An efficient noninvasive method for discriminating among faeces of sympatric North American canids

Molecular technology can identify species noninvasively from faeces found in the field. We describe a fast and reliable genetic method that differentiates faeces of five potentially sympatric North American canids without using multiple primer sets or restriction enzyme digestion. Our primer set amplifies a short fragment (237–288 bp) of the mitochondrial d-loop that is a different length in each species: kit fox (Vulpes macrotis), red fox (Vulpes vulpes), gray fox (Urocyon cinereoargenteus), coyote (Canis latrans), and dog (Canis familiaris). We extensively tested our technique using published and novel d-loop sequences and then applied it to two large faecal data sets collected in California and Virginia. It provides an efficient tool for noninvasively distinguishing sympatric canids in diverse regions of North America.     

Rapid genetic identification of brown bullhead (Ameiurus nebulosus), black bullhead (Ameiurus melas) and their hybrids

Hybridization can complicate the effective conservation of cryptic and morphologically similar species. Here we provide a rapid genetic method for the identification of pure-strain brown bullhead (Ameiurus nebulosus) and black bullhead (Ameiurus melas) using nuclear and mitochondrial DNA restriction fragment length polymorphism assays. We confirm earlier reports of introgressive hybridization among A. nebulosus and A. melas in Lake Erie, and reveal the presence of introgressed hybrids in Detroit River. Our method provides a prime example of the utility of internet-based genetic databases for the development of molecular tools in biological and environmental research, and suggests hybridization plays a larger role in the genetic structure of bullheads.     

A novel multiplex PCR assay for species identification in the Chinese Egret (Egretta eulophotes) and Little Egret (E. garzetta)

A multiplex PCR assay is described herein for the species identification of juvenile egrets, Chinese Egret (Egretta eulophotes) and Little Egret (E. garzetta), which colonially breed in a heronary and share similar exterior characteristics. In this new assay, the primers were designed to yield to a 101?bp positive control amplicon of partial ND6 and tRNA^Glu gene, and the species-specific primers were designed for species identification in E. eulophotes and E. garzetta (partial ND6 and Control Region gene, about 278 and 302?bp, respectively). Using simple agarose gel electrophoresis the PCR products discriminates the two species unambiguously indicated that our new assay could be used for accurate and convenient species identification in E. eulophotes and E. garzetta.     

Hybridization and polymorphic microsatellite markers for two lagomorph species (Genus Sylvilagus): implications for conservation

Eastern cottontails (Sylvilagus floridanus) are a prolific and popular game species in North America but have declined within the core of their range. Swamp rabbits (S. aquaticus) are sympatric with eastern cottontails and are a species of concern at the northern fringe of their range in Illinois. Although hybridization between these species may be a potential factor affecting their declines, no studies have yet been conducted to address this issue. We examined the potential for hybridization between sympatric eastern cottontails and swamp rabbits in Illinois by sequencing a portion of the mitochondrial 12S gene in 17 individuals of each species. Additionally, we developed six microsatellite markers for the eastern cottontail (7–13 alleles/locus) that also amplify in swamp rabbits (3–5 alleles/locus). We found fixed differences between species with haplotypes forming well-differentiated clades indicating a lack of hybridization. Hybridization between eastern cottontails and swamp rabbits does not appear to be affecting their declines. Misidentification due to ambiguous morphological characteristics of the two species occurred in an earlier study and in our own. We suggest the mitochondrial 12S gene may be a convenient molecular marker for the resolution of species identity questions. Information on microsatellites, occurrence of natural hybridization, and a molecular marker for species resolution will be useful for developing management plans for lagomorphs and focusing further research on factors affecting population status.     

Microsatellite repeat motif and amplicon length affect amplification success of degraded faecal DNA

Degradation reduces DNA quality and quantity in faecal samples and leads to low amplification success. We investigated the influence of repeat motif and amplicon length by comparing the amplification success of five dinucleotide, five tetranucleotide, and two compound microsatellite markers for African elephant (Loxodonta africana) faecal DNA samples. We found that both repeat motif and amplicon length influenced amplification success, and suggest the use of simple microsatellite markers containing alleles with small amplicon sizes to maximise amplification success of degraded DNA.     

