Construction and characterization of the IGF Arabidopsis BAC library

A bacterial artificial chromosome (BAC) library has been established for Arabidopsis thaliana (ecotype Col-0) covering about seven haploid nuclear genome equivalents. This library, called the Institut für Genbiologische Forschung (IGF) BAC library, consists of 10?752 recombinant clones carrying inserts (generated by partial EcoRI digestion) of an average size of about 100?kb in a modified BAC vector, pBeloBAC-Kan. Hybridization with organellar DNA and nuclear repetitive DNA elements revealed the presence of 1.1% clones with mitochondrial DNA, 0.2% clones with plastid DNA, 3.2% clones with the 180?bp paracentromeric repeat, 1.6% clones with 5S rDNA, and 10.8% clones with the 18S-25S rDNA repeat. With its extensive genome coverage, its rather uniformly sized inserts (80?kb?<85% <120?kb) and low contamination with organellar DNA, this library provides an excellent resource for A. thaliana genomic mapping, map-based gene cloning, and genome sequencing.     

Construction of a BAC library and identification of Dmrt1 gene of the rice field eel, Monopterus albus

A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from the rice field eel (Monopterus albus). The BAC library consists of a total of 33,000 clones with an average insert size of 115 kb. Based on the rice field eel haploid genome size of 600 Mb, the BAC library is estimated to contain approximately 6.3 genome equivalents and represents 99.8% of the genome of the rice field eel. This is first BAC library constructed from this species. To estimate the possibility of isolating a specific clone, high-density colony hybridization-based library screening was performed using Dmrt1 cDNA of the rice field eel as a probe. Both library screening and PCR identification results revealed three positive BAC clones which were overlapped, and formed a contig covering the Dmrt1 gene of 195 kb. By sequence comparisons with the Dmrt1 cDNA and sequencing of first four intron-exon junctions, Dmrt1 gene of the rice field eel was predicted to contain four introns and five exons. The sizes of first and second intron are 1.5 and 2.6 kb, respectively, and the sizes of last two introns were predicted to be about 20 kb. The Dmrt1 gene structure was conserved in evolution. These results also indicate that the BAC library is a useful resource for BAC contig construction and molecular isolation of functional genes.     

Construction and characterization of the IGF Arabidopsis BAC library

A bacterial artificial chromosome (BAC) library has been established for Arabidopsis thaliana (ecotype Col-0) covering about seven haploid nuclear genome equivalents. This library, called the Institut für Genbiologische Forschung (IGF) BAC library, consists of 10 752 recombinant clones carrying inserts (generated by partial EcoRI digestion) of an average size of about 100 kb in a modified BAC vector, pBeloBAC-Kan. Hybridization with organellar DNA and nuclear repetitive DNA elements revealed the presence of 1.1% clones with mitochondrial DNA, 0.2% clones with plastid DNA, 3.2% clones with the 180 bp paracentromeric repeat, 1.6% clones with 5S rDNA, and 10.8% clones with the 18S-25S rDNA repeat. With its extensive genome coverage, its rather uniformly sized inserts (80 kb <85% <120 kb) and low contamination with organellar DNA, this library provides an excellent resource for A. thaliana genomic mapping, map-based gene cloning, and genome sequencing. Received: 26 November 1997 / Accepted: 19 February 1998     

A bacterial artificial chromosome library for the Chinese alligator (Alligator sinensis)

Chinese alligator (Alligator sinensis) is a rare and endangered species endemic to China. To better understand genetic details of the Chinese alligator genomic structure, a highly redundant bacterial artificial chromosome (BAC) library was constructed. This library consists of 216,238 clones with an average insert size of about 90kb, indicating that the library contains 6.8-fold genome equivalents. Subsequently, we constructed a 516kb contig map for the Chinese alligator olfactory receptor (OR) genes, which spans nine BAC clones, and subjected the BACs to full sequencing. The sequence analysis revealed that this contig contained 16 OR functional genes and meanwhile demonstrated that the nine BACs, which constituted the contig, overlapped correctly, proving the usability of this genome library. As a result, this BAC library could provide a useful platform for physical mapping, genome sequencing or complex analysis of targeted genomic regions for this rare species.     

