Set Of Nuclear Markers Research Articles

A Small Set of Nuclear Markers for Reliable Differentiation of the Two Closely Related Oak Species Quercus Robur and Q. Petraea.

Quercus robur and Q. petraea are, in addition to Fagus sylvatica, the main economically used deciduous tree species in Europe. Identification of these two species is crucial because they differ in their ecological demands. Because of a changing climate, foresters must know more than ever which species will perform better under given environmental conditions. The search for differentiating molecular markers between these two species has already lasted for decades. Until now, differentiation has only been possible in approaches with a combination of several molecular markers and a subsequent statistical analysis to calculate the probability of being one or the other species. Here, we used MiSeq Illumina data from pools of Q. robur and Q. petraea specimens and identified nuclear SNPs and small InDels versus the Q. robur reference genome. Selected sequence variants with 100% allele frequency difference between the two pools were further validated in an extended set of Q. robur and Q. petraea specimens, and then the number of markers was deliberately reduced to the smallest possible set for species differentiation. A combination of six markers from four nuclear regions is enough to identify Q. robur, Q. petraea or hybrids between these two species quite well and represents a marker set that is cost-efficient and useable in every laboratory.

Standardized nuclear markers improve and homogenize species delimitation in Metazoa

Abstract Species are the fundamental units of life and evolution. Their recognition is essential for science and society. Molecular methods have been increasingly used for the identification of animal species, despite several challenges. Here, we explore with genomic data from nine animal lineages a set of nuclear markers, namely metazoan‐level universal single‐copy orthologs (metazoan USCOs), for their use in species delimitation. Our data sets include arthropods and vertebrates. We use various data assembly strategies and use coalescent‐based species inference as well as population admixture analyses and phenetic methods. We demonstrate that metazoan USCOs distinguish well closely related morphospecies and consistently outperform classical mitochondrial DNA barcoding in discriminating closely related species in different animal taxa, as judged by comparison with morphospecies delimitations. USCOs overcome the general shortcomings of mitochondrial DNA barcodes, and due to standardization across Metazoa, also those of other approaches. They accurately assign samples not only to lower but also to higher taxonomic levels. Metazoan USCOs provide a powerful and unifying framework for DNA‐based species delimitation and taxonomy in animals and their employment could result in a more efficient use of research data and resources.

Evidence of mitochondrial capture in Australian glass shrimp (

Context The Australian glass shrimp (Paratya australiensis) has been identified as a cryptic species complex and several lineages occur sympatrically in eastern Australia. In south-eastern Queensland, the predominant lineages are ‘4’ and ‘6’, although the only known area of sympatry in the Conondale Range is the result of a translocation. Aim Our aim was to determine any evidence of natural sympatry between the two lineages in the Mary River catchment area using polymerase chain reatction–restriction fragment length polymorphism (PCR-RFLP) analysis. Methods Of six sites sampled, only one site showed sympatry between Lineage 4 and 6, so 39 individuals from this site were sequenced using fragment of the mtDNA COI gene and a neighbour joining tree revealed the existence of two lineages. Results On the basis of two sets of nuclear markers (allozymes and microsatellites), there was no evidence of two species, because all loci conformed to Hardy–Weinberg expectations and only Lineage 4 alleles were identified. Conclusion These results led to the inference that the existence of two mtDNA lineages could be due to mitochondrial capture. Implication The current situation in the Broken Bridge High site could be due to historical interbreeding between the two lineages reflected in the mtDNA data but not evident in the nuclear data.

Rewinding the molecular clock in the genus Carabus (Coleoptera: Carabidae) in light of fossil evidence and the Gondwana split: A reanalysis.

