Identification and validation of microsatellite markers in strawberry tree (Arbutusunedo L.)

Strawberry tree (Arbutus unedo L.), an evergreen shrub/small tree of the family Ericaceae, is a main constituent of the Mediterranean basin flora; although it is also found in southwestern Prance, Macaronesia, and Ireland. The small fruits are edible but mostly used for preparation of preserves and jams, and for liquors such as the Portuguese traditional "aguardente de medronho". Traditionally cultivated by small farmers, often in consociation with Quercus sp., strawberry tree is presently emerging as a new important fruit crop cultivated in large orchards by modern export-oriented enterprises. This change of paradigm requires a growing role of plant breeding, upstream of the production process. Genomic tools for this species are mostly limited to the chloroplast genome sequence and to genomic data described in this work. In order to identify strawberry tree microsatellite (SSR) loci we performed partial genome next-generation sequencing using the Ion Torrent technology. The sequenced similar to 24.6M nucleotides resulted in the identification of 1185 microsatellite markers mostly constituted by dinucleotide motifs. The relative amount of microsatellite dinucleotide motifs (AG/CT - 71.7%, AC/GT - 20.5%, AT/AT - 2.9%, and CG/CG - 0.3%) is similar to the one observed in other Ericaceae species. Among a tested sample of 40 SSR primer pairs, 20 amplified well-defined PCR products, 12 (30%) were validated as polymorphic. Used in our collaborative project for molecular identification of selected and improved clones, the identified SSR loci constitute a strong tool for a large panoply of applied and fundamental studies of this emerging fruit crop.Pluriannual Funding Program of the Portuguese National Foundation for Science and Technologyinfo:eu-repo/semantics/publishedVersio

Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants

BACKGROUND: DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants. RESULTS: We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1). After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes. CONCLUSION: This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction

Manual on application of molecular tools in aquaculture and inland fisheries management. Part 2. Laboratory protocols and data analysis

The aim of this manual is to provide a comprehensive practical tool for the generation and analysis of genetic data for subsequent application in aquatic resources management in relation to genetic stock identification in inland fisheries and aquaculture. The material only covers general background on genetics in relation to aquaculture and fisheries resource management, the techniques and relevant methods of data analysis that are commonly used to address questions relating to genetic resource characterisation and population genetic analyses. No attempt is made to include applications of genetic improvement techniques e.g. selective breeding or producing genetically modified organisms (GMOs). The manual includes two ‘stand-alone’ parts, of which this is the second volume: Part 1 – Conceptual basis of population genetic approaches: will provide a basic foundation on genetics in general, and concepts of population genetics. Issues on the choices of molecular markers and project design are also discussed. Part 2 – Laboratory protocols, data management and analysis: will provide step-by-step protocols of the most commonly used molecular genetic techniques utilised in population genetics and systematic studies. In addition, a brief discussion and explanation of how these data are managed and analysed is also included. This manual is expected to enable NACA member country personnel to be trained to undertake molecular genetic studies in their own institutions, and as such is aimed at middle and higher level technical grades. The manual can also provide useful teaching material for specialised advanced level university courses in the region and postgraduate students. The manual has gone through two development/improvement stages. The initial material was tested at a regional workshop and at the second stage feedback from participants was used to improve the contents

Molecular biology techniques as a tool for detection and characterisation of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of paratuberculosis, also known as Johne’s disease, a chronic intestinal infection in cattle and other ruminants. Paratuberculosis is characterised by diarrhea and weight loss that occurs after a period of a few months up to several years without any clinical signs. The considerable economic losses to dairy and beef cattle producers are caused by reduced milk production and poor reproduction performance in subclinically infected animals. Early diagnosis of infected cattle is essential to prevent the spread of the disease. Efforts have been made to eradicate paratuberculosis by using a detection and cull strategy, but eradication is hampered by the lack of suitable and sensitive diagnostic methods. This thesis, based on five scientific investigations, describes the development of different DNA amplification strategies for detection and characterisation of M. paratuberculosis. Various ways to pre-treat bacterial cultures, tissue specimens and fecal samples prior to PCR analysis were investigated. Internal positive PCR control molecules were developed and used in PCR analyses to improve the reliability and to facilitate the interpretation of the results. The sensitivity of the ultimate methods was found to be approximate that of culture and allowed detection of low numbers of M. paratuberculosis expected to be found in subclinically infected animals. Genomic DNA of a Swedish mycobacterial isolate, incorrectly identified by PCR as M. paratuberculosis was characterised. The isolate was closely related to M. cookii and harboured one copy of a DNA segment with 94% similarity to IS900, the target sequence used in diagnostic PCR for detection of M. paratuberculosis. This finding highlighted the urgency of developing or evaluating PCR systems based on genes other than IS900. A PCR-based fingerprinting method using primers targeting the enterobacterial intergenic consensus sequence (ERIC) and the IS900 sequence was developed and successfully used to distinguish M. paratuberculosis from closely related mycobacteria, including the above mentioned mycobacterial isolate. In conclusion, the molecular biology techniques developed in these studies have proved useful for accelerating the diagnostic detection and characterisation of M. paratuberculosis

