Species-specific DNA Sequences Research Articles

Genomic Signature in Evolutionary Biology: A Review

Organisms are unique physical entities in which information is stored and continuously processed. The digital nature of DNA sequences enables the construction of a dynamic information reservoir. However, the distinction between the hardware and software components in the information flow is crucial to identify the mechanisms generating specific genomic signatures. In this work, we perform a bibliometric analysis to identify the different purposes of looking for particular patterns in DNA sequences associated with a given phenotype. This study has enabled us to make a conceptual breakdown of the genomic signature and differentiate the leading applications. On the one hand, it refers to gene expression profiling associated with a biological function, which may be shared across taxa. This signature is the focus of study in precision medicine. On the other hand, it also refers to characteristic patterns in species-specific DNA sequences. This interpretation plays a key role in comparative genomics, identifying evolutionary relationships. Looking at the relevant studies in our bibliographic database, we highlight the main factors causing heterogeneities in genome composition and how they can be quantified. All these findings lead us to reformulate some questions relevant to evolutionary biology.

Catch crop mixtures have higher potential for nutrient carry-over than pure stands under changing environments

Winter catch crops are grown to scavenge nutrients over a period of unfavorable growth conditions and to conserve nutrients for subsequent release to the following main crop. Since environmental conditions have a strong impact on the growth and nutrient capture in roots and shoots of individual catch crop species, we anticipated that mixtures will be more durable and efficient in nutrient capture due to compensatory effects among component species. We tested this hypothesis and determined the nitrogen and phosphorus accumulation in the shoots and roots of four catch crop species grown in pure vs. mixed stands at two sites for two or three years. Element concentrations were determined in the root and shoot biomass of each species and used to calculate the nutrient pool fixed in the root or shoot biomass. A qPCR-based technique was applied to quantify the root biomass of individual species based on species-specific DNA sequences. Despite considerable variation across environments, the overall plant biomass of white mustard (Sinapis alba), lacy phacelia (Phacelia tanacetifolia) and bristle oat (Avena strigosa) was similar and higher than that of Egyptian clover (Trifolium alexandrinum). While pure stands varied 6- to 24-fold in shoot biomass depending on environmental conditions, the variation was only ~3-fold for catch crop mixtures, with less pronounced variation in the root biomass. In general, the root biomass was comparable to the shoot biomass in each species. Roots contributed 26–46% of the nitrogen and 36–48% of the phosphorus to the total accumulation of these nutrients in the catch crop biomass, thus emphasizing the importance of plant roots as belowground nutrient pool for potential carry-over of nutrients to the subsequent crop. Although the mixture was mostly dominated by two of the four species, namely mustard and phacelia, it captured similar or even larger amounts of nutrients than the best-performing pure stand under any growth condition. This was the case for shoot- and for root-bound nutrients. Our results indicate that catch crop mixtures have higher durability than pure cultures to environmental variations. The amount of nitrogen captured by the mixture meets the average postharvest nitrogen that is left over by a wide range of cash crops, thus emphasizing that catch crop mixtures represent an efficient nutrient management tool in crop rotations.

In depth search of the Sequence Read Archive database reveals global distribution of the emerging pathogenic fungus Scedosporium aurantiacum.

Scedosporium species are emerging opportunistic fungal pathogens causing various infections mainly in immunocompromised patients, but also in immunocompetent individuals, following traumatic injuries. Clinical manifestations range from local infections, such as subcutaneous mycetoma or bone and joint infections, to pulmonary colonization and severe disseminated diseases. They are commonly found in soil and other environmental sources. To date S. aurantiacum has been reported only from a handful of countries. To identify the worldwide distribution of this species we screened publicly available sequencing data from fungal metabarcoding studies in the Sequence Read Archive (SRA) of The National Centre for Biotechnology Information (NCBI) by multiple BLAST searches. S. aurantiacum was found in 26 countries and two islands, throughout every climatic region. This distribution is like that of other Scedosporium species. Several new environmental sources of S. aurantiacum including human and bovine milk, chicken and canine gut, freshwater, and feces of the giant white-tailed rat (Uromys caudimaculatus) were identified. This study demonstrated that raw sequence data stored in the SRA database can be repurposed using a big data analysis approach to answer biological questions of interest.Lay summaryTo understand the distribution and natural habitat of S. aurantiacum, species-specific DNA sequences were searched in the SRA database. Our large-scale data analysis illustrates that S. aurantiacum is more widely distributed than previously thought and new environmental sources were identified.