Molecular clocks have become powerful tools given increasing sequencing and fossil resources. However, calibration analyses outcomes depend on the choice of priors. Here, we revisited the seminal dating study published by Andújar and coworkers of the genus Carabus proposing that prior choices need re-evaluation. We hypothesized that reflecting fossil evidence and the Gondwanan split properly significantly rewinds the molecular clock. We re-used the dataset including five mitochondrial and four nuclear DNA fragments with a total length of 7888 nt. Fossil evidence for Oligocene occurrence of Calosoma was considered. Root age was set based on the fossil evidence of Harpalinae ground beetles in the Upper Cretaceous. Paleogene divergence of the outgroup taxa Ceroglossini and Pamborini is introduced as a new prior based on current paleontological and geological literature. The ultrametric time-calibrated tree of the extended nd5 dataset resulted in a median TMRCA Carabus of 53.92 Ma (HPD 95% 45.01–63.18 Ma), roughly 30 Ma older than in the Andújar study. The splits among C. rugosus and C. morbillosus (A), C. riffensis from the European Mesocarabus (B), and Eurycarabus and Nesaeocarabus (C) were dated to 17.58 (12.87–22.85), 24.14 (18.02–30.58), and 21.6 (16.44–27.43) Ma. They were decidedly older than those previously reported (7.48, 10.93, and 9.51 Ma). These changes were driven almost entirely by constraining the Carabidae time-tree root with a Harpalinae amber fossil at ~99 Ma. Utilizing the nd5 dating results of three well-supported Carabus clades as secondary calibration points for the complete MIT-NUC dataset led to a TMRCA of Carabus of 44.72 (37.54–52.22) Ma, compared with 25.16 Ma (18.41–33.04 Ma) in the previous study. Considering fossil evidence for Oligocene Calosoma and Late Cretaceous Harpalini together with the Gondwanan split as a new prior, our new approach supports the origin of genus Carabus in the Eocene. Our results are preliminary because of the heavy reliance on the nd5 gene, and thus will have to be tested with a sufficient set of nuclear markers. Additionally, uncertainties due to dating root age of the tree based on a single fossil and outgroup taxon affect the results. Improvement of the fossil database, particularly in the supertribe Carabitae, is needed to reduce these uncertainties in dating Carabus phylogeny.

Development and characterization of novel polymorphic microsatellite markers for the Korean freshwater snail Semisulcospira coreana and cross-species amplification using next-generation sequencing

Korean freshwater snails of the genus Semisulcospira are widely distributed across East Asia. It has been a very popular nutritional food in Korea, and is an ecologically important water quality indicator because it lives only in clean water. However, no microsatellite markers have been generated to study the population genetic diversity of this genus. In the present study, we developed and characterized 18 novel microsatellite loci from Semisulcospira coreana genomic DNA. The microsatellites were isolated using 454 GS-FLX titanium sequencing and 18 markers were used for genotyping in S. coreana. In addition, we also tested the cross-species transferability of the microsatellite markers in four additional Semisulcospira spp. We identified 18 polymorphic loci and the number of alleles per loci, and their polymorphism information content values ranged from 2 to 17 and 0.203 to 0.902, respectively. The observed and expected heterozygosities of the loci ranged from 0.063 to 0.924 and 0.226 to 0.924, respectively. According to the analysis of the cross-species transferability of these markers, four species, S. forticosta, S. gottschei, S. tegulata, and S. libertina, showed a very high transferability (80%–85%). These results show that this set of nuclear markers could be useful for population genetics studies of this species and closely related species.

Development of nuclear microsatellite markers in Stizophyllum (Bignoniaceae) using next-generation sequencing

AbstractHere we developed the first set of nuclear microsatellite markers (nSSRs) for Stizophyllum riparium (Bignoniaceae), a widespread species of Neotropical liana. Thirty-two sets of primers were isolated from a genomic dataset obtained with an Illumina HiSeq Platform, and characterized for 57 individuals of S. riparium from three populations. Nine nSSRs were polymorphic and the number of alleles ranged from eight to 29. The unbiased expected heterozygosity per locus ranged from 0.724 to 0.952 and the polymorphic information content values ranged from 0.717 to 0.944. All nine primer pairs also amplified for two closely related species (S. inaequilaterum and S. perforatum). The new set of nuclear markers will be useful for population genetics studies of Stizophyllum as a whole.

Multiple processes drive genetic structure of humpback whale (Megaptera novaeangliae) populations across spatial scales.

Elucidating patterns of population structure for species with complex life histories, and disentangling the processes driving such patterns, remains a significant analytical challenge. Humpback whale (Megaptera novaeangliae) populations display complex genetic structures that have not been fully resolved at all spatial scales. We generated a data set of nuclear markers for 3575 samples spanning the seven breeding stocks and substocks found in the South Atlantic and western and northern Indian Oceans. For the total sample, and males and females separately, we assessed genetic diversity, tested for genetic differentiation between putative populations and isolation by distance, estimated the number of genetic clusters without a priori population information and estimated rates of gene flow using maximum-likelihood and Bayesian approaches. At the ocean basin scale, structure is governed by geographical distance (IBD P<0.05) and female fidelity to breeding areas, in line with current understanding of the drivers of broadscale population structure. Consistent with previous studies, the Arabian Sea breeding stock was highly genetically differentiated (FST 0.034-0.161; P<0.01 for all comparisons). However, the breeding stock boundary between west South Africa and east Africa was more porous than expected based on genetic differentiation, cluster and geneflow analyses. Instances of male fidelity to breeding areas and relatively high rates of dispersal for females were also observed between the three substocks in the western Indian Ocean. The relationships between demographic units and current management boundaries may have ramifications for assessments of the status and continued protections of populations still in recovery from commercial whaling.

