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Analyses of high-throughput transcriptome sequences of non-model organisms are based on two main approaches: de novo assembly and genome-guided assembly using mapping to assign reads prior to assembly. Given the limits of mapping reads to a reference when it is highly divergent, as is frequently the case for non-model species, we evaluate whether using blastn would outperform mapping methods for read assignment in such situations (>15% divergence). We demonstrate its high performance by using simulated reads of lengths corresponding to those generated by the most common sequencing platforms, and over a realistic range of genetic divergence (0% to 30% divergence). Here we focus on gene identification and not on resolving the whole set of transcripts (i.e. the complete transcriptome). For simulated datasets, the transcriptome-guided assembly based on blastn recovers 94.8% of genes irrespective of read length at 0% divergence; however, assignment rate of reads is negatively correlated with both increasing divergence level and reducing read lengths. Nevertheless, we still observe 92.6% of recovered genes at 30% divergence irrespective of read length. This analysis also produces a categorization of genes relative to their assignment, and suggests guidelines for data processing prior to analyses of comparative transcriptomics and gene expression to minimize potential inferential bias associated with incorrect transcript assignment. We also compare the performances of de novo assembly alone vs in combination with a transcriptome-guided assembly based on blastn both via simulation and empirically, using data from a cyprinid fish species and from an oak species. For any simulated scenario, the transcriptome-guided assembly using blastn outperforms the de novo approach alone, including when the divergence level is beyond the reach of traditional mapping methods. Combining de novo assembly and a related reference transcriptome for read assignment also addresses the bias/error in contigs caused by the dependence on a related reference alone. Empirical data corroborate these findings when assembling transcriptomes from the two non-model organisms: Parachondrostoma toxostoma (fish) and Quercus pubescens (plant). For the fish species, out of the 31,944 genes known from D. rerio, the guided and de novo assemblies recover respectively 20,605 and 20,032 genes but the performance of the guided assembly approach is much higher for both the contiguity and completeness metrics. For the oak, out of the 29,971 genes known from Vitis vinifera, the transcriptome-guided and de novo assemblies display similar performance, but the new guided approach detects 16,326 genes where the de novo assembly only detects 9,385 genes. [ABSTRACT FROM AUTHOR]

Gilles, A., Costedoat, C., Barascud, B., Voisin, A., Banarescu, P., Bianco, P. G., Economidis, P. S., Marić, D. & Chappaz, R. (2010). Speciation pattern of Telestes souffia complex (Teleostei, Cyprinidae) in Europe using morphological and molecular markers.- Zoologica Scripta, 39, 225-242. It is notorious that many species boundaries are erroneously defined. When molecular markers are used, misleading evidence can notably be due to the characteristics inherent to mitochondrial DNA and quantity of markers used but also because of a limited range distribution sampling. European cyprinids biodiversity inventory is still an ongoing task and surprising phylogeographic patterns and phylogenetic relationships are still recovered on account of methodological evolution. This is particularly obvious for the Telestes souffia complex. This species occurs in a fragmented range and species boundaries is greatly debated. In this study, we provide an updated delimitation of the different evolutionary entities constituting this T. souffia complex and propose a taxonomic revision. Morphological and molecular analyses were carried out on 520 specimens coming from 19 localities representing the complete geographical range of this species complex and six related sister species that could potentially interact with it (outgroup 'sensu lato'). Phylogenetic reconstructions and multivariate analyses demonstrated that the T. souffia complex is constituted of at least three species ( T. souffia, Telestes muticellus and Telestes montenigrinus). Data also suggested that T. souffia comprises three subspecies ( T. s. souffia, T. s. agassii; T. s. rysela). We also confirm the splitting of the T. souffia sister species Telestes pleurobipunctatus into two distinct species. The Peloponnesian lineage will be referred as Telestes alfiensis and the continental lineage as T. pleurobipunctatus. Morphological and molecular markers displayed some degree of incongruence within T. souffia suggesting that introgressive hybridization has played a role in the evolution of the Telestes genus. However, discordance among data sets could also result from heterogeneous rate of morphological evolution. Finally, we demonstrated that T. muticellus was implicated in two categories of hybridization: an inter-species hybridization (between T. muticellus and T. souffia) and an inter-generic hybridization (between T. muticellus and Squalius lucumonis), a phenomenon rarely observed for a same species. [ABSTRACT FROM AUTHOR]

Background: Cyprinids display the most abundant and widespread species among the European freshwater Teleostei and are known to hybridize quite commonly. Nevertheless, a limited number of markers for conducting comparative differentiation, evolutionary and hybridization dynamics studies are available to date. Findings: Five multiplex PCR sets were optimized in order to assay 41 cyprinid-specific polymorphic microsatellite loci (including 10 novel loci isolated from Chondrostoma nasus nasus, Chondrostoma toxostoma toxostoma and Leuciscus leuciscus) for 503 individuals (440 purebred specimens and 63 hybrids) from 15 European cyprinid species. The level of genetic diversity was assessed in Alburnus alburnus, Alburnoides bipunctatus, C. genei, C. n. nasus, C. soetta, C. t. toxostoma, L. idus, L. leuciscus, Pachychilon pictum, Rutilus rutilus, Squalius cephalus and Telestes souffia. The applicability of the markers was also tested on Abramis brama, Blicca bjoerkna and Scardinius erythrophtalmus specimens. Overall, between 24 and 37 of these markers revealed polymorphic for the investigated species and 23 markers amplified for all the 15 European cyprinid species. Conclusions: The developed set of markers demonstrated its performance in discriminating European cyprinid species. Furthermore, it allowed detecting and characterizing hybrid individuals. These microsatellites will therefore be useful to perform comparative evolutionary and population genetics studies dealing with European cyprinids, what is of particular interest in conservation issues and constitutes a tool of choice to conduct hybridization studies. [ABSTRACT FROM AUTHOR]