discomark: nuclear marker discovery from orthologous sequences using draft genome data.

High-throughput sequencing has laid the foundation for fast and cost-effective development of phylogenetic markers. Here we present the program discomark, which streamlines the development of nuclear DNA (nDNA) markers from whole-genome (or whole-transcriptome) sequencing data, combining local alignment, alignment trimming, reference mapping and primer design based on multiple sequence alignments to design primer pairs from input orthologous sequences. To demonstrate the suitability of discomark, we designed markers for two groups of species, one consisting of closely related species and one group of distantly related species. For the closely related members of the species complex of Cloeon dipterum s.l. (Insecta, Ephemeroptera), the program discovered a total of 78 markers. Among these, we selected eight markers for amplification and Sanger sequencing. The exon sequence alignments (2526 base pairs) were used to reconstruct a well-supported phylogeny and to infer clearly structured haplotype networks. For the distantly related species, we designed primers for the insect order Ephemeroptera, using available genomic data from four sequenced species. We developed primer pairs for 23 markers that are designed to amplify across several families. The discomark program will enhance the development of new nDNA markers by providing a streamlined, automated approach to perform genome-scale scans for phylogenetic markers. The program is written in Python, released under a public licence (GNU GPL version 2), and together with a manual and example data set available at: https://github.com/hdetering/discomark.

An analysis of synteny of Arachis with Lotus and Medicago sheds new light on the structure, stability and evolution of legume genomes.

BACKGROUND: Most agriculturally important legumes fall within two sub-clades of the Papilionoid legumes: the Phaseoloids and Galegoids, which diverged about 50 Mya. The Phaseoloids are mostly tropical and include crops such as common bean and soybean. The Galegoids are mostly temperate and include clover, fava bean and the model legumes Lotus and Medicago (both with substantially sequenced genomes). In contrast, peanut (Arachis hypogaea) falls in the Dalbergioid clade which is more basal in its divergence within the Papilionoids. The aim of this work was to integrate the genetic map of Arachis with Lotus and Medicago and improve our understanding of the Arachis genome and legume genomes in general. To do this we placed on the Arachis map, comparative anchor markers defined using a previously described bioinformatics pipeline. Also we investigated the possible role of transposons in the patterns of synteny that were observed. RESULTS: The Arachis genetic map was substantially aligned with Lotus and Medicago with most synteny blocks presenting a single main affinity to each genome. This indicates that the last common whole genome duplication within the Papilionoid legumes predated the divergence of Arachis from the Galegoids and Phaseoloids sufficiently that the common ancestral genome was substantially diploidized. The Arachis and model legume genomes comparison made here, together with a previously published comparison of Lotus and Medicago allowed all possible Arachis-Lotus-Medicago species by species comparisons to be made and genome syntenies observed. Distinct conserved synteny blocks and non-conserved regions were present in all genome comparisons, implying that certain legume genomic regions are consistently more stable during evolution than others. We found that in Medicago and possibly also in Lotus, retrotransposons tend to be more frequent in the variable regions. Furthermore, while these variable regions generally have lower densities of single copy genes than the more conserved regions, some harbor high densities of the fast evolving disease resistance genes. CONCLUSION: We suggest that gene space in Papilionoids may be divided into two broadly defined components: more conserved regions which tend to have low retrotransposon densities and are relatively stable during evolution; and variable regions that tend to have high retrotransposon densities, and whose frequent restructuring may fuel the evolution of some gene families.

Legume anchor markers link syntenic regions between Phaseolus vulgaris, Lotus japonicus, Medicago truncatula and Arachis.

We have previously described a bioinformatics pipeline identifying comparative anchor-tagged sequence (CATS) loci, combined with design of intron-spanning primers. The derived anchor markers defining the linkage position of homologous genes are essential for evaluating genome conservation among related species and facilitate transfer of genetic and genome information between species. Here we validate this global approach in the common bean and in the AA genome complement of the allotetraploid peanut. We present the successful conversion of approximately 50% of the bioinformatics-defined primers into legume anchor markers in bean and diploid Arachis species. One hundred and four new loci representing single-copy genes were added to the existing bean map. These new legume anchor-marker loci enabled the alignment of genetic linkage maps through corresponding genes and provided an estimate of the extent of synteny and collinearity. Extensive macrosynteny between Lotus and bean was uncovered on 8 of the 11 bean chromosomes and large blocks of macrosynteny were also found between bean and Medicago. This suggests that anchor markers can facilitate a better understanding of the genes and genetics of important traits in crops with largely uncharacterized genomes using genetic and genome information from related model plants.