DNA extraction from fresh-frozen and formalin-fixed, paraffinembedded human brain tissue

Both fresh-frozen and formalin-fixed,paraffinembedded（FFPE）human brain tissues are invaluable resources for molecular genetic studies of central nervous system diseases,especially neurodegenerative disorders.To identify the optimal method for DNA extraction from human brain tissue,we compared methods on differently-processed tissues.Fragments of LRRK2 and MAPT（257 bp and 483 bp/245 bp）were amplified for evaluation.We found that for FFPE samples,the success rate of DNA extraction was greater when using a commercial kit than a laboratory-based method（successful DNA extraction from 76%versus 33%of samples）.PCR amplicon size and storage period were key factors influencing the success rate of DNA extraction from FFPE samples.In the fresh-frozen samples,the DNA extraction success rate was 100%using either a commercial kit（QIAamp DNA Micro）or a laboratorybased method（sample boiling in 0.1 mol/L NaOH,followed by proteinase K digestion,and then DNA extraction using Chelex-100）regardless of PCR amplicon length or tissue storage time.Although the present results demonstrate that PCR-amplifiable genomic DNA can be extracted from both fresh-frozen and FFPE samples,fresh brain tissue is recommended for DNA extraction in future neuropathological studies.     

Microsatellite loci for two threatened dragonfly (Odonata: Anisoptera) species: Oxygastra curtisii (Dale, 1834) and Macromia splendens (Pictet, 1843)

Twenty one polymorphic microsatellite loci were isolated from two species of dragonfly (Odonata: Anisoptera), Macromia splendens (n = 8 loci) and Oxygastra curtisii (n = 13 loci). Both species have their main distribution areas in southwestern Europe, with records in the north of Africa in the case of O. curtisii. M. splendens is listed as vulnerable by IUCN, while O. curtisii is regarded as near threatened. Genetic diversity was assessed in samples from the Iberian Peninsula representing two populations for each species. Number of alleles per locus ranged from 5 to 11 (O. curtisii) and between 4 and 16 (M. splendens), while mean expected heterozygosity varied between 0.118–0.745 (O. curtisii) and 0.130–0.849 (M. splendens). Five loci (four for O. curtisii and one for M. splendens) showed significant deviations (P < 0.05) from expected Hardy–Weinberg equilibrium conditions, with the locus from M. splendens experiencing null alleles. These loci are currently being used to assess spatial genetic structure in these protected species.     

A blocking primer increases specificity in environmental DNA detection of bull trout (Salvelinus confluentus)

Environmental DNA (eDNA) is increasingly applied as a highly sensitive way to detect aquatic animals non-invasively. However, distinguishing closely related taxa can be particularly challenging. Previous studies of ancient DNA and genetic diet analysis have used blocking primers to enrich target template in the presence of abundant, non-target DNA. Here we apply a blocking primer to increase the specificity of a TaqMan assay for eDNA detection of rare and endangered bull trout (Salvelinus confluentus) in the presence of the closely related (Salvelinus namaycush). We found that addition of a blocking primer substantially increased assay specificity without compromising sensitivity or quantification ability.     

Discrimination of PCR products by colour and size improves the accuracy of sex-typing in avian species

Gender determination of endangered birds with no sexual dimorphism is needed in captive-breeding and conservation projects. Several molecular approaches have been developed, focusing on different species. In this study, we successfully tested a protocol for sex identification, based on the amplification of fluorescently labeled products in 63 species listed in the Convention of Washington (CITES) Appendix I, and belonging to Psittaciformes, Accipitriformes, Falconiformes, Strigiformes, Pelecaniformes, Ciconiiformes, Gruiformes, Passeriformes, and Sphenisciformes. We designed a multiplexed PCR that produces a single amplicon in males and two distinct amplicons, respectively coloured in blue and green, in females. This procedure generates digitally coloured electropherograms that yield a reliable and quick sexing.     

