Species-specific Single Nucleotide Polymorphisms Research Articles

IdentiFLY: The Development and Validation of a 15-Plex SNP Assay for Forensic Identification of UK Blowfly Species (Calliphoridae)

Members of the blowfly family (Calliphoridae) are usually the first insect species to arrive at a corpse, using the body as an oviposition site, and, as such, they are the most important group of insects used to estimate the post-mortem interval (PMI). PMI estimations are based on species-specific developmental timings; therefore, accurate species identification is crucial. Current identification methods are based on morphological characteristics, which are time-consuming and difficult to perform on damaged, immature specimens and closely related species. Advances have led to specimens being identified via a host of molecular techniques, mainly DNA sequencing. Although molecular identification is becoming increasingly more common, there is currently a lack of genetic data regarding UK Calliphoridae species. This study aimed to address this issue. We present the development and validation of an identification assay capable of differentiating six UK species (Calliphora vicina, Calliphora vomitoria, Lucilia sericata, Lucilia illustris, Lucilia caesar, and Protophormia terranovae). The sequencing of six genes, including both nuclear (28S rRNA and Elongation factor 1 alpha) and mitochondrial markers (Cytochrome oxidase I and II, Cytochrome b and 16S rRNA) identified 298 species-specific single nucleotide polymorphisms (SNPs). Fifteen SNPs from six genes were chosen for inclusion in a SNaPshot™ multiplex assay. The developed assay is capable of differentiating the species based on between 4 and 12 SNPs. Validation following guidelines by the International Society of Forensic Genetics (ISFG) demonstrated the assay to be accurate, reproducible, sensitive, and specific.

Rapid detection, quantification and speciation of Leishmania using real-time PCR and DNA sequencing at the rRNA Internal Transcribed Spacer 2.

The ability to discriminate infection between closely related Leishmania species within the Viannia species complex, specifically L. braziliensis, L. guyanensis and L. panamensis is critical to inform the clinical diagnosis and determine the most efficacious treatment modality. We designed a nested primer set targeting the rRNA Internal Transcribed Spacer 2 (ITS2), located on Chromosome 27, to distinguish among all human infective Leishmania species. Separate nested and single primer pairs were developed for conventional and quantitative PCR approaches respectively. Species-specific single nucleotide polymorphisms and indels located within the PCR products were identified by Sanger sequencing. This single locus approach provides a sensitive and specific platform to identify the species of Leishmania causing infection.

Species-specific SNP arrays for non-invasive genetic monitoring of a vulnerable bat

Genetic tagging from scats is one of the minimally invasive sampling (MIS) monitoring approaches commonly used to guide management decisions and evaluate conservation efforts. Microsatellite markers have traditionally been used but are prone to genotyping errors. Here, we present a novel method for individual identification in the Threatened ghost bat Macroderma gigas using custom-designed Single Nucleotide Polymorphism (SNP) arrays on the MassARRAY system. We identified 611 informative SNPs from DArTseq data from which three SNP panels (44–50 SNPs per panel) were designed. We applied SNP genotyping and molecular sexing to 209 M. gigas scats collected from seven caves in the Pilbara, Western Australia, employing a two-step genotyping protocol and identifying unique genotypes using a custom-made R package, ScatMatch. Following data cleaning, the average amplification rate was 0.90 ± 0.01 and SNP genotyping errors were low (allelic dropout 0.003 ± 0.000) allowing clustering of scats based on one or fewer allelic mismatches. We identified 19 unique bats (9 confirmed/likely males and 10 confirmed/likely females) from a maternity and multiple transitory roosts, with two male bats detected using roosts, 9 km and 47 m apart. The accuracy of our SNP panels enabled a high level of confidence in the identification of individual bats. Targeted SNP genotyping is a valuable tool for monitoring and tracking of non-model species through a minimally invasive sampling approach.