DATFAP: a database of primers and homology alignments for transcription factors from 13 plant species.

BACKGROUND: Many well-known transcription factor databases do not provide PCR primers for the sequences. Often, transcription factors from the same family have a very high sequence similarity, and so specific primer sets that only amplify their targets and none of the other family members may be hard to design manually. Also, it may often be useful to have one general primer set targeting the slightly different homologs of some transcription factor from several species. RESULTS: DATFAP is a free, web-based, very user-friendly browsing tool based on a new database of more than 55,000 EST sequences from 13 plant species, classified as transcription factors. Further, the database offers primers designed for RealTime-PCR as well as homology alignments and phylogenies for the sequences. The provided PCR primers are designed so that they have a perfect sequence alignment to their target only and not to any other sequences in the database from the same species. Via a direct link to a helper tool, the user may also design a general primer set targeting all sequences in any hand-picked group of homologs. A sophisticated search facility enables the user to find exactly the relevant sequences which subsequently may be easily downloaded. All homologies among the more than one billion possible pairwise sequence comparisons of DATFAP have been charted in advance. Thus, the user may quickly display the alignment of any sequence and all its database homologs. CONCLUSION: Because of the comprehensive homology analysis, it is very easy to find related transcription factors from different species, i.e. to navigate the network of inter-related transcription factors from the different species, and to find specific or general primers for them. DATFAP is found at the project homepage.

Automated group assignment in large phylogenetic trees using GRUNT: GRouping, Ungrouping, Naming Tool.

BACKGROUND: Accurate taxonomy is best maintained if species are arranged as hierarchical groups in phylogenetic trees. This is especially important as trees grow larger as a consequence of a rapidly expanding sequence database. Hierarchical group names are typically manually assigned in trees, an approach that becomes unfeasible for very large topologies. RESULTS: We have developed an automated iterative procedure for delineating stable (monophyletic) hierarchical groups to large (or small) trees and naming those groups according to a set of sequentially applied rules. In addition, we have created an associated ungrouping tool for removing existing groups that do not meet user-defined criteria (such as monophyly). The procedure is implemented in a program called GRUNT (GRouping, Ungrouping, Naming Tool) and has been applied to the current release of the Greengenes (Hugenholtz) 16S rRNA gene taxonomy comprising more than 130,000 taxa. CONCLUSION: GRUNT will facilitate researchers requiring comprehensive hierarchical grouping of large tree topologies in, for example, database curation, microarray design and pangenome assignments. The application is available at the greengenes website 1.

Primique: automatic design of specific PCR primers for each sequence in a family.

BACKGROUND: In many contexts, researchers need specific primers for all sequences in a family such that each primer set amplifies only its target sequence and none of the others, e.g. to detect which transcription factor out of a family of very similar proteins that is present in a sample, or to design diagnostic assays for the identification of pathogen strains. RESULTS: This paper presents primique, a new graphical, user-friendly, fast, web-based tool which solves the problem: It designs specific primers for each sequence in an uploaded set. Further, a secondary set of sequences not to be amplified by any primer pair may be uploaded. Primers with high sequence similarity to non-target sequences are selected against. Lastly, the suggested primers may be checked against the National Center for Biotechnology Information databases for possible mis-priming. CONCLUSION: Results are presented in interactive tables, and various primer properties are listed and displayed graphically. Any close match alignments can be displayed. Given 30 sequences, the running time of primique is about 20 seconds.primique can be reached via this web address: http://cgi-www.daimi.au.dk/cgi-chili/primique/front.py.

A general pipeline for the development of anchor markers for comparative genomics in plants.