A rapid and reproducible method for isolating genomic DNA from a few crop plants suitable for polymerase chain reaction-based genotyping

As most of the molecular markers in crop molecular breeding programmes are successful based on polymerase chain reaction (PCR), the isolated genomic DNA must be suitable for the same. Though PCR is a robust method and in most of the cases requires only a minute amount of genomic DNA as template, removal of potential PCR-inhibitory factors is quite important. The present work reports the optimization of a rapid genomic DNA isolation method, suitable for PCR-based genotyping of plants. As very minute amount of the genomic DNA isolated in this rapid method was found to be sufficient for PCR, a researcher is capable to go for several hundred independent PCR from single isolation. The method was validated in 4 different crops (wheat, tomato, brinjal and cauliflower) using different PCR-based molecular markers. In case of wheat, genomic DNA isolated in this method was found to be suitable PCR using the specific marker for the detection of the Lr34 gene. For tomato, genomic DNA isolated in this method was successfully used with the molecular markers for the detection of resistance alleles for yellow leaf curl disease and root knot disease. In case of brinjal, the isolated genomic DNA was found to be suitable for simple sequence repeat (SSR) marker assay. In a similar way, genomic DNA isolated in this method from cauliflower leaves was observed to be suitable for amplifying a gene of ~1.5 kb length. Thus, this method will be quite helpful to expedite marker assisted selection of plants in plant molecular breeding programmes

A simple, rapid and efficient method for the extraction of genomic DNA from Allium roseum L. (Alliaceae)

The isolation of intact, high-molecular-mass genomic DNA is essential for many molecular biology applications including long range PCR, endonuclease restriction digestion, southern blot analysis, and genomic library construction. Many protocols are available for the extraction of DNA from plant material, but obtain it is difficult in many plants because of metabolites that interfere with DNA isolation procedures and subsequent applications. With frame of the present work, we developed the first reliable and efficient method for isolatingAllium roseum L. genomic DNA that is free from polysaccharides and polyphenols. This protocol uses 100 mM Tris-HCl (pH 8), 20 mM EDTA, 1.4 M NaCl, 3% PVP (polyvinylpyrrolidone 40.000), 3% mercaptoethanol, and an incubation at 65°C for 1 h. The purity of isolated genomic DNA was confirmed by spectrophotometric analyses (A260/230 ratio of 1.947, A260/280 of 1.804). DNA was obtained in the amount of 189  Allium roseum L. genomic DNA that is free from polysaccharides and polyphenols. This protocol uses 100 mM Tris-HCl (pH 8), 20 mM EDTA, 1.4 M NaCl, 3% PVP (polyvinylpyrrolidone 40.000), 3% mercaptoethanol, and an incubation at 65°C for 1 h. The purity of isolated genomic DNA was confirmed by spectrophotometric analyses (A260/230 ratio of 1.947, A260/280 of 1.804). DNA was obtained in the amount of 189 Allium roseum L. genomic DNA that is free from polysaccharides and polyphenols. This protocol uses 100 mM Tris-HCl (pH 8), 20 mM EDTA, 1.4 M NaCl, 3% PVP (polyvinylpyrrolidone 40.000), 3% mercaptoethanol, and an incubation at 65°C for 1 h. The purity of isolated genomic DNA was confirmed by spectrophotometric analyses (A260/230 ratio of 1.947, A260/280 of 1.804). DNA was obtained in the amount of 189 g per gram of leaf material, and it proved amenable to restriction digestion

