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.     

Construction and Identification of Bacterial Artificial Chromosome Library for 0-613-2R in Upland Cotton

A bacterial artificial chromosome （BAC） library containing a large genomlc DNA insert is an important tool for genome physical mapping, map-based cloning, and genome sequencing. To Isolate genes via a map-based cloning strategy and to perform physical mapping of the cotton genome, a high-quality BAC library containing large cotton DNA Inserts Is needed. We have developed a BAC library of the restoring line 0-613-2R for Isolating the fertility restorer （Rf1） gene and genomic research in cotton （Gossypium hirsutum L.）. The BAC library contains 97 825 clones stored In 255 pieces of a 384-well mlcrotiter plate. Random samples of BACs digested with the Notl enzyme Indicated that the average Insert size Is approximately 130 kb, with a range of 80-275 kb, and 95.7% of the BAC clones in the library have an average insert size larger than 100 kb. Based on a cotton genome size of 2 250 Mb, library coverage is 5.7 × haploid genome equivalents. Four clones were selected randomly from the library to determine the stability of the BAC clones. There were no different fingerprints for 0 and 100 generations of each clone digested with Notl and Hlndiii enzymes. Thus, the atabiiity of a single BAC clone can be sustained at iesat for 100 generations. Eight simple sequence repeat （SSR） markers flanking the Rf; gene were chosen to screen the BAC library by pool using PCR method and 25 positive clones were identified with 3.1 positive clones per SSR marker.     

Direct targeting and rapid isolation of BAC clones spanning a defined chromosome region

To isolate genes of interest in plants, it is essential to construct bacterial artificial chromosome (BAC) libraries from specific genotypes. Construction and organisation of BAC libraries is laborious and costly, especially from organisms with large and complex genomes. In the present study, we developed the pooled BAC library strategy that allows rapid and low cost generation and screening of genomic libraries from any genotype of interest. The BAC library is constructed, directly organised into a few pools and screened for BAC clones of interest using PCR and hybridisation steps, without requiring organization into individual clones. As a proof of concept, a pooled BAC library of approximately 177,000 recombinant clones has been constructed from the barley cultivar Cebada Capa that carries the Rph7 leaf rust resistance gene. The library has an average insert size of 140 kb, a coverage of six barley genome equivalents and is organised in 138 pools of about 1,300 clones each. We rapidly established a single contig of six BAC clones spanning 230 kb at the Rph7 locus on chromosome 3HS. The described low-cost cloning strategy is fast and will greatly facilitate direct targeting of genes and large-scale intra- and inter-species comparative genome analysis.Edwige Isidore and Beatrice Scherrer contributed equally to the work.     

Construction and Characterization of Two Bacterial Artificial Chromosome Libraries of Grass Carp

Bacterial artificial chromosome (BAC) library is an important tool in genomic research. We constructed two libraries from the genomic DNA of grass carp (Ctenopharyngodon idellus) as a crucial part of the grass carp genome project. The libraries were constructed in the EcoRI and HindIII sites of the vector CopyControl pCC1BAC. The EcoRI library comprised 53,000 positive clones, and approximately 99.94% of the clones contained grass carp nuclear DNA inserts (average size, 139.7 kb) covering 7.4× haploid genome equivalents and 2% empty clones. Similarly, the HindIII library comprised 52,216 clones with approximately 99.82% probability of finding any genomic fragments containing single-copy genes; the average insert size was 121.5 kb with 2.8% insert-empty clones, thus providing genome coverage of 6.3× haploid genome equivalents of grass carp. We selected gene-specific probes for screening the target gene clones in the HindIII library. In all, we obtained 31 positive clones, which were identified for every gene, with an average of 6.2 BAC clones per gene probe. Thus, we succeeded in constructing the desired BAC libraries, which should provide an important foundation for future physical mapping and whole-genome sequencing in grass carp.     

