Characterization of microsatellite markers for Moricandia moricandioides (Brassicaceae) and related species

PREMISE OF THE STUDY: Polymorphic microsatellite markers were developed to study population structure and mating patterns of the monocarpic herb Moricandia moricandioides (Brassicaceae). METHODS AND RESULTS: Illumina MiSeq sequencing was used to develop a panel of 15 polymorphic microsatellite markers that were tested across 77 individuals from three populations on the Iberian Peninsula. All markers were polymorphic in at least two studied populations, and the number of alleles ranged from one to 11 per locus. The levels of observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.153 to 0.865, respectively. Nine and 11 loci were successfully amplified in the congeneric species M. arvensis and M. foetida, respectively. CONCLUSIONS: The 15 microsatellite markers will be useful for population genetic studies of the genus Moricandia. These markers will serve as a useful tool for exploring population structure and mating patterns of M. moricandioides.This work received funding from the European Union’s Horizon 2020 (Marie Skłodowska-Curie No. 655653), Fundación BBVA (PR17-ECO- 0021), and the Spanish Ministerio de Economia y Competitividad (CGL2017-86626- C2- 1- P)

Automatic identification of species-specific repetitive DNA sequences and their utilization for detecting microbial organisms

Motivation: The concentration of pathogen DNA in biological samples is often very low. Therefore, the sensitivity of diagnostic tests is always a critical factor

UniPrime: a workflow-based platform for improved universal primer design

UniPrime is an open-source software (http://uniprime.batlab.eu), which automatically designs large sets of universal primers by simply inputting a gene ID reference. UniPrime automatically retrieves and aligns homologous sequences from GenBank, identifies regions of conservation within the alignment and generates suitable primers that can amplify variable genomic regions. UniPrime differs from previous automatic primer design programs in that all steps of primer design are automated, saved and are phylogenetically limited. We have experimentally verified the efficiency and success of this program by amplifying and sequencing four diverse genes (AOF2, EFEMP1, LRP6 and OAZ1) across multiple Orders of mammals. UniPrime is an experimentally validated, fully automated program that generates successful cross-species primers that take into account the biological aspects of the PCR

Wolbachia in the flesh: symbiont intensities in germ-line and somatic tissues challenge the conventional view of Wolbachia transmission routes

Symbionts can substantially affect the evolution and ecology of their hosts. The investigation of the tissue-specific distribution of symbionts (tissue tropism) can provide important insight into host-symbiont interactions. Among other things, it can help to discern the importance of specific transmission routes and potential phenotypic effects. The intracellular bacterial symbiont Wolbachia has been described as the greatest ever panzootic, due to the wide array of arthropods that it infects. Being primarily vertically transmitted, it is expected that the transmission of Wolbachia would be enhanced by focusing infection in the reproductive tissues. In social insect hosts, this tropism would logically extend to reproductive rather than sterile castes, since the latter constitute a dead-end for vertically transmission. Here, we show that Wolbachia are not focused on reproductive tissues of eusocial insects, and that non-reproductive tissues of queens and workers of the ant Acromyrmex echinatior, harbour substantial infections. In particular, the comparatively high intensities of Wolbachia in the haemolymph, fat body, and faeces, suggest potential for horizontal transmission via parasitoids and the faecal-oral route, or a role for Wolbachia modulating the immune response of this host. It may be that somatic tissues and castes are not the evolutionary dead-end for Wolbachia that is commonly thought

The clonal relationships between pre-cancer and cancer revealed by ultra-deep sequencing

The study of the relationships between pre-cancer and cancer and identification of early driver mutations is becoming increasingly important as the value of molecular markers of early disease and personalised drug targets is recognized, especially now the extent of clonal heterogeneity in fully invasive disease is being realized. It has been assumed that pre-cancerous lesions exhibit a fairly passive progression to invasive disease; the degree to which they, too, are heterogeneous is unknown. We performed ultra-deep sequencing of thousands of selected mutations, together with copy number analysis, from multiple, matched pre-invasive lesions, primary tumours and metastases from five patients with oral cancer, some with multiple primary tumours presenting either synchronously or metachronously, totalling 75 samples. This allowed the clonal relationships between the samples to be observed for each patient. We expose for the first time the unexpected variety and complexity of the relationships between this group of oral dysplasias and their associated carcinomas and, ultimately, the diversity of processes by which tumours are initiated, spread and metastasize. Instead of a series of genomic precursors of their adjacent invasive disease, we have shown dysplasia to be a distinct dynamic entity, refuting the belief that pre-cancer and invasive tumours with a close spatial relationship always have linearly related genomes. We show that oral pre-cancer exhibits considerable subclonal heterogeneity in its own right, that mutational changes in pre-cancer do not predict the onset of invasion, and that the genomic pathway to invasion is neither unified nor predictable. Sequence data from this study have been deposited in the European Nucleotide Archive, Accession No. PRJEB6588

Characterization of Species-Specific Repeats in 613 Prokaryotic Species

Prokaryotes are in general believed to possess small, compactly organized genomes, with repetitive sequences forming only a small part of them. Nonetheless, many prokaryotic genomes in fact contain species-specific repeats (>85 bp long genomic sequences with less than 60% identity to other species) as we have previously demonstrated. However, it is not known at present how frequent such species-specific repeats are and what their functional roles in bacterial genomes may be. Therefore, we have conducted a comprehensive survey of prokaryotic species-specific repeats and characterized them to examine as to whether there are functional classes among different repeats or not and how they are mutually related to each other. Of the 613 distinct prokaryotic species analyzed, 97% were found to contain at least one species-specific repeats. It seems interesting to note that the species-specific repeats thus identified appear to be functionally variable in different genomes: in some genomes, they are mostly associated with duplicated protein-coding genes, whereas in some other genomes with rRNA and tRNA genes. Contrary to what may be expected, only one-fourth of the species-specific repeats were found to be associated with mobile genetic elements

