Fingerprint Contigs Research Articles

Development of a Sequence-Based Reference Physical Map of Pea (Pisum sativum L.).

Whole genome profiling (WGP) is a sequence-based physical mapping technology and uses sequence tags generated by next generation sequencing for construction of bacterial artificial chromosome (BAC) contigs of complex genomes. The physical map provides a framework for assembly of genome sequence and information for localization of genes that are difficult to find through positional cloning. To address the challenges of accurate assembly of the pea genome (∼4.2 GB of which approximately 85% is repetitive sequences), we have adopted the WGP technology for assembly of a pea BAC library. Multi-dimensional pooling of 295,680 BAC clones and sequencing the ends of restriction fragments of pooled DNA generated 1,814 million high quality reads, of which 825 million were deconvolutable to 1.11 million unique WGP sequence tags. These WGP tags were used to assemble 220,013 BACs into contigs. Assembly of the BAC clones using the modified Fingerprinted Contigs (FPC) program has resulted in 13,040 contigs, consisting of 213,719 BACs, and 6,294 singleton BACs. The average contig size is 0.33 Mbp and the N50 contig size is 0.62 Mbp. WGPTM technology has proved to provide a robust physical map of the pea genome, which would have been difficult to assemble using traditional restriction digestion based methods. This sequence-based physical map will be useful to assemble the genome sequence of pea. Additionally, the 1.1 million WGP tags will support efficient assignment of sequence scaffolds to the BAC clones, and thus an efficient sequencing of BAC pools with targeted genome regions of interest.

Integrating a newly developed BAC-based physical mapping resource for Lolium perenne with a genome-wide association study across a L. perenne European ecotype collection identifies genomic contexts associated with agriculturally important traits.

Background and Aims Lolium perenne (perennial ryegrass) is the most widely cultivated forage and amenity grass species in temperate areas worldwide and there is a need to understand the genetic architectures of key agricultural traits and crop characteristics that deliver wider environmental services. Our aim was to identify genomic regions associated with agriculturally important traits by integrating a bacterial artificial chromosome (BAC)-based physical map with a genome-wide association study (GWAS).MethodsBAC-based physical maps for L. perenne were constructed from ~212 000 high-information-content fingerprints using Fingerprint Contig and Linear Topology Contig software. BAC clones were associated with both BAC-end sequences and a partial minimum tiling path sequence. A panel of 716 L. perenne diploid genotypes from 90 European accessions was assessed in the field over 2 years, and genotyped using a Lolium Infinium SNP array. The GWAS was carried out using a linear mixed model implemented in TASSEL, and extended genomic regions associated with significant markers were identified through integration with the physical map.Key ResultsBetween ~3600 and 7500 physical map contigs were derived, depending on the software and probability thresholds used, and integrated with ~35 k sequenced BAC clones to develop a resource predicted to span the majority of the L. perenne genome. From the GWAS, eight different loci were significantly associated with heading date, plant width, plant biomass and water-soluble carbohydrate accumulation, seven of which could be associated with physical map contigs. This allowed the identification of a number of candidate genes.ConclusionsCombining the physical mapping resource with the GWAS has allowed us to extend the search for candidate genes across larger regions of the L. perenne genome and identified a number of interesting gene model annotations. These physical maps will aid in validating future sequence-based assemblies of the L. perenne genome.

Physical Mapping of Bread Wheat Chromosome 5A: An Integrated Approach.

The huge size, redundancy, and highly repetitive nature of the bread wheat [Triticum aestivum (L.)] genome, makes it among the most difficult species to be sequenced. To overcome these limitations, a strategy based on the separation of individual chromosomes or chromosome arms and the subsequent production of physical maps was established within the frame of the International Wheat Genome Sequence Consortium (IWGSC). A total of 95,812 bacterial artificial chromosome (BAC) clones of short-arm chromosome 5A (5AS) and long-arm chromosome 5A (5AL) arm-specific BAC libraries were fingerprinted and assembled into contigs by complementary analytical approaches based on the FingerPrinted Contig (FPC) and Linear Topological Contig (LTC) tools. Combined anchoring approaches based on polymerase chain reaction (PCR) marker screening, microarray, and sequence homology searches applied to several genomic tools (i.e., genetic maps, deletion bin map, neighbor maps, BAC end sequences (BESs), genome zipper, and chromosome survey sequences) allowed the development of a high-quality physical map with an anchored physical coverage of 75% for 5AS and 53% for 5AL with high portions (64 and 48%, respectively) of contigs ordered along the chromosome. In the genome of grasses, Brachypodium [Brachypodium distachyon (L.) Beauv.], rice (Oryza sativa L.), and sorghum [Sorghum bicolor (L.) Moench] homologs of genes on wheat chromosome 5A were separated into syntenic blocks on different chromosomes as a result of translocations and inversions during evolution. The physical map presented represents an essential resource for fine genetic mapping and map-based cloning of agronomically relevant traits and a reference for the 5A sequencing projects.