Construction and Characterization of a Human Chromosome 2-Specific BAC Library

We have constructed a human chromosome 2-specific bacterial artificial chromosome (BAC) library using DNA from the somatic cell hybrid GM10826. The average size of the clones is about 63 kb. The coverage and distribution of the library were estimated by screening with known polymorphic genetic markers and fluorescence in situ hybridization (FISH). Twentyone markers tested positive when DNA pools prepared from approximately one-sixth of the library were screened with 33 known markers. This is consistent with the theoretical calculation of 63% coverage at one genomic equivalent. This suggested that the coverage of the library is approximately 5-6×. FISH analysis with 54 BACs revealed single site hybridization to chromosome 2, and the clones were distributed randomly on the chromosome. We have also performed direct sequencing of the BAC insert ends to generate sequence-tagged sites suitable for mapping and chromosome walking. This is the first reported human chromosome 2-specific BAC library and should provide a resource for physical mapping and disease searching for this chromosome.     

A large-insert porcine library with sevenfold genome coverage: a tool for positional cloning of candidate genes for major quantitative traits

A porcine genomic bacterial artificial chromosome (BAC) library was constructed by cloning partial EcoRI-digested high-molecular-weight DNA from a Korean native boar into the EcoRI site of the pBACe3.6 vector. The library consists of about 165,000 clones with an average insert size of 125 kb, representing about seven genome equivalents of coverage. About 130,000 clones (corresponding to fivefold genome coverage) were arrayed in 14 superpools which were organized as four dimensional pools. The library was further characterized by PCR screening of 38 microsatellite probes. An average of 4.84 positive clones were selected per marker. This indicates that the library is unbiased and will be useful for initiating fine scale physical mapping of major QTL in pigs. The library is being used to isolate specific clones by screening with type I and type II marker clones located in the QTL region affecting intramuscular fat content on SSC6.     

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.     

Construction of a bacterial artificial chromosome library, determination of genome size, and characterization of an Hsp70 gene family in Phytophthora nicotianae

The oomycete plant pathogen Phytophthora nicotianae causes diseases on a wide range of plant species. To facilitate isolation and functional characterization of pathogenicity genes, we have constructed a large-insert bacterial artificial chromosome (BAC) library using nuclear DNA from P. nicotianae H1111. The library contains 10,752 clones with an average insert size of 90 kb and is free of mitochondrial DNA. The quality of the library was verified by hybridization with 37 genes, all of which resulted in the identification of multiple positive clones. The library is estimated to be 10.6 haploid genome equivalents based on hybridization of 23 single-copy genes and the genome size of P. nicotianae was estimated to be 95.5 Mb. Hybridization with a nuclear repetitive DNA probe revealed that 4.4% of clones in the library contained 28S rDNA. Hybridization of total genomic DNA to the library indicated that at least 39% of the BAC library contains repetitive DNA sequences. A BAC pooling strategy was developed for efficient library screening. The library was used to identify and characterize BAC clones containing an Hsp70 gene family whose four members were identified to be clustered within approximately 18 kb in the P. nicotianae genome based on the physical mapping of eight BACs spanning a genomic region of approximately 186 kb. The BAC library created provides an invaluable resource for the isolation of P. nicotianae genes and for comparative genomics studies.     

A sunflower BAC library suitable for PCR screening and physical mapping of targeted genomic regions

A sunflower BAC library consisting of 147,456 clones with an average size of 118 kb has been constructed and characterized. It represents approximately 5× sunflower haploid genome equivalents. The BAC library has been arranged in pools and superpools of DNA allowing screening with various PCR-based markers. Each of the 32 superpools contains 4,608 clones and corresponds to a 36 matrix pools. Thus, the screening of the entire library could be accomplished in less than 80 PCR reactions including positive and negative controls. As a demonstration of the feasibility of the concept, a set of 24 SSR markers covering about 36 cM in the sunflower SSR map (Tang et al. in Theor Appl Genet 105:1124–1136, 2002) have been used to screen the BAC library. About 125 BAC clones have been identified and then organized in 23 contigs by HindIII digestion. The contigs are anchored on the SSR map and thus constitutes a first-generation physical map of this region. The utility of this BAC library as a genomic resource for physical mapping and map-based cloning in sunflower is discussed.     

