CRISPR/Cas9-mediated mutagenesis of CAROTENOID CLEAVAGE DIOXYGENASE 8 in tomato provides resistance against the parasitic weed Phelipanche aegyptiaca.

Broomrapes (Phelipanche aegyptiaca and Orobanche spp.) are obligate plant parasites that cause extreme damage to crop plants. The parasite seeds have strict requirements for germination, involving preconditioning and exposure to specific chemicals strigolactones [SLs] exuded by the host roots. SLs are plant hormones derived from plant carotenoids via a pathway involving the Carotenoid Cleavage Dioxygenase 8 (CCD8). Having no effective means to control parasitic weeds in most crops, and with CRISPR/Cas9 being an effective gene-editing tool, here we demonstrate that CRISPR/Cas9-mediated mutagenesis of the CCD8 gene can be used to develop host resistance to the parasitic weed P. aegyptiaca. Cas9/single guide (sg) RNA constructs were targeted to the second exon of CCD8 in tomato (Solanum lycopersicum L.) plants. Several CCD8Cas9 mutated tomato lines with variable insertions or deletions in CCD8 were obtained with no identified off-targets. Genotype analysis of T1 plants showed that the introduced CCD8 mutations are inherited. Compared to control tomato plants, the CCD8Cas9 mutant had morphological changes that included dwarfing, excessive shoot branching and adventitious root formation. In addition, SL-deficient CCD8Cas9 mutants showed a significant reduction in parasite infestation compared to non-mutated tomato plants. In the CCD8Cas9 mutated lines, orobanchol (SL) content was significantly reduced but total carotenoids level and expression of genes related to carotenoid biosynthesis were increased, as compared to control plants. Taking into account, the impact of plant parasitic weeds on agriculture and difficulty to constitute efficient control methods, the current study offers insights into the development of a new, efficient method that could be combined with various collections of resistant tomato rootstocks

Evaluating whole transcriptome amplification for gene profiling experiments using RNA-Seq

BACKGROUND: RNA-Seq has enabled high-throughput gene expression profiling to provide insight into the functional link between genotype and phenotype. Low quantities of starting RNA can be a severe hindrance for studies that aim to utilize RNA-Seq. To mitigate this bottleneck, whole transcriptome amplification (WTA) technologies have been developed to generate sufficient sequencing targets from minute amounts of RNA. Successful WTA requires accurate replication of transcript abundance without the loss or distortion of specific mRNAs. Here, we test the efficacy of NuGEN’s Ovation RNA-Seq V2 system, which uses linear isothermal amplification with a unique chimeric primer for amplification, using white adipose tissue from standard laboratory rats (Rattus norvegicus). Our goal was to investigate potential biological artifacts introduced through WTA approaches by establishing comparisons between matched raw and amplified RNA libraries derived from biological replicates. RESULTS: We found that 93% of expressed genes were identical between all unamplified versus matched amplified comparisons, also finding that gene density is similar across all comparisons. Our sequencing experiment and downstream bioinformatic analyses using the Tuxedo analysis pipeline resulted in the assembly of 25,543 high-quality transcripts. Libraries constructed from raw RNA and WTA samples averaged 15,298 and 15,253 expressed genes, respectively. Although significant differentially expressed genes (P < 0.05) were identified in all matched samples, each of these represents less than 0.15% of all shared genes for each comparison. CONCLUSIONS: Transcriptome amplification is efficient at maintaining relative transcript frequencies with no significant bias when using this NuGEN linear isothermal amplification kit under ideal laboratory conditions as presented in this study. This methodology has broad applications, from clinical and diagnostic, to field-based studies when sample acquisition, or sample preservation, methods prove challenging. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12896-015-0155-7) contains supplementary material, which is available to authorized users

Integrating isotopes and documentary evidence : dietary patterns in a late medieval and early modern mining community, Sweden

We would like to thank the Archaeological Research Laboratory, Stockholm University, Sweden and the Tandem Laboratory (Ångström Laboratory), Uppsala University, Sweden, for undertaking the analyses of stable nitrogen and carbon isotopes in both human and animal collagen samples. Also, thanks to Elin Ahlin Sundman for providing the δ13C and δ15N values for animal references from Västerås. This research (Bäckström’s PhD employment at Lund University, Sweden) was supported by the Berit Wallenberg Foundation (BWS 2010.0176) and Jakob and Johan Söderberg’s foundation. The ‘Sala project’ (excavations and analyses) has been funded by Riksens Clenodium, Jernkontoret, Birgit and Gad Rausing’s Foundation, SAU’s Research Foundation, the Royal Physiographic Society of Lund, Berit Wallenbergs Foundation, Åke Wibergs Foundation, Lars Hiertas Memory, Helge Ax:son Johnson’s Foundation and The Royal Swedish Academy of Sciences.Peer reviewedPublisher PD