Development of 31 polymorphic microsatellite markers for guinea fowl (Numida meleagris) using next-generation sequencing technology

Though efforts to domesticate guinea fowl (Numida meleagris) have been productive, both feral and wild populations continue to decline due to habitat fragmentation and heavy hunting, increasing the need to monitor and sustain this species. This study reports the first 31 original polymorphic microsatellite markers for guinea fowl by the next-generation sequencing technology. Observed (H _O ) and expected heterozygosities (H _E ) ranged from 0.033 to 1.000 (mean 0.396) and 0.033 to 0.799 (mean 0.419), respectively. Number of alleles ranged between 2 and 9 per locus (mean 3.387). Nine loci significantly deviated from Hardy–Weinberg Equilibrium (p < 0.05), after Bonferroni correction. Null allele frequency was less than 0.2 in all loci except locus Nmg22 (0.3). Results from this study can serve as baseline information for genetic diversity studies and also inform breeding strategies for the improvement and conservation in both domestic and wild populations of the species.     

Eleven novel microsatellite loci for Japanese whiting (Sillago japonica) and cross amplification in the endangered small-scale sillago (Sillago parvisquamis)

Japanese whiting (Sillago japonica) are a relatively common species that inhabit coastal shallow waters in Japan and are the target species in an important recreational fishery. We isolated eleven candidate microsatellite loci from a small insert genomic DNA library of S. japonica. We screened for polymorphisms in the eleven loci using wild individuals (n = 48) collected from Suounada Sound, in the Seto Inland Sea, Japan. The number of alleles per locus ranged from 6 to 26 with no evidence of linkage disequilibrium. Observed heterozygosity ranged from 0.58 to 0.98 with one locus exhibiting a significant departure from Hardy–Weinberg equilibrium. A test for cross-amplification using the closely related species, Sillago parvisquamis yielded scoreable peaks and a high level of polymorphism in four loci. These polymorphic microsatellites can be used to identify population structure in S. japonica and provide potential markers for the endangered S. parvisquamis.     

Microsatellite loci for testing temporal changes in the population genetics of the Florida mouse (Podomys floridanus)

The Florida mouse (Podomys floridanus) is endemic to Florida and is considered vulnerable by the IUCN due to extensive habitat loss. Here, we describe a panel of 76 microsatellite DNA markers for population genetic studies of this species. Fourteen of these loci were examined to estimate genetic diversity for 39 specimens collected in Florida in 1957 and 2006. DNA extractions from the 1957 specimens were carried out from snips of skin taken from museum voucher skins, whereas the 2006 samples were extracted from fresh liver tissue. The use of museum skins for reconstructing the genetics of historical populations is becoming a critical part of understanding past, present, and future genetic trends for threatened species. Mean expected heterozygosity for the two populations was 0.73 and mean observed heterozygosity was 0.75. The number of alleles per locus ranged from 2 to 15. All microsatellites were polymorphic, and exhibited variability across time.     

Microsatellite loci development for rare Colorado Sclerocactus (Cactaceae)

The genus Sclerocactus (Cactaceae) consists of 15 species, which have a confused taxonomic history due to morphological similarities and distribution overlap. Habitat loss and hybridization are of concern and have established cause for genetic investigations to further understand the genus and develop conservation strategies. Thirteen variable microsatellite loci were identified using S. glaucus and S. parviflorus and were tested in three additional species: S. wetlandicus, S. brevispinus and S. cloverii. The mean number of alleles per locus ranged from 2 to 11, with an average of 6.6. The observed and expected mean heterozygosity ranged from 0.00 to 0.90 and 0.26 to 0.90, respectively. These loci will aid in determining levels of hybridization, diversity and taxonomy of Sclerocactus.     

Translin-Associated Factor X Gene (TSNAX) may be Associated with Female major Depressive Disorder in the Japanese Population

Several investigations have reported that the translin-associated factor X gene (TSNAX)/disrupted-in-schizophrenia-1 gene (DISC1) was associated with major psychiatric disorders including schizophrenia, bipolar disorder (BP), and major depressive disorder (MDD). TSNAX is located immediately upstream of DISC1, and has been shown to undergo intergenic splicing with DISC1. It thus may also be influenced by translocation. To our knowledge, there are no reported gene-based association analyses between TSNAX and mood disorders in the Japanese population. We conducted a case–control study of Japanese samples (158 bipolar patients, 314 major depressive disorder patients, and 811 controls) with three tagging SNPs in TSNAX, selected using HapMap database. In addition, we performed an association analysis between TSNAX and the efficacy of fluvoxamine treatment in 120 Japanese patients with MDD. The MDD patients in this study had scores of 12 or higher on the 17 items of the Structured Interview Guide for Hamilton Rating Scale for Depression (SIGH-D). We defined a clinical response as a decrease of more than 50% in baseline SIGH-D within 8 weeks, and clinical remission as an SIGH-D score of less than 7 at 8 weeks. We found an association between rs766288 in TSNAX and female MDD in the allele/genotype analysis. However, we did not find any association between TSNAX and BP or the fluvoxamine therapeutic response in MDD in the allele/genotype analysis or haplotype analysis. Our results suggest that rs766288 in TSNAX may play a role in the pathophysiology of female MDD in the Japanese population. A replication study using larger samples may be required for conclusive results, since our sample size was small.     

