Cross-species bacterial artificial chromosome (BAC) library screening via overgo-based hybridization and BAC-contig mapping of a yield enhancement quantitative trait locus (QTL) yld1.1 in the Malaysian wild rice Oryza rufipogon

The construction of BAC-contig physical maps is an important step towards a partial or ultimate genome sequence analysis. Here, we describe our initial efforts to apply an overgo approach to screen a BAC library of the Malaysian wild rice species, Oryza rufipogon. Overgo design is based on repetitive element masking and sequence uniqueness, and uses short probes (approximately 40 bp), making this method highly efficient and specific. Pairs of 24-bp oligos that contain an 8-bp overlap were developed from the publicly available genomic sequences of the cultivated rice, O. sativa, to generate 20 overgo probes for a 1-Mb region that encompasses a yield enhancement QTL yld1.1 in O. rufipogon. The advantages of a high similarity in melting temperature, hybridization kinetics and specific activities of overgos further enabled a pooling strategy for library screening by filter hybridization. Two pools of ten overgos each were hybridized to high-density filters representing the O. rufipogon genomic BAC library. These screening tests succeeded in providing 69 PCR-verified positive hits from a total of 23,040 BAC clones of the entire O. rufipogon library. A minimal tilling path of clones was generated to contribute to a fully covered BAC-contig map of the targeted 1-Mb region. The developed protocol for overgo design based on O. sativa sequences as a comparative genomic framework, and the pooled overgo hybridization screening technique are suitable means for high-resolution physical mapping and the identification of BAC candidates for sequencing.     

Identification of Citrus sinensis BAC clones containing genes relevant to fruit quality by two-dimensional overgo hybridization

As a complement to whole-genome sequencing efforts, we are comparing targeted genomic regions among sweet orange cultivars to identify coding and conserved noncoding regions, including regulatory elements, responsible for biological features unique to this species. Here, we report the identification of 1,018 bacterial artificial chromosome (BAC) clones containing genes relevant to fruit quality from a Citrus sinensis cv. “Vaniglia” 19.3X BAC library by two-dimensional 9?×?9 overgo hybridization. To design the overgo probes, we used the “C38” expressed sequence tag assembly (http://harvest.ucr.edu/) and OligoSpawn software (http://138.23.178.42). For BAC library screening, we selected 81 overgo probes associated with unigenes that putatively code for enzymes relevant to fruit quality (flavonol, anthocyanin, carotenoid, cellulose, starch, ascorbic acid, aromatic amino acid, and lignin biosynthesis; sucrose catabolism; glycolysis; oxidative/nonoxidative pentose phosphate pathway; fatty acid biosynthesis and oxidation; Krebs cycle). Hybridization probes were pooled and hybridized in groups of intersecting rows and columns to high-density BAC filters, followed by a deconvolution process that established BAC-probe addresses. BAC addresses were obtained for 75 of the 81 overgo probes initially selected, for a total of 1,018 BAC clones, a number consistent with the depth of coverage of the BAC library. BAC end sequencing was carried out, and end-sequence pairs were mapped to their best location in the Citrus clementina genome sequence assembly using the comparative genomic database Phytozome (http://www.phytozome.net/). The BAC clones corresponding to each probe were mapped within the same scaffold as the target gene, demonstrating that the approach we used was successful in isolating the targeted genomic regions.     

Integration of animal linkage and BAC contig maps using overgo hybridization

The alignment of genome linkage maps, defined primarily by segregation of sequence-tagged site (STS) markers, with BAC contig physical maps and full genome sequences requires high throughput mechanisms to identify BAC clones that contain specific STS. A powerful technique for this purpose is multi-dimensional hybridization of "overgo" probes. The probes are chosen from available STS sequence data by selecting unique probe sequences that have a common melting temperature. We have hybridized sets of 216 overgo probes in subset pools of 36 overgos at a time to filter-spotted chicken BAC clone arrays. A four-dimensional pooling strategy, including one degree of redundancy, has been employed. This requires 24 hybridizations to completely assign BACs for all 216 probes. Results to date are consistent with about a 10% failure rate in overgo probe design and a 15-20% false negative detection rate within a group of 216 markers. Three complete rounds of overgo hybridization, each to sets of about 39,000 BACs (either BAMHI or ECORI partial digest inserts) generated a total of 1853 BAC alignments for 517 mapped chicken genome STS markers. These data are publicly available, and they have been used in the assembly of a first generation BAC contig map of the chicken genome.     