Development of the first polymorphic microsatellite markers for the Roman snail Helix pomatia L., 1758 (Helicidae) and cross-species amplification within the genus Helix.

The terrestrial snail Helix pomatia (Gastropoda: Stylommatophora: Helicidae) is one of the largest gastropod species in Europe. This species is strictly protected in some European Union countries; however, at the same time, it is also farmed and commercialized for human consumption. Here, we describe 11 microsatellite markers that are very useful in population genetic studies for assessing the status of both wild and farmed populations of this species of community interest. The microsatellites were isolated using 454 pyrosequencing technologies and 11 primer pairs were selected and used for genotyping an H. pomatia population and also checked for cross-species amplification on H. lucorum and H. lutescens specimens. The number of alleles per locus ranged from 3 to 13 and observed heterozygosity was between 0.458 and 0.917. Seven of these loci were polymorphic in H. lucorum, and four in H. lutescens. This set of nuclear markers provides a powerful tool for population genetic studies of this species of community interest, and also for closely related species. The described microsatellite markers should also facilitate the identification of populations of conservation concern.

A tribal level phylogeny of Lake Tanganyika cichlid fishes based on a genomic multi-marker approach

The species-flocks of cichlid fishes in the East African Great Lakes Victoria, Malawi and Tanganyika constitute the most diverse extant adaptive radiations in vertebrates. Lake Tanganyika, the oldest of the lakes, harbors the morphologically and genetically most diverse assemblage of cichlids and contains the highest number of endemic cichlid genera of all African lakes. Based on morphological grounds, the Tanganyikan cichlid species have been grouped into 12–16 distinct lineages, so-called tribes. While the monophyly of most of the tribes is well established, the phylogenetic relationships among the tribes remain largely elusive. Here, we present a new tribal level phylogenetic hypothesis for the cichlid fishes of Lake Tanganyika that is based on the so far largest set of nuclear markers and a total alignment length of close to 18kb. Using next-generation amplicon sequencing with the 454 pyrosequencing technology, we compiled a dataset consisting of 42 nuclear loci in 45 East African cichlid species, which we subjected to maximum likelihood and Bayesian inference phylogenetic analyses. We analyzed the entire concatenated dataset and each marker individually, and performed a Bayesian concordance analysis and gene tree discordance tests. Overall, we find strong support for a position of the Oreochromini, Boulengerochromini, Bathybatini and Trematocarini outside of a clade combining the substrate spawning Lamprologini and the mouthbrooding tribes of the ‘H-lineage’, which are both strongly supported to be monophyletic. The Eretmodini are firmly placed within the ‘H-lineage’, as sister-group to the most species-rich tribe of cichlids, the Haplochromini. The phylogenetic relationships at the base of the ‘H-lineage’ received less support, which is likely due to high speciation rates in the early phase of the radiation. Discordance among gene trees and marker sets further suggests the occurrence of past hybridization and/or incomplete lineage sorting in the cichlid fishes of Lake Tanganyika.

Green turtle population structure in the Pacific: new insights from single nucleotide polymorphisms and microsatellites