Comprehensive Analysis of Chloroplast Genome of Hibiscus sinosyriacus: Evolutionary Studies in Related Species and Genera

The Hibiscus genus of the Malvaceae family is widely distributed and has diverse applications. Hibiscus sinosyriacus is a valuable ornamental tree, but it has not been extensively researched. This study aimed to complete the chloroplast genome of H. sinosyriacus and elucidate its evolutionary relationship with closely related species and genera. The complete chloroplast genome of H. sinosyriacus was found to be 160,892 bp in length, with annotations identifying 130 genes, including 85 coding genes, 37 tRNAs, and 8 rRNAs. Interspecific variations in the Hibiscus spp. were explored, and H. sinosyriacus has species-specific single-nucleotide polymorphisms in four genes. Genome structure analysis and visualization revealed that in the Abelmoschus genus, parts of the large single-copy region, including rps19, rpl22, and rps3, have been incorporated into the inverted repeat region, leading to a duplication and an increase in the number of genes. Furthermore, within the Malvales order, the infA gene remains in some genera. Phylogenetic analysis using the whole genome and coding sequences established the phylogenetic position of H. sinosyriacus. This research has further advanced the understanding of the phylogenetic relationships of Hibiscus spp. and related genera, and the results of the structural and variation studies will be helpful for future research.

Development of a tetra-primer ARMS–PCR for identification of sika and red deer and their hybrids

Accurate identification of deer-derived components is significant in food and drug authenticity. Over the years, several methods have been developed to authenticate these products; however, identifying whether female deer products are hybrids is challenging. In this study, the zinc finger protein X-linked (ZFX) gene sequences of sika deer (Cervus nippon), red deer (Cervus elaphus) and their hybrid offspring were amplified and sequenced, the X221 and X428 species-specific single nucleotide polymorphisms (SNP) loci were verified, and a tetra-primer amplification refractory mutation system (T-ARMS–PCR) assay was developed to identify the parent-of-origin of female sika deer, red deer, and their hybrid deer. The T-ARMS–PCR developed based on the X221 locus could identify sika deer, red deer, and their hybrid offspring according to the presence or absence of PCR product sizes of 486 bp, 352 bp, and 179 bp, respectively, just as X428 locus could identify sika deer, red deer, and their hybrid offspring according to the presence or absence of PCR product sizes of 549 bp, 213 bp, and 383 bp, respectively. Forty products labeled deer-derived ingredients randomly purchased were tested using this assay, and the results showed that the identification results based on the two SNP loci were utterly consistent with the actual sources. In addition, this method was found to be accurate, simple, convenient, and with high specificity, thus providing an essential technical reference for deer product species identification. It is also an important supplement to the identification methods of the original ingredients of existing deer products.Graphical abstract

Development and validation of novel SNP markers for the rapid identification of natural hybrids of the 11 closely related pufferfish species (Takifugu spp.) distributed in Japan

Accurate identification of interspecific hybrids is vital for breeding programs in hybridizing taxa, as well as for the use of hybrids in aquaculture and fisheries. The use of species-specific single nucleotide polymorphisms (SNPs) has become the standard for the molecular identification of hybrids. However, it remains challenging to use these SNPs to identify hybrids involving multiple candidate parental species. Here, we developed and validated SNP markers for the identification of hybrids of the 11 closely related pufferfish species (genus Takifugu) distributed in Japan; these species have different aquacultural characteristics, such as growth rate, physiology, behavior, and toxicity. In total, 1232 species-specific SNPs were identified through double-digest restriction-site-associated DNA sequencing. The species had 11 (T. flavipterus) to 295 (T. chrysops) species-specific SNPs. We validated 33 genome-wide species-specific SNPs using TaqMan® genotyping assays for the 11 species and their hybrids, which were identified using amplified fragment length polymorphism analyses. No false negative or positive assignments were observed in 77 and 30 specimens of pure species and F1 hybrids, respectively. This marker combination allows rapid and reliable identification of natural hybrids, which will be useful for pufferfish aquaculture and will enhance our understanding of the natural hybridization between various Takifugu species.