Assessing the Barley Genome Zipper and Genomic Resources for Breeding Purposes.

The aim of this study was to estimate the accuracy and convergence of newly developed barley (Hordeum vulgare L.) genomic resources, primarily genome zipper (GZ) and population sequencing (POPSEQ), at the genome-wide level and to assess their usefulness in applied barley breeding by analyzing seven known loci. Comparison of barley GZ and POPSEQ maps to a newly developed consensus genetic map constructed with data from 13 individual linkage maps yielded an accuracy of 97.8% (GZ) and 99.3% (POPSEQ), respectively, regarding the chromosome assignment. The percentage of agreement in marker position indicates that on average only 3.7% GZ and 0.7% POPSEQ positions are not in accordance with their centimorgan coordinates in the consensus map. The fine-scale comparison involved seven genetic regions on chromosomes 1H, 2H, 4H, 6H, and 7H, harboring major genes and quantitative trait loci (QTL) for disease resistance. In total, 179 GZ loci were analyzed and 64 polymorphic markers were developed. Entirely, 89.1% of these were allocated within the targeted intervals and 84.2% followed the predicted order. Forty-four markers showed a match to a POPSEQ-anchored contig, the percentage of collinearity being 93.2%, on average. Forty-four markers allowed the identification of twenty-five fingerprinted contigs (FPCs) and a more clear delimitation of the physical regions containing the traits of interest. Our results demonstrate that an increase in marker density of barley maps by using new genomic data significantly improves the accuracy of GZ. In addition, the combination of different barley genomic resources can be considered as a powerful tool to accelerate barley breeding.

Integrated physical, genetic and genome map of chickpea (Cicer arietinum L.).

Physical map of chickpea was developed for the reference chickpea genotype (ICC 4958) using bacterial artificial chromosome (BAC) libraries targeting 71,094 clones (~12× coverage). High information content fingerprinting (HICF) of these clones gave high-quality fingerprinting data for 67,483 clones, and 1,174 contigs comprising 46,112 clones and 3,256 singletons were defined. In brief, 574 Mb genome size was assembled in 1,174 contigs with an average of 0.49 Mb per contig and 3,256 singletons represent 407 Mb genome. The physical map was linked with two genetic maps with the help of 245 BAC-end sequence (BES)-derived simple sequence repeat (SSR) markers. This allowed locating some of the BACs in the vicinity of some important quantitative trait loci (QTLs) for drought tolerance and reistance to Fusarium wilt and Ascochyta blight. In addition, fingerprinted contig (FPC) assembly was also integrated with the draft genome sequence of chickpea. As a result, ~965 BACs including 163 minimum tilling path (MTP) clones could be mapped on eight pseudo-molecules of chickpea forming 491 hypothetical contigs representing 54,013,992 bp (~54 Mb) of the draft genome. Comprehensive analysis of markers in abiotic and biotic stress tolerance QTL regions led to identification of 654, 306 and 23 genes in drought tolerance “QTL-hotspot” region, Ascochyta blight resistance QTL region and Fusarium wilt resistance QTL region, respectively. Integrated physical, genetic and genome map should provide a foundation for cloning and isolation of QTLs/genes for molecular dissection of traits as well as markers for molecular breeding for chickpea improvement.Electronic supplementary materialThe online version of this article (doi:10.1007/s10142-014-0363-6) contains supplementary material, which is available to authorized users.

Integration of the Draft Sequence and Physical Map as a Framework for Genomic Research in Soybean (Glycine max (L.) Merr.) and Wild Soybean (Glycine soja Sieb. and Zucc.)

