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A high-density genetic linkage map is essential for plant genetics and genomics research. However, due to the deficiency of genomic data and high-quality molecular markers, no genetic map has been published for Prince Rupprecht's larch ( Larix principis-rupprechtii Mayr), a conifer species with high ecological and commercial value in northern China. In this study, 145 F1 progeny individuals from an intraspecific cross between two elite clones of L . principis-rupprechtii and their parents were employed to construct the first genetic map in this important tree species using specific-locus amplified fragment sequencing (SLAF-seq). After preprocessing, the procedure yielded 300.20 Gb of raw data containing 1501.22 M pair-end reads. A total of 324,352 SNP markers were detected and 122,785 of them were polymorphic, with a polymorphism rate of 37.86%. Ultimately, 6099 SNPs were organized into a genetic map containing 12 linkage groups, consistent with the haploid chromosome number of larch and most other species in the Pinaceae family. The linkage map spanned 2415.58 cM and covered 99.6% of the L. principis-rupprechtii genome with an average of 0.4 cM between adjacent markers. To the best of our knowledge, this map is the first reference map for L . principis-rupprechtii , as well as the densest one obtained in larch species thus far. The genome-wide SNPs and the high-resolution genetic map will provide a foundation for future quantitative trait loci mapping, map-based cloning, marker-assisted selection, comparative genomics, and genome sequence assembly for larch trees., Competing Interests: The authors declare that they have no conflict of interest.

Background: 5,10-Methylentetrahydrofolate reductase (MTHFR) C677T polymorphism is one of the most studied genetic variations in the human genome. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is one of the most used techniques to characterize the point mutations in genomic sequences because of its suitability and low cost. The most widely used method for the MTHFR C677T polymorphism characterization was developed by Frosst et al. (1995) but appears to have some technical limitations. The aim of this study was to propose a novel PCR-RFLP method for the detection of this polymorphism., Methods: In order to retrieve all published articles possibly describing any PCR-RFLP methods useful to analyze MTHFR C677T polymorphism, we performed systematic queries on PubMed, using a combination of Boolean operators (AND/OR) and MeSH terms. Amplify software was used in order to design a new primer pair following the optimal standard criteria. Primer-BLAST software was used to check primer pair's biological specificity., Results: The analysis of previous literature showed that PCR-RFLP method remains the most used technique. None of the 108 primer pairs described was ideal with regard to main accepted primer pair biochemical technical parameters. The new primer pair amplifies a DNA-fragment of 513 base pair (bp) that, in the presence of the polymorphism, is cut by Hinf I enzyme in two pieces of 146 bp and 367 bp and clearly visible on 2% agarose gel. The level of expertise and the materials required are minimal and the protocol takes one day to carry out., Conclusion: Our original PCR-RFLP strategy, specifically designed to make the analysis optimal with respect to PCR primers and gel analysis, fits the ideal criteria compared to the widely used strategy by Frosst et al (1995) as well as any other PCR-RFLP strategies proposed for MTHFR C677T polymorphism genotyping to date., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

The amplified fragment length polymorphisms (AFLP) method has become an attractive tool in phylogenetics due to the ease with which large numbers of characters can be generated. In contrast to sequence-based phylogenetic approaches, AFLP data consist of anonymous multilocus markers. However, potential artificial amplifications or amplification failures of fragments contained in the AFLP data set will reduce AFLP reliability especially in phylogenetic inferences. In the present study, we introduce a new automated scoring approach, called "AMARE" (AFLP MAtrix REduction). The approach is based on replicates and makes marker selection dependent on marker reproducibility to control for scoring errors. To demonstrate the effectiveness of our approach we record error rate estimations, resolution scores, PCoA and stemminess calculations. As in general the true tree (i.e. the species phylogeny) is not known, we tested AMARE with empirical, already published AFLP data sets, and compared tree topologies of different AMARE generated character matrices to existing phylogenetic trees and/or other independent sources such as morphological and geographical data. It turns out that the selection of masked character matrices with highest resolution scores gave similar or even better phylogenetic results than the original AFLP data sets.