Expression and trans-specific polymorphism of self-incompatibility RNases in Coffea (Rubiaceae)

Self-incompatibility (SI) is widespread in the angiosperms, but identifying the biochemical components of SI mechanisms has proven to be difficult in most lineages. Coffea (coffee; Rubiaceae) is a genus of old-world tropical understory trees in which the vast majority of diploid species utilize a mechanism of gametophytic self-incompatibility (GSI). The S-RNase GSI system was one of the first SI mechanisms to be biochemically characterized, and likely represents the ancestral Eudicot condition as evidenced by its functional characterization in both asterid (Solanaceae, Plantaginaceae) and rosid (Rosaceae) lineages. The S-RNase GSI mechanism employs the activity of class III RNase T2 proteins to terminate the growth of "self" pollen tubes. Here, we investigate the mechanism of Coffea GSI and specifically examine the potential for homology to S-RNase GSI by sequencing class III RNase T2 genes in populations of 14 African and Madagascan Coffea species and the closely related self-compatible species Psilanthus ebracteolatus. Phylogenetic analyses of these sequences aligned to a diverse sample of plant RNase T2 genes show that the Coffea genome contains at least three class III RNase T2 genes. Patterns of tissue-specific gene expression identify one of these RNase T2 genes as the putative Coffea S-RNase gene. We show that populations of SI Coffea are remarkably polymorphic for putative S-RNase alleles, and exhibit a persistent pattern of trans-specific polymorphism characteristic of all S-RNase genes previously isolated from GSI Eudicot lineages. We thus conclude that Coffea GSI is most likely homologous to the classic Eudicot S-RNase system, which was retained since the divergence of the Rubiaceae lineage from an ancient SI Eudicot ancestor, nearly 90 million years ago.United States National Science Foundation [0849186]; Society of Systematic Biologists; American Society of Plant Taxonomists; Duke University Graduate Schoolinfo:eu-repo/semantics/publishedVersio

Incongruence between genetic and morphological diversity in Microcebus griseorufus of Beza Mahafaly

BACKGROUND: The past decade has seen a remarkable increase in the number of recognized mouse lemur species (genus Microcebus). As recently as 1994, only two species of mouse lemur were recognized according to the rules of zoological nomenclature. That number has now climbed to as many as fifteen proposed species. Indeed, increases in recognized species diversity have also characterized other nocturnal primates – galagos, sportive lemurs, and tarsiers. Presumably, the movement relates more to a previous lack of information than it does to any recent proclivity for taxonomic splitting. Due to their nocturnal habits, one can hypothesize that mouse lemurs will show only minimal variation in pelage coloration as such variation should be inconsequential for the purposes of mate and/or species recognition. Even so, current species descriptions for nocturnal strepsirrhines place a good deal of emphasis on relatively fine distinctions in pelage coloration. RESULTS: Here, we report results from a multi-year study of mouse lemur populations from Beza Mahafaly in southern Madagascar. On the basis of morphological and pelage variation, we initially hypothesized the presence of up to three species of mouse lemurs occurring sympatrically at this locality, one of which appeared to be undescribed. Genetic analysis reveals definitively, however, that all three color morphs belong to a single recognized species, Microcebus griseorufus. Indeed, in some cases, the three color morphs can be characterized by identical mitochondrial haplotypes. CONCLUSION: Given these results, we conclude that investigators should always proceed with caution when using a single data source to identify novel species. A synthetic approach that combines morphological, genetic, geographic, and ecological data is most likely to reveal the true nature of species diversity

Species delimitation in lemurs: multiple genetic loci reveal low levels of species diversity in the genus Cheirogaleus