Soybean is a model for the legume research community because of its importance as a crop, densely populated genetic maps, and the availability of a genome sequence. Even though a whole-genome shotgun sequence and bacterial artificial chromosome (BAC) libraries are available, a high-resolution, chromosome-based physical map linked to the sequence assemblies is still needed for whole-genome alignments and to facilitate map-based gene cloning. Three independent G. max BAC libraries combined with genetic and gene-based markers were used to construct a minimum tiling path (MTP) of BAC clones. A total of 107,214 clones were assembled into 1355 FPC (FingerPrinted Contigs) contigs, incorporating 4628 markers and aligned to the G. max reference genome sequence using BAC end-sequence information. Four different MTPs were made for G. max that covered from 92.6% to 95.0% of the soybean draft genome sequence (gmax1.01). Because our purpose was to pick the most reliable and complete MTP, and not the MTP with the minimal number of clones, the FPC map and draft sequence were integrated and clones with unpaired BES were added to build a high-quality physical map with the fewest gaps possible (http://soybase.org). A physical map was also constructed for the undomesticated ancestor (G. soja) of soybean to explore genome variation between G. max and G. soja. 66,028 G. soja clones were assembled into 1053 FPC contigs covering approximately 547 Mbp of the G. max genome sequence. These physical maps for G. max and its undomesticated ancestor, G. soja, will serve as a framework for ordering sequence fragments, comparative genomics, cloning genes, and evolutionary analyses of legume genomes.

A high-resolution comparative map of porcine chromosome 4 (SSC4)

We used the IMNpRH2(12,000-rad) RH and IMpRH(7,000-rad) panels to integrate 2019 transcriptome (RNA-seq)-generated contigs with markers from the porcine genetic and radiation hybrid (RH) maps and bacterial artificial chromosome finger-printed contigs, into 1) parallel framework maps (LOD ≥ 10) on both panels for swine chromosome (SSC) 4, and 2) a high-resolution comparative map of SSC4, thus and human chromosomes (HSA) 1 and 8. A total of 573 loci were anchored and ordered on SSC4 closing gaps identified in the porcine sequence assembly Sscrofa9. Alignment of the SSC4 RH with the genetic map identified five microsatellites incorrectly mapped around the centromeric region in the genetic map. Further alignment of the RH and comparative maps with the genome sequence identified four additional regions of discrepancy that are also suggestive of errors in assembly, three of which were resolved through conserved synteny with blocks on HSA1 and HSA8.

LTC: a novel algorithm to improve the efficiency of contig assembly for physical mapping in complex genomes

BackgroundPhysical maps are the substrate of genome sequencing and map-based cloning and their construction relies on the accurate assembly of BAC clones into large contigs that are then anchored to genetic maps with molecular markers. High Information Content Fingerprinting has become the method of choice for large and repetitive genomes such as those of maize, barley, and wheat. However, the high level of repeated DNA present in these genomes requires the application of very stringent criteria to ensure a reliable assembly with the FingerPrinted Contig (FPC) software, which often results in short contig lengths (of 3-5 clones before merging) as well as an unreliable assembly in some difficult regions. Difficulties can originate from a non-linear topological structure of clone overlaps, low power of clone ordering algorithms, and the absence of tools to identify sources of gaps in Minimal Tiling Paths (MTPs).ResultsTo address these problems, we propose a novel approach that: (i) reduces the rate of false connections and Q-clones by using a new cutoff calculation method; (ii) obtains reliable clusters robust to the exclusion of single clone or clone overlap; (iii) explores the topological contig structure by considering contigs as networks of clones connected by significant overlaps; (iv) performs iterative clone clustering combined with ordering and order verification using re-sampling methods; and (v) uses global optimization methods for clone ordering and Band Map construction. The elements of this new analytical framework called Linear Topological Contig (LTC) were applied on datasets used previously for the construction of the physical map of wheat chromosome 3B with FPC. The performance of LTC vs. FPC was compared also on the simulated BAC libraries based on the known genome sequences for chromosome 1 of rice and chromosome 1 of maize.ConclusionsThe results show that compared to other methods, LTC enables the construction of highly reliable and longer contigs (5-12 clones before merging), the detection of "weak" connections in contigs and their "repair", and the elongation of contigs obtained by other assembly methods.