Fluorescent amplified fragment length polymorphism (FAFLP) analysis was applied to genetically fingerprint 'working culture control strains' used by accredited food microbiology laboratories. A working culture control strain is defined as a subculture from a strain initially obtained from an authenticated source [such as the National Collection of Type Cultures (NCTC)] that is maintained for use with routine testing within the laboratory. Working culture control strains from eight food examination laboratories, representing four bacterial species, were analysed by FAFLP; these were Salmonella Nottingham, Staphylococcus aureus, Listeria monocytogenes and Bacillus cereus. The resultant FAFLP profiles of the eight working culture control strains for each of these species were compared against the appropriate freeze-dried ampoules obtained directly from NCTC. FAFLP results demonstrated that within 50% of working cultures analysed, several laboratories were routinely using working cultures that were genetically different from the original reference NCTC strains. This study highlights the need for laboratories to review the protocols used to process and maintain control strains and working cultures, with a potential view to utilize single-use quality control materials., (2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.)

Several methods of molecular analysis of microbial diversity, including terminal restriction fragment length polymorphism (T-RFLP) analysis are based on measurement of the DNA fragment length. Significant variation between sequence-determined and measured length of restriction fragments (drift) has been observed, which can affect the efficiency of the identification of microorganisms in the analyzed communities. In the past, this variation has been attributed to varying fragment length and purine content. In this study, principal component analysis and multiple regression analysis were applied to find the contributions of those and several other fragment characteristics. We conclude that secondary structure melting point and G+C nucleotide content, besides the fragment length, contribute to the variation observed, whereas the contribution of purine content is less important. Incomplete denaturation of the sample at the start of electrophoretic separation of fragments has been excluded as a major cause of the variation observed. Our regression model explains the observed drift variation by approximately 56%, with standard deviation of the prediction equal to approximately 1.3 bp., ((c) 2010 Elsevier B.V. All rights reserved.)

A reliable data set is a fundamental prerequisite for consistent results and conclusions in population genetic studies. However, marker scoring of genetic fingerprints such as amplified fragment length polymorphisms (AFLPs) is a highly subjective procedure, inducing inconsistencies owing to personal or laboratory-specific criteria. We applied two alternative marker selection algorithms, the newly developed script scanAFLP and the recently published AFLPScore, to a large AFLP genome scan to test how population genetic parameters and error rates were affected. These results were confronted with replicated random selections of marker subsets. We show that the newly developed marker selection criteria reduced the mismatch error rate and had a notable influence on estimates of genetic diversity and differentiation. Both effects are likely to influence biological inference. For example, genetic diversity (HS) was 29% lower while genetic differentiation (FST) was 8% higher when applying scanAFLP compared with AFLPScore. Likewise, random selections of markers resulted in substantial deviations of population genetic parameters compared with the data sets including specific selection criteria. These randomly selected marker sets showed surprisingly low variance among replicates. We conclude that stringent marker selection and phenotype calling reduces noise in the data set while retaining patterns of population genetic structure.

The variability of terminal restriction fragment polymorphism analysis applied to complex microbial communities was assessed statistically. Recent technological improvements were implemented in the successive steps of the procedure, resulting in a standardized procedure which provided a high level of reproducibility.

Since rapid diagnosis is critical in control of tuberculosis, nucleic acid amplification techniques have been widely used. The purpose of the present study was to assess the performance of Amplified Mycobacterium tuberculosis Direct Test (Amplified MTD Test, Gen-Probe) for the diagnosis of pulmonary and extrapulmonary tuberculosis in our laboratory. A total of 267 specimens (170 pulmonary and 97 extrapulmonary) were tested in the Clinical Mycobacteriology Laboratory of Manisa (a province located in Aegean part of Turkey) University Hospital from September 2001 to March 2005. When Amplified MTD (AMTD) test results were compared to the culture results taken as the gold standard, the sensitivity, specificity, positive and negative predictive values (PPV, NPV) for pulmonary specimens were found to be 84%, 96%, 73%, and 98%, respectively. When AMTD test positive, culture negative discrepant results were evaluated against the clinical history of the patients, these rates were detected as; 88%, 100%, 100%, and 98%, respectively. For 97 extrapulmonary specimens, sensitivity, specificity, PPV and NPV of AMTD test were 60%, 100%, 100%, and 98%, respectively. In conclusion, the results of the AMTD assay were reliable for the rapid diagnosis of pulmonary tuberculosis; if the results were evaluated together with the clinical status of patients, the performance of the test would be increased. However, even though the culture positive extrapulmonary specimens were sparse in our study (5%), the sensitivity of the AMTD test in extrapulmonary specimens was found less than that in pulmonary specimens. Therefore it is thought that AMTD test results should be evaluated carefully for the diagnosis of extrapulmonary tuberculosis.