Anchoring 9,371 maize expressed sequence tagged unigenes to the bacterial artificial chromosome contig map by two-dimensional overgo hybridization

Our goal is to construct a robust physical map for maize (Zea mays) comprehensively integrated with the genetic map. We have used a two-dimensional 24 x 24 overgo pooling strategy to anchor maize expressed sequence tagged (EST) unigenes to 165,888 bacterial artificial chromosomes (BACs) on high-density filters. A set of 70,716 public maize ESTs seeded derivation of 10,723 EST unigene assemblies. From these assemblies, 10,642 overgo sequences of 40 bp were applied as hybridization probes. BAC addresses were obtained for 9,371 overgo probes, representing an 88% success rate. More than 96% of the successful overgo probes identified two or more BACs, while 5% identified more than 50 BACs. The majority of BACs identified (79%) were hybridized with one or two overgos. A small number of BACs hybridized with eight or more overgos, suggesting that these BACs must be gene rich. Approximately 5,670 overgos identified BACs assembled within one contig, indicating that these probes are highly locus specific. A total of 1,795 megabases (Mb; 87%) of the total 2,050 Mb in BAC contigs were associated with one or more overgos, which are serving as sequence-tagged sites for single nucleotide polymorphism development. Overgo density ranged from less than one overgo per megabase to greater than 20 overgos per megabase. The majority of contigs (52%) hit by overgos contained three to nine overgos per megabase. Analysis of approximately 1,022 Mb of genetically anchored BAC contigs indicates that 9,003 of the total 13,900 overgo-contig sites are genetically anchored. Our results indicate overgos are a powerful approach for generating gene-specific hybridization probes that are facilitating the assembly of an integrated genetic and physical map for maize.     

A physical map of the Chinese chestnut (Castanea mollissima) genome and its integration with the genetic map

Three Chinese chestnut bacterial artificial chromosome (BAC) libraries were developed and used for physical map construction. Specifically, high information content fingerprinting was used to assemble 126,445 BAC clones into 1,377 contigs and 12,919 singletons. Integration of the dense Chinese chestnut genetic map with the physical map was achieved via high-throughput hybridization using overgo probes derived from sequence-based genetic markers. A total of 1,026 probes were anchored to the physical map including 831 probes corresponding to 878 expressed sequence tag-based markers. Within the physical map, three BAC contigs were anchored to the three major fungal blight-resistant quantitative trait loci on chestnut linkage groups B, F, and G. A subset of probes corresponding to orthologous genes in poplar showed only a limited amount of conserved gene order between the poplar and chestnut genomes. The integrated genetic and physical map of Chinese chestnut is available at www.fagaceae.org/physical_maps.     

An ordered BAC contig map of the equine major histocompatibility complex

A physical map of ordered bacterial artificial chromosome (BAC) clones was constructed to determine the genetic organization of the horse major histocompatibility complex. Human, cattle, pig, mouse, and rat MHC gene sequences were compared to identify highly conserved regions which served as source templates for the design of overgo primers. Thirty-five overgo probes were designed from 24 genes and used for hybridization screening of the equine USDA CHORI 241 BAC library. Two hundred thirty-eight BAC clones were assembled into two contigs spanning the horse MHC region. The first contig contains the MHC class II region and was reduced to a minimum tiling path of nine BAC clones that span approximately 800 kb and contain at least 20 genes. A minimum tiling path of a second contig containing the class III/I region is comprised of 14 BAC clones that span approximately 1.6 Mb and contain at least 34 genes. Fluorescence in situ hybridization (FISH) using representative clones from each of the three regions of the MHC localized the contigs onto ECA20q21 and oriented the regions relative to one another and the centromere. Dual-colored FISH revealed that the class I region is proximal to the centromere, the class II region is distal, and the class III region is located between class I and II. These data indicate that the equine MHC is a single gene-dense region similar in structure and organization to the human MHC and is not disrupted as in ruminants and pigs.     

Construction and utility of 10-kb libraries for efficient clone-gap closure for rice genome sequencing