Comparative Genomics Identifies Candidate Genes for Infectious Salmon Anemia (ISA) Resistance in Atlantic Salmon (Salmo salar)

Infectious salmon anemia (ISA) has been described as the hoof and mouth disease of salmon farming. ISA is caused by a lethal and highly communicable virus, which can have a major impact on salmon aquaculture, as demonstrated by an outbreak in Chile in 2007. A quantitative trait locus (QTL) for ISA resistance has been mapped to three microsatellite markers on linkage group (LG) 8 (Chr 15) on the Atlantic salmon genetic map. We identified bacterial artificial chromosome (BAC) clones and three fingerprint contigs from the Atlantic salmon physical map that contains these markers. We made use of the extensive BAC end sequence database to extend these contigs by chromosome walking and identified additional two markers in this region. The BAC end sequences were used to search for conserved synteny between this segment of LG8 and the fish genomes that have been sequenced. An examination of the genes in the syntenic segments of the tetraodon and medaka genomes identified candidates for association with ISA resistance in Atlantic salmon based on differential expression profiles from ISA challenges or on the putative biological functions of the proteins they encode. One gene in particular, HIV-EP2/MBP-2, caught our attention as it may influence the expression of several genes that have been implicated in the response to infection by infectious salmon anemia virus (ISAV). Therefore, we suggest that HIV-EP2/MBP-2 is a very strong candidate for the gene associated with the ISAV resistance QTL in Atlantic salmon and is worthy of further study.Electronic supplementary materialThe online version of this article (doi:10.1007/s10126-010-9284-0) contains supplementary material, which is available to authorized users.

A Single Molecule Scaffold for the Maize Genome

About 85% of the maize genome consists of highly repetitive sequences that are interspersed by low-copy, gene-coding sequences. The maize community has dealt with this genomic complexity by the construction of an integrated genetic and physical map (iMap), but this resource alone was not sufficient for ensuring the quality of the current sequence build. For this purpose, we constructed a genome-wide, high-resolution optical map of the maize inbred line B73 genome containing >91,000 restriction sites (averaging 1 site/∼23 kb) accrued from mapping genomic DNA molecules. Our optical map comprises 66 contigs, averaging 31.88 Mb in size and spanning 91.5% (2,103.93 Mb/∼2,300 Mb) of the maize genome. A new algorithm was created that considered both optical map and unfinished BAC sequence data for placing 60/66 (2,032.42 Mb) optical map contigs onto the maize iMap. The alignment of optical maps against numerous data sources yielded comprehensive results that proved revealing and productive. For example, gaps were uncovered and characterized within the iMap, the FPC (fingerprinted contigs) map, and the chromosome-wide pseudomolecules. Such alignments also suggested amended placements of FPC contigs on the maize genetic map and proactively guided the assembly of chromosome-wide pseudomolecules, especially within complex genomic regions. Lastly, we think that the full integration of B73 optical maps with the maize iMap would greatly facilitate maize sequence finishing efforts that would make it a valuable reference for comparative studies among cereals, or other maize inbred lines and cultivars.

Refined localization of the Escherichia coli F4ab/F4ac receptor locus on pig chromosome 13

Diarrhoea in newborn and weaned pigs caused by enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbriae leads to considerable losses in pig production. In this study, we refined the mapping of the receptor locus for ETEC F4ab/F4ac adhesion (F4bcR) by joint analysis of Nordic and Swiss data. A total of 236 pigs from a Nordic experimental herd, 331 pigs from a Swiss experimental herd and 143 pigs from the Swiss performing station were used for linkage analysis. Genotyping data of six known microsatellite markers, two newly developed markers (MUC4gt and HSA125gt) and an intronic SNP in MUC4 (MUC4-8227) were used to create the linkage map. The region for F4bcR was refined to the interval SW207-S0075 on pig chromosome 13. The most probable position of F4bcR was in the SW207-MUC4 region. The order of six markers was supported by physical mapping on the BAC fingerprint contig from the Wellcome Trust Sanger Institute. Thus, the region for F4bcR could be reduced from 26 to 14 Mb.