A bacterial artificial chromosome library for sugarcane

Modern cultivated sugarcane is a complex aneuploid polyploid with an estimated genome size of 3000 Mb. Although most traits in sugarcane show complex inheritance, a rust locus showing monogenic inheritance has been documented. In order to facilitate cloning of the rust locus, we have constructed a bacterial artificial chromosome (BAC) library for the cultivar R570. The library contains 103,296 clones providing 4.5 sugarcane genome equivalents. A random sampling of 240 clones indicated an average insert size of 130 kb allowing a 98% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a Genetix Q-BOT in a 4 × 4 double-spotted array on 22.5-cm² filters. Each set of five filters provides a genome coverage of 4x with 18,432 clones represented per filter. Screening of the library with three different barley chloroplast gene probes indicated an exceptionally low chloroplast DNA content of less than 1%. To demonstrate the library’s potential for map-based cloning, single-copy RFLP sugarcane mapping probes anchored to nine different linkage groups and three different gene probes were used to screen the library. The number of positive hybridization signals resulting from each probe ranged from 8 to 60. After determining addresses of the signals, clones were evaluated for insert size and HindIII-fingerprinted. The fingerprints were then used to determine clone relationships and assemble contigs. For comparison with other monocot genomes, sugarcane RFLP probes were also used to screen a Sorghum bicolor BAC library and two rice BAC libraries. The rice and sorghum BAC clones were characterized for insert size and fingerprinted, and the results compared to sugarcane. The library was screened with a rust resistance RFLP marker and candidate BAC clones were subjected to RFLP fragment matching to identify those corresponding to the same genomic region as the rust gene. Received: 12 September 1998 / Accepted: 12 March 1999     

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.     

A bacterial artificial chromosome library for barley (Hordeum vulgare L.) and the identification of clones containing putative resistance genes

Modern cultivated barley is an important cereal crop with an estimated genome size of 5000 Mb. To develop the resources for positional cloning and structural genomic analyses in barley, we constructed a bacterial artificial chromosome (BAC) library for the cultivar Morex using the cloning enzyme HindIII. The library contains 313344 clones (816 384-well plates). A random sampling of 504 clones indicated an average insert size of 106 kbp (range=30–195 kbp) and 3.4% empty vectors. Screening the colony filters for chloroplast DNA content indicated an exceptionally low 1.5% contamination with chloroplast DNA. Thus, the library provides 6.3 haploid genome equivalents allowing a >99% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a Genetix Q-BOT in a 4×4 double-spotted array on 22.5-cm² filters. Each set of 17 filters allows the entire library to be screened with 18432 clones represented per filter. Screening the library with 40 single copy probes identified an average 6.4 clones per probe, with a range of 1–13 clones per probe. A set of resistance-gene analog (RGA) sequences identified 121 RGA-containing BAC clones representing 20 different regions of the genome with an average of 6.1 clones per locus. Additional screening of the library with a P-loop disease resistance primer probe identified 459 positive BAC clones. These data indicate that this library is a valuable resource for structural genomic applications in barley. Received: 20 September 1999 / Accepted: 25 March 2000     

Construction of a Bacterial Artificial Chromosome Library of TM-1, a Standard Line for Genetics and Genomics in Upland Cotton

A bacterial artificial chromosome (BAC) library was constructed for Gossyplum hirsutum acc. TM-1, a genetic and genomic standard line for Upland cotton. The library consists of 147 456 clones with an average insert size of 122.8 kb ranging from 97 to 240 kb. About 96.0% of the clones have inserts over 100 kb. Therefore, this library represents theoretically 7.4 haploid genome equivalents based on an AD genome size of 2 425 Mb. Clones were stored in 384 384- well plates and arrayed into multiplex pools for rapid and reliable library screening. BAC screening was carded out by four-round polymerase chain reactions using 23 simple sequence repeats (SSR) markers, three sequence-related amplified polymorphism markers and one pair of pdmere for a gene associated with fiber development to test the quality of the library. Correspondingly, in total 92 positive BAC clones were Identified with an average four positive clones per SSR marker, ranging from one to eight hits. Additionally, since these SSR markers have been localized to chromosome 12 (A12) and 26 (D12) according to the genetic map, these BAC clonee are expected to serve as seeds for the physical mapping of these two homologous chromosomes, sequentially map-based cloning of quantitative trait loci or genes associated with Important agronomic traits.     