Rice is an important crop and a model system for monocot genomics, and is a target for whole genome sequencing by the International Rice Genome Sequencing Project (IRGSP). The IRGSP is using a clone by clone approach to sequence rice based on minimum tiles of BAC or PAC clones. For chromosomes 10 and 3 we are using an integrated physical map based on two fingerprinted and end-sequenced BAC libraries to identifying a minimum tiling path of clones. In this study we constructed and tested two rice genomic libraries with an average insert size of 10 kb (10-kb library) to support the gap closure and finishing phases of the rice genome sequencing project. The HaeIII library contains 166,752 clones covering approximately 4.6x rice genome equivalents with an average insert size of 10.5 kb. The Sau3AI library contains 138,960 clones covering 4.2x genome equivalents with an average insert size of 11.6 kb. Both libraries were gridded in duplicate onto 11 high-density filters in a 5 x 5 pattern to facilitate screening by hybridization. The libraries contain an unbiased coverage of the rice genome with less than 5% contamination by clones containing organelle DNA or no insert. An efficient method was developed, consisting of pooled overgo hybridization, the selection of 10-kb gap spanning clones using end sequences, transposon sequencing and utilization of in silico draft sequence, to close relatively small gaps between sequenced BAC clones. Using this method we were able to close a majority of the gaps (up to approximately 50 kb) identified during the finishing phase of chromosome-10 sequencing. This method represents a useful way to close clone gaps and thus to complete the entire rice genome.     

Construction of a California condor BAC library and first-generation chicken-condor comparative physical map as an endangered species conservation genomics resource

To support genomic analysis of the endangered California condor (Gymnogyps californianus), a BAC library (CHORI-262) was generated using DNA from the blood of a female. The library consists of 89,665 recombinant BAC clones providing approximately 14-fold coverage of the presumed approximately 1.48-Gb genome. Taking advantage of recent progress in chicken genomics, we developed a first-generation comparative chicken-condor physical map using an overgo hybridization approach. The overgos were derived from chicken (164 probes) and New World vulture (8 probes) sequences. Screening a 2.8x subset of the total library resulted in 236 BAC-gene assignments with 2.5 positive BAC clones per successful probe. A preliminary comparative chicken-condor BAC-based map included 93 genes. Comparison of selected condor BAC sequences with orthologous chicken sequences suggested a high degree of conserved synteny between the two avian genomes. This work will aid in identification and characterization of candidate loci for the chondrodystrophy mutation to advance genetic management of this disease.     

Cross-species overgo hybridization and comparative physical mapping within avian genomes

The chicken genome sequence facilitates comparative genomics within other avian species. We performed cross-species hybridizations using overgo probes designed from chicken genomic and zebra finch expressed sequence tags (ESTs) to turkey and zebra finch BAC libraries. As a result, 3772 turkey BACs were assigned to 336 markers or genes, and 1662 zebra finch BACs were assigned to 164 genes. As expected, cross-hybridization was more successful with overgos within coding sequences than within untranslated region, intron or flanking sequences and between chicken and turkey, when compared with chicken-zebra finch or zebra finch-turkey cross-hybridization. These data contribute to the comparative alignment of avian genome maps using a 'one sequence, multiple genomes' strategy.     

Construction and Characterization of a Bacterial Artificial Chromosome (BAC) Library of Pacific White Shrimp, Litopenaeus vannamei

The pacific white shrimp (Litopenaeus vannamei) is one of the most economically important marine aquaculture species in the world. To facilitate gene cloning and characterization, genome analysis, physical mapping, and molecular selection breeding of marine shrimp, we have developed the techniques to isolate high-quality megabase-sized DNA from hemocyte nuclear DNA of female shrimp and constructed a bacterial artificial chromosome (BAC) genomic library for the species. The library was constructed in the Hind III site of the vector pECBAC1, consisting of 101,760 clones arrayed in 265 384-well microtiter plates, with an average insert size of 101 kb, and covering the genome approximately fivefold. To characterize the library, 92,160 clones were spotted onto high-density nylon filters for hybridization screening. A set of 18 pairs of overgo probes designed from eight cDNA sequences of L. vannamei genes were used in hybridization screening, and 35 positive clones were identified. These results suggest that the shrimp BAC libraries will provide a useful resource for screening of genomic regions of interest candidate genes, gene families, or large-sized synthetic DNA region and promote future works on comparative genomics, physical mapping, and large-scale genome sequencing in the species.     

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.     

A BAC-based physical map of the channel catfish genome

Catfish is the major aquaculture species in the United States. To enhance its genome studies involving genetic linkage and comparative mapping, a bacterial artificial chromosome (BAC) contig-based physical map of the channel catfish (Ictalurus punctatus) genome was generated using four-color fluorescence-based fingerprints. Fingerprints of 34,580 BAC clones (5.6x genome coverage) were generated for the FPC assembly of the BAC contigs. A total of 3307 contigs were assembled using a cutoff value of 1x10(-20). Each contig contains an average of 9.25 clones with an average size of 292 kb. The combined contig size for all contigs was 0.965 Gb, approximately the genome size of the channel catfish. The reliability of the contig assembly was assessed by both hybridization of gene probes to BAC clones contained in the fingerprinted assembly and validation of randomly selected contigs using overgo probes designed from BAC end sequences. The presented physical map should greatly enhance genome research in the catfish, particularly aiding in the identification of genomic regions containing genes underlying important performance traits.     