Construction, characterization and FISH mapping of a bacterial artificial chromosome library of Chinese pangolin (Manis pentadactyla)

Chinese pangolins as a representative species in the order Pholidota have highly specified morphological characters and occupy an important place in the mammalian phylogenetic tree. To obtain genomic data for this species, we have constructed a bacterial artificial chromosome (BAC) library of Chinese pangolin. The library contains 208,272 clones with an average insert size of 122.1 kb and represents approximately eight times the Chinese pangolin haploid genome (if we assume that the Chinese pangolins have a genome size similar to human). One hundred and twenty randomly-selected BAC clones were mapped onto Chinese pangolin chromosomes by fluorescence in situ hybridization (FISH), showing a largely unbiased chromosomal distribution. Several clones containing repetitive DNA and ribosomal DNA genes were also found. The BAC library and FISH mapped BAC clones are useful resources for comparative genomics and cytogenetics of mammals and in particular, the ongoing genome sequencing project of Chinese pangolins.     

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.     

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.     

BAC FISH analysis in Allium cepa.

Onion (Allium cepa L.; 1C=15,000 Mb) is an agriculturally important plant. The genome of onion has been extensively studied at the conventional cytogenetic level, but molecular analyses have lagged behind due to its large genome size. To overcome this bottleneck, a partial bacterial artificial chromosome (BAC) library of onion was constructed. The average insert size of the BAC library was about 100 kb. A total of 48,000 clones, corresponding to 0.32 genome equivalent, were obtained. Fluorescent in situ hybridization (FISH) screening resulted in identification of BAC clones localized on centromeric, telomeric, or several limited interstitial chromosomal regions, although most of the clones hybridized with entire chromosomes. The partial BAC library proved to be a useful resource for molecular cytogenetic studies of onion, and should be useful for further mapping and sequencing studies of important genes of this plant. BAC FISH screening is a powerful method for identification of molecular cytogenetic markers in large-genome plants.     

Construction and Characterization of the BAC Library for Common Carp <Emphasis Type="Italic">Cyprinus Carpio</Emphasis> L. And Establishment of Microsynteny with Zebrafish <Emphasis Type="Italic">Danio Rerio</Emphasis>

A bacterial artificial chromosome (BAC) library of common carp Cyprinus carpio L. was constructed as a part of ongoing common carp genome project, which is aiming assembly of common carp genome. The library, containing a total of 92,160 BAC clones with an average insert size of 141 kb, was constructed into the restriction site of Hind III on BAC vector CopyControl pCC1BAC, covering 7.7 X haploid genome equivalents. Three dimension pools and superpools of the BAC library were established and 23 positive clones of 14 targets were identified from one-fifth of the BAC library. Pilot project of BAC end sequencing was conducted on 2,688 BAC ends from 1,344 clones and harvested 2,522 high-quality Q20 sequences with average length of 677 bp. The sequencing success rate was 93.8% and pair-end success rate was 92.3%. A total of 212 microsyntenies had been established between common carp and zebrafish genomes as a trial for genome-wide comparative genomics in these two closely related species.     

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 bacterial artificial chromosome (BAC) library for citrus and identification of BAC contigs containing resistance gene candidates

A BAC library was constructed from the genomic DNA of an intergeneric Citrus and Poncirus hybrid. The library consists of 24,576 clones with an average insert size of 115 kb, representing approximately seven haploid genome equivalents and is able to give a greater than 99% probability of isolating single-copy citrus DNA sequences from this library. High-density colony hybridization-based library screening was performed using DNA markers linked to the citrus tristeza virus (CTV) resistance gene and citrus disease resistance gene candidate (RGC) sequences. Between four and eight clones were isolated with each of the CTV resistance gene-linked markers, which agrees with the library’s predicted genome coverage. Three hundred and twenty-two clones were identified using 13 previously cloned citrus RGC sequences as probes in library screening. One to four fragments in each BAC were shown to hybridize with RGC sequences. One hundred and nine of the RGC BAC clones were fingerprinted using a sequencing gel-based procedure. From the fingerprints, 25 contigs were assembled, each having a size of 120–250 kb and consisting of 2–11 clones. These results indicate that the library is a useful resource for BAC contig construction and molecular isolation of disease resistance genes. Received: 22 May 2000 / Accepted: 25 September 2000     

Construction of a hexaploid wheat (Triticum aestivum L.) bacterial artificial chromosome library for cloning genes for stripe rust resistance.