A multiplex PCR method based on nuclear and cytoplasmic inheritance to identify the horse and donkey-derived components of Asini Colla Corii and the hide

To identify the original components of Asini Colla Corii and its raw material hides provides a guarantee for authenticity of Asini Colla Corii. It is urgent for Asini Colla Corii production enterprises and market supervision departments to develop effective identification methods of Asini Colla Corii and hides derived from horses, donkeys, mules and hinnies. This study screened species-specific DNA sequences of nuclear and mitochondrial genomes as detection targets, designed horse and donkey specific primers and established multiple PCR identification methods for identifying the animal hides (including the horse, donkey, mule and hinny) and Asini Colla Corii containing horse-derived and donkey-derived components. Our method can identify the horse, donkey, mule and hinny hides and horse, donkey-derived components of Asini Colla Corii with high species specificity (no crossed amplification was observed ). The limit of detection was 0.2 ng DNA. The method developed in this study provides technical support for Asini Colla Corii production enterprises and market supervision departments.

DNA barcoding: a way forward to obtain deep insights about the realistic diversity of living organisms

DNA barcoding is an innovative tool to rapidly identify and classify organisms based on highly conserved species-specific DNA sequences. This technique has developed in parallel with other genomic-based investigations, which share special emphasis on the acquisition of data pertaining to hotspots regions of DNA (usually short sequence of DNA up to 400–800 bps). These molecular techniques helped answer many queries that remain unanswered by the traditional approaches to accurately identify organisms across the kingdoms of life. Barcoding has narrowed the gap created between diverse range of ecological and traditional studies previously left by the traditional taxonomists. These internal DNA sequence tags, which serve as molecular operational taxonomic units, originate from both nuclear as well as cytoplasmic DNA, making it further convenient to barcode organisms from lower to higher organisms. The most commonly used DNA regions in molecular taxonomic approaches include the internal transcribed spacer regions in nuclear DNA, Cox1 region in mitochondrial DNA, rbcL and mat K gene in chloroplast DNA. Further, many other DNA regions suitable for barcoding have also been identified. It is increasingly evident from a large number of investigations that DNA barcoding has made Linnaean taxonomic system more accessible and convenient to other traditional biologists. Current understanding of the DNA barcoding will further help to understand the basic mechanism and also reveal applications of this approach in different fields. Primarily these approaches offer traditional taxonomists a great opportunity to expand the realistic inventory of the diversity of flora and fauna on planet earth. This review has been written to have better understanding of the mechanism of DNA barcoding, knowledge of DNA regions that can be used as targets, applications and implications of DNA barcoding.

A multiplex PCR method for detection of five animal species in processed meat products using novel species-specific nuclear DNA sequences

The continuous development of fast and simple new methods to identify animal-derived ingredients is very important for the authentication of meat products. This study intended to develop a multiplex PCR method using new species-specific nuclear DNA (nDNA) sequences for the detection of ingredients derived from sheep/goat, bovine, chicken, duck and pig in meat products. Sequence alignment analysis in 53 species showed high specificity of species-specific nDNA. Species-specific primers were designed on the conservative region of each species-specific nDNA sequence. The specificity and conservation of the sequences and primers were verified by PCR reaction and sequencing with the limit of detection down to 0.5 ng. Then, a species-specific multiplex PCR method was developed and optimized to simultaneously detect sheep/goat (237 bp), bovine (223 bp), chicken (192 bp), duck (168 bp) and pig (154 bp) in one reaction. Various processed meat products containing one or more animal-derived ingredients were detected by the developed multiplex PCR method, and the results were consistent with their labeled meat species. Our study provides a fast and simple detection method for regulating labeling of animal-derived ingredients in meat products.