Development of SNP markers for Cucurbita species discrimination

There are five domesticated species in the genus Cucurbita. These are Cucurbita. pepo, Cucurbita maxima, Cucurbita moschata, Cucurbita ficifolia, and Cucurbita argyrosperma. Cucurbita has a large genetic diversity, with various growth forms, sizes, shapes, colors, compositions, tastes, and resistance to biotic and abiotic factors. Maintaining the biological diversity of genetic resources and managing the right historical information are important first steps in the development of new varieties with desirable traits. This study presents the classification of Cucurbita species using DNA barcodes and the development of powerful single nucleotide polymorphism (SNP) markers that can distinguish the five Cucurbita species. A total of 54 accessions from five Cucurbita species (C. pepo, C. maxima, C. moschata, C. ficifolia, and C. argyrosperma) were used for SNP marker development. The chloroplast DNA was extracted, and the three universal barcoding regions (matK, trnH-psbA, and rbcL) were sequenced. Neighbor-joining (NJ) trees were constructed using the nucleotide sequence of the cpDNA barcodes. The rbcL and trnH-psbA barcodes failed to differentiate between C. moschata and C. argyrosperma, whereas the matK region efficiently differentiated with 87% bootstrap support. Moreover, the matK region showed a species identification rate of 100%, making it an efficient marker for identifying the five domesticated Cucurbita species. Multiplex SNaPShot (mini sequencing) analysis of four SNPs in the matK region revealed four species-specific SNPs that effectively classify Cucurbita species into five groups. When all four markers are used together, they are effective. These four species specific SNP markers were validated using known Cucurbita germplasm (111 accessions) and applied for the species identification of unknown Cucurbita germplasm (370 accessions) available at the RDA gene bank in the Republic of Korea. These markers were 100% effective for identifying the five Cucurbita species. Therefore, these SNP markers can be used for effective management of Cucurbita genetic resources in genebanks around the world.

Construction and analysis of the chromosome-level haplotype-resolved genomes of two Crassostrea oyster congeners: Crassostrea angulata and Crassostrea gigas.

The Portuguese oyster Crassostrea angulata and the Pacific oyster C. gigas are two major Crassostrea species that are naturally distributed along the Northwest Pacific coast and possess great ecological and economic value. Here, we report the construction and comparative analysis of the chromosome-level haplotype-resolved genomes of the two oyster congeners. Based on a trio-binning strategy, the PacBio high-fidelity and Illumina Hi-C reads of the offspring of the hybrid cross C. angulata (♂) × C. gigas (♀) were partitioned and independently assembled to construct two chromosome-level fully phased genomes. The assembly size (contig N50 size, BUSCO completeness) of the two genomes were 582.4M (12.8M, 99.1%) and 606.4M (5.46M, 98.9%) for C. angulata and C. gigas, respectively, ranking at the top of mollusk genomes with high contiguity and integrity. The general features of the two genomes were highly similar, and 15,475 highly conserved ortholog gene pairs shared identical gene structures and similar genomic locations. Highly similar sequences can be primarily identified in the coding regions, whereas most noncoding regions and introns of genes in the same ortholog group contain substantial small genomic and/or structural variations. Based on population resequencing analysis, a total of 2,756 species-specific single-nucleotide polymorphisms and 1,088 genes possibly under selection were identified. This is the first report of trio-binned fully phased chromosome-level genomes in marine invertebrates. The study provides fundamental resources for the research on mollusk genetics, comparative genomics, and molecular evolution.

A SNP marker to discriminate the european brook lamprey (Lampetra planeri), river lamprey (L. fluviatilis) and their hybrids.

The European River lamprey Lampetra fluviatilis and the brook lamprey L. planeri are two closely related species that are also considered as partially reproductively isolated ecotypes. At the larval stage, they cannot be distinguished morphologically or genetically by mitochondrial DNA. We aimed at developing a molecular tool to identify early life stages of L. fluviatilis and L. planeri. We first identified Single Nucleotide Polymorphism (SNP) markers with distinct alleles between L. fluviatilis and L. planeri based on RADseq data from 186 individuals collected in France. Then, we developed a quantitative PCR protocol to genotype a species-specific SNP, which was tested on 270 samples including larvae and adults from both species and 17 sites from Western Europe. Results were consistent with morphological identification in all cases except for samples from the Rhône drainage and the Loch Lomond in Scotland. This marker allows the identification of larval stages of L. fluviatilis, L. planeri as well as hybrids. The two cases where the marker was not diagnostic might be explained by an isolated and distinct L. planeri lineage in the Rhône drainage, and high levels of admixture among L. fluviatilis, L. planeri and the lake-parasitic form in Loch Lomond.