A bacterial artificial chromosome library for soybean PI 437654 and identification of clones associated with cyst nematode resistance

We have constructed a soybean bacterial artificial chromosome (BAC) library using the plant introduction (PI) 437654. The library contains 73728 clones stored in 192384-well microtiter plates. A random sampling of 230 BACs indicated an average insert size of 136 kb with a range of 20 to 325 kb, and less than 4% of the clones do not contain inserts. Ninety percent of BAC clones in the library have an average insert size greater than 100 kb. Based on a genome size of 1115 Mb, library coverage is 9 haploid genome equivalents. Screening the BAC library colony filters with cpDNA sequences showed that contamination of the genomic library with chloroplast clones was low (1.85%). Library screening with three genomic RFLP probes linked to soybean cyst nematode (SCN) resistance genes resulted in an average of 18 hits per probe (range 7 to 30). Two separate pools of forward and reverse suppression subtractive cDNAs obtained from SCN-infected and uninfected roots of PI 437654 were hybridized to the BAC library filters. The 488 BACs identified from positive signals were fingerprinted and analyzed using FPC software (version 4.0) resulting in 85 different contigs. Contigs were grouped and analyzed in three categories: (1) contigs of BAC clones which hybridized to forward subtracted cDNAs, (2) contigs of BAC clones which hybridized to reverse subtracted cDNAs, and (3) contigs of BAC clones which hybridized to both forward and reverse subtracted cDNAs. This protocol provides an estimate of the number of genomic regions involved in early resistance response to a pathogenic attack.     

Construction of a watermelon BAC library and identification of SSRs anchored to melon or Arabidopsis genomes

A bacterial artificial chromosome (BAC) library was constructed for watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus) with an average insert-size of 106 kb, providing 21 haploid genome equivalents. The library was used to identify BAC clones that are anchored to probes evenly distributed on the genomes of melon or Arabidopsis. Twenty eight probes (representing 66% of the tested probes) from melon and 30 probes (65%) from Arabidopsis identified positive BAC clones. Two methods were implemented to identify SSRs from the positively hybridizing BAC clones. First, analysis of BAC end sequences revealed 37 SSRs. For the second method, pooled DNA of BACs identified by the melon probes was used to develop a shotgun library. The library was then screened with synthetic SSR oligonucleotides by hybridization. Sequence analysis of positively hybridizing shotgun clones revealed 142 different SSRs. Thirty eight SSRs were characterized using three watermelon cultivars, five plant introduction (PI) accessions of C. lanatus var lanatus and four PIs of C. lanatus var citroides. Of these, 36 (95%) were found to be polymorphic with up to six alleles per marker. Polymorphism information content values for polymorphic markers varied between 0.22 and 0.79 with an average of 0.53. The methods described herein will be valuable for the construction of a watermelon linkage map with SSRs evenly distributed on its genome that is anchored to the genomes of melon and Arabidopsis.     

An improved method to identify BAC clones using pooled overgos

Hybridization using overgo probes is an established approach for screening arrayed bacterial artificial chromosome (BAC) libraries. We have improved the use of overgos by increasing the yield of positive clones using reduced levels of radioisotopes and enzyme. The strategy involves labeling with all four radiolabeled nucleotides in a hot pulse followed by a cold nucleotide chase and then extending the exposure time to compensate for reduced specific activity of the probes. The resulting cost savings and reduced human exposure to radiation make the use of highly pooled overgo probes a more attractive approach for screening of BAC libraries from organisms with large genomes.     

Consolidation of the genetic and cytogenetic maps of turbot (Scophthalmus maximus) using FISH with BAC clones