ESR Endangered Species Research Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials ESR 20:227-234 (2013) - DOI: https://doi.org/10.3354/esr00500 Green turtle population structure in the Pacific: new insights from single nucleotide polymorphisms and microsatellites Suzanne E. Roden1,*, Phillip A. Morin1, Amy Frey1, George H. Balazs2, Patricia Zarate3, I-Jiunn Cheng4, Peter H. Dutton1 1Marine Mammal and Turtle Division, Southwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 8901 La Jolla Shores Drive, La Jolla, California 92037, USA 2Marine Turtle Research Program, Pacific Islands Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2570 Dole Street, Honolulu, Hawaii 96822, USA 3Marine Science Program, Charles Darwin Foundation, Santa Cruz Island, Galapagos Islands, Ecuador 4Institute of Marine Biology, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 20224, Taiwan, ROC *Email: suzanne.roden@noaa.gov ABSTRACT: A set of nuclear single nucleotide polymorphisms (SNPs) and microsatellite markers was used to detect genetic stock structure among 5 Pacific green turtle Chelonia mydas nesting populations. We sampled populations in the Galapagos Islands, Ecuador (n = 57), Colola, Mexico (n = 75), French Frigate Shoals, Hawaii (n = 141), Yap, Micronesia (n = 73), and Wan-an, Taiwan (n = 57), to represent eastern, central, and western Pacific regions. A combination of 29 single independent SNPs and linked SNPs combined as haplotypes were used for a total of 20 independent markers. In addition, 8 polymorphic microsatellite markers were applied to the same sample set. Both sets of nuclear markers confirmed significant differentiation between all sampled populations in the 3 Pacific regions (p ≤ 0.001). The use of these SNPs and microsatellites resulted in sufficient power to detect small population differences not seen in previous studies using smaller numbers of nuclear markers. Our results suggest that male-mediated gene flow between regional nesting stocks is more limited than previously believed, allowing the potential to delineate stocks more clearly. Finally, we discuss the value of SNP markers as an alternative or complement to other nuclear markers such as microsatellites for the examination of stock structure. KEY WORDS: Green turtle · Chelonia mydas · Single nucleotide polymorphism · SNP · Microsatellite · Sea turtle · Genetics · Conservation Full text in pdf format PreviousNextCite this article as: Roden SE, Morin PA, Frey A, Balazs GH, Zarate P, Cheng IJ, Dutton PH (2013) Green turtle population structure in the Pacific: new insights from single nucleotide polymorphisms and microsatellites. Endang Species Res 20:227-234. https://doi.org/10.3354/esr00500 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in ESR Vol. 20, No. 3. Online publication date: May 31, 2013 Print ISSN: 1863-5407; Online ISSN: 1613-4796 Copyright © 2013 Inter-Research.

Characterization of cell death inducing Phytophthora capsici CRN effectors suggests diverse activities in the host nucleus

Plant-Microbe interactions are complex associations that feature recognition of Pathogen Associated Molecular Patterns by the plant immune system and dampening of subsequent responses by pathogen encoded secreted effectors. With large effector repertoires now identified in a range of sequenced microbial genomes, much attention centers on understanding their roles in immunity or disease. These studies not only allow identification of pathogen virulence factors and strategies, they also provide an important molecular toolset suited for studying immunity in plants. The Phytophthora intracellular effector repertoire encodes a large class of proteins that translocate into host cells and exclusively target the host nucleus. Recent functional studies have implicated the CRN protein family as an important class of diverse effectors that target distinct subnuclear compartments and modify host cell signaling. Here, we characterized three necrosis inducing CRNs and show that there are differences in the levels of cell death. We show that only expression of CRN20_624 has an additive effect on PAMP induced cell death but not AVR3a induced ETI. Given their distinctive phenotypes, we assessed localization of each CRN with a set of nuclear markers and found clear differences in CRN subnuclear distribution patterns. These assays also revealed that expression of CRN83_152 leads to a distinct change in nuclear chromatin organization, suggesting a distinct series of events that leads to cell death upon over-expression. Taken together, our results suggest diverse functions carried by CRN C-termini, which can be exploited to identify novel processes that take place in the host nucleus and are required for immunity or susceptibility.

Effects of current and historic habitat fragmentation on the genetic structure of the sand goby Pomatoschistus minutus (Osteichthys, Gobiidae)

Habitat fragmentation is a major force that will influence the evolution of a species and its distribution range. Pomatoschistus minutus, the sand goby, has a North Atlantic–Mediterranean distribution and shows various level of habitat fragmentation along its geographic repartition. The use of mitochondrial sequences of the cytochrome b (cyt b) gene and two co-dominant sets of nuclear markers (introns and microsatellites) allowed us to describe the relationships between P. minutus populations belonging to several different geographical regions of Europe and to assess the structure of populations inhabiting the Golfe du Lion, along the French Mediterranean coast. The present study confirms that the taxon located in the Adriatic Sea (Venice) should be considered as a distinct species, separated approximately 1.75 Mya. The comparison of P. minutus between the Atlantic and western Mediterranean coasts using polymorphic co-dominant markers revealed that they belong to two demographically independent units, and thus could be considered as well as distinct species, more recently separated (0.3 Mya). The Pleistocene glaciations seem therefore to have played an important role in the diversification of this complex. Finally, at a regional scale in the Golfe du Lion, P. minutus appears to form a single huge homogeneous population.

