BAC-HAPPY Mapping (BAP Mapping): A New and Efficient Protocol for Physical Mapping

Physical and linkage mapping underpin efforts to sequence and characterize the genomes of eukaryotic organisms by providing a skeleton framework for whole genome assembly. Hitherto, linkage and physical “contig” maps were generated independently prior to merging. Here, we develop a new and easy method, BAC HAPPY MAPPING (BAP mapping), that utilizes BAC library pools as a HAPPY mapping panel together with an Mbp-sized DNA panel to integrate the linkage and physical mapping efforts into one pipeline. Using Arabidopsis thaliana as an exemplar, a set of 40 Sequence Tagged Site (STS) markers spanning ∼10% of chromosome 4 were simultaneously assembled onto a BAP map compiled using both a series of BAC pools each comprising 0.7x genome coverage and dilute (0.7x genome) samples of sheared genomic DNA. The resultant BAP map overcomes the need for polymorphic loci to separate genetic loci by recombination and allows physical mapping in segments of suppressed recombination that are difficult to analyze using traditional mapping techniques. Even virtual “BAC-HAPPY-mapping” to convert BAC landing data into BAC linkage contigs is possible.     

Endosperm Tolerance of Paternal Aneuploidy Allows Radiation Hybrid Mapping of the Wheat D-Genome and a Measure of γ Ray-Induced Chromosome Breaks

Physical mapping and genome sequencing are underway for the ≈17 Gb wheat genome. Physical mapping methods independent of meiotic recombination, such as radiation hybrid (RH) mapping, will aid precise anchoring of BAC contigs in the large regions of suppressed recombination in Triticeae genomes. Reports of endosperm development following pollination with irradiated pollen at dosages that cause embryo abortion prompted us to investigate endosperm as a potential source of RH mapping germplasm. Here, we report a novel approach to construct RH based physical maps of all seven D-genome chromosomes of the hexaploid wheat ‘Chinese Spring’, simultaneously. An 81-member subset of endosperm samples derived from 20-Gy irradiated pollen was genotyped for deletions, and 737 markers were mapped on seven D-genome chromosomes. Analysis of well-defined regions of six chromosomes suggested a map resolution of ∼830 kb could be achieved; this estimate was validated with assays of markers from a sequenced contig. We estimate that the panel contains ∼6,000 deletion bins for D-genome chromosomes and will require ∼18,000 markers for high resolution mapping. Map-based deletion estimates revealed a majority of 1–20 Mb interstitial deletions suggesting mutagenic repair of double-strand breaks in pollen provides a useful resource for RH mapping and map based cloning studies.     

Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics

Mitochondrial genome sequences are important markers for phylogenetics but taxon sampling remains sporadic because of the great effort and cost required to acquire full-length sequences. Here, we demonstrate a simple, cost-effective way to sequence the full complement of protein coding mitochondrial genes from pooled samples using the 454/Roche platform. Multiplexing was achieved without the need for expensive indexing tags (‘barcodes’). The method was trialled with a set of long-range polymerase chain reaction (PCR) fragments from 30 species of Coleoptera (beetles) sequenced in a 1/16th sector of a sequencing plate. Long contigs were produced from the pooled sequences with sequencing depths ranging from ∼10 to 100× per contig. Species identity of individual contigs was established via three ‘bait’ sequences matching disparate parts of the mitochondrial genome obtained by conventional PCR and Sanger sequencing. This proved that assembly of contigs from the sequencing pool was correct. Our study produced sequences for 21 nearly complete and seven partial sets of protein coding mitochondrial genes. Combined with existing sequences for 25 taxa, an improved estimate of basal relationships in Coleoptera was obtained. The procedure could be employed routinely for mitochondrial genome sequencing at the species level, to provide improved species ‘barcodes’ that currently use the cox1 gene only.     