A BAC-based physical map of Zhikong scallop (Chlamys farreri Jones et Preston)

Zhikong scallop (Chlamys farreri) is one of the most economically important aquaculture species in China. Physical maps are crucial tools for genome sequencing, gene mapping and cloning, genetic improvement and selective breeding. In this study, we have developed a genome-wide, BAC-based physical map for the species. A total of 81,408 clones from two BAC libraries of the scallop were fingerprinted using an ABI 3130xl Genetic Analyzer and a fingerprinting kit developed in our laboratory. After data processing, 63,641 (～5.8× genome coverage) fingerprints were validated and used in the physical map assembly. A total of 3,696 contigs were assembled for the physical map. Each contig contained an average of 10.0 clones, with an average physical size of 490 kb. The combined total physical size of all contigs was 1.81 Gb, equivalent to approximately 1.5 fold of the scallop haploid genome. A total of 10,587 BAC end sequences (BESs) and 167 markers were integrated into the physical map. We evaluated the physical map by overgo hybridization, BAC-FISH (fluorescence in situ hybridization), contig BAC pool screening and source BAC library screening. The results have provided evidence of the high reliability of the contig physical map. This is the first physical map in mollusc; therefore, it provides an important platform for advanced research of genomics and genetics, and mapping of genes and QTL of economical importance, thus facilitating the genetic improvement and selective breeding of the scallop and other marine molluscs.     

Chemiluminescent detection of sequential DNA hybridizations to high-density, filter-arrayed cDNA libraries: a subtraction method for novel gene discovery.

A chemiluminescent approach for sequential DNA hybridizations to high-density filter arrays of cDNAs, using a biotin-based random priming method followed by a streptavidin/alkaline phosphatase/CDP-Star detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid high-density filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently "deplexed" into individual components for specific probe localizations.     

Locus-specific contig assembly in highly-duplicated genomes, using the BAC-RF method

Polyploidy, the presence of multiple sets of chromosomes that are similar but not identical, complicates both chromosome walking and assembly of sequence-ready contigs for many plant taxa including a large number of economically-significant crops. Traditional ‘dot-blot hybridization’ or PCR-based assays for identifying BAC clones corresponding to a mapped DNA landmark usually do not provide sufficient information to distinguish between allelic and non-allelic loci. A restriction fragment matching method using pools of BAC DNA in combination with dot-blots reveals the locus specificity of individual BACs that correspond to multi-locus DNA probes, in a manner that can efficiently be applied on a large scale. This approach also provides an alternative means of mapping DNA loci that exploits many advantages of ‘radiation hybrid’ mapping in taxa for which such hybrids are not available. The BAC-RF method is a practical and reliable approach for using high-density RFLP maps to anchor sequence-ready BAC contigs in highly-duplicated genomes, provides an alternative to high-density robotic gridding for screening BAC libraries when the necessary equipment is not available, and permits the expedient isolation of individual members of multigene or repetitive DNA families for a wide range of genetic and evolutionary investigations.     

A BAC clone-based physical map of ovine major histocompatibility complex

An ovine bacterial artificial chromosome (BAC) library containing 190,000 BAC clones was constructed and subsequently screened to construct a BAC-based physical map for the ovine major histocompatibility complex (MHC). Two hundred thirty-three BAC clones were selected by 84 overgo probes designed on human, mouse, and swine MHC sequence homologies. Ninety-four clones were ordered by DNA fingerprinting to form contigs I, II, and III that correspond to ovine MHC class I-class III, class IIa, and class IIb. The minimum tiling paths of contigs I, II, and III are 15, 4, and 4 BAC clones, spanning approximately 1900, 400, and 300 kb, respectively. The order and orientation of most BAC clones in each contig were confirmed by BAC-end sequencing. An open gap exists between class IIa and class III. This work helps to provide a foundation for detailed study of ovine MHC genes and of evolution of MHCs in mammals.     

Characterization of three maize bacterial artificial chromosome libraries toward anchoring of the physical map to the genetic map using high-density bacterial artificial chromosome filter hybridization

Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73. High-density filter sets from all three libraries, made using different restriction enzymes (HindIII, EcoRI, and MboI, respectively), were evaluated with a set of complex probes including the 185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage lambda. The results indicate that the libraries are of high quality with low contamination by organellar and lambda-sequences. The use of libraries from multiple enzymes increased the chance of recovering each region of the genome. Ninety maize restriction fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6x coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 x Mo17 genetic map and to mark the bin boundaries on the physical map. All of the clones used as hybridization probes detected at least three BACs. Twenty-two single-copy number core markers identified an average of 7.4 +/- 3.3 positive clones, consistent with the expectation of six clones. This information is integrated into fingerprinting data generated by the Arizona Genomics Institute to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction.     