Paper-based DNA biosensor for food authenticity testing

A paper-based DNA biosensor was developed for food authenticity testing using dairy products as a model. DNA from milk-based samples was isolated, and species-specific DNA sequences were amplified and identified by the biosensor using specific DNA probes. The properties of gold nanoparticles were exploited for visual detection. The biosensor was applied for detection of three species, namely cow, sheep and goat, while as low as 1.6 fmol for cow and goat, and 3.1 fmol for sheep PCR product were detected. Moreover, adulteration down to 0.01% could be detected, based on binary mixtures of cows', ewes' and goats' milk yogurt, containing 0.01 to 5% of cows' yogurt in ewes' and goats' yogurts, respectively. The proposed paper-based DNA biosensor offered 10 times higher detectability than other methods, good specificity and reproducibility, and could be applied easily for the detection of other adulterated food products, such as meat, olive oil and legumes.

Database establishment for the secondary fungal DNA barcode translational elongation factor 1α (TEF1α) 1.

With new or emerging fungal infections, human and animal fungal pathogens are a growing threat worldwide. Current diagnostic tools are slow, non-specific at the species and subspecies levels, and require specific morphological expertise to accurately identify pathogens from pure cultures. DNA barcodes are easily amplified, universal, short species-specific DNA sequences, which enable rapid identification by comparison with a well-curated reference sequence collection. The primary fungal DNA barcode, ITS region, was introduced in 2012 and is now routinely used in diagnostic laboratories. However, the ITS region only accurately identifies around 75% of all medically relevant fungal species, which has prompted the development of a secondary barcode to increase the resolution power and suitability of DNA barcoding for fungal disease diagnostics. The translational elongation factor 1α (TEF1α) was selected in 2015 as a secondary fungal DNA barcode, but it has not been implemented into practice, due to the absence of a reference database. Here, we have established a quality-controlled reference database for the secondary barcode that together with the ISHAM-ITS database, forms the ISHAM barcode database, available online at http://its.mycologylab.org/ . We encourage the mycology community for active contributions.

Lateral flow test for meat authentication with visual detection

A new lateral flow assay has been developed for meat authentication. The assay is based on gold nanoparticles and enables visual detection, by naked eye, of meat species-specific DNA sequences. The procedure includes DNA isolation from fresh meat samples, amplification of specific DNA sequences and detection by the lateral flow assay. The proposed assay is rapid and has been applied for the identification of four animal species: horse, pork, beef and sheep. The detection is completed within 25–30 min after amplification. The assay offers high detectability and good selectivity and reproducibility. As low as 0.01% of horse and 0.02% of pork DNA were detectable in binary mixtures by the reported lateral flow device.

Candida species biotypes in the oral cavity of infants and children with orofacial clefts under surgical rehabilitation

Patients with orofacial clefts present various risk factors for oral infectious diseases, resulting from anatomical and physiological changes and those resulting from rehabilitating therapeutic interventions. The incidence of Candida species in groups of babies and children with orofacial clefts, during pre- and post-operative periods and until return to first consultation, and the profiles for antifungal sensitivity and virulence in vitro were investigated. Oral samples were collected at different times over the surgical procedures and post-surgical clinical consultation and seeded in chromogenic culture media CHROMagar Candida®. Candida biotypes were identified by accessing species-specific genomic DNA sequences by PCR techniques and electrophoretic procedures. Antifungal susceptibility testing was performed by the method of microdilution in broth using the antifungals amphotericin B (AP), nystatin (NYS) and fluconazole (FLC). SAP and PL exoenzyme activities were determined by classical microbiological methods. Some orofacial clefts occurred preferentially in male or female. Low incidence (39.1%) of oral colonization by Candida species (C. albicans, C. krusei, C. tropicalis and Candida spp.) was reported in patient admission to surgical ward, with no correlation to orofacial cleft types or surgical history. Significant reduction in frequencies of Candida and changes of species, over sampling periods, showed dynamic patterns of oral colonization: elimination, maintenance or neocolonization of the biotypes. These biotypes showed sensitivity to AP (100%), partial resistance to FLC (<10%) and variable MICs for NYS (0.125–4 μg/mL), in addition to strong exoenzyme activities, especially for SAP. Clinical and therapeutic conducts for surgical rehabilitation, anatomical and physiological characteristics of patients with orofacial clefts, and cultural behavior and regionalism of the patient population served could influence the frequencies and dynamics of oral colonization by Candida species. The data showed Candida biotypes resistant to FLC and sensitive (AP) or clinically compatible (NYS) to polyenes, especially C. albicans, in the oral cavity of patients predisposed to oral colonization and candidiases, contributing to clinical conducts in possible antifungal therapies. These biotypes were considered potentially virulent and able to partially modulate their virulence factors, especially SAP, under the conditions favored by host.