PrimerHunter: a primer design tool for PCR-based virus subtype identification

Rapid and reliable virus subtype identification is critical for accurate diagnosis of human infections, effective response to epidemic outbreaks and global-scale surveillance of highly pathogenic viral subtypes such as avian influenza H5N1. The polymerase chain reaction (PCR) has become the method of choice for virus subtype identification. However, designing subtype-specific PCR primer pairs is a very challenging task: on one hand, selected primer pairs must result in robust amplification in the presence of a significant degree of sequence heterogeneity within subtypes, on the other, they must discriminate between the subtype of interest and closely related subtypes. In this article, we present a new tool, called PrimerHunter, that can be used to select highly sensitive and specific primers for virus subtyping. Our tool takes as input sets of both target and nontarget sequences. Primers are selected such that they efficiently amplify any one of the target sequences, and none of the nontarget sequences. PrimerHunter ensures the desired amplification properties by using accurate estimates of melting temperature with mismatches, computed based on the nearest neighbor model via an efficient fractional programming algorithm. Validation experiments with three avian influenza HA subtypes confirm that primers selected by PrimerHunter have high sensitivity and specificity for target sequences

Comparing mutation calls in fixed tumour samples between the Affymetrix OncoScan® Array and PCR based next-generation sequencing

Background: The importance of accurate and affordable mutation calling in fixed pathology samples is becoming increasingly important as we move into the era of personalised medicine. The Affymetrix OncoScan® Array platform is designed to produce actionable mutation calls in archival material. Methods: We compared calls made using the OncoScan platform with calls made using a custom designed PCR panel followed by next-generation sequencing (NGS), in order to benchmark the sensitivity and specificity of the OncoScan calls in a large cohort of fixed tumour samples. 392 fixed, clinical samples were sequenced, encompassing 641 PCR regions, 403 putative positive calls and 1528 putative negative calls. Results: A small number of mutations could not be validated, either due to large indels or pseudogenes impairing parts of the NGS pipeline. For the remainder, if calls were filtered according to simple quality metrics, both sensitivity and specificity for the OncoScan platform were over 98%. This applied even to samples with poorer sample quality and lower variant allele frequency (5–10%) than product claims indicated. Conclusions: This benchmarking study will be useful to users and potential users of this platform, who wish to compare technologies or interpret their own results

PrimerHunter: a primer design tool for PCR-based virus subtype identification

Rapid and reliable virus subtype identification is critical for accurate diagnosis of human infections, effective response to epidemic outbreaks and global-scale surveillance of highly pathogenic viral subtypes such as avian influenza H5N1. The polymerase chain reaction (PCR) has become the method of choice for virus subtype identification. However, designing subtype-specific PCR primer pairs is a very challenging task: on one hand, selected primer pairs must result in robust amplification in the presence of a significant degree of sequence heterogeneity within subtypes, on the other, they must discriminate between the subtype of interest and closely related subtypes. In this article, we present a new tool, called PrimerHunter, that can be used to select highly sensitive and specific primers for virus subtyping. Our tool takes as input sets of both target and nontarget sequences. Primers are selected such that they efficiently amplify any one of the target sequences, and none of the nontarget sequences. PrimerHunter ensures the desired amplification properties by using accurate estimates of melting temperature with mismatches, computed based on the nearest neighbor model via an efficient fractional programming algorithm. Validation experiments with three avian influenza HA subtypes confirm that primers selected by PrimerHunter have high sensitivity and specificity for target sequences

Climate clever clovers: New paradigm to reduce the environmental footprint of ruminants by breeding low methanogenic forages utilizing haplotype variation

© 2017 Kaur, Appels, Bayer, Keeble-Gagnere, Wang, Hirakawa, Shirasawa, Vercoe, Stefanova, Durmic, Nichols, Revell, Isobe, Edwards and Erskine. Mitigating methane production by ruminants is a significant challenge to global livestock production. This research offers a new paradigm to reduce methane emissions from ruminants by breeding climate-clever clovers. We demonstrate wide genetic diversity for the trait methanogenic potential in Australia’s key pasture legume, subterranean clover (Trifolium subterraneum L.). In a bi-parental population the broadsense heritability in methanogenic potential was moderate (H2D 0.4) and allelic variation in a region of Chr 8 accounted for 7.8% of phenotypic variation. In a genome-wide association study we identified four loci controlling methanogenic potential assessed by an in vitro fermentation system. Significantly, the discovery of a single nucleotide polymorphism (SNP) on Chr 5 in a defined haplotype block with an upstream putative candidate gene from a plant peroxidase-like superfamily (TSub_g18548) and a downstream lectin receptor protein kinase (TSub_g18549) provides valuable candidates for an assay for this complex trait. In this way haplotype variation can be tracked to breed pastures with reduced methanogenic potential. Of the quantitative trait loci candidates, the DNA-damage-repair/toleration DRT100-like protein (TSub_g26967), linked to avoid the severity of DNA damage induced by secondary metabolites, is considered central to enteric methane production, as are disease resistance (TSub_g26971, TSub_g26972, and TSub_g18549) and ribonuclease proteins (TSub_g26974, TSub_g26975). These proteins are good pointers to elucidate the genetic basis of in vitro microbial fermentability and enteric methanogenic potential in subterranean clover. The genes identified allow the design of a suite of markers for marker-assisted selection to reduce rumen methane emission in selected pasture legumes. We demonstrate the feasibility of a plant breeding approach without compromising animal productivity to mitigate enteric methane emissions, which is one of the most significant challenges to global livestock production