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The presence of cryptic species can hinder effective conservation planning and implementation, as has been the case for speciose groups of freshwater fishes that are difficult to differentiate due to conserved morphologies. The West Texas shiner Notropis megalops and the Texas shiner Notropis amabilis are a cryptic pair of leuciscids (minnows) that co‐occur in spring‐fed tributaries of the Rio Grande in Texas and Mexico. Both N. megalops and N. amabilis are listed as Species of Greatest Conservation Need by the Texas Parks and Wildlife Department. Notropis amabilis is widespread and listed as apparently secure by Texas Parks and Wildlife Department whereas N. megalops has a very limited distribution and has not been ranked by Texas Parks and Wildlife Department because of data deficiency. Morphological differences between these species have been described; however, proper identification in situ remains problematic. Furthermore, given their range of overlap there is potential for hybridization, and limited genetic data have been collected comparing the species. Therefore, reduced representation genomic and mitochondrial sequencing data were used to reassess the distinctness of the species, screen for hybridization, and characterize their relative frequencies throughout their range of overlap. Genomic analyses recovered two distinct genetic groups corresponding to the species (F'CT = 0.89) with no evidence of admixture or introgression. The species were found to co‐occur at three sampling locations, two in the Devils River and one in the Pecos, but not in equal frequencies. Overall, these results provide data and tools for further research on N. megalops needed for accurate conservation policies and management practices. [ABSTRACT FROM AUTHOR]

The MiniMUGA genotyping array is a popular tool for genetic quality control of laboratory mice and genotyping samples from most experimental crosses involving laboratory strains, particularly for reduced complexity crosses. The content of the production version of the MiniMUGA array is fixed; however, there is the opportunity to improve the array's performance and the associated report's usefulness by leveraging thousands of samples genotyped since the initial description of MiniMUGA. Here, we report our efforts to update and improve marker annotation, increase the number and the reliability of the consensus genotypes for classical inbred strains and substrains, and increase the number of constructs reliably detected with MiniMUGA. In addition, we have implemented key changes in the informatics pipeline to identify and quantify the contribution of specific genetic backgrounds to the makeup of a given sample, remove arbitrary thresholds, include the Y Chromosome and mitochondrial genome in the ideogram, and improve robust detection of the presence of commercially available substrains based on diagnostic alleles. Finally, we have updated the layout of the report to simplify the interpretation and completeness of the analysis and added a section summarizing the ideogram in table format. These changes will be of general interest to the mouse research community and will be instrumental in our goal of improving the rigor and reproducibility of mouse-based biomedical research. [ABSTRACT FROM AUTHOR]

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research.

Abstract Background Phalaris species (Poaceae) occupy diverse environments throughout all continents except Antarctica. Phalaris arundinacea is an important forage, ornamental, wetland restoration and biofuel crop grown globally as well as being a wetland invasive. The nuclear ribosomal internal transcribed spacer (ITS) region has been used for Phalaris barcoding as a DNA region with high nucleotide diversity for Phalaris species identification. Recent findings that P. arundinacea populations in Minnesota USA are most likely native and not European prompted this analysis to determine whether Eurasian vs. native North American P. arundinacea differed in ITS regions. Our objectives were to amplify and compare ITS regions (ITS1 and ITS2) of historic herbaria (1882–2001) and extant (fresh) Phalaris specimens; analyze ITS regions for species-specific polymorphisms (diagnostic SNPs) and compare ITS regions of historic Phalaris specimens with known, extant Phalaris species. Results We obtained complete ITS1 and ITS2 sequences from 31 Phalaris historic (herbaria samples, 1908 to 2001) and five extant (fresh) specimens. Herbaria Phalaris specimens did not produce new SNPs (single nucleotide polymorphisms) not present in extant specimens. Diagnostic SNPs were identified in 8/12 (66.6%) Phalaris species. This study demonstrates the use of herbaria tissue for barcoding as a means for improved species identification of Phalaris herbaria specimens. No significant correlation between specimen age and genomic DNA concentration was found. Phalaris arundinacea showed high SNP variation within its clade, with the North American being distinctly different than other USA and most Eurasian types, potentially allowing for future identification of specific SNPs to geographic origin. Conclusions While not as efficient as extant specimens to obtain DNA, Phalaris herbaria specimens can produce high quality ITS sequences to evaluate historic genetic resources and facilitate identification of new species-specific barcodes. No correlation between DNA concentration and age of historic samples (119 year range) occurred. Considerable polymorphism was exhibited in the P. arundinacea clade with several N. American accessions being distinct from Eurasian types. Further development of within species- and genus-specific barcodes could contribute to designing PCR primers for efficient and accurate identification of N. American P. arundinacea. Our finding of misidentified Phalaris species indicates the need to exercise stringent quality control measures on newly generated sequence data and to approach public sequence databases in a critical way.