ComboScreen facilitates the multiplex hybridization-based screening of high-density clone arrays

MOTIVATION: The construction of physical maps based on bacterial clones [e.g. bacterial artificial chromosomes (BACs)] is valuable for a number of molecular genetics applications, including the high-resolution mapping of genomic regions of interest and the identification of clones suitable for systematic sequencing. A common approach for large-scale screening of bacterial clone libraries involves the hybridization of high-density arrays of immobilized, lysed colonies with collections of DNA probes. The use of a multiplex hybridization screening strategy, whereby pooled probes are analysed en masse, simplifies the effort by reducing the total number of parallel experiments required. However, this approach generates large amounts of hybridization-based data that must be carefully analysed, assimilated, and disambiguated in a careful but efficient manner. RESULTS: To facilitate the screening of high-density clone arrays by a multiplex hybridization approach, we have written a program called ComboScreen. This program provides an organizational framework and analytical tools required for the high-throughput hybridization screening of clone arrays with pools of probes. We have used this program extensively for constructing mouse sequence-ready BAC contig maps.     

BAC representation of two low‐copy regions of the genome of Arabidopsis thaliana

Two regions of Arabidopsis chromosome 4, totalling 4.7 Mb, were assayed for representation in the TAMU and IGF BAC libraries. A directed approach to BAC identification was developed. Gel-purified DNA samples of YACs selected from the YAC-based physical map of chromosome 4 were used to probe high-density colony arrays of the BAC libraries. Strategies were developed that allowed the efficient construction of restriction maps and BAC contigs. Four hundred and sixty-four BACs were mapped, assembled into two complete contigs and used to analyse genomic representation. These BACs provided a mean of 9.4-fold redundant coverage, with a range of 2- to 22-fold. The representation provided by the two libraries showed almost coincident peaks and troughs, with a periodicity of approximately 200 kb. These results demonstrate that, provided both TAMU and IGF libraries are used in their entirety, BACs should provide an excellent resource for both physical mapping and sequencing of the Arabidopsis genome.     

AFLP-derived SCARs facilitate construction of a 1.1 Mb sequence-ready map of a region that spans the Vf locus in the apple genome

The availability of high-density anchored markers is a prerequisite for reliable construction of a deep coverage BAC contig, which leads to creation of a sequence-ready map in the target chromosomal region. Unfortunately, such markers are not available for most plant species, including woody perennial plants. Here, we report on an efficient approach to build a megabase-size sequence-ready map in the apple genome for the Vf region containing apple scab resistance gene(s) by targeting AFLP-derived SCAR markers to this specific genomic region. A total of 11 AFLP-derived SCAR markers, previously tagged to the Vf locus, along with three other Vf-linked SCAR markers have been used to screen two apple genome BAC libraries. A single BAC contig which spans the Vf region at a physical distance of approximately 1,100 kb has been constructed by assembling the recovered BAC clones, followed by closure of inter-contig gaps. The contig is 4 ×deep, and provides a minimal tiling path of 16 contiguous and overlapping BAC clones, thus generating a sequence-ready map. Within the Vf region, duplication events have occurred frequently, and the Vf locus is restricted to the ca. 290 kb region covered by a minimum of three overlapping BAC clones.     

A high resolution physical and RH map of pig chromosome 6q1.2 and comparative analysis with human chromosome 19q13.1

Background

The generation of BAC/PAC contigs in targeted genome regions is a powerful method to establish high-resolution physical maps. In domestic animal species the generation of such contigs is typically initiated with the screening of libraries with probes derived from human genes that are expected to be located in the region of interest by comparative mapping. However, in many instances the available gene-derived probes are too far apart to allow the cloning of BAC/PAC contigs larger than a few hundred kb. High resolution physical mapping allows to estimate the sizes of gaps and to control the orientation of the individual sub-contigs, which helps to avoid errors during the assembly of smaller contigs into final Mb-sized contigs. The recently constructed porcine IMNpRH2 panel allowed us to use this approach for the construction of high-resolution physical maps of SSC 6q1.2.