Establish an allele-specific real-time PCR for Leishmania species identification

BackgroundLeishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites.MethodsA probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation < 0.5 and inter-assay coefficients of variability < 5%. The specificity of this assay was tested with DNA samples obtained from Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi. Total 42 clinical specimens were used to evaluate the ability of this assay for Leishmania species identification. The phylogenetic tree was constructed using HGPRT and SPDSYN gene fragments to validate the performance of this assay.ResultsThis new method was able to detect 3 and 12 parasites/reaction for VL and CL respectively, and exhibited no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania. Twenty-two samples from VL patients were identified as L. donovani (n = 3) and L. infantum (n = 19), and 20 specimens from CL patients were identified as L. major (n = 20), providing an agreement of 100% compared with sequencing results. For further validation, 29 sequences of HGPRT fragment from nine Leishmania species and 22 sequences from VL patients were used for phylogenetic analysis, which agreed with the results of this new method. Similar results were obtained with 43 sequences of SPDSYN fragment from 18 Leishmania species and 20 sequences from CL patients.ConclusionsOur assay provides a rapid and accurate tool for Leishmania species identification which is applicable for species-adapted therapeutic schedule and patient management.Graphical

Ultrasensitive Detection of Bacillus anthracis by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts.

The anthrax pathogen Bacillus anthracis poses a significant threat to human health. Identification of B. anthracis is challenging because of the bacterium’s close genetic relationship to other Bacillus cereus group species. Thus, molecular detection is founded on species-specific PCR targeting single-copy genes. Here, we validated a previously recognized multi-copy target, a species-specific single nucleotide polymorphism (SNP) present in 2–5 copies in every B. anthracis genome analyzed. For this, a hydrolysis probe-based real-time PCR assay was developed and rigorously tested. The assay was specific as only B. anthracis DNA yielded positive results, was linear over 9 log10 units, and was sensitive with a limit of detection (LoD) of 2.9 copies/reaction. Though not exhibiting a lower LoD than established single-copy PCR targets (dhp61 or PL3), the higher copy number of the B. anthracis–specific 16S rRNA gene alleles afforded ≤2 unit lower threshold (Ct) values. To push the detection limit even further, the assay was adapted for reverse transcription PCR on 16S rRNA transcripts. This RT-PCR assay was also linear over 9 log10 units and was sensitive with an LoD of 6.3 copies/reaction. In a dilution series of experiments, the 16S RT-PCR assay achieved a thousand-fold higher sensitivity than the DNA-targeting assays. For molecular diagnostics, we recommend a real-time RT-PCR assay variant in which both DNA and RNA serve as templates (thus, no requirement for DNase treatment). This can at least provide results equaling the DNA-based implementation if no RNA is present but is superior even at the lowest residual rRNA concentrations.

A sequencing-free assay for foliose Ulva species identification, hybrid detection and bulk biomass characterisation

Sea Lettuce (Ulva spp. Ulvophyceae, Ulvaceae) has tremendous ecological and industrial impacts, from the negative effects of green tide events to the industrial production of food, feed, and value-added products. Due to the morphological similarities between Ulva species, their identification requires the use of “barcoding”, which relies on the sequencing of short fragments of DNA and the comparison of the obtained sequence to that of sequences present in public repositories. However, Sanger sequencing can be costly when hundreds of samples need to be analysed. In addition, “barcoding”, which uses Sanger sequencing, cannot be applied directly on bulk biomass, and requires independent assessment of the individuals within, which is often not possible in commercial products. Here, we describe a novel “sequencing-free” method for species identification of foliose Ulva species that by-passes the drawbacks associated with Sanger sequencing. The assay uses restriction digestion enzymes which target species-specific Single Nucleotide Polymorphisms (SNPs) present within the ITS1 sequence. Digestion of the ITS1 PCR product with two enzyme mixtures allows for the discrimination of the main foliose Ulva species, as well as U. compressa, which can have a foliose morphotype. Of the species tested, only U. pseudorotundata and U. arasakii show the same digestion pattern. Since those two Ulva species are allopatric, we expect this issue to be of limited impact for the users. In addition to species identification, we demonstrate that the assay can be used for hybrid detection, which can have interests for Ulva breeding and species delimitation. Importantly, the assay can be used to detect at once the different Ulva species present within a bulk of Ulva biomass, which could allow for traceability and characterisation of the purity of Ulva commercial products. This study provides a new quick and cost-effective method for foliose Ulva species identification that could readily be extended to other species.