Wildlife forensic DNA and lowland tapir (Tapirus terrestris) poaching

DNA molecular analyses were used in a forensic investigation involving illegal wildlife poaching in central Brazil. The Brazilian environmental agency (IBAMA) sent us two samples of confiscated meat from illegal poaching for species and individual identification. Comparative analysis of cytochrome b sequences (1,070 bp) provided incontestable evidence that the meat samples were from the lowland tapir, Tapirus terrestris. The analysis of nine microsatellites indicated that the two seized meat samples shared the same genotypes, implying that meat from only one individual was present. The lowland tapir is the last representative of mega-mammals in South America and is a vulnerable species, locally extinct in several regions. The present paper reports a successful genetic investigation of an illegal hunting crime, in which DNA-based molecular analyses of seized ground meat samples were the only tool able to identify the species and individual, proving to be highly useful for wildlife crime investigations.     

TaqMan assays for species identification of the red squirrel (Sciurus vulgaris) and the grey squirrel (Sciurus carolinensis)

We have developed TaqMan based assays for species-specific identification of two species of squirrel found in the British Isles, the native red squirrel (Sciurus vulgaris) and the introduced north American grey squirrel (Sciurus carolinensis). These assays correctly identified tissue and hair samples of both species and there was no cross-species amplification. This is a useful method for non-invasive surveys to help conserve the red squirrel and manage the spread of the grey squirrel in the UK and Ireland.     

A rapid molecular method to detect the invasive golden apple snail Pomacea canaliculata (Lamarck, 1822)

The golden apple snail, Pomacea canaliculata has become a major agricultural and environmental pest across Asia. Here, using the mitochondrial cytochrome oxidase I as a diagnostic, we develop a multiplex PCR, that discriminates P. canaliculata from the most common non-invasive Pomacea species, thereby providing a fast and reliable diagnostic tool.     

Characterization of eight polymorphic microsatellite loci for the endangered Alabama red-bellied turtle (Pseudemys alabamensis; Emydidae)

The federally endangered Alabama red-bellied turtle (ARBT; Pseudemys alabamensis) occurs in an isolated range in southern Alabama and Mississippi. Several distinct nesting populations of ARBT exist within this range. Here we identify eight microsatellite markers to be used for a genetic comparison of members of these populations. Previously isolated microsatellite loci in the related species Pseudemys floridana amplified cleanly from DNA extracted from ARBT tissue samples. Alleles at all eight loci were polymorphic. Alleles ranged in size from 136 to 295 base pairs in length. The total number of alleles at each locus ranged from 2 to 11. Loci showed no evidence of linkage disequilibrium or variation from Hardy–Weinberg equilibrium. Observed and expected heterozygosities ranged from 0.13 to 0.88 and 0.12 to 0.84 respectively. Some variation in alleles was observed among members of different nesting populations suggesting that these eight loci will be an important tool in further assessing population structure of Pseudemys alabamensis.     

A novel molecular method for noninvasive sex identification of order Carnivora

Noninvasive sampling can provide an efficient means of genetically monitoring mammals. Due to the fragmented quality of DNA derived from such samples, few methods span taxonomic groups beyond species. Here, we describe a more universal protocol for the molecular sex identification of order Carnivora. PCR amplification of a 176 bp segment of the SRY gene indicates a male, whereas no amplification indicates a female. A 245 bp segment of the ZFX gene serves as a positive control. This method is internally consistent, and multiple trials confer confidence in results. It applies to at least three families (Canidae, Felidae, and Phocidae) in both carnivore suborders (Feliformia and Caniformia), rendering this protocol the first sex identification method with amplicon sizes appropriate for noninvasive samples suitable for multiple families within order Carnivora.