A hexaploid wheat (Triticum aestivum L.) bacterial artificial chromosome (BAC) library was constructed for cloning Yr5 and other genes conferring resistance to stripe rust (Puccinia striiformis f. sp. tritici). Intact nuclei from a Yr5 near-isogenic line were used to isolate high molecular weight DNA, which was partially cleaved with HindIII and cloned into pECBAC1 and pIndigoBAC-5 vectors. The wheat BAC library consisted of 422,400 clones arrayed in 1100 micro-titer plates (each plate with 384 wells). Random sampling of 300 BAC clones indicated an average insert size of 140 kb, with a size range from 25 to 365 kb. Ninety percent of the clones in the library had an insert size greater than 100 kb and fewer than 5% of the clones did not contain inserts. Based on an estimated genome size of 15,966 Mb for hexaploid wheat, the BAC library was estimated to have a total coverage of 3.58x wheat genome equivalents, giving approximately 96% probability of identifying a clone representing any given wheat DNA sequence. Twelve BAC clones containing an Yr5 locus-specific marker (Yr5STS7/8) were successfully selected by PCR screening of 3-dimensional BAC pools. The results demonstrated that the T. aestivum BAC library is a valuable genomic resource for positional cloning of Yr5. The library also should be useful in cloning other genes for stripe rust resistance and other traits of interest in hexaploid wheat.     

Construction of a BAC library for Chinese amphioxus Branchiostoma belcheri and identification of clones containing Amphi-Pax genes

Amphioxus is a crucial organism for the study of vertebrate evolution. Although a genomic BAC library of Branchiostoma floridae has been constructed, we report here another BAC library construction of its distant relative species Branchiostoma belcheri. The amphioxus BAC library established in present study consists of 45,312 clones arrayed in one hundred and eighteen 384-well plates. The average insert fragment size was 120 kb estimated by Pulsed Field Gel Electrophoresis (PFGE) analysis of 318 randomly selected clones. The representation of the library is about 12 equivalent to the genome, allowing a 99.9995% probability of recovering any specific sequence of interest. We further screened the library with 4 single copied Amphi-Pax genes and identified total of 26 positive clones with average of 6.5 clones for each gene. The result indicates this library is well suited for many applications and should also serve as a useful complemental resource for the scientific community.     

A BAC library of the East African haplochromine cichlid fish Astatotilapia burtoni

A BAC library was constructed from Astatotilapia burtoni, a haplochromine cichlid that is found in Lake Tanganyika, East Africa, and its surrounding rivers. The library was generated from genomic DNA of blood cells and comprises 96,768 individual clones. Its median insert size is 150 kb and the coverage is expected to represent about 14 genome equivalents. The coverage evaluation was based on genome size estimates that were obtained by flow cytometry. In addition, hybridization screens with five probes largely corroborate the above coverage estimate, although the number of clones ranged from 5 to 22 authenticated clones per single copy probe. The BAC library described here is expected to be useful to the scientific community interested in cichlid genomics as an important resource to gain new insights into the rapid evolution of the great species diversity of haplochromine cichlid fishes.     

Construction and Characterization of Two Bacterial Artificial Chromosome Libraries of Zhikong Scallop, Chlamys farreri Jones et Preston, and Identification of BAC Clones Containing the Genes Involved in Its Innate Immune System

Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis, and physical mapping of scallop, two BAC libraries were constructed from nuclear DNA of Zhikong scallop, Chlamys farreri Jones et Preston. The libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99% of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0x haploid genome equivalents. Similarly, the MboI library consists of 7680 clones, with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 5.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping, and large-scale sequencing in the species.     

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 BAC library for the molecular dissection of a single wild beet centromere and sugar beet (Beta vulgaris) genome analysis.

We have constructed a sugar beet bacterial artificial chromosome (BAC) library of the chromosome mutant PRO1. This Beta vulgaris mutant carries a single chromosome fragment of 6-9 Mbp that is derived from the wild beet Beta procumbens and is transmitted efficiently in meiosis and mitosis. The library consists of 50,304 clones, with an average insert size of 125 kb. Filter hybridizations revealed that approximately 3.1% of the clones contain mitochondrial or chloroplast DNA. Based on a haploid genome size of 758 Mbp, the library represents eight genome equivalents. Thus, there is a greater than 99.96% probability that any sequence of the PROI genome can be found in the library. Approximately 0.2% of the clones hybridized with centromeric sequences of the PRO1 minichromosome. Using the identified BAC clones in fluorescence in situ hybridization experiments with PRO1 and B. procumbens chromosome spreads, their wild-beet origin and centromeric localization were demonstrated. Comparative Southern hybridization of pulsed-field separated PROI DNA and BAC inserts indicate that the centromeric region of the minichromosome is represented by overlapping clones in the library. Therefore, the PRO1 BAC library provides a useful tool for the characterization of a single plant centromere and is a valuable resource for sugar beet genome analysis.     