Monitoring of parasitic diseases in productive animals in the Samara region

The purpose of the research: to monitor the epizootic situation on parasitic diseases in productive animals in the Samara region using a database. materials and methods. Research was performed at the Department of invasive diseases of Samara Research Veterinary Station Samara RVS FSBSI. Fecal samples from productive animals were examined by Fuelleborn’s method using polymerase chain reaction to visualize species-specific DNA sequences, and an immune chromatographic method in detecting of antigens Dirofilaria immitis in serum, plasma and whole blood of dogs and oocysts Giardia duodenalis in dogs’ feces. The extensity of invasion was estimated with regard to the number of analyses of biomaterial from animals whose owners consulted veterinary physicians. For the monitoring, we used the database “Parasitic diseases in productive animals and small domestic animals in the Samara region” developed in Samara RVS in 2013 which enables to monitor the health status of the animal during its lifetime as well as epizootic situation on parasitic diseases in single localities, economies, districts and in Samara region in general. Results and discussion. Research results revealed that Strongylata had been registered in 17 districts at extensity of invasion from 5 to 70%, Strongyloides, Moniezia, Trichocephala - in 10 districts (EI 10-80%), Skrjabinema - in 7 districts ( EI 5-10%), Nematodirus - in 5 districts (EI 20-80%), Parascaris, Ascaris, Paramphistomum - in 3 districts (EI 5-40%), Fasciola, Coccidia, Dictyocaulus - in 2 districts (EI 5-15%). The biggest species diversity of helminths (7 species) were found in Bolsheglushitsky, Kinelsky and Borsky districts of the Samara region. The monitoring showed the decrease in worm species diversity in 2016 in all districts of Samara region compared to 2015. Parasitic protozoans, Eimeria, were found in all districts of the Samara region. A significant reduction of worm species diversity was associated with permanent planned treatment of productive animals with anthelmintics. The results obtained were used in a number of livestock farms of the Samara region to control the epizootic situation as well as for treatment and prevention of diseases in productive animals.

Colletotrichum coccodes in potato and tomato leaves in Russia

Colletotrichum coccodes is a plant pathogenic fungus affecting different organs of potato, tomato, and some other plants. The leaf infection with C. coccodes may result in a rapid development of infection nidus and high losses caused by the infection of tomato fruits and potato tubers during harvesting. Our study showed the presence of C. coccodes on tomato and potato leaves with dry necrotic lesions. The presence of a DNA region specific for C. coccodes was detected in DNA samples isolated from infected tomato leaves collected in the Rostov region (7 and 1 samples from two distinct fields) and the Krasnodar Territory (5 samples from one field). In the case of potato leaf samples, the frequency of C. coccodes detection was lower than in tomato leaves. This fungus was revealed only in five potato leaf samples: one from the Northern Ossetia, one from the Kostroma region, and three from the Mariy El Republic. Pure cultures of two isolates were isolated from a naturally infected potato leaf. According to morphological criteria, size of spores and sclerotia, habitus of colonies, and species-specific DNA sequences, both strains completely corresponded to C. coccodes.

Multiplex-PCR As a Rapid and Sensitive Method for Identification of Meat Species in Halal-Meat Products.

Species identification and authentication in meat products are important subjects for ensuring the health of consumers. The multiplex-PCR amplification and species- specific primer set were used for the identification of horse, donkey, pig and other ruminants in raw and processed meat products. Oligonucleotid primers were designed and patented for amplification of species-specific mitochondrial DNA sequences of each species and samples were prepared from binary meat mixtures. The results showed that meat species were accurately determined in all combinations by multiplex-PCR, and the sensitivity of this method was 0.001 ng, rendering this technique open to and suitable for use in industrial meat products. It is concluded that more fraud is seen in lower percentage industrial meat products than in higher percentage ones. There was also more fraud found in processed products than in raw ones. This rapid and useful test is recommended for quality control firms for applying more rigorous controls over industrial meat products, for the benefit of target consumers.

Subtelomeres constitute a safeguard for gene expression and chromosome homeostasis.