Optimization of DNA Extraction for RAPD and ISSR Analysis of Arbutus unedo L. Leaves

Genetic analysis of plants relies on high yields of pure DNA. For the strawberry tree (Arbutus unedo) this represents a great challenge since leaves can accumulate large amounts of polysaccharides, polyphenols and secondary metabolites, which co-purify with DNA. For this specie, standard protocols do not produce efficient yields of high-quality amplifiable DNA. Here, we present for the first time an improved leaf-tissue protocol, based on the standard cetyl trimethyl ammonium bromide protocol, which yields large amounts of high-quality amplifiable DNA. Key steps in the optimized protocol are the addition of antioxidant compounds—namely polyvinyl pyrrolidone (PVP), 1,4-dithiothreitol (DTT) and 2-mercaptoethanol, in the extraction buffer; the increasing of CTAB (3%, w/v) and sodium chloride (2M) concentration; and an extraction with organic solvents (phenol and chloroform) with the incubation of samples on ice. Increasing the temperature for cell lyses to 70 °C also improved both DNA quality and yield. The yield of DNA extracted was 200.0 ± 78.0 μg/μL and the purity, evaluated by the ratio A260/A280, was 1.80 ± 0.021, indicative of minimal levels of contaminating metabolites. The quality of the DNA isolated was confirmed by random amplification polymorphism DNA and by inter-simple sequence repeat amplification, proving that the DNA can be amplified via PCR

Molecular studies on intraspecific diversity and phylogenetic position of Coniothyrium minitans

Simple sequence repeat (SSR)±PCR amplification using a microsatellite primer (GACA)% and ribosomal RNA gene sequencing were used to examine the intraspecific diversity in the mycoparasite Coniothyrium minitans based on 48 strains, representing eight colony types, from 17 countries world-wide. Coniothyrium cerealis, C. fuckelii and C. sporulosum were used for interspecific comparison. The SSR±PCR technique revealed a relatively low level of polymorphism within C. minitans but did allow some differentiation between strains. While there was no relationship between SSR±PCR profiles and colony type, there was some limited correlation between these profiles and country of origin. Sequences of the ITS 1 and ITS 2 regions and the 5±8S gene of rRNA genes were identical in all twenty-four strains of C. minitans examined irrespective of colony type and origin. These results indicate that C. minitans is genetically not very variable despite phenotypic differences. ITS and 5±8S rRNA gene sequence analyses showed that C. minitans had similarities of 94% with C. fuckelii and C. sporulosum (which were identical to each other) and only 64% with C. cerealis. Database searches failed to show any similarity with the ITS 1 sequence for C. minitans although the 5±8S rRNA gene and ITS 2 sequences revealed an 87% similarity with Aporospora terricola. The ITS sequence including the 5±8S rRNA gene sequence of Coniothyrium cerealis showed 91% similarity to Phaeosphaeria microscopica. Phylogenetic analyses using database information suggest that C. minitans, C. sporulosum, C. fuckelii and A. terricola cluster in one clade, grouping with Helminthosporium species and 'Leptosphaeria' bicolor. Coniothyrium cerealis grouped with Ampelomyces quisqualis and formed a major cluster with members of the Phaeosphaeriacae and Phaeosphaeria microscopica

Patent applications for using DNA technologies to authenticate medicinal herbal material

Herbal medicines are used in many countries for maintaining health and treating diseases. Their efficacy depends on the use of the correct materials, and life-threatening poisoning may occur if toxic adulterants or substitutes are administered instead. Identification of a medicinal material at the DNA level provides an objective and powerful tool for quality control. Extraction of high-quality DNA is the first crucial step in DNA authentication, followed by a battery of DNA techniques including whole genome fingerprinting, DNA sequencing and DNA microarray to establish the identity of the material. New or improved technologies have been developed and valuable data have been collected and compiled for DNA authentication. Some of these technologies and data are patentable. This article provides an overview of some recent patents that cover the extraction of DNA from medicinal materials, the amplification of DNA using improved reaction conditions, the generation of DNA sequences and fingerprints, and the development of high-throughput authentication methods. It also briefly explains why these patents have been granted