Construction and characterization of a bacterial artificial chromosome library from chili pepper

A library of the bacterial artificial chromosome (BAC) that consisted of a total of 78,336 clones with an average insert size of 80 kb was constructed from Capsicum annuum, 'CM334', which is resistant to Phytophthora capsici and PVY. Based on a haploid genome size of pepper of 2,702 Mbp/C, the BAC library was estimated to contain approximately three genome equivalents and represented at least 90% of the pepper genome. In order to determine the percentage of BAC clones that contained mitochondrial DNAs, the entire library was screened with probes of chili pepper mitochondrial DNAs. The result showed that only twenty-five clones, which is 0.03% of the total BAC clones, were hybridized to mitochondrial gene probes. This indicates that the library is comprised predominantly of the nuclear sequences. The library was also tested for isolating specific clones by screening with a few known genes from the chili pepper, phytoene synthase gene, and two MADS genes--HpMADS1 and HpMADS3. The result showed that the three clones for phytoene synthase and the two clones for each MADS gene were positively hybridized to the specific probes. This indicates that the library is highly reliable and represents a resource for initiating map-based cloning and contig mapping in chili pepper.     

Construction, characterization, and preliminary BAC-end sequencing analysis of a bacterial artificial chromosome library of white clover (Trifolium repens L.).

White clover (Trifolium repens L.) is a forage legume widely used in combination with grass in pastures because of its ability to fix nitrogen. We have constructed a bacterial artificial chromosome (BAC) library of an advanced breeding line of white clover. The library contains 37 248 clones with an average insert size of approximately 85 kb, representing an approximate 3-fold coverage of the white clover genome based on an estimated genome size of 960 Mb. The BAC library was pooled and screened by polymerase chain reaction (PCR) amplification using both white clover microsatellites and PCR-based markers derived from Medicago truncatula, resulting in an average of 6 hits per marker; this supports the estimated 3-fold genome coverage in this allotetraploid species. PCR-based screening of 766 clones with a multiplex set of chloroplast primers showed that only 0.5% of BAC clones contained chloroplast-derived inserts. The library was further evaluated by sequencing both ends of 724 of the clover BACs. These were analysed with respect to their sequence content and their homology to the contents of a range of plant gene, expressed sequence tag, and repeat element databases. Forty-three microsatellites were discovered in the BAC-end sequences (BESs) and investigated as potential genetic markers in white clover. The BESs were also compared with the partially sequenced genome of the model legume M. truncatula with the specific intention of identifying putative comparative-tile BACs, which represent potential regions of microsynteny between the 2 species; 14 such BACs were discovered. The results suggest that a large-scale BAC-end sequencing strategy has the potential to anchor a significant proportion of the genome of white clover onto the gene-space sequence of M. truncatula.     

A bacterial artificial chromosome (BAC) library of Malus floribunda 821 and contig construction for positional cloning of the apple scab resistance gene Vf.

The apple scab resistance gene Vf, originating from the wild species Malus floribunda 821, has been incorporated into a wide variety of apple cultivars through a classical breeding program. With the aim of isolating the Vf gene, a bacterial artificial chromosome (BAC) library consisting of 31 584 clones has been constructed from M. floribunda 821. From the analysis of 88 randomly selected BAC clones, the average insert size is estimated at 125 kb. If it is assumed that the genome size of M. floribunda 821 is 769 Mb/haploid, the library represents about 5x haploid genome equivalents. This provides a 99% probability of finding any specific sequence from this library. PCR-based screening of the library has been carried out using eight random genomic sequence-characterized amplified regions (SCARs), chloroplast- and mitochondria-specific SCARs, and 13 high-density Vf-linked SCAR markers. An average of five positive BAC clones per random SCAR has been obtained, whereas less than 1% of BAC clones are derived from the chloroplast or mitochondrial genomes. Most BAC clones identified with Vf-linked SCAR markers are physically linked. Three BAC contigs along the Vf region have been obtained by assembling physically linked BAC clones based on their fingerprints. The overlapping relatedness of BAC clones has been further confirmed by cytogenetic mapping using fiber fluorescence in situ hybridization (fiber-FISH). The M. floribunda 821 BAC library provides a valuable genetic resource not only for map-based cloning of the Vf gene, but also for finding many other important genes for improving the cultivated apple.