The subtelomere, a telomere-adjacent chromosomal domain, contains species-specific homologous DNA sequences, in addition to various genes. However, the functions of subtelomeres, particularly subtelomeric homologous (SH) sequences, remain elusive. Here, we report the first comprehensive analyses of the cellular functions of SH sequences in the fission yeast, Schizosaccharomyces pombe. Complete removal of SH sequences from the genome revealed that they are dispensable for mitosis, meiosis and telomere length control. However, when telomeres are lost, SH sequences prevent deleterious inter-chromosomal end fusion by facilitating intra-chromosomal circularization. Surprisingly, SH-deleted cells sometimes survive telomere loss through inter-chromosomal end fusions via homologous loci such as LTRs, accompanied by centromere inactivation of either chromosome. Moreover, SH sequences function as a buffer region against the spreading of subtelomeric heterochromatin into the neighboring gene-rich regions. Furthermore, we found a nucleosome-free region at the subtelomeric border, which may be a second barrier that blocks heterochromatin spreading into the subtelomere-adjacent euchromatin. Thus, our results demonstrate multiple defense functions of subtelomeres in chromosome homeostasis and gene expression.

Molecular Characterization, Morphological Characteristics, Virulence, and Geographic Distribution of Rhizoctonia spp. in Washington State.

Rhizoctonia root rot and bare patch, caused by Rhizoctonia solani anastomosis group (AG)-8 and R. oryzae, are chronic and important yield-limiting diseases of wheat and barley in the Inland Pacific Northwest (PNW) of the United States. Major gaps remain in our understanding of the epidemiology of these diseases, in part because multiple Rhizoctonia AGs and species can be isolated from the same cereal roots from the field, contributing to the challenge of identifying the causal agents correctly. In this study, a collection totaling 498 isolates of Rhizoctonia was assembled from surveys conducted from 2000 to 2009, 2010, and 2011 over a wide range of cereal production fields throughout Washington State in the PNW. To determine the identity of the isolates, PCR with AG- or species-specific primers and/or DNA sequence analysis of the internal transcribed spacers was performed. R. solani AG-2-1, AG-8, AG-10, AG-3, AG-4, and AG-11 comprised 157 (32%), 70 (14%), 21 (4%), 20 (4%), 1 (0.2%), and 1 (0.2%), respectively, of the total isolates. AG-I-like binucleate Rhizoctonia sp. comprised 44 (9%) of the total; and 53 (11%), 80 (16%), and 51 (10%) were identified as R. oryzae genotypes I, II, and III, respectively. Isolates of AG-2-1, the dominant Rhizoctonia, occurred in all six agronomic zones defined by annual precipitation and temperature within the region sampled. Isolates of AG-8 also were cosmopolitan in their distribution but the frequency of isolation varied among years, and they were most abundant in zones of low and moderate precipitation. R. oryzae was cosmopolitan, and collectively the three genotypes comprised 37% of the isolates. Only isolates of R. solani AG-8 and R. oryzae genotypes II and III (but not genotype I) caused symptoms typically associated with Rhizoctonia root rot and bare patch of wheat. Isolates of AG-2-1 caused only mild root rot and AG-I-like binucleate isolates and members of groups AG-3, AG-4, and AG-11 showed only slight or no discoloration of the roots. However, all isolates of AG-2-1 caused severe damping-off of canola, resulting in 100% mortality. Isolates of Rhizoctonia AG-8, AG-2-1, AG-10, AG-I-like binucleate Rhizoctonia, and R. oryzae genotypes I, II, and III could be distinguished by colony morphology on potato dextrose agar, by PCR with specific primers, or by the type and severity of disease on wheat and canola seedlings, and results of these approaches correlated completely. Based on cultured isolates, we also identified the geographic distribution of all of these Rhizoctonia isolates in cereal-based production systems throughout Washington State.

A DNA Mini-Barcoding System for Authentication of Processed Fish Products.