BACKGROUND: Complete or near-complete genomic sequence information is presently only available for a few plant species representing a large phylogenetic diversity among plants. In order to effectively transfer this information to species lacking sequence information, comparative genomic tools need to be developed. Molecular markers permitting cross-species mapping along co-linear genomic regions are central to comparative genomics. These "anchor" markers, defining unique loci in genetic linkage maps of multiple species, are gene-based and possess a number of features that make them relatively sparse. To identify potential anchor marker sequences more efficiently, we have established an automated bioinformatic pipeline that combines multi-species Expressed Sequence Tags (EST) and genome sequence data. RESULTS: Taking advantage of sequence data from related species, the pipeline identifies evolutionarily conserved sequences that are likely to define unique orthologous loci in most species of the same phylogenetic clade. The key features are the identification of evolutionarily conserved sequences followed by automated design of intron-flanking Polymerase Chain Reaction (PCR) primer pairs. Polymorphisms can subsequently be identified by size- or sequence variation of PCR products, amplified from mapping parents or populations. We illustrate our procedure in legumes and grasses and exemplify its application in legumes, where model plant studies and the genome- and EST-sequence data available have a potential impact on the breeding of crop species and on our understanding of the evolution of this large and diverse family. CONCLUSION: We provide a database of 459 candidate anchor loci which have the potential to serve as map anchors in more than 18,000 legume species, a number of which are of agricultural importance. For grasses, the database contains 1335 candidate anchor loci. Based on this database, we have evaluated 76 candidate anchor loci with respect to marker development in legume species with no sequence information available, demonstrating the validity of this approach.

GeMprospector--online design of cross-species genetic marker candidates in legumes and grasses.

The web program GeMprospector (URL: http://cgi-www.daimi.au.dk/cgi-chili/GeMprospector/main) allows users to automatically design large sets of cross-species genetic marker candidates targeting either legumes or grasses. The user uploads a collection of ESTs from one or more legume or grass species, and they are compared with a database of clusters of homologous EST and genomic sequences from other legumes or grasses, respectively. Multiple sequence alignments between submitted ESTs and their homologues in the appropriate database form the basis of automated PCR primer design in conserved exons such that each primer set amplifies an intron. The only user input is a collection of ESTs, not necessarily from more than one species, and GeMprospector can boost the potential of such an EST collection by combining it with a large database to produce cross-species genetic marker candidates for legumes or grasses.

PHY.FI: fast and easy online creation and manipulation of phylogeny color figures.

BACKGROUND: The need to depict a phylogeny, or some other kind of abstract tree, is very frequently experienced by researchers from a broad range of biological and computational disciplines. Thousands of papers and talks include phylogeny figures, and often during everyday work, one would like to quickly get a graphical display of, e.g., the phylogenetic relationship between a set of sequences as calculated by an alignment program such as ClustalW or the phylogenetic package Phylip. A wealth of software tools capable of tree drawing exists; most are comprehensive packages that also perform various types of analysis, and hence they are available only for download and installing. Some online tools exist, too. RESULTS: This paper presents an online tool, PHY.FI, which encompasses all the qualities of existing online programs and adds functionality to hopefully eliminate the need for post-processing the phylogeny figure in some other general-purpose graphics program. PHY.FI is versatile, easy-to-use and fast, and supports comprehensive graphical control, several download image formats, and the possibility of dynamically collapsing groups of nodes into named subtrees (e.g. "Primates"). The user can create a color figure from any phylogeny, or other kind of tree, represented in the widely used parenthesized Newick format. CONCLUSION: PHY.FI is fast and easy to use, yet still offers full color control, tree manipulation, and several image formats. It does not require any downloading and installing, and thus any internet user regardless of computer skills, and computer platform, can benefit from it. PHY.FI is free for all and is available from this web address: http://cgi-www.daimi.au.dk/cgi-chili/phyfi/go.

PriFi: using a multiple alignment of related sequences to find primers for amplification of homologs.

Using a comparative approach, the web program PriFi (http://cgi-www.daimi.au.dk/cgi-chili/PriFi/main) designs pairs of primers useful for PCR amplification of genomic DNA in species where prior sequence information is not available. The program works with an alignment of DNA sequences from phylogenetically related species and outputs a list of possibly degenerate primer pairs fulfilling a number of criteria, such that the primers have a maximal probability of amplifying orthologous sequences in other phylogenetically related species. Operating on a genome-wide scale, PriFi automates the first steps of a procedure for developing general markers serving as common anchor loci across species. To accommodate users with special preferences, configuration settings and criteria can be customized.