Construction and characterization of bacterial artificial chromosome libraries from the silkworm, Bombyx mori

Two bacterial artificial chromosome (BAC) libraries were constructed using nuclear DNA from posterior silkglands of the silkworm (Bombyx mori) strains p50 and C108. The libraries contain a total of 36,864 clones, or approximately 9 genome equivalents. The average insert sizes in the libraries were 134.5?kb and 120.8?kb, respectively. PCR-based screening was performed on the p50 library using probes for 34 sequence-tagged sites (STSs). Between 3 and 11 (6.1 hits on average) clones were isolated with each STS, in good agreement with the library size, 5.8 genome equivalents. The previously reported close linkage between the Bombyx homologs of the invected (Bm in) and engrailed (Bm en) genes was confirmed by construction of a BAC contig that contained both. Moreover, screening revealed novel information about the chromosomal organization of the sericin-1 and DH-PBAN genes, which were localized within a 22-kb interval and are divergently oriented. These results show that it is possible to construct contigs and analyze chromosome organization using these libraries.     

An Anchored Framework BAC Map of Mouse Chromosome 11 Assembled Using Multiplex Oligonucleotide Hybridization

Despite abundant library resources for many organisms, physical mapping of these organisms has been seriously limited due to lack of efficient library screening techniques. We have developed a highly efficient strategy for large-scale screening of genomic libraries based on multiplex oligonucleotide hybridization on high-density genomic filters. We have applied this strategy to generate a bacterial artificial chromosome (BAC) anchored map of mouse chromosome 11. Using the MIT mouse SSLP data, 320 pairs of oligonucleotide probes were designed with an “overgo” computer program that selects new primer sequences that avoid the microsatellite repeat. BACs identified by these probes are automatically anchored to the chromosome. Ninety-two percent of the probes identified positive clones from a 5.9-fold coverage mouse BAC library with an average of 7 positive clones per marker. An average of 4.2 clones was confirmed for 204 markers by PCR. Our data show that a large number of clones can be efficiently isolated from a large genomic library using this strategy with minimal effort. This strategy will have wide application for large-scale mapping and sequencing of human and other large genomes.     

Construction and characterization of a soybean bacterial artificial chromosome library and use of multiple complementary libraries for genome physical mapping

Two plant-transformation-competent large-insert binary clone bacterial artificial chromosome (hereafter BIBAC) libraries were previously constructed for soybean cv. Forrest, using BamHI or HindIII. However, they are not well suited for clone-based genomic sequencing due to their larger ratio of vector to insert size (27.6 kbp:125 kbp). Therefore, we developed a larger-insert bacterial artificial chromosome (BAC) library for the genotype in a smaller vector (pECBAC1), using EcoRI. The BAC library contains 38,400 clones; about 99.1% of the clones have inserts; the average insert size is 157 kbp; and the ratio of vector to insert size is much smaller (7.5 kbp:157 kbp). Colony hybridization with probes derived from several chloroplast and mitochondrial genes showed that 0.89% and 0.45% of the clones were derived from the chloroplast and mitochondrial genomes, respectively. Considering these data, the library represents 5.4 haploid genomes of soybean. The library was hybridized with six RFLP marker probes, 5S rDNA and 18S-5.8S-25S rDNA, respectively. Each RFLP marker hybridized to about six clones, and the 5S and 18S-5.8S-25S rDNA probes collectively hybridized to 402 BACs—about 1.05% of the clones in the library. The BAC library complements the existing soybean Forrest BIBAC libraries by using different restriction enzymes and vector systems. Together, the BAC and BIBAC libraries encompass 13.2 haploid genomes, providing the most comprehensive clone resource for a single soybean genotype for public genome research. We show that the BAC library has enhanced the development of the soybean whole-genome physical map and use of three complementary BAC libraries improves genome physical mapping by fingerprint analysis of most of the clones of the library. The rDNA-containing clones were also fingerprinted to evaluate the feasibility of constructing contig maps of the rDNA regions. It was found that physical maps for the rDNA regions could not be readily constructed by fingerprint analysis, using one or two restriction enzymes. Additional data to fingerprints and/or different fingerprinting methods are needed to build contig maps for such highly tandem repetitive regions and thus, the physical map of the entire soybean genome.