Genetic Identification Methods Research Articles

Genetic Identification of an Unknown <I>Rhagoletis</I> Fruit Fly (Diptera: Tephritidae) Infesting Chinese Crabapple: Implications for Apple Pest Management

The apple maggot, Rhagoletis pomonella (Walsh) (Diptera: Tephritidae), is a serious introduced quarantine pest in the apple (Malus spp.)-growing regions of central Washington and Oregon. In August 2011, seven fly larvae of unknown origin were discovered infesting fruit of an exotic Chinese crabapple, Malus spectabilis (Aiton) Borkhausen, in Kennewick, Benton County, WA. If confirmed, Chinese crabapple would have represented a new host for R. pomonella in Washington and triggered quarantine measures in a surrounding three-county region of the state. Here, we establish, based on five microsatellite loci, the identity of the crabapple-infesting larvae as the western cherry fruit fly, Rhagoletis indifferens Curran, representing a new host record for the fly. Morphological analysis of six flies reared to adulthood confirmed the genetic identification. The results demonstrate the utility of integrating rapid genetic identification methods with field surveys of economic pests, which decreased detection times by months, and avoided enacting costly quarantine measures that saved local and federal bodies > US$0.5 million in monitoring, inspection, and control costs. We discuss current ongoing efforts to develop rapid, accurate, and inexpensive on site DNA-based detection tools for R. pomonella that would have general applicability for the control of pest insects.

Genetic Differentiation and Efficient Sex-specific Marker Development of a Pair of Y- and X-linked Markers in Yellow Catfish

Pf62-Y and Pf62-X is a pair of allelic Y chromosome-linked and X chromosome-linked markers, and have been used to identify YY super-males, XY males and XX females for commercial production of all-male populations in yellow catfish (Pelteobagrus fulvidraco). However, the SCAR primers used previously have only two nucleotide difference, which restricts the wide utility because of nucleotide polymorphism. In this study, a continuous 8102 bp Pf62-Y sequence and a 5362 bp Pf62-X sequence have been cloned by genome walking, and significant genetic differentiation has been revealed between the corresponding X and Y chromosome allele sequences. Moreover, three pairs of primers were designed to efficiently identify YY super-males, XY males and XX females in an artificial breeding population, and to distinguish XY males and XX females in various wild populations. Together, the three new sex-specific genetic markers develop a highly stable and efficient method for genetic sex identification and sex control application in sustainable aquaculture of all-male yellow catfish.

Ensuring seafood identity: Grouper identification by real-time nucleic acid sequence-based amplification (RT-NASBA)

Grouper are one of the most economically important seafood products in the state of Florida and their popularity as a high-end restaurant dish is increasing across the U.S. There is an increased incidence rate of the purposeful, fraudulent mislabeling of less costly and more readily available fish species as grouper in the U.S., particularly in Florida. This is compounded by commercial quotas on grouper becoming increasingly more restrictive, which continues to drive both wholesale and restaurant prices higher each year. Currently, the U.S. Food and Drug Administration recognize 56 species of fish that can use “grouper” as an acceptable market name for interstate commerce. This group of fish includes species from ten different genera, making accurate taxonomic identification difficult especially if distinguishing features such as skin, head, and tail have been removed. This is leading regulatory agencies to employ genetic identification methods which tend to have much higher species-level resolution than phenotypic methods. Standard genetic identification methods are highly technical and require expensive lab-based equipment to perform, which often leads to longer turnover times. We have developed a generic grouper assay that detects the majority of the grouper species listed on the 2011 FDA Seafood List, including all of the species found in Florida waters. This assay is based upon real-time nucleic acid sequence-based amplification (RT-NASBA) targeting mitochondrial 16S rRNA for the accurate detection of grouper. This assay can be performed in fewer than 90 min with little potential for cross-reactivity from non-target species.

The Genus Corynebacterium and Other Medically Relevant Coryneform-Like Bacteria

Catalase-positive Gram-positive bacilli, commonly called "diphtheroids" or "coryneform" bacteria were historically nearly always dismissed as contaminants when recovered from patients, but increasingly have been implicated as the cause of significant infections. These taxa have been underreported, and the taxa were taxonomically confusing. The mechanisms of pathogenesis, especially for newly described taxa, were rarely studied. Antibiotic susceptibility data were relatively scant. In this minireview, clinical relevance, phenotypic and genetic identification methods, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) evaluations, and antimicrobial susceptibility testing involving species in the genus Corynebacterium and other medically relevant Gram-positive rods, collectively called coryneforms, are described.