Background: Phalaris species (Poaceae) occupy diverse environments throughout all continents except Antarctica. Phalaris arundinacea is an important forage, ornamental, wetland restoration and biofuel crop grown globally as well as being a wetland invasive. The nuclear ribosomal internal transcribed spacer (ITS) region has been used for Phalaris barcoding as a DNA region with high nucleotide diversity for Phalaris species identification. Recent findings that P. arundinacea populations in Minnesota USA are most likely native and not European prompted this analysis to determine whether Eurasian vs. native North American P. arundinacea differed in ITS regions. Our objectives were to amplify and compare ITS regions (ITS1 and ITS2) of historic herbaria (1882–2001) and extant (fresh) Phalaris specimens; analyze ITS regions for species-specific polymorphisms (diagnostic SNPs) and compare ITS regions of historic Phalaris specimens with known, extant Phalaris species. Results: We obtained complete ITS1 and ITS2 sequences from 31 Phalaris historic (herbaria samples, 1908 to 2001) and five extant (fresh) specimens. Herbaria Phalaris specimens did not produce new SNPs (single nucleotide polymorphisms) not present in extant specimens. Diagnostic SNPs were identified in 8/12 (66.6%) Phalaris species. This study demonstrates the use of herbaria tissue for barcoding as a means for improved species identification of Phalaris herbaria specimens. No significant correlation between specimen age and genomic DNA concentration was found. Phalaris arundinacea showed high SNP variation within its clade, with the North American being distinctly different than other USA and most Eurasian types, potentially allowing for future identification of specific SNPs to geographic origin. Conclusions: While not as efficient as extant specimens to obtain DNA, Phalaris herbaria specimens can produce high quality ITS sequences to evaluate historic genetic resources and facilitate identification of new species-specific barcodes. No correlation between DNA concentration and age of historic samples (119 year range) occurred. Considerable polymorphism was exhibited in the P. arundinacea clade with several N. American accessions being distinct from Eurasian types. Further development of within species- and genus-specific barcodes could contribute to designing PCR primers for efficient and accurate identification of N. American P. arundinacea. Our finding of misidentified Phalaris species indicates the need to exercise stringent quality control measures on newly generated sequence data and to approach public sequence databases in a critical way. [ABSTRACT FROM AUTHOR]

The single lynx species present in the Iberian Peninsula—Lynx pardinus (Temmink, 1827)—is one of the most threatened felines in the world so, any biological indication of its presence is significant. However, it is a very elusive species and physical evidence through direct visualization or camera trapping is difficult to obtain. Genetic noninvasive methods, in particular, molecular scatology, are a reliable alternative to unequivocally identify the presence of a species in a given geographical area. We have developed a highly specific and sensitive molecular method to identify the presence of this critically endangered lynx species from severely deteriorated fecal samples. The method consists in a double specific nested PCR of mitochondrial D-loop sequences and two diagnostic SNPs detected by a multiplex primer extension reaction. [ABSTRACT FROM AUTHOR]