Import of Palmer amaranth (Amaranthus palmeri S. Wats.) seed with sweet potato (Ipomoea batatas (L.) Lam) slips

Palmer amaranth is one of the most economically important and widespread weeds of arable land in the United States. Although no populations are currently known to exist in Canada, its distribution has expanded northward such that it is present in many of the states bordering Canada and multiple pathways exist for its introduction. In this short communication, we report on the transport of viable Palmer amaranth seed on imported sweet potato slips. A reproductive pair of Palmer amaranth seedlings were identified from soil accompanying imported sweet potato slips in 2018. Identification was confirmed using species-specific single nucleotide polymorphisms.

Development of a SNP-based tool for the identification and discrimination of Melolontha melolontha and Melolontha hippocastani.

The European (Melolontha melolontha L.) and Forest (M. hippocastani F.) cockchafer are widespread pests throughout Central Europe. Both species exhibit a 3-5-year life cycle and occur in temporally shifted populations, which have been monitored and documented for more than 100 years. Visual identification of adults and larvae belonging to these morphologically similar species requires expertise and, particularly in the case of larvae, is challenging and equivocal. The goal of the study was the development of an efficient and fast molecular genetic tool for the identification and discrimination of M. melolontha and M. hippocastani. We established a collection of both species from Switzerland, Austria and Northern Italy in 2016, 2017 and 2018. An approximately 1550 bp long fragment of the cytochrome c oxidase subunit 1 (CO1) mitochondrial gene was amplified and sequenced in 13 M. melolontha and 13 M. hippocastani beetles. Alignment of the new sequences with reference sequences (NCBI GenBank and BOLDSYSTEMS databases) and subsequent phylogenetic analysis revealed consistent clustering of the two species. After the identification of M. melolontha and M. hippocastani species-specific single nucleotide polymorphisms (SNPs) in the CO1 alignment, we developed an effective SNP tool based on the ABI PRISM® SNaPshot™ Multiplex Kit for the rapid and accurate species discrimination of adults and larvae.

Molecular confirmation of Dicrocoelium dendriticum in the Himalayan ranges of Pakistan.

Lancet liver flukes of the genus Dicrocoelium (Trematoda: Digenea) are recognised parasites of domestic and wild herbivores. The aim of the present study was to confirm the species identity of Dicrocoeliid flukes collected from the Chitral valley in the Himalayan ranges of Pakistan. The morphology of 48 flukes belonging to eight host populations was examined; but overlapping traits prevented accurate species designation. Phylogenetic comparison of published D. dendriticum ribosomal cistron DNA, and cytochrome oxidase-1 (COX-1) mitochondrial DNA sequences with those from D. chinensis was performed to assess within and between species variation and re-affirm the use of species-specific single nucleotide polymorphism markers. PCR and sequencing of 34 corresponding fragments of ribosomal DNA and 14 corresponding fragments of mitochondrial DNA from the Chitral valley flukes, revealed 10 and 4 unique haplotypes, respectively. These confirmed for the first time the molecular species identity of Pakistani lancet liver flukes as D. dendriticum. This work provides a preliminary illustration of a phylogenetic approach that could be developed to study the ecology, biological diversity, and epidemiology of Dicrocoeliid lancet flukes when they are identified in new settings.