Detecting metabolic activities in single cells, with emphasis on nanoSIMS

Investigating the contribution of microbial populations to biochemical processes of global significance is challenging as there are few approaches that can detect microbial metabolic activities on single-cell level. Given the widespread distribution and importance of microorganisms in elemental transformations, improved methods for measuring microbial activities in naturally occurring microbial communities is essential. In this article, microautoradiography (MAR), Raman microspectroscopy, and Secondary Ion Mass Spectrometry (SIMS) and their combination with isotope labeling and molecular genetic methods for cell identification (i.e. FISH and related methods) are reviewed. We focus our review on the application of MAR-FISH, Raman-FISH, and FISH-SIMS to environmental samples, with a more detailed description of the use of nanoSIMS-based methodologies to identify, quantify, and visualize the incorporation of labeled substrates of single microorganisms in complex microbial communities. We highlight examples from the marine habitat. In addition, relevant technical aspects as well as important considerations concerning sample preparation and handling are presented. We conclude with a perspective on the usefulness of such tools to study the role of microorganisms in biogeochemical cycling from micron to global scales.

Development of a FINS- based method for the identification of skates species of commercial interest

Rajidae family is one of the most diverse families within Batoidea superorder with more of 220 described species, of which many skates are particularly vulnerable to overfishing. This fact makes IUCN (International Union for Conservation of Nature) Red List of Threatened Species contains 209 of these skate species.The main marketing formats of these species are fresh and frozen wings. This makes impossible their identification on the basis of their morphological characters. For these reasons, in the present study a method for genetic identification of different skate species has been developed. The technique is based on sequencing of a fragment of 555 bp from amplified DNA, COI (Cytochrome Oxidase subunit 1) gene, by PCR and subsequent phylogenetic analysis (FINS: Forensically Informative Nucleotide Sequencing). The technique allows the genetic identification of more than 40 skate species in skate products.The main novelty of this work lies in the fact that up to now there is no work about the genetic identification that includes so many skate species. Therefore, this molecular tool is appropriate to clarify questions related with the correct labeling of skate commercial products, the traceability of raw materials, and the control of imported skates, and also can be applied to questions linked to the control of skate fisheries.

Virulence Characteristics of Sucrose-fermentingVibrio vulnificusStrains

We identified 6 sucrose-fermenting Vibrio vulnificus strains and examined their virulence characteristics. They were all encapsulated, motile, capable of producing toxins and utilizing transferrin-bound iron, cytotoxic to cultured cells, and virulent enough to kill mice. They could be definitely identified only by genetic identification methods such as PCR, and not by conventional culture-based identification methods such as API 20E (bioMérieux, France). These results indicate that it is essential to adopt genetic approaches as early as possible in order to avoid misdiagnosis of such strains, especially in clinical situations.

Species and stock identification of prey consumed by endangered southern resident killer whales in their summer range

Recovery plans for endangered southern resident killer whales Orcinus orca have iden- tified reduced prey availability as a risk to the population. In order to better assess this risk, we stud- ied prey selection from 2004 to 2008 in 2 regions of the whales' summer range: San Juan Islands, Washington and the western Strait of Juan de Fuca, British Columbia. Following the whales in a small boat, we collected fish scales and tissue remains from predation events, and feces, using a fine mesh net. Visual fish scale analysis and molecular genetic methods were used to identify the species con- sumed. Chinook salmon, a relatively rare species, was by far the most frequent prey item, confirming previous studies. For Chinook salmon prey, we used genetic identification methods to estimate the spawning region of origin. Of the Chinook salmon sampled, 80 to 90% were inferred to have origi- nated from the Fraser River, and only 6 to 14% were inferred to have originated from Puget Sound area rivers. Within the Fraser River, the Upper Fraser, Middle Fraser, South Thompson River and Lower Fraser stocks were inferred to currently be sequentially important sources of Chinook salmon prey through the summer. This information will be of significant value in guiding management actions to recover the southern resident killer whale population.

FishPopTrace—Developing SNP-based population genetic assignment methods to investigate illegal fishing

The FAO estimates that 80% of marine fish stocks are fully or overexploited worldwide. Illegal unreported and unregulated (IUU) fishing contributes vastly to this condition, and poses a severe threat to marine ecosystems. Controlling for compliance and enforcing fishing regulations is hampered by difficulties in identifying the geographic origin of fish and fish products, at point of landing and further down the food supply chain. While forensic genetic species identification methods are routinely employed to investigate commercial fraud, there are at present no validated methods for identifying the geographic origin of marine fish. FishPopTrace is an international project, funded by the EU framework programme (FP7), aiming to generate forensically validated panels of SNP markers for geographic origin assignment in four commercially important fish species, cod ( Gadus morhua), hake ( Merluccius merluccius), herring ( Clupea harengus) and common sole ( Solea solea). 454-sequencing with sample tagging is employed to generate large numbers of population informative candidate SNP loci in each species. Selected SNPs are subsequently genotyped using Illumina 1536-arrays across populations to provide high resolution maps of genetic variation. Panels comprising subsets of these markers will ultimately be validated for traceability and enforcement applications.