Results

Two sequence-ready BAC/PAC contigs of the gene-rich region on porcine chromosome 6q1.2 (SSC 6q1.2) containing the RYRl gene were constructed. The two contigs spanned about 1.2 Mb and 2.0 Mb respectively. The construction of these contigs was monitored by the results provided by the mapping of 15 markers on the IMpRH_7000rad and 35 markers on the IMNpRH2_12000rad radiation hybrid panels. Analyses on the IMpRH panel allowed us to globally link and orientate preliminary smaller contigs, whereas analyses on the high resolution IMNpRH2 panel allowed us to finally identify the order of genes and markers.

Conclusions

A framework map of 523 cR₁₂₀₀₀ was established covering the whole studied region. The order of markers on the framework 1000:1 RH map was found totally consistent with the data deduced from the contig map. The kb/cR ratio was very constant in the whole region, with an average value of 6.6 kb/cR. We estimate that the size of the remaining gap between the two contigs is of about 300 kb. The integrated physical and RH map of the investigated region on SSC 6q1.2 was used for a comparative analysis with respect to the syntenic regions on HSA 19q13.1 and MMU 7 and revealed a perfectly conserved gene order across the entire studied interval.

     

Characterization of a new BAC library for rainbow trout: evidence for multi-locus duplication

A 10X rainbow trout bacterial artificial chromosome (BAC) library was constructed to aid in the physical and genetic mapping efforts of the rainbow trout genome. The library was derived from the Swanson clonal line (YY male) and consists of 184,704 clones with an average insert size of 137,500 bp (PFGE) or 118,700 bp (DNA fingerprinting). The clones were gridded onto 10 large nylon membranes to produce high-density arrays for screening the library by hybridization. The library was probed with 11 cDNAs from the NCCCWA EST project chosen because of interest in their homology to known gene sequences, seven known genes, and a Y-specific sex marker. Putative positive clones identified by hybridization were re-arrayed and gridded for secondary confirmation. FPC analysis of HindIII and EcoRV DNA fingerprinting was used to estimate the level of redundancy in the library, to construct BAC contigs and to detect duplicated loci in the semi-duplicated rainbow trout genome. A good correlation (R2 = 0.7) was found between the number of hits per probe and the number of contigs that were assembled from the positive BACs. The average number of BACs per contig was 9.6, which is in good agreement with 10X genome coverage of the library. Two-thirds of the loci screened were predicted to be duplicated as the positive BACs for those genes were assembled into two or three different contigs, which suggests that most of the rainbow trout genome is duplicated.     

Rational design of landmark probes for quantitative DNA fiber mapping (QDFM)

Rapid construction of high-resolution physical maps requires accurate information about overlap between DNA clones and the size of gaps between clones or clone contigs. We recently developed a procedure termed ‘quantitative DNA fiber mapping’ (QDFM) to help construct physical maps by measuring the overlap between clones or the physical distance between non-overlapping contigs. QDFM is based on hybridization of non-isotopically labeled probes onto DNA molecules that were bound to a solid support and stretched homogeneously to ~2.3 kb/µm. In this paper, we describe the design of probes that bind specifically to the cloning vector of DNA recombinants to facilitate physical mapping. Probes described here delineate the most frequently used cloning vectors such as BACs, P1s, PACs and YACs. As demonstrated in representative hybridizations, vector-specific probes provide valuable information about molecule integrity, insert size and orientation as well as localization of hybridization domains relative to specifically-marked vector sequences.     

Homology-driven assembly of a sequence-ready mouse BAC contig map spanning regions related to the 46-Mb gene-rich euchromatic segments of human chromosome 19