Species substitution is a form of seafood fraud for the purpose of economic gain. DNA barcoding utilizes species-specific DNA sequence information for specimen identification. Previous work has established the usability of short DNA sequences—mini-barcodes—for identification of specimens harboring degraded DNA. This study aims at establishing a DNA mini-barcoding system for all fish species commonly used in processed fish products in North America. Six mini-barcode primer pairs targeting short (127–314 bp) fragments of the cytochrome c oxidase I (CO1) DNA barcode region were developed by examining over 8,000 DNA barcodes from species in the U.S. Food and Drug Administration (FDA) Seafood List. The mini-barcode primer pairs were then tested against 44 processed fish products representing a range of species and product types. Of the 44 products, 41 (93.2%) could be identified at the species or genus level. The greatest mini-barcoding success rate found with an individual primer pair was 88.6% compared to 20.5% success rate achieved by the full-length DNA barcode primers. Overall, this study presents a mini-barcoding system that can be used to identify a wide range of fish species in commercial products and may be utilized in high throughput DNA sequencing for authentication of heavily processed fish products.

Breed-Specific Detection of Mangalica Meat in Food Products

A fast and reliable diagnostic system has been developed for the detection of Mangalica meat in foods. This qualitative test is based on a recombinase polymerase amplification which can be performed on the field, in situ, where it may be necessary to determine Mangalica content in food products at once. The required equipments for the procedure are pipettes, a portable homogenizer and a portable thermostat. DNA amplification is carried out at a constant temperature, and the detection is based on antibody reaction. The detection limit is one copy of the target sequence in 1 μl reaction volume. The test can be used for uncovering falsification of local brands on the spot within a very short (25–45 min) period of time. The present approach can be adopted for the detection of other food ingredients, if the species-specific target DNA sequence is known, e.g. in case of chicken, turkey, horse, and cattle.

Application of Quadruple Multiplex PCR Detection for Beef, Duck, Mutton and Pork in Mixed Meat

In this study, we have developed quadruple multiplex PCR assay for meat (beef, duck, mutton and pork) in processed meat products. By mixing four primers in appropriate ratios could be identified by the PCR. A forward primer was designed on a conserved DNA sequence in the mitochondrial ND2 and 16S rDNA gene in sheep and duck genes, and reverse primers on species-specific DNA sequences for each species. PCR primers were designed to give different length fragments from the four meats. The products showed species-specific DNA fragments of 116, 212, 177 and 322 bp from beef, pork, mutton and duck. Optimal PCR conditions were established. The assay sensitivity under these conditions was 0.1ng, and its specificity was 100 %. The results of the study suggest that PCR represents a simple, efficient test method as a practical alternative for the rapid detection and identification of meat.

A multiplex PCR-based approach for reliable identification of different meat species and possible adulteration in meat markets in Chittagong, Bangladesh

Food authenticity issues in the form of adulteration and improper description have been around for a long time in Bangladesh. The adulterated food often enters the supply chain and jeopardizes the credibility of the sellers as well as health of the consumers. Molecular detection has been a reliable tool for animal species identification since many days. Polymerase chain reaction (PCR) is quite useful tool that is highly accurate and relatively faster in differentiating specimens derived from different animal species. The present study was carried out to validate previously described methods to identify different meat species by a multiplex PCR method and a preliminary molecular survey was performed on 60 meat samples for identification of possible adulteration in butcher shop in Chittagong. A multiplex PCR was developed and evaluated on the mitochondrial cytochrome b gene to differentiate in six meat species (chicken, duck, sheep, goat, cattle and buffalo). A common forward primer was designed on a conserved DNA sequence of the cytochrome b gene and reverse primers on species-specific DNA sequences from each species. By multiplex PCR, DNA bands of 157 bp, 227 bp, 274 bp, 331 bp, 398 bp, 439 bp were visualized that corresponds to meat samples derived from goat, chicken, cattle, sheep, duck and buffalo, respectively. There were no adulterated meat identified among the samples examined during this study, indicating fair economic business in the study area.

Structural features of Chi recognition in AddAB with implications for RecBCD

AddAB and RecBCD-type helicase-nuclease complexes control the first stage of bacterial homologous recombination (HR) – the resection of double strand DNA breaks. A switch in the activities of the complexes to initiate repair by HR is regulated by a short, species-specific DNA sequence known as a Crossover Hotspot Instigator (Chi) site. It has been shown that, upon encountering Chi, AddAB and RecBCD pause translocation before resuming at a reduced rate. Recently, the structure of B.subtilis AddAB in complex with its regulatory Chi sequence revealed the nature of Chi binding and the paused translocation state. Here the structural features associated with Chi binding are described in greater detail and discussed in relation to the related E.coli RecBCD system.