Pasi Rastas

DNA-binding specificities of human transcription factors.

Although the proteins that read the gene regulatory code, transcription factors (TFs), have been largely identified, it is not well known which sequences TFs can recognize. We have analyzed the sequence-specific binding of human TFs using high-throughput SELEX and ChIP sequencing. A total of 830 binding profiles were obtained, describing 239 distinctly different binding specificities. The models represent the majority of human TFs, approximately doubling the coverage compared to existing systematic studies. Our results reveal additional specificity determinants for a large number of factors for which a partial specificity was known, including a commonly observed A- or T-rich stretch that flanks the core motifs. Global analysis of the data revealed that homodimer orientation and spacing preferences, and base-stacking interactions, have a larger role in TF-DNA binding than previously appreciated. We further describe a binding model incorporating these features that is required to understand binding of TFs to DNA.

The Glanville fritillary genome retains an ancient karyotype and reveals selective chromosomal fusions in Lepidoptera

Previous studies have reported that chromosome synteny in Lepidoptera has been well conserved, yet the number of haploid chromosomes varies widely from 5 to 223. Here we report the genome (393 Mb) of the Glanville fritillary butterfly (Melitaea cinxia; Nymphalidae), a widely recognized model species in metapopulation biology and eco-evolutionary research, which has the putative ancestral karyotype of n=31. Using a phylogenetic analyses of Nymphalidae and of other Lepidoptera, combined with orthologue-level comparisons of chromosomes, we conclude that the ancestral lepidopteran karyotype has been n=31 for at least 140 My. We show that fusion chromosomes have retained the ancestral chromosome segments and very few rearrangements have occurred across the fusion sites. The same, shortest ancestral chromosomes have independently participated in fusion events in species with smaller karyotypes. The short chromosomes have higher rearrangement rate than long ones. These characteristics highlight distinctive features of the evolutionary dynamics of butterflies and moths.

MOODS: fast search for position weight matrix matches in DNA sequences

Summary: MOODS (MOtif Occurrence Detection Suite) is a software package for matching position weight matrices against DNA sequences. MOODS implements state-of-the-art online matching algorithms, achieving considerably faster scanning speed than with a simple brute-force search. MOODS is written in C++, with bindings for the popular BioPerl and Biopython toolkits. It can easily be adapted for different purposes and integrated into existing workflows. It can also be used as a C++ library. Availability: The package with documentation and examples of usage is available at http://www.cs.helsinki.fi/group/pssmfind. The source code is also available under the terms of a GNU General Public License (GPL). Contact: janne.h.korhonen@helsinki.fi

Lep-MAP: fast and accurate linkage map construction for large SNP datasets

MOTIVATION Current high-throughput sequencing technologies allow cost-efficient genotyping of millions of single nucleotide polymorphisms (SNPs) for hundreds of samples. However, the tools that are currently available for constructing linkage maps are not well suited for large datasets. Linkage maps of large datasets would be helpful in de novo genome assembly by facilitating comprehensive genome validation and refinement by enabling chimeric scaffold detection, as well as in family-based linkage and association studies, quantitative trait locus mapping, analysis of genome synteny and other complex genomic data analyses. RESULTS We describe a novel tool, called Lepidoptera-MAP (Lep-MAP), for constructing accurate linkage maps with ultradense genome-wide SNP data. Lep-MAP is fast and memory efficient and largely automated, requiring minimal user interaction. It uses simultaneously data on multiple outbred families and can increase linkage map accuracy by taking into account achiasmatic meiosis, a special feature of Lepidoptera and some other taxa with no recombination in one sex (no recombination in females in Lepidoptera). We demonstrate that Lep-MAP outperforms other methods on real and simulated data. We construct a genome-wide linkage map of the Glanville fritillary butterfly (Melitaea cinxia) with over 40 000 SNPs. The data were generated with a novel in-house SOLiD restriction site-associated DNA tag sequencing protocol, which is described in the online supplementary material. AVAILABILITY AND IMPLEMENTATION Java source code under GNU general public license with the compiled classes and the datasets are available from http://sourceforge.net/users/lep-map.

Genome-wide SNP identification for the construction of a high-resolution genetic map of Japanese flounder (Paralichthys olivaceus): applications to QTL mapping of Vibrio anguillarum disease resistance and comparative genomic analysis

High-resolution genetic maps are essential for fine mapping of complex traits, genome assembly, and comparative genomic analysis. Single-nucleotide polymorphisms (SNPs) are the primary molecular markers used for genetic map construction. In this study, we identified 13,362 SNPs evenly distributed across the Japanese flounder (Paralichthys olivaceus) genome. Of these SNPs, 12,712 high-confidence SNPs were subjected to high-throughput genotyping and assigned to 24 consensus linkage groups (LGs). The total length of the genetic linkage map was 3,497.29 cM with an average distance of 0.47 cM between loci, thereby representing the densest genetic map currently reported for Japanese flounder. Nine positive quantitative trait loci (QTLs) forming two main clusters for Vibrio anguillarum disease resistance were detected. All QTLs could explain 5.1–8.38% of the total phenotypic variation. Synteny analysis of the QTL regions on the genome assembly revealed 12 immune-related genes, among them 4 genes strongly associated with V. anguillarum disease resistance. In addition, 246 genome assembly scaffolds with an average size of 21.79 Mb were anchored onto the LGs; these scaffolds, comprising 522.99 Mb, represented 95.78% of assembled genomic sequences. The mapped assembly scaffolds in Japanese flounder were used for genome synteny analyses against zebrafish (Danio rerio) and medaka (Oryzias latipes). Flounder and medaka were found to possess almost one-to-one synteny, whereas flounder and zebrafish exhibited a multi-syntenic correspondence. The newly developed high-resolution genetic map, which will facilitate QTL mapping, scaffold assembly, and genome synteny analysis of Japanese flounder, marks a milestone in the ongoing genome project for this species.

