCl2 treatment, there was methylation variation in T0 generation seedlings, and partial site changes maintained in T1 generation, which suggested that the effects of NiCl2 on DNA methylation could be heritable in offspring. (2) NiCl2 brought deformity and damage to nucleolar structure in Arabidopsis root tip cells, and the damage was positively correlated with the NiCl2 concentration. 3. In the nucleolus, there was an increased cytosine methylation in 18S rDNA. The plant nucleolus variation and 18S rDNA methylation may be used as an examination indicator for Ni pollution in soil or plant. Conclusions NiCl2 application caused variation of DNA methylation of the Arabidopsis genomic and offspring's. NiCl2 also resulted in nucleolar injury and deformity of root tip cells. The methylation rate of 18S rDNA also changed by adding NiCl2.

Establishing a 29 Y-STR Loci Multiplex PCR System / 法医学杂志

OBJECTIVE@#To establish a 29 Y-STR loci multiplex PCR system for investigating the genetic polymorphisms and to assess its application value in forensic science.@*METHODS@#A multiplex PCR system was established using a five color fluorescence labeling 29 Y-STR loci (DYS456, DYS389 I , DYS437, DYS447, DYS389 11, DYS438, DYS522, DYS460, DYS458, DYS622, DYS390, DYS392, DYS448, DYS449, DYS391, Y-GA TA-H4, DYS388, DYS19, DYS385a/b, DYS527a/b, DYS393, DYS459a/b, DYS635, DYS439, DYS570 and DYS627) for multiple amplification and capillary electrophoresis. And its applicability was validated with genetic polymorphism data of 29 Y-STR of unrelated 2,000 male samples in Shandong Han population.@*RESULTS@#A total of 1,981 different haplotypes of 2,000 individuals showed genotype diver- sity between 0.370 0 and 0.965 4. The system provided stable and accurate typing with high sensitivity of 0.05 ng. It satisfied the needs of variety of routine biological samples.@*CONCLUSION@#The 29 Y-STR loci multiplex PCR system could be applied for actual cases and establishment of Y-STR database. In addition, it has great significance in forensic science practices and related research.

DNA Extraction of Cast-off Cells of Fingerprints from 502 Glue Fumigated Contact Samples / 法医学杂志

OBJECTIVE@#To establish a method of fingerprint position, sample transfer and fingerprint DNA extraction in contact samples.@*METHODS@#Sixty-six cases were visualized by 502 glue fingerprint fumigation. Two methods, ordinary wipe and acetone wipe, were used to transfer cast-off cells of fingerprints from testing samples, respectively. DNA was extracted and purified by ultramicro magnetic bead kit. The data was resolved on genetic analysis after amplification.@*RESULTS@#In 33 samples, 30 samples got better STR analysis by acetone wipe method. The peak range was 1,000-4,000 RFU and peak shapes were equable. It was hard to get ideal STR typing by ordinary wipe method.@*CONCLUSION@#The samples are visualized by 502 glue fingerprint fumigation and the case-off cells are transferred by acetone wipe method. The method shows better STR analysis result, which might be a better method for forensic science practice.