A genotyping assay for the Ion Torrent Ion PGM platform was developed for fast and cost-effective targeted genotyping of key single nucleotide polymorphisms ( SNPs) earlier identified using a genome-wide SNP array in Atlantic salmon Salmo salar. The method comprised a simple primer design step for multiplex-polymerase chain reaction ( PCR), followed by two rounds of Ion Torrent Ion PGM sequencing to empirically evaluate marker efficiency in large multiplexes and to optimise or exclude them when necessary. Of 282 primer pairs initially tested, 217 were successfully amplified, indicating good amplification success (>75%). These markers included the sdy partial gene product to determine genetic sex, as well as three additional modules comprising SNPs for assessing neutral genetic variation ( NSNP = 150), examining functional genetic variation associated with sea age at maturity ( NSNP = 5), and for performing genetic subpopulation assignment ( NSNP = 61). The assay was primarily developed to monitor long-term genetic changes in S. salar from the Teno River, but modules are likely suitable for application in a wide range of S. salar populations. Furthermore, the fast and versatile assay development pipeline offers a strategy for developing targeted sequencing assays in any species. [ABSTRACT FROM AUTHOR]

Background: Rapid and reliable identification of quarantine pests is essential for plant inspection services to prevent introduction of invasive species. For insects, this may be a serious problem when dealing with morphologically similar cryptic species complexes and early developmental stages that lack distinctive characters useful for taxonomic identification. DNA based barcoding could solve many of these problems. The standard barcode fragment, an approx. 650 base pairs long sequence of the 5'end of the mitochondrial cytochrome oxidase I (COI), enables differentiation of a very wide range of arthropods. However, problems remain in some taxa, such as Tephritidae, where recent genetic differentiation among some of the described species hinders accurate molecular discrimination. Results: In order to explore the full species discrimination potential of COI, we sequenced the barcoding region of the COI gene of a range of economically important Tephritid species and complemented these data with all GenBank and BOLD entries for the systematic group available as of January 2012. We explored the limits of species delimitation of this barcode fragment among 193 putative Tephritid species and established operational taxonomic units (OTUs), between which discrimination is reliably possible. Furthermore, to enable future development of rapid diagnostic assays based on this sequence information, we characterized all single nucleotide polymorphisms (SNPs) and established "near-minimal" sets of SNPs that differentiate among all included OTUs with at least three and four SNPs, respectively. Conclusions: We found that although several species cannot be differentiated based on the genetic diversity observed in COI and hence form composite OTUs, 85% of all OTUs correspond to described species. Because our SNP panels are developed based on all currently available sequence information and rely on a minimal pairwise difference of three SNPs, they are highly reliable and hence represent an important resource for developing taxonspecific diagnostic assays. For selected cases, possible explanations that may cause composite OTUs are discussed [ABSTRACT FROM AUTHOR]

Cockles are highly appreciated mollusks and provide important services in coastal areas. The two European species, edible (Cerastoderma edule) and lagoon (Cerastoderma glaucum) cockles, are not easily distinguishable, especially when young. Interestingly, the species show different resistance to Marteilia cochillia, the parasite responsible for marteiliosis outbreaks, which is devastating cockle production in some areas. C. edule is severely affected by the parasite, while C. glaucum seems to be resistant, although underlying reasons are still unknown. Hybrids between both species might be interesting to introgress allelic variants responsible for tolerance, either naturally or through artificial selection, from lagoon into edible cockle. Here, we used 2b restriction site-associated DNA sequencing (2b–RAD) to identify single nucleotide polymorphisms (SNP) diagnostic for cockle discrimination (fixed for alternative allelic variants). Among the nine diagnostic SNPs selected, seven were validated using a SNaPshot assay in samples covering most of the distribution range of both species. The validated SNPs were used to check cockles that were suggested to be hybrids by a claimed diagnostic tool based on the internal transcribed spacers of the ribosomal RNA. Although these were shown to be false positives, we cannot rule out the fact that hybrids can occur and be viable. The SNP tool here developed will be valuable for their identification and management. [ABSTRACT FROM AUTHOR]