A new naturalized plant in Korea: Carex molestiformis Reznicek and Rothrock (Cyperaceae)

국내 ë¯¸ê¸°ë¡ì¢ ê°€ëŠ”íƒ€ëž˜ì‚¬ì´ˆ(Carex molestiformis Reznik and Rothrock)를 강원도 평창군 ë²½íŒŒë ¹ 일대 에서 처음으로 발견하였다. 본 분류군의 원산지는 북아메리카 미국의 남부지역(Missouri, Oklahoma, Arkansas등 11개 주)으로, 자생지에서는 주로 개활지나 강둑, 범람원 등에서 무리지어 밀생한다. íƒ€ëž˜ì‚¬ì´ˆì ˆ (sect. Ovales)에 속하며, ìžì› ì„± 소수를 갖는 ë‹¤ë „ìƒ 초본으로, 한 화서에 달리는 소수의 수가 2–4개로 ì ê³ , 과낭의 폭이 2.6–3.4 mm로 넓으며, 과낭 배면의 맥이 6–9개로 많아 국내에 자생하는 ê·¼ì—°ì¢ ì¸ 타래사초(C. maackii Maxim.) 및 ê·€í™”ì¢ ì¸ 한석사초(C. scoparia)와 ëª í™•ížˆ 구별된다. êµ­ëª ì€ 본 분류군이 í˜•íƒœì ìœ¼ë¡œ 타 래사초와 ìœ ì‚¬í•˜ë©°, 식물체가 ì „ì²´ì ìœ¼ë¡œ 가늘어 ‘가는타래사초’로 ì‹ ì¹­í•˜ì˜€ê³ , 분류군에 대한 기재문, 화상 자료 및 도해 ê·¸ë¦¬ê³ ìœ ì‚¬ì¢ ì— 대한 검색표를 ì œì‹œí•˜ì˜€ë‹¤. 또한, 가는타래사초와 ê·¼ì—°ì¢ ì˜ ìœ ì „ì  차이를 확 ì¸í•˜ê³ ìž 3개(chloroplast DNA matK, ndhF, nuclear ribosomal DNA internal transcribed spacer) DNA barcode 지역의 부분 염기서열을 비교하여, 총 8개의 가는타래사초의 ì¢ íŠ¹ì´ì  단일염기다형성(single nucleotide polymorphisms)을 확인하였다. 주요어: ë¯¸ê¸°ë¡ì¢ , 가는타래사초, íƒ€ëž˜ì‚¬ì´ˆì ˆ, 사초과

Single Nucleotide Polymorphism Assay for Genetic Identification of Lophophora williamsii.

Lophophora is a member of the Cactaceae family, which contains two species: Lophophora williamsii and L.diffusa. Lophophora williamsii is an illegal plant containing mescaline, a hallucinogenic alkaloid. In this study, a novel method based on a single nucleotide polymorphism (SNP) assay was developed for identifying L.williamsii; this assay reliably detects SNPs within chloroplast DNA (rbcL, matK, and trnL-trnF IGS) and was validated for identifying Lophophora and L.williamsii simultaneously. The chloroplast DNA sequences from four L.williamsii and three L.diffusa plants were obtained and compared using DNA sequence data from approximately 300 other Cactaceae species available in GenBank. From this sequence data, a total of seven SNPs were determined to be suitable for identifying L.williamsii. A multiplex assay was constructed using the ABI PRISM® SNaPshot™ Multiplex Kit (Applied Biosystems, Forster City, CA) to analyze species-specific SNPs. Using this multiplex assay, we clearly distinguished the Lophophora among 19 species in the Cactaceae family. Additionally, L.williamsii was distinguished from L.diffusa. These results suggest that the newly developed assay may help resolve crimes related to illegal distribution and use. This multiplex assay will be useful for the genetic identification of L.williamsii and can complement conventional methods of detecting mescaline.

Genotyping-by-Sequencing Derived Single Nucleotide Polymorphisms Provide the First Well-Resolved Phylogeny for the Genus Triticum (Poaceae).