A compartmentalized approach to the assembly of physical maps

BackgroundPhysical maps have been historically one of the cornerstones of genome sequencing and map-based cloning strategies. They also support marker assisted breeding and EST mapping. The problem of building a high quality physical map is computationally challenging due to unavoidable noise in the input fingerprint data.ResultsWe propose a novel compartmentalized method for the assembly of high quality physical maps from fingerprinted clones. The knowledge of genetic markers enables us to group clones into clusters so that clones in the same cluster are more likely to overlap. For each cluster of clones, a local physical map is first constructed using FingerPrinted Contigs (FPC). Then, all the individual maps are carefully merged into the final physical map. Experimental results on the genomes of rice and barley demonstrate that the compartmentalized assembly produces significantly more accurate maps, and that it can detect and isolate clones that would induce "chimeric" contigs if used in the final assembly.ConclusionThe software is available for download at

An integrated RH map of porcine chromosome 10

BackgroundWhole genome radiation hybrid (WG-RH) maps serve as "scaffolds" to significantly improve the orientation of small bacterial artificial chromosome (BAC) contigs, order genes within the contigs and assist assembly of a sequence-ready map for virtually any species. Here, we report the construction of a porcine: human comparative map for pig (Sus scrofa) chromosome 10 (SSC10) using the IMNpRH212,000-rad porcine WG-RH panel, integrated with the IMpRH7000-rad WG-RH, genetic and BAC fingerprinted contig (FPC) maps.ResultsMap vectors from the IMNpRH212,000-rad and IMpRH7,000-rad panels were merged to construct parallel framework (FW) maps, within which FW markers common to both panels have an identical order. This strategy reduced map discrepancies between the two panels and significantly improved map accuracy. A total of 216 markers, including 50 microsatellites (MSs), 97 genes and ESTs, and 69 BAC end sequences (BESs), were ordered within two linkage groups at two point (2 pt) LOD score of 8. One linkage group covers SSC10p with accumulated map distances of 738.2 cR7,000 and 1814.5 cR12,000, respectively. The second group covers SSC10q at map distances of 1336.9 cR7,000 and 3353.6 cR12,000, yielding an overall average map resolution of 16.4 kb/cR12,000 or 393.5 kb per marker on SSC10. This represents a ~2.5-fold increase in map resolution over the IMpRH7,000-rad panel. Based on 127 porcine markers that have homologous sequences in the human genome, a detailed comparative map between SSC10 and human (Homo sapiens) chromosome (HSA) 1, 9 and 10 was built.ConclusionThis initial comparative RH map of SSC10 refines the syntenic regions between SSC10 and HSA1, 9 and 10. It integrates the IMNpRH212,000-rad and IMpRH7,000-rad, genetic and BAC FPC maps and provides a scaffold to close potential gaps between contigs prior to genome sequencing and assembly. This map is also useful in fine mapping of QTLs on SSC10.

Genomic Organization and Evolution of the Vomeronasal Type 2 Receptor-Like (OlfC) Gene Clusters in Atlantic Salmon, Salmo salar

There are three major multigene superfamilies of olfactory receptors (OR, V1R, and V2R) in mammals. The ORs are expressed in the main olfactory organ, whereas the V1Rs and V2Rs are located in the vomeronasal organ. Fish only possess one olfactory organ in each nasal cavity, the olfactory rosette; therefore, it has been proposed that their V2R-like genes be classified as olfactory C family G protein-coupled receptors (OlfC). There are large variations in the sizes of OR gene repertoires. Previous studies have shown that fish have between 12 and 46 functional V2R-like genes, whereas humans have lost all functional V2Rs, and frog sp. have more than 240. Pseudogenization of V2R genes is a prevalent event across species. In the mouse and frog genomes, there are approximately double the number of pseudogenes compared with functional genes. An oligonucleotide probe was designed from a conserved sequence from four Atlantic salmon OlfC genes and used to screen the Atlantic salmon bacterial artificial chromosome (BAC) library. Hybridization-positive BACs were matched to fingerprint contigs, and representative BACs were shotgun cloned and sequenced. We identified 55 OlfC genes. Twenty-nine of the OlfC genes are classified as putatively functional genes and 26 as pseudogenes. The OlfC genes are found in two genomic clusters on chromosomes 9 and 20. Phylogenetic analysis revealed that the OlfC genes could be divided into 10 subfamilies, with nine of these subfamilies corresponding to subfamilies found in other teleosts and one being salmon specific. There is also a large expansion in the number of OlfC genes in one subfamily in Atlantic salmon. Subfamily gene expansions have been identified in other teleosts, and these differences in gene number reflect species-specific evolutionary requirements for olfaction. Total RNA was isolated from the olfactory epithelium and other tissues from a presmolt to examine the expression of the odorant genes. Several of the putative OlfC genes that we identified are expressed only in the olfactory epithelium, consistent with these genes encoding odorant receptors.