Complete Khoisan and Bantu genomes from southern Africa

The genetic structure of the indigenous hunter-gatherer peoples of southern Africa, the oldest known lineage of modern human, is important for understanding human diversity. Studies based on mitochondrial and small sets of nuclear markers have shown that these hunter-gatherers, known as Khoisan, San, or Bushmen, are genetically divergent from other humans. However, until now, fully sequenced human genomes have been limited to recently diverged populations. Here we present the complete genome sequences of an indigenous hunter-gatherer from the Kalahari Desert and a Bantu from southern Africa, as well as protein-coding regions from an additional three hunter-gatherers from disparate regions of the Kalahari. We characterize the extent of whole-genome and exome diversity among the five men, reporting 1.3 million novel DNA differences genome-wide, including 13,146 novel amino acid variants. In terms of nucleotide substitutions, the Bushmen seem to be, on average, more different from each other than, for example, a European and an Asian. Observed genomic differences between the hunter-gatherers and others may help to pinpoint genetic adaptations to an agricultural lifestyle. Adding the described variants to current databases will facilitate inclusion of southern Africans in medical research efforts, particularly when family and medical histories can be correlated with genome-wide data.

Identification of endangered Hawaiian ducks (Anas wyvilliana), introduced North American mallards (A. platyrhynchos) and their hybrids using multilocus genotypes

Hawaiian ducks (Anas wyvilliana), or koloa, are endemic to the Hawaiian Islands and are listed as a federal and state endangered species. Hybridization between koloa and introduced mallards (A. platyrhynchos) is believed to be a primary threat to the recovery of koloa. We evaluated the utility of two sets of nuclear markers (microsatellite loci and amplified fragment length polymorphisms) and a variable portion of the mitochondrial DNA control region to distinguish among koloa, mallards, and hybrids. We show that microsatellite and AFLP markers can be used to distinguish between koloa and mallard-koloa hybrids with a high degree of confidence. For all but one of the putative koloa in our sample, the posterior probability of belonging to the koloa category was >0.90. Similarly all but one of the mallard-koloa hybrids were assigned to the hybrid category with posterior probabilities >0.98. Subsets of markers led to poorer resolution among koloa, mallard and hybrid categories. Among a sample of 61 koloa, hybrids and mallards, we found 25 different mtDNA haplotypes, belonging to two groups of haplotypes (A and B) identified previously in mallards and their relatives. All putative koloa samples exhibited group B haplotypes, of which 65% comprised one haplotype, while the rest were divided among four haplotypes. All Hawai’i mallard samples exhibited haplotypes that belonged to group A. Hybrids and California mallards exhibited haplotypes belonging to both groups, but a majority were of group A, suggesting that hybridization may more commonly involve mating between Hawai’i mallard females and koloa males.

Geographic Structure of Mitochondrial and Nuclear Gene Polymorphisms in Australian Green Turtle Populations and Male-Biased Gene Flow

The genetic structure of green turtle (Chelonia mydas) rookeries located around the Australian coast was assessed by (1) comparing the structure found within and among geographic regions, (2) comparing microsatellite loci vs. restriction fragment length polymorphism analyses of anonymous single copy nuclear DNA (ascnDNA) loci, and (3) comparing the structure found at nuclear DNA markers to that of previously analyzed mitochondrial (mtDNA) control region sequences. Significant genetic structure was observed over all regions at both sets of nuclear markers, though the microsatellite data provided greater resolution in identifying significant genetic differences in pairwise tests between regions. Inferences about population structure and migration rates from the microsatellite data varied depending on whether statistics were based on the stepwise mutation or infinite allele model, with the latter being more congruent with geography. Estimated rates of gene flow were generally higher than expected for nuclear DNA (nDNA) in comparison to mtDNA, and this difference was most pronounced in comparisons between the northern and southern Great Barrier Reef (GBR). The genetic data combined with results from physical tagging studies indicate that the lack of nuclear gene divergence through the GBR is likely due to the migration of sGBR turtles through the courtship area of the nGBR population, rather than male-biased dispersal. This example highlights the value of combining comparative studies of molecular variation with ecological data to infer population processes.