High genetic load in an old isolated butterfly population

We investigated inbreeding depression and genetic load in a small (Ne ∼ 100) population of the Glanville fritillary butterfly (Melitaea cinxia), which has been completely isolated on a small island [Pikku Tytärsaari (PT)] in the Baltic Sea for at least 75 y. As a reference, we studied conspecific populations from the well-studied metapopulation in the Åland Islands (ÅL), 400 km away. A large population in Saaremaa, Estonia, was used as a reference for estimating genetic diversity and Ne. We investigated 58 traits related to behavior, development, morphology, reproductive performance, and metabolism. The PT population exhibited high genetic load (L = 1 − WPT/WÅL) in a range of fitness-related traits including adult weight (L = 0.12), flight metabolic rate (L = 0.53), egg viability (L = 0.37), and lifetime production of eggs in an outdoor population cage (L = 0.70). These results imply extensive fixation of deleterious recessive mutations, supported by greatly reduced diversity in microsatellite markers and immediate recovery (heterosis) of egg viability and flight metabolic rate in crosses with other populations. There was no significant inbreeding depression in most traits due to one generation of full-sib mating. Resting metabolic rate was significantly elevated in PT males, which may be related to their short lifespan (L = 0.25). The demographic history and the effective size of the PT population place it in the part of the parameter space in which models predict mutation accumulation. This population exemplifies the increasingly common situation in fragmented landscapes, in which small and completely isolated populations are vulnerable to extinction due to high genetic load.

Construction of Ultradense Linkage Maps with Lep-MAP2: Stickleback F2 Recombinant Crosses as an Example

High-density linkage maps are important tools for genome biology and evolutionary genetics by quantifying the extent of recombination, linkage disequilibrium, and chromosomal rearrangements across chromosomes, sexes, and populations. They provide one of the best ways to validate and refine de novo genome assemblies, with the power to identify errors in assemblies increasing with marker density. However, assembly of high-density linkage maps is still challenging due to software limitations. We describe Lep-MAP2, a software for ultradense genome-wide linkage map construction. Lep-MAP2 can handle various family structures and can account for achiasmatic meiosis to gain linkage map accuracy. Simulations show that Lep-MAP2 outperforms other available mapping software both in computational efficiency and accuracy. When applied to two large F2-generation recombinant crosses between two nine-spined stickleback (Pungitius pungitius) populations, it produced two high-density (∼6 markers/cM) linkage maps containing 18,691 and 20,054 single nucleotide polymorphisms. The two maps showed a high degree of synteny, but female maps were 1.5–2 times longer than male maps in all linkage groups, suggesting genome-wide recombination suppression in males. Comparison with the genome sequence of the three-spined stickleback (Gasterosteus aculeatus) revealed a high degree of interspecific synteny with a low frequency (<5%) of interchromosomal rearrangements. However, a fairly large (ca. 10 Mb) translocation from autosome to sex chromosome was detected in both maps. These results illustrate the utility and novel features of Lep-MAP2 in assembling high-density linkage maps, and their usefulness in revealing evolutionarily interesting properties of genomes, such as strong genome-wide sex bias in recombination rates.

A hidden markov technique for haplotype reconstruction

We give a new algorithm for the genotype phasing problem. Our solution is based on a hidden Markov model for haplotypes. The model has a uniform structure, unlike most solutions proposed so far that model recombinations using haplotype blocks. In our model, the haplotypes can be seen as a result of iterated recombinations applied on a few founder haplotypes. We find maximum likelihood model of this type by using the EM algorithm. We show how to solve the subtleties of the EM algorithm that arise when genotypes are generated using a haplotype model. We compare our method to the well-known currently available algorithms (phase, hap, gerbil) using some standard and new datasets. Our algorithm is relatively fast and gives results that are always best or second best among the methods compared.

Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.

Flight‐induced changes in gene expression in the Glanville fritillary butterfly

Insect flight is one of the most energetically demanding activities in the animal kingdom, yet for many insects flight is necessary for reproduction and foraging. Moreover, dispersal by flight is essential for the viability of species living in fragmented landscapes. Here, working on the Glanville fritillary butterfly (Melitaea cinxia), we use transcriptome sequencing to investigate gene expression changes caused by 15 min of flight in two contrasting populations and the two sexes. Male butterflies and individuals from a large metapopulation had significantly higher peak flight metabolic rate (FMR) than female butterflies and those from a small inbred population. In the pooled data, FMR was significantly positively correlated with genome‐wide heterozygosity, a surrogate of individual inbreeding. The flight experiment changed the expression level of 1513 genes, including genes related to major energy metabolism pathways, ribosome biogenesis and RNA processing, and stress and immune responses. Males and butterflies from the population with high FMR had higher basal expression of genes related to energy metabolism, whereas females and butterflies from the small population with low FMR had higher expression of genes related to ribosome/RNA processing and immune response. Following the flight treatment, genes related to energy metabolism were generally down‐regulated, while genes related to ribosome/RNA processing and immune response were up‐regulated. These results suggest that common molecular mechanisms respond to flight and can influence differences in flight metabolic capacity between populations and sexes.