<p>Abstract</p> <p>Background</p> <p>Species are viewed as the fundamental unit in most subdisciplines of biology. To conservationists this unit represents the currency for global biodiversity assessments. Even though Madagascar belongs to one of the top eight biodiversity hotspots of the world, the taxonomy of its charismatic lemuriform primates is not stable. Within the last 25 years, the number of described lemur species has more than doubled, with many newly described species identified among the nocturnal and small-bodied cheirogaleids. Here, we characterize the diversity of the dwarf lemurs (genus <it>Cheirogaleus</it>) and assess the status of the seven described species, based on phylogenetic and population genetic analysis of mtDNA (<it>cytb </it>+ <it>cox2</it>) and three nuclear markers (<it>adora3</it>, <it>fiba </it>and <it>vWF</it>).</p> <p>Results</p> <p>This study identified three distinct evolutionary lineages within the genus <it>Cheirogaleus</it>. Population genetic cluster analyses revealed a further layer of population divergence with six distinct genotypic clusters.</p> <p>Conclusion</p> <p>Based on the general metapopulation lineage concept and multiple concordant data sets, we identify three exclusive groups of dwarf lemur populations that correspond to three of the seven named species: <it>C. major</it>, <it>C. medius </it>and <it>C. crossleyi</it>. These three species were found to be genealogically exclusive in both mtDNA and nDNA loci and are morphologically distinguishable. The molecular and morphometric data indicate that <it>C. adipicaudatus </it>and <it>C. ravus </it>are synonymous with <it>C. medius </it>and <it>C. major</it>, respectively. <it>Cheirogaleus sibreei </it>falls into the <it>C. medius </it>mtDNA clade, but in morphological analyses the membership is not clearly resolved. We do not have sufficient data to assess the status of <it>C. minusculus</it>. Although additional patterns of population differentiation are evident, there are no clear subdivisions that would warrant additional specific status. We propose that ecological and more geographic data should be collected to confirm these results.</p

Concatenation and Concordance in the Reconstruction of Mouse Lemur Phylogeny: An Empirical Demonstration of the Effect of Allele Sampling in Phylogenetics

The systematics and speciation literature is rich with discussion relating to the potential for gene tree/species tree discordance. Numerous mechanisms have been proposed to generate discordance, including differential selection, longbranch attraction, gene duplication, genetic introgression, and/or incomplete lineage sorting. For speciose clades in which divergence has occurred recently and rapidly, recovering the true species tree can be particularly problematic due to incomplete lineage sorting. Unfortunately, the availability of multilocus or “phylogenomic” data sets does not simply solve the problem, particularly when the data are analyzed with standard concatenation techniques. In our study, we conduct a phylogenetic study for a nearly complete species sample of the dwarf and mouse lemur clade, Cheirogaleidae. Mouse lemurs (genus, Microcebus) have been intensively studied over the past decade for reasons relating to their high level of cryptic species diversity, and although there has been emerging consensus regarding the evolutionary diversity contained within the genus, there is no agreement as to the inter-specific relationships within the group. We attempt to resolve cheirogaleid phylogeny, focusing especially on the mouse lemurs, by employing a large multilocus data set. We compare the results of Bayesian concordance methods with those of standard gene concatenation, finding that though concatenation yields the strongest results as measured by statistical support, these results are found to be highly misleading. By employing an approach where individual alleles are treated as operational taxonomic units, we show that phylogenetic results are substantially influenced by the selection of alleles in the concatenation process. Includes supplementary materials

The utility of PacBio circular consensus sequencing for characterizing complex gene families in non-model organisms

BACKGROUND: Molecular characterization of highly diverse gene families can be time consuming, expensive, and difficult, especially when considering the potential for relatively large numbers of paralogs and/or pseudogenes. Here we investigate the utility of Pacific Biosciences single molecule real-time (SMRT) circular consensus sequencing (CCS) as an alternative to traditional cloning and Sanger sequencing PCR amplicons for gene family characterization. We target vomeronasal gene receptors, one of the most diverse gene families in mammals, with the goal of better understanding intra-specific V1R diversity of the gray mouse lemur (Microcebus murinus). Our study compares intragenomic variation for two V1R subfamilies found in the mouse lemur. Specifically, we compare gene copy variation within and between two individuals of M. murinus as characterized by different methods for nucleotide sequencing. By including the same individual animal from which the M. murinus draft genome was derived, we are able to cross-validate gene copy estimates from Sanger sequencing versus CCS methods. RESULTS: We generated 34,088 high quality circular consensus sequences of two diverse V1R subfamilies (here referred to as V1RI and V1RIX) from two individuals of Microcebus murinus. Using a minimum threshold of 7× coverage, we recovered approximately 90% of V1RI sequences previously identified in the draft M. murinus genome (59% being identical at all nucleotide positions). When low coverage sequences were considered (i.e. < 7× coverage) 100% of V1RI sequences identified in the draft genome were recovered. At least 13 putatively novel V1R loci were also identified using CCS technology. CONCLUSIONS: Recent upgrades to the Pacific Biosciences RS instrument have improved the CCS technology and offer an alternative to traditional sequencing approaches. Our results suggest that the Microcebus murinus V1R repertoire has been underestimated in the draft genome. In addition to providing an improved understanding of V1R diversity in the mouse lemur, this study demonstrates the utility of CCS technology for characterizing complex regions of the genome. We anticipate that long-read sequencing technologies such as PacBio SMRT will allow for the assembly of multigene family clusters and serve to more accurately characterize patterns of gene copy variation in large gene families, thus revealing novel micro-evolutionary patterns within non-model organisms. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-720) contains supplementary material, which is available to authorized users