Bacterial artificial chromosomes (BAC) have been widely used for fluorescence in situ hybridization (FISH) mapping of chromosome landmarks in different organisms, including a few in teleosts. In this study, we used BAC-FISH to consolidate the previous genetic and cytogenetic maps of the turbot (Scophthalmus maximus), a commercially important pleuronectiform. The maps consisted of 24 linkage groups (LGs) but only 22 chromosomes. All turbot LGs were assigned to specific chromosomes using BAC probes obtained from a turbot 5× genomic BAC library. It consisted of 46,080 clones with inserts of at least 100 kb and <5 % empty vectors. These BAC probes contained gene-derived or anonymous markers, most of them linked to quantitative trait loci (QTL) related to productive traits. BAC clones were mapped by FISH to unique marker-specific chromosomal positions, which showed a notable concordance with previous genetic mapping data. The two metacentric pairs were cytogenetically assigned to LG2 and LG16, and the nucleolar organizer region (NOR)-bearing pair was assigned to LG15. Double-color FISH assays enabled the consolidation of the turbot genetic map into 22 linkage groups by merging LG8 with LG18 and LG21 with LG24. In this work, a first-generation probe panel of BAC clones anchored to the turbot linkage and cytogenetical map was developed. It is a useful tool for chromosome traceability in turbot, but also relevant in the context of pleuronectiform karyotypes, which often show small hardly identifiable chromosomes. This panel will also be valuable for further integrative genomics of turbot within Pleuronectiformes and teleosts, especially for fine QTL mapping for aquaculture traits, comparative genomics, and whole-genome assembly.     

Construction and characterization of a common bean bacterial artificial chromosome library

We have constructed a common bean (Phaseolus vulgaris L.) bacterial artificial chromosome (BAC) library consisting of 33 792 clones and an estimated 3- to 5-fold coverage of the common bean genome. Leaf nuclei were used as the source for high-molecular-weight DNA, and an endonuclease/methylase competition assay was employed to partially cleave the DNA. The library was screened with a number of nuclear and mitochondrial probes. Each nuclear probe identified at least two BACs with an average insert size of ca. 100 kb. Only 26 clones were identified after hybridizing with mitochondrial probes, indicating contamination with organellar sequences is low. Numerous clones could be identified after screening the library with two repetitive probes flanking the nuclear fertility restorer Fr. Intriguingly, 12 clones appeared to hybridize to both markers, and restriction analysis of these clones revealed that they can be assembled into maximally four contigs, suggesting that these repetitive probes may be useful for the physical mapping of the Fr locus.     

Construction of a bacterial artificial chromosome (BAC) library and identification of overlapping BAC clones with chromosome 4-specific RFLP markers in rice

 To facilitate construction of physical map of the rice genome, a bacterial artificial chromosome (BAC) library of IR64 genomic DNA was constructed. It consists of 18 432 clones and contains 3.28 rice genomic equivalents. The insert size ranged from 37 to 364 kb with an average of 107 kb. We used 31 RFLP markers on chromosome 4 to screen the library by colony hybridization. Sixty eight positive clones were identified with 2.2 positive clones per RFLP marker. The positive clones were analyzed to generate 29 contigs whose sizes ranged from 50 to 384 kb with an average of 145.6 kb. Chromosome walking was initiated for ten contigs linked to resistance genes. Thirty eight BAC clones were obtained and two contigs were integrated. Altogether, they covered 5.65 Mb (15.1%) of chromosome 4. These contigs may be used as landmarks for physical mapping of chromosome 4, and as starting points for chromosome walking towards the map-based cloning of disease resistance genes which were located nearby. Received: 15 November 1996 / Accepted: 24 January 1997     

FISH physical mapping with barley BAC clones

Fluorescence in situ hybridization (FISH) is a useful technique for physical mapping of genes, markers, and other single- or low-copy sequences. Since clones containing less than 10 kb of single-copy DNA do not reliably produce detectable signals with current FISH techniques in plants, a bacterial artificial chromosome (BAC) partial library of barley was constructed and a FISH protocol for detecting unique sequences in barley BAC clones was developed. The library has a 95 kb average barley insert, representing about 20% of a barley genome. Two BAC clones containing hordein gene sequences were identified and partially characterized. FISH using these two BAC clones as probes showed specific hybridization signals near the end of the short arm of one pair of chromosomes. Restriction digests of these two BAC clones were compared with restriction patterns of genomic DNA; all fragments contained in the BAC clones corresponded to bands present in the genomic DNA, and the two BAC clones were not identical. The barley inserts contained in these two BAC clones were faithful copies of the genomic DNA. FISH with four BAC clones with inserts varying from 20 to 150 kb, showed distinct signals on paired chromatids. Physical mapping of single- or low-copy sequences in BAC clones by FISH will help to correlate the genetic and physical maps. FISH with BAC clones also provide an additional approach for saturating regions of interest with markers and for constructing contigs spanning those regions.