Draft sequence derived from the 46-Mb gene-rich euchromatic portion of human chromosome 19 (HSA19) was utilized to generate a sequence-ready physical map spanning homologous regions of mouse chromosomes. Sequence similarity searches with the human sequence identified more than 1000 individual orthologous mouse genes from which 382 overgo probes were developed for hybridization. Using human gene order and spacing as a model, these probes were used to isolate and assemble bacterial artificial chromosome (BAC) clone contigs spanning homologous mouse regions. Each contig was verified, extended, and joined to neighboring contigs by restriction enzyme fingerprinting analysis. Approximately 3000 mouse BACs were analyzed and assembled into 44 contigs with a combined length of 41.4 Mb. These BAC contigs, covering 90% of HSA19-related mouse DNA, are distributed throughout 15 homology segments derived from different regions of mouse chromosomes 7, 8, 9, 10, and 17. The alignment of the HSA19 map with the ordered mouse BAC contigs revealed a number of structural differences in several overtly conserved homologous regions and more precisely defined the borders of the known regions of HSA19-syntenic homology. Our results demonstrate that given a human draft sequence, BAC contig maps can be constructed quickly for comparative sequencing without the need for preestablished mouse-specific genetic or physical markers and indicate that similar strategies can be applied with equal success to genomes of other vertebrate species.     

Footprints in time: comparative quantitative trait loci mapping of the pitcher-plant mosquito,Wyeomyia smithii

Identifying regions of the genome contributing to phenotypic evolution often involves genetic mapping of quantitative traits. The focus then turns to identifying regions of ‘major’ effect, overlooking the observation that traits of ecological or evolutionary relevance usually involve many genes whose individual effects are small but whose cumulative effect is large. Herein, we use the power of fully interfertile natural populations of a single species of mosquito to develop three quantitative trait loci (QTL) maps: one between two post-glacially diverged populations and two between a more ancient and a post-glacial population. All demonstrate that photoperiodic response is genetically a highly complex trait. Furthermore, we show that marker regressions identify apparently ‘non-significant’ regions of the genome not identified by composite interval mapping, that the perception of the genetic basis of adaptive evolution is crucially dependent upon genetic background and that the genetic basis for adaptive evolution of photoperiodic response is highly variable within contemporary populations as well as between anciently diverged populations.     

Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic ‘Touch DNA’ Evidence

DNA profiles can be obtained from ‘touch DNA’ evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a ‘blind-swabbing’ approach will co-sample cellular material from the different individuals, even if the individuals’ cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim’s DNA may be found in significant excess thus masking any potential perpetrator’s DNA.In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, ‘smart analysis’ method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 µl microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., “clumps”) bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.     

Human and mouse oligonucleotide-based array CGH

Array-based comparative genomic hybridization is a high resolution method for measuring chromosomal copy number changes. Here we present a validated protocol using in-house spotted oligonucleotide libraries for array comparative genomic hybridization (CGH). This oligo array CGH platform yields reproducible results and is capable of detecting single copy gains, multi-copy amplifications as well as homozygous and heterozygous deletions as small as 100 kb with high resolution. A human oligonucleotide library was printed on amine binding slides. Arrays were hybridized using a hybstation and analysed using BlueFuse feature extraction software, with >95% of spots passing quality control. The protocol allows as little as 300 ng of input DNA and a 90% reduction of Cot-1 DNA without compromising quality. High quality results have also been obtained with DNA from archival tissue. Finally, in addition to human oligo arrays, we have applied the protocol successfully to mouse oligo arrays. We believe that this oligo-based platform using ‘off-the-shelf’ oligo libraries provides an easy accessible alternative to BAC arrays for CGH, which is cost-effective, available at high resolution and easily implemented for any sequenced organism without compromising the quality of the results.     

Piggy-BACing the human genome II. A high-resolution, physically anchored, comparative map of the porcine autosomes

Using the INRA-Minnesota porcine radiation hybrid panel, we have constructed a human-pig comparative map composed of 2274 loci, including 206 ESTs and 2068 BAC-end sequences, assigned to 34 linkage groups. The average spacing between comparative anchor loci is 1.15 Mb based on human genome sequence coordinates. A total of 51 conserved synteny groups that include 173 conserved segments were identified. This radiation hybrid map has the highest resolution of any porcine map to date and its integration with the porcine linkage map (reported here) will greatly facilitate the positional cloning of genes influencing complex traits of both agricultural and biomedical interest. Additionally, this map will provide a framework for anchoring contigs generated through BAC fingerprinting efforts and assist in the selection of a BAC minimal tiling path and assembly of the first sequence-ready map of the porcine genome.     