Cloning, characterization of two female-specific AFLP markers and development of PCR-based sex identification method for the half-smooth tongue sole Cynoglossus semilaevis

Abstract Two female-specific AFLP (amplified fragment length polymorphism) markers (named CseF464 and CseF136) were isolated by using one selective primer combination (E-AGC/M-CTG) from the genomic DNA of 20 females and 20 males of the half-smooth tongue sole Cynoglossus semilaevis. Both the markers were re-amplified, recovered from the agarose gels, cloned and sequenced. Bioinformatics analysis indicated that the length of the two markers were 468 bp and 134 bp, respectively, and the sequences showed no similarity to each other, as well as to the known sequences deposited in the GenBank database using BLASTn. Two pairs of SCAR (sequence characterized amplified regions) primers were designed based on the sequences of the two female-specific markers. Furthermore, PCR-based genetic sex identification method was developed in Cynoglossus semilaevis. A specific fragment was amplified in all females but not in any males by using these SCAR primers on the initial 20 female and 20 male individuals of Cynoglossus semilaevis. The feasibility of the two SCAR primer pairs was confirmed in additional 100 individuals (50 females and 50 males). This allowed for reliable, rapid molecular identification of genetic sex of the species, genetic mapping on the sex chromosomes and better understanding of the sex determination and sex differentiation in the half smooth tongue sole.

Integration of genetic and genomic methods for identification of genes and gene variants encoding QTLs in the nonhuman primate

We have developed an integrated approach, using genetic and genomic methods, in conjunction with resources from the Southwest National Primate Research Center (SNPRC) baboon colony, for the identification of genes and their functional variants that encode quantitative trait loci (QTL). In addition, we use comparative genomic methods to overcome the paucity of baboon specific reagents and to augment translation of our findings in a nonhuman primate (NHP) to the human population. We are using the baboon as a model to study the genetics of cardiovascular disease (CVD). A key step for understanding gene–environment interactions in cardiovascular disease is the identification of genes and gene variants that influence CVD phenotypes. We have developed a sequential methodology that takes advantage of the SNPRC pedigreed baboon colony, the annotated human genome, and current genomic and bioinformatic tools. The process of functional polymorphism identification for genes encoding QTLs involves comparison of expression profiles for genes and predicted genes in the genomic region of the QTL for individuals discordant for the phenotypic trait mapping to the QTL. After comparison, genes of interest are prioritized, and functional polymorphisms are identified in candidate genes by genotyping and quantitative trait nucleotide analysis. This approach reduces the time and labor necessary to prioritize and identify genes and their polymorphisms influencing variation in a quantitative trait compared with traditional positional cloning methods.

Genetic identification of edible birds’ nest based on mitochondrial DNA sequences

Abstract Edible bird’s nest (EBN) is a functional food constructed with swiftlets’ salivary glue. Counterfeit EBN products have been found in the market due to limited supply and high price of genuine EBN. In this article, a method for genetic identification of EBN was developed. The technique is based on sequence of cytochrome b gene in mitochondrial DNA. The sample sequences together with the sequences of swiftlets in GenBank were used to construct phylogenetic trees for genetic identification of samples. This method was applied to 11 EBN samples, one instant EBN soup product from Indonesia, and Huaiji EBN, a counterfeit EBN in some regions of China. Results showed that all the EBN samples and the instant EBN soup were from Aerodramus fuciphagus while the Huaiji EBN sample was from Apus nipalensis. This was consistent with identification based on morphology of the samples. Therefore, this method is a promising tool to identify the species of bird producing a given sample of EBN, and thus could be used to authenticate—that is, distinguish authentic from counterfeit—EBN.

Identification of the endangered small red brocket deer (Mazama bororo) using noninvasive genetic techniques (Mammalia; Cervidae)

The small red brocket deer Mazama bororo is one of the most endangered deer in the Neotropics. The great morphological similarities with three other sympatric brocket deer species, coupled with the fact that they inhabit densely forested habitats complicate detection and prevent the use of traditional methodologies for accurate identification of species. The ability to determine the presence of this endangered species in an area is crucial for estimating its distribution range, and is critical for establishing conservation management strategies. Here we describe a fast and reliable noninvasive genetic method for species identification of Mazama species from faeces. We designed a primer set that amplifies a short 224-bp fragment of the cytochrome b and demonstrate its effectiveness in successful amplification of DNA isolated from both tissue and faecal samples. This fragment contains a BSTNI/ECORII digestion site that is unique to the endangered M. bororo. The digested polymerase chain reaction products yielded a 160-bp fragment that is clearly visible in a 2% agarose gel. Two other diagnostic sites were identified to differentiate the other three sympatric species, SspI (M. gouazoubira) and AflIII (M. americana, and M. nana).