Wheat (Triticum spp.) has been an important staple food crop for mankind since the beginning of agriculture. The genus Triticum L. is composed of diploid, tetraploid, and hexaploid species, majority of which have not yet been discriminated clearly, and hence their phylogeny and classification remain unresolved. Genotyping-by-sequencing (GBS) is an easy and affordable method that allows us to generate genome-wide single nucleotide polymorphism (SNP) markers. In this study, we used GBS to obtain SNPs covering all seven chromosomes from 283 accessions of Triticum-related genera. After filtering low-quality and redundant SNPs based on haplotype information, the GBS assay provided 14,188 high-quality SNPs that were distributed across the A (71%), B (26%), and D (2.4%) genomes. Cluster analysis and discriminant analysis of principal components (DAPC) allowed us to distinguish six distinct groups that matched well with Triticum species complexity. We constructed a Bayesian phylogenetic tree using 14,188 SNPs, in which 17 Triticum species and subspecies were discriminated. Dendrogram analysis revealed that the polyploid wheat species could be divided into groups according to the presence of A, B, D, and G genomes with strong nodal support and provided new insight into the evolution of spelt wheat. A total of 2,692 species-specific SNPs were identified to discriminate the common (T. aestivum) and durum (T. turgidum) wheat cultivar and landraces. In principal component analysis grouping, the two wheat species formed individual clusters and the SNPs were able to distinguish up to nine groups of 10 subspecies. This study demonstrated that GBS-derived SNPs could be used efficiently in genebank management to classify Triticum species and subspecies that are very difficult to distinguish by their morphological characters.

SNP genotyping reveals substructuring in weakly differentiated populations of Atlantic cod (Gadus morhua) from diverse environments in the Baltic Sea

Atlantic cod (Gadus morhua) is one of the most important fish species in northern Europe for several reasons including its predator status in marine ecosystems, its historical role in fisheries, its potential in aquaculture and its strong public profile. However, due to over-exploitation in the North Atlantic and changes in the ecosystem, many cod populations have been reduced in size and genetic diversity. Cod populations in the Baltic Proper, Kattegat and North Sea have been analyzed using a species specific single nucleotide polymorphism (SNP) array. Using a subset of 8,706 SNPs, moderate genetic differences were found between subdivisions in three traditionally delineated cod management stocks: Kattegat, western and eastern Baltic. However, an FST measure of population differentiation based on allele frequencies from 588 outlier loci for 2 population groups, one including 5 western and the other 4 eastern Baltic populations, indicated high genetic differentiation. In this paper, differentiation has been demonstrated not only between, but also within western and eastern Baltic cod stocks for the first time, with salinity appearing to be the most important environmental factor influencing the maintenance of cod population divergence between the western and eastern Baltic Sea.

Genome-informed integrative taxonomic description of three cryptic species in the earthworm genus Carpetania (Oligochaeta, Hormogastridae)

Research on cryptic species complexes has reached a consensus on the necessity of integrating multiple sources of evidence. Reduced representation library techniques such as Genotyping-by-Sequencing (GBS) have proven useful to study these groups. Both integrative taxonomy and genome-wide single nucleotide polymorphism (SNP) data remain to be widely applied to earthworms, an animal group with widespread presence of cryptic diversity. The genus Carpetania (formerly the Hormogaster elisae species complex) was found to contain six deeply divergent genetic lineages and some inconspicuous morphological differentiation based on a handful of Sanger-sequenced markers. Marchán et al. (submitted) delimited three well-supported species-level clades on the basis of a genome-wide SNP dataset and geometric morphometric analyses, highlighting the necessity of a formal taxonomic description of these taxa. In this work, further analyses are applied to the SNP data and a thorough morphological study is performed in order to provide an integrative description of two new species and to redescribe Carpetania elisae. Species-specific SNPs are identified and used as diagnostic characters, and genome-wide and cytochrome oxidase C subunit 1 (COI) genetic distances are compared finding a strong correlation between them. The taxonomic description of these three cryptic species provides a useful tool to include them effectively in ecological studies and biodiversity conservation actions.www.zoobank.org/urn:lsid:zoobank.org:act:19CC41D7-BD25-4337-BED5-CDB04CAFB537www.zoobank.org/urn:lsid:zoobank.org:act:206EE090-4AF5-4D12-82CA-F4A6494E90D4www.zoobank.org/urn:lsid:zoobank.org:pub:2797FDDF-EA3D-4347-96C6-E632DC26CDA3