Target DNA enrichment combined with high-throughput sequencing technologies is a powerful approach to probing a large number of loci in genomes of interest. However, software algorithms that explicitly consider nucleotide sequence information of target loci in multiple reference species for optimizing design of target enrichment baits to be applicable across a wide range of species have not been developed. Here we present an algorithm that infers target DNA enrichment baits from multiple nucleotide sequence alignments. By applying clustering methods and the combinatorial 1-center sequence optimization to bait design, we are able to minimize the total number of baits required to efficiently probe target loci in multiple species. Consequently, more loci can be probed across species with a given number of baits. Using transcript sequences of 24 apoid wasps (Hymenoptera: Crabronidae, Sphecidae) from the 1KITE project and the gene models of Nasonia vitripennis, we inferred 57,650, 120-bp-long baits for capturing 378 coding sequence sections of 282 genes in apoid wasps. Illumina reduced-representation library sequencing confirmed successful enrichment of the target DNA when applying these baits to DNA of various apoid wasps. The designed baits furthermore enriched a major fraction of the target DNA in distantly related Hymenoptera, such as Formicidae and Chalcidoidea, highlighting the baits' broad taxonomic applicability. The availability of baits with broad taxonomic applicability is of major interest in numerous disciplines, ranging from phylogenetics to biodiversity monitoring. We implemented our new approach in a software package, called BaitFisher, which is open source and freely available at https://github.com/cmayer/BaitFisher-package.git.

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Freshwater sponges include six extant families which belong to the suborder Spongillina (Porifera). The taxonomy of freshwater sponges is problematic and their phylogeny and evolution are not well understood. Sequences of the ribosomal internal transcribed spacers (ITS1 and ITS2) of 11 species from the family Lubomirskiidae, 13 species from the family Spongillidae, and 1 species from the family Potamolepidae were obtained to study the phylogenetic relationships between endemic and cosmopolitan freshwater sponges and the evolution of sponges in Lake Baikal. The present study is the first one where ITS1 sequences were successfully aligned using verified secondary structure models and, in combination with ITS2, used to infer relationships between the freshwater sponges. Phylogenetic trees inferred using maximum likelihood, neighbor-joining, and parsimony methods and Bayesian inference revealed that the endemic family Lubomirskiidae was monophyletic. Our results do not support the monophyly of Spongillidae because Lubomirskiidae formed a robust clade with E. muelleri, and Trochospongilla latouchiana formed a robust clade with the outgroup Echinospongilla brichardi (Potamolepidae). Within the cosmopolitan family Spongillidae the genera Radiospongilla and Eunapius were found to be monophyletic, while Ephydatia muelleri was basal to the family Lubomirskiidae. The genetic distances between Lubomirskiidae species being much lower than those between Spongillidae species are indicative of their relatively recent radiation from a common ancestor. These results indicated that rDNA spacers sequences can be useful in the study of phylogenetic relationships of and the identification of species of freshwater sponges.

SummaryThe recent identification of phage-type RNA polymerases encoded in the nuclear genome of higher plants has provided circumstantial evidence for functioning of these polymerases in the transcription of the mitochondrial and plastid genomes, as demonstrated by sequence analysis and in vitro import experiments. To determine the subcellular localization of the phage-type organellar RNA polymerases in planta, the putative transit peptides of the RNA polymerases RpoT;1 and RpoT;3 from Arabidopsis thaliana and RpoT from Chenopodium album were fused to the coding sequence of a green fluorescent protein (GFP). The constructs were used to stably transform A. thaliana. Transgenic plants were examined for green fluorescence with epifluorescence and confocal laser scanning microscopy. Plants expressing the GFP fusions under control of the CaMV35S promoter exhibited a distinct subcellular localization of the GFP fluorescence for each of the fusion constructs. In plants expressing GFP fusions with the putative transit peptides of ARAth;RpoT;1 and CHEal;RpoT, fluorescence was found exclusively in mitochondria, both in root and leaf cells. In contrast, GFP fluorescence in plants expressing the ARAth;RpoT;3-GFP construct accumulated in chloroplasts of leaf cells and nongreen plastids (leucoplasts) of root cells. By demonstrating targeting in planta, the data add substantial evidence for the phage-type RNA polymerases from C. album and A. thaliana to function in the transcriptional machinery of mitochondria and plastids.