Field Evaluation of Mortality from Hemolymph Extraction as a Source of DNA, and Application to PCR-RFLP Identification of Threatened Freshwater Mussel Species

Evolutionary convergence and plasticity of shell characters creates great confusion in freshwater mussel systematics and complicates field census efforts. Genetic identification offers a powerful alternative. But methods involving DNA sequencing are expensive and require tissue sampling, whose effects on survival and health of animals from natural populations have seldom been assessed. We used hemolymph sampling as a nonlethal source of tissue for DNA extraction, and developed genetic identification methods using Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis as an alternative for field surveys. We focused on two morphologically similar endemic species in Lake Waccamaw, North Carolina: the abundant Elliptio waccamawensis and the less common Lampsilis fullerkati. These served as models for surveyors who are often faced with conducting accurate census of cryptic rare species that co-occur with more common forms. Hemolymph-sampled and control individuals of these two species and Leptodea ochracea were caged together in enclosures in Lake Waccamaw. Eight-week survival was 100% and we detected no significant effect of hemolymph extraction on shell growth. PCR-RFLP analysis of the 16s rRNA gene reliably identified species and detected cases of morphological misidentification, both in these collections and from belt transects in the lake. We also developed PCR-RFLP markers that distinguished pairs of cryptic taxa from surveys of streams in southeastern North Carolina. Our results show how nonlethal tissue sampling and PCR-RFLP assays designed for a regional fauna can be useful tools in freshwater mussel conservation programs.

A simple genetic identification method for Northeast Atlantic smoothhound sharks (Mustelus spp.)

Abstract Farrell, E. D., Clarke, M. W., and Mariani, S. 2009. A simple genetic identification method for Northeast Atlantic smoothhound sharks (Mustelus spp.). – ICES Journal of Marine Science, 66: 561–565. Considerable ambiguity exists in the identification of the commercially valuable smoothhound sharks (Mustelus spp.) in the Northeast (NE) Atlantic. The lack of a clear and accurate method of identification prevents the collation of reliable species-specific landings and survey data for these fish and hinders the accurate delineation of the distribution ranges of species and stock boundaries, making it impossible to apply sound species-specific conservation and management strategies. This paper reports on the development of a multiplex PCR reaction that utilizes a set of mtDNA primers for the identification of Mustelus asterias, Mustelus mustelus, and Galeorhinus galeus. The high throughput method allows for the rapid and cost-effective identification of large numbers of samples; its application to 431 fish collected between 2006 and 2008 also raises important questions regarding the biogeography of the genus Mustelus in the NE Atlantic.

Genetic Stock Composition of Subyearling Chinook Salmon in Seasonal Floodplain Wetlands of the Lower Willamette River, Oregon

AbstractWe used genetic identification methods to examine the stock composition of subyearling Chinook salmon Oncorhynchus tshawytscha in floodplain wetland and main‐stem habitats of the lower Willamette River, Oregon. Using a microsatellite DNA baseline of 13 standardized loci and 30 Columbia River basin populations, we analyzed 280 subyearlings collected in winter and spring 2005‐2006 from wetland and main‐stem river sites. Genetic stock identification analysis indicated that spring Chinook salmon originating from the Willamette River made up a substantial proportion of the samples and contributed 16‐71% to sample mixtures representing the wetland habitat sites. Fall Chinook salmon from lower Columbia River sources were also present and contributed 58% of winter samples. Spring Chinook salmon from lower Columbia River populations were present in both wetland (17%) and river (16%) samples in spring 2005, and subyearlings from summer‐fall‐run populations in the middle and upper Columbia River contributed to spring wetland samples in 2006 (26%). The results suggest that floodplain restoration projects intended to improve fish habitats during winter and spring periods in the lower Willamette River may benefit Chinook salmon populations from the upper Willamette River, lower Columbia River, and upper Columbia River summer‐fall evolutionarily significant units.