Identification of genetically linked RGAs by BAC screening in maize and implications for gene cloning,mapping and MAS

The resistance gene analogue (RGA) pic19 in maize, a candidate for sugarcane mosaic virus (SCMV) resistance gene (R gene) Scmv1, was used to screen a maize BAC library to identify homologous sequences in the maize genome and to investigate their genomic organisation. Fifteen positive BAC clones were identified and could be classified into five physically independent contigs consisting of overlapping clones. Genetic mapping clustered three contigs into the same genomic region as Scmv1 on chromosome 6S. The two remaining contigs mapped to the same region as a QTL for SCMV resistance on chromosome 1. Thus, RGAs mapping to a target region can be successfully used to identify further-linked candidate sequences. The pic19 homologous sequences of these clones revealed a sequence similarity of 94-98% on the nucleotide level. The high sequence similarity reveals potential problems for the use of RGAs as molecular markers. Their application in marker-assisted selection (MAS) and the construction of high-density genetic maps is complicated by the existence of closely linked homologues resulting in 'ghost' marker loci analogous to 'ghost' QTLs. Therefore, implementation of genomic library screening, including genetic mapping of potential homologues, seems necessary for the safe application of RGA markers in MAS and gene isolation.     

Identification of the Genomic Insertion Site of Pmel-1 TCR α and β Transgenes by Next-Generation Sequencing

The pmel-1 T cell receptor transgenic mouse has been extensively employed as an ideal model system to study the mechanisms of tumor immunology, CD8⁺ T cell differentiation, autoimmunity and adoptive immunotherapy. The ‘zygosity’ of the transgene affects the transgene expression levels and may compromise optimal breeding scheme design. However, the integration sites for the pmel-1 mouse have remained uncharacterized. This is also true for many other commonly used transgenic mice created before the modern era of rapid and inexpensive next-generation sequencing. Here, we show that whole genome sequencing can be used to determine the exact pmel-1 genomic integration site, even with relatively ‘shallow’ (8X) coverage. The results were used to develop a validated polymerase chain reaction-based genotyping assay. For the first time, we provide a quick and convenient polymerase chain reaction method to determine the dosage of pmel-1 transgene for this freely and publically available mouse resource. We also demonstrate that next-generation sequencing provides a feasible approach for mapping foreign DNA integration sites, even when information of the original vector sequences is only partially known.     

Optimization of linkage mapping strategy and construction of a high-density American lotus linkage map

Background

Lotus is a diploid plant with agricultural, medicinal, and ecological significance. Genetic linkage maps are fundamental resources for genome and genetic study, and also provide molecular markers for breeding in agriculturally important species. Genotyping by sequencing revolutionized genetic mapping, the restriction-site associated DNA sequencing (RADseq) allowed rapid discovery of thousands of SNPs markers, and a crucial aspect of the sequence based mapping strategy is the reference sequences used for marker identification.

Results

We assessed the effectiveness of linkage mapping using three types of references for scoring markers: the unmasked genome, repeat masked genome, and gene models. Overall, the repeat masked genome produced the optimal genetic maps. A high-density genetic map of American lotus was constructed using an F₁ population derived from a cross between Nelumbo nucifera ‘China Antique’ and N. lutea ‘AL1’. A total of 4,098 RADseq markers were used to construct the American lotus ‘AL1’ genetic map, and 147 markers were used to construct the Chinese lotus ‘China Antique’ genetic map. The American lotus map has 9 linkage groups, and spans 494.3 cM, with an average distance of 0.7 cM between adjacent markers. The American lotus map was used to anchor scaffold sequences in the N. nucifera ‘China Antique’ draft genome. 3,603 RADseq markers anchored 234 individual scaffold sequences into 9 megascaffolds spanning 67% of the 804 Mb draft genome.

Conclusions

Among the unmasked genome, repeat masked genome and gene models, the optimal reference sequences to call RADseq markers for map construction is repeat masked genome. This high density genetic map is a valuable resource for genomic research and crop improvement in lotus.