Integrating sequence with FPC fingerprint maps

Recent advances in both clone fingerprinting and draft sequencing technology have made it increasingly common for species to have a bacterial artificial clone (BAC) fingerprint map, BAC end sequences (BESs) and draft genomic sequence. The FPC (fingerprinted contigs) software package contains three modules that maximize the value of these resources. The BSS (blast some sequence) module provides a way to easily view the results of aligning draft sequence to the BESs, and integrates the results with the following two modules. The MTP (minimal tiling path) module uses sequence and fingerprints to determine a minimal tiling path of clones. The DSI (draft sequence integration) module aligns draft sequences to FPC contigs, displays them alongside the contigs and identifies potential discrepancies; the alignment can be based on either individual BES alignments to the draft, or on the locations of BESs that have been assembled into the draft. FPC also supports high-throughput fingerprint map generation as its time-intensive functions have been parallelized for Unix-based desktops or servers with multiple CPUs. Simulation results are provided for the MTP, DSI and parallelization. These features are in the FPC V9.3 software package, which is freely available.

Microsatellite discovery from BAC end sequences and genetic mapping to anchor the soybean physical and genetic maps

Whole-genome sequencing of the soybean (Glycine max (L.) Merr. 'Williams 82') has made it important to integrate its physical and genetic maps. To facilitate this integration of maps, we screened 3290 microsatellites (SSRs) identified from BAC end sequences of clones comprising the 'Williams 82' physical map. SSRs were screened against 3 mapping populations. We found the AAT and ACT motifs produced the greatest frequency of length polymorphisms, ranging from 17.2% to 32.3% and from 11.8% to 33.3%, respectively. Other useful motifs include the dinucleotide repeats AG, AT, and AG, with frequency of length polymorphisms ranging from 11.2% to 18.4% (AT), 12.4% to 20.6% (AG), and 11.3% to 16.4% (GT). Repeat lengths less than 16 bp were generally less useful than repeat lengths of 40-60 bp. Two hundred and sixty-five SSRs were genetically mapped in at least one population. Of the 265 mapped SSRs, 60 came from BAC singletons not yet placed into contigs of the physical map. One hundred and ten originated in BACs located in contigs for which no genetic map location was previously known. Ninety-five SSRs came from BACs within contigs for which one or more other BACs had already been mapped. For these fingerprinted contigs (FPC) a high percentage of the mapped markers showed inconsistent map locations. A strategy is introduced by which physical and genetic map inconsistencies can be resolved using the preliminary 4x assembly of the whole genome sequence of soybean.

Integrated cytogenetic map of mitotic metaphase chromosome 9 of maize: resolution, sensitivity, and banding paint development

To study the correlation of the sequence positions on the physical DNA finger print contig (FPC) map and cytogenetic maps of pachytene and somatic maize chromosomes, sequences located along the chromosome 9 FPC map approximately every 10 Mb were selected to place on maize chromosomes using fluorescent in situ hybridization (FISH). The probes were produced as pooled polymerase chain reaction products based on sequences of genetic markers or repeat-free portions of mapped bacterial artificial chromosome (BAC) clones. Fifteen probes were visualized on chromosome 9. The cytological positions of most sequences correspond on the pachytene, somatic, and FPC maps except some probes at the pericentromeric regions. Because of unequal condensation of mitotic metaphase chromosomes, being lower at pericentromeric regions and higher in the arms, probe positions are displaced to the distal ends of both arms. The axial resolution of FISH on somatic chromosome 9 varied from 3.3 to 8.2 Mb, which is 12-30 times lower than on pachytene chromosomes. The probe collection can be used as chromosomal landmarks or as a "banding paint" for the physical mapping of sequences including transgenes and BAC clones and for studying chromosomal rearrangements.