Outbreak of subclinical mastitis in a flock of dairy goats associated with atypicalStaphylococcus haemolyticus

Staphylococcus haemolyticus is a pathogen frequently isolated from dairy cows and small ruminants. However, it always appears in only a few animals and not as a major pathogen. Recently, in a dairy goat herd of approximately 250 milking animals, 25.6% (46/180 goats) had milk cultures with atypical highly mucoid colonies accompanied by elevated somatic cell counts. The isolates were identified as Staph. haemolyticus. The present study describes the steps used in an attempt to identify the bacterium and to compare it with other coagulase-negative staphylococci (CNS) including Staph. haemolyticus. Species identification performed with the API STAPH-IDENT 32 kit showed >99.4% identity confirmed by 16S rDNA sequencing tests. Microscopically the atypical Staph. haemolyticus strains showed unique cuboidal tetrad clusters reminiscent of those of the genus Sarcina. The outbreak caused by an atypical CNS underlines the need for accurate biochemical and genetic methods for ultimate identification of CNS to the species level.

Development of a Method for the Genetic Identification of Flatfish Species on the Basis of Mitochondrial DNA Sequences

In the present study a method for genetic identification of flatfish species was developed. The technique is based on DNA sequencing of amplified DNA by PCR and subsequent phylogenetic analysis ( FINS). A phylogenetic tree using the cytochrome oxidase subunit I (COI) was constructed and the bootstrap values calculated. The mentioned technique allows the genetic identification of more than 50 flatfish species in fresh, frozen, and precooked products. This analytical system was validated and subsequently applied to 30 commercial samples, obtaining 13 that were incorrectly labeled (43%). Four of the mislabeled samples were whole fish (31%), and nine were fillets (69%). The species with the higher rate of incorrect labeling were Pleuronectes platessa (17%) and Solea solea (10%). Other species incorrectly labeled were Hipoglossus hipoglossus (7%), Reinharditus hippoglossoides, Limanda ferruginea, and Microstomus kitt (3% each species). Therefore, this molecular tool is appropriate to clarify questions related with the correct labeling of commercial products, the traceability of raw materials, and the control of imported flatfish, and also can be applied to questions linked to the control of fisheries.

A simple genetic method for identification of mycotoxigenic fungi-Development of heteroduplex panel analysis and its field application-

A novel method for heteroduplex panel analysis (HPA) utilizing fragments of the PCR amplified ITS regions of rDNA was developed. The method involves formation of heteroduplexes with a set of reference fragments amplified from A. flavus, A. parasiticus, A. tamarii and A. nomius, and subsequent minislab gel electrophoresis. The test panel is compared with species-specific standard panels (F-1, P-1, T-1 and N-1), generated by pairwise reannealing among four reference fragments. Application of HPA to a field study demonstrated that a new genotype of FP-1 has been predominantly distributed throughout sugarcane field soil in the southernmost islands of Japan. The high prevalence of type FP-1 may reflect its adaptation to the soil environment. The type FP-1 isolates were also isolated from the sugarcane fields in Vietnam, but not find them in our culture collection derived from other sources. CBS 108.30 originated from sugarcane in Egypt was the only one strain identified as type FP-1. These results imply that type FP-1 may be associated with sugarcane fields in Asia. Type FP-1 is consisted of the morphologically intergrading isolates between A. parasiticus and A. flavus. The phylogenetic analysis based on the ITS sequences indicated that type FP-1 formed an independent clade positioned between A. parasiticus and A. flavus, and was more closely related to the former species. HPA also demonstrated that A. nomius has been distributed in sugarcane field soil and sugarcane stems in Japan and Vietnam. All of the tested isolates of A. nomius and type FP-1 were able to produce aflatoxins B and G. These fungi are probably responsible for aflatoxin contamination of crude sugar produced from sugarcane.

In vitro and in vivo gastrointestinal survival, antibiotic susceptibility and genetic identification of Propionibacterium freudenreichii ssp. shermanii JS

The in vitro tolerance of Propionibacterium freudenreichii strains to low pH and high bile concentration, their antibiotic susceptibility and enzyme activity profiles were assessed. The gastrointestinal survival of the P. freudenreichii ssp. shermanii JS strain was also examined when administered to healthy adult volunteers in whey-based fruit juice. For tracking the strain JS in faecal samples, a genetic identification method for the strain was developed. No loss of viability was observed in any of the P. freudenreichii strains during the three-hour treatment at pH 4 or 3, whereas at pH 2 the cell counts of all strains decreased remarkably. Bile salt at 0.5% concentration had no apparent effect on the viability of the strains. No atypical antibiotic resistance was detected. P. freudenreichii ssp. shermanii JS strain was recovered in high numbers in faecal samples during the consumption of whey-based fruit juice enriched with the JS strain, and with few exceptions, it was not detected after the 2-week follow-up period.