Genomic organization and characterization of two vomeronasal 1 receptor-like genes ( ora1 and ora2) in Atlantic salmon Salmo salar

Olfactory receptors are encoded by three large multigene superfamilies (OR, V1R and V2R) in mammals. Fish do not possess a vomeronasal system; therefore, it has been proposed that their V1R-like genes be classified as olfactory receptors related to class A G protein-coupled receptors ( ora). Unlike mammalian genomes, which contain more than a hundred V1R genes, the five species of teleost fish that have been investigated to date appear to have six ora genes ( ora1-6) except for pufferfish that have lost ora1. The common ancestor of salmonid fishes is purported to have undergone a whole genome duplication. As salmonids have a life history that requires the use of olfactory cues to navigate back to their natal habitats to spawn, we set out to determine if ora1 or ora2 is duplicated in a representative species, Atlantic salmon ( Salmo salar). We used an oligonucleotide probe designed from a conserved sequence of several teleost ora2 genes to screen an Atlantic salmon BAC library (CHORI-214). Hybridization-positive BACs belonged to a single fingerprint contig of the Atlantic salmon physical map. All were also positive for ora2 by PCR. One of these BACs was chosen for further study, and shotgun sequencing of this BAC identified two V1R-like genes, ora1 and ora2, that are in a head-to-head conformation as is seen in some other teleosts. The gene products, ora1 and ora2, are highly conserved among teleosts. We only found evidence for a single ora1-2 locus in the Atlantic salmon genome, which was mapped to linkage group 6. Fluorescent in situ hybridization (FISH) analysis placed ora1-2 on chromosome 12. Conserved synteny was found surrounding the ora1 and ora2 genes in Atlantic salmon, medaka and three-spined stickleback, but not zebrafish.

Physical mapping in large genomes: accelerating anchoring of BAC contigs to genetic maps through in silico analysis

Anchored physical maps represent essential frameworks for map-based cloning, comparative genomics studies, and genome sequencing projects. High throughput anchoring can be achieved by polymerase chain reaction (PCR) screening of bacterial artificial chromosome (BAC) library pools with molecular markers. However, for large genomes such as wheat, the development of high dimension pools and the number of reactions that need to be performed can be extremely large making the screening laborious and costly. To improve the cost efficiency of anchoring in such large genomes, we have developed a new software named Elephant (electronic physical map anchoring tool) that combines BAC contig information generated by FingerPrinted Contig with results of BAC library pools screening to identify BAC addresses with a minimal amount of PCR reactions. Elephant was evaluated during the construction of a physical map of chromosome 3B of hexaploid wheat. Results show that a one dimensional pool screening can be sufficient to anchor a BAC contig while reducing the number of PCR by 384-fold thereby demonstrating that Elephant is an efficient and cost-effective tool to support physical mapping in large genomes.

A BAC pooling strategy combined with PCR-based screenings in a large, highly repetitive genome enables integration of the maize genetic and physical maps

BackgroundMolecular markers serve three important functions in physical map assembly. First, they provide anchor points to genetic maps facilitating functional genomic studies. Second, they reduce the overlap required for BAC contig assembly from 80 to 50 percent. Finally, they validate assemblies based solely on BAC fingerprints. We employed a six-dimensional BAC pooling strategy in combination with a high-throughput PCR-based screening method to anchor the maize genetic and physical maps.ResultsA total of 110,592 maize BAC clones (~ 6x haploid genome equivalents) were pooled into six different matrices, each containing 48 pools of BAC DNA. The quality of the BAC DNA pools and their utility for identifying BACs containing target genomic sequences was tested using 254 PCR-based STS markers. Five types of PCR-based STS markers were screened to assess potential uses for the BAC pools. An average of 4.68 BAC clones were identified per marker analyzed. These results were integrated with BAC fingerprint data generated by the Arizona Genomics Institute (AGI) and the Arizona Genomics Computational Laboratory (AGCoL) to assemble the BAC contigs using the FingerPrinted Contigs (FPC) software and contribute to the construction and anchoring of the physical map. A total of 234 markers (92.5%) anchored BAC contigs to their genetic map positions. The results can be viewed on the integrated map of maize [1,2].ConclusionThis BAC pooling strategy is a rapid, cost effective method for genome assembly and anchoring. The requirement for six replicate positive amplifications makes this a robust method for use in large genomes with high amounts of repetitive DNA such as maize. This strategy can be used to physically map duplicate loci, provide order information for loci in a small genetic interval or with no genetic recombination, and loci with conflicting hybridization-based information.