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 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 Sorghum propinquum BAC library,suitable for cloning genes associated with loss-of-function mutations during crop domestication

A large insert Sorghum propinquum BAC library has been constructed to analyze the physical organization of the sorghum genome and to facilitate positional cloning of sorghum genes and QTLs associated with the early stages of grain crop domestication. This library was established from 12 different ligations using high-molecular-weight DNA generated from either one cycle or two cycles of size selection. This library consists of 38 016 BAC clones with an estimated average insert size of 126 kb and coverage of 6.6 genome equivalents. The 6.6 genome-equivalent BAC library of S. propinquum provides a 99.7% probability of finding one or more BACs that contain genes of interest. Twenty mapped DNA probes, ten homologous and ten heterologous, were used to screen the library, and 121 positive clones were identified, 6.05 per locus or 6.37 per probe.     

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     

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.     

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.     

Bacterial artificial chromosome library of Finegoldia magna ATCC 29328 for genetic mapping and comparative genomics

We constructed a bacterial artificial chromosome (BAC) library of Finegoldia magna ATCC 29328 DNA to facilitate further genome analysis of F. magna. The BAC library contained 385 clones with an average insert size of 55 kb, representing a 10.1-fold genomic coverage. Repeated DNA hybridization using primer sets designed on the basis of BAC-end sequences yielded nine contigs covering 95% of the chromosome and two contigs covering 98% of the plasmid. The contigs were localized on the physical map of F. magna ATCC 29328 DNA. A total of 121 BAC-end sequences revealed 103 unique genes, which had not been previously reported for F. magna. The homolog ORF of albumin-binding protein (urPAB), one of the known virulence factors from F. magna, was sequenced and localized on the physical map. Homology analysis of 121 BAC-end sequences revealed that F. magna is most closely related to clostridia, particularly Clostridium tetani. This close relationship is consistent with the recent classification of peptostreptococci based on 16S rRNA sequence analysis. The BAC library constructed here will be useful for the whole genome sequencing project and other postgenomic applications.     

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 and characterization of a gridded cattle BAC library

A bovine genomic large-insert bacterial artificial chromosome (BAC) library has been constructed from leukocytes of a Holstein-Friesian male. Size fractionated DpnII-digested genomic DNA was ligated to the dephosphorylated BamH1 ends of a pBACe3.6 vector. Approximately 8.3 x 10(4) individual BAC clones were picked into 384-well plates. Two-hundred and sixty-seven randomly chosen clones were characterized by pulsed-field gel electrophoresis (PFGE). The average insert size was 104 kb with a frequency of clones without inserts of 5.5%. Thirty-four BAC clones were mapped by fluorescence in situ hybridization (FISH) to cattle chromosomes. Three showed signals at more than one location, one of them on the centromeric regions of all autosomes, indicating that the clone contains centromeric repeats. A subset of these BAC clones was used for the development of sequence tagged sites. Both subcloning and direct sequencing of the BACs were used for generating sequence tagged site information. The clones from the library were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Membranes and superpools are available through the Resource Centre of the German Human Genome Project in Berlin (http:// www.rzpd.de).     

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.     

Channel catfish BAC-end sequences for marker development and assessment of syntenic conservation with other fish species

In the present study, 25 195 BAC ends for channel catfish (Ictalurus punctatus) were sequenced, generating 20 366 clean BAC-end sequences (BES), with an average read length of 557 bp after trimming. A total of 11 414 601 bp were generated, representing approximately 1.2% of the catfish genome. Based on this survey, the catfish genome was found to be highly AT-rich, with 60.7% A+T and 39.3% G+C. Approximately 12% of the catfish genome consisted of dispersed repetitive elements, with the Tc1/mariner transposons making up the largest percentage by base pair (4.57%). Microsatellites were detected in 17.5% of BES. Catfish BACs were anchored to the zebrafish and Tetraodon genome sequences by BLASTN, generating 16% and 8.2% significant hits (E < e(-5)) respectively. A total of 1074 and 773 significant hits were unique to the zebrafish and Tetraodon genomes, respectively, of which 417 and 406, respectively, were identified as known genes in other species, providing a major genome resource for comparative genomic mapping.     

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.     

Development of a BAC library for yellow-poplar (Liriodendron tulipifera) and the identification of genes associated with flower development and lignin biosynthesis

Liriodendron tulipifera L., a member of the Magnoliaceae, occupies an important phylogenetic position as a basal angiosperm that has retained numerous putatively ancestral morphological characters, and thus has often been used in studies of the evolution of flowering plants and of specific gene families. However, genomic resources for these early branching angiosperm lineages are very limited. In this study, we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from L. tulipifera. Flow cytometry estimates that this nuclear genome is approximately 1,802 Mbp per haploid genome (±16 SD). The BAC library contains 73,728 clones, a 4.8-fold genome coverage, with an average insert size of 117 kb, a chloroplast DNA content of 0.2%, and little to no bacterial sequences nor empty vector content clones. As a test of the utility of this BAC library, we screened the library with six single/low-copy genic probes. We obtained at least two positive clones for each gene and confirmed the clones by DNA sequencing. A total of 182 paired end sequences were obtained from 96 of the BAC clones. Using BLAST searches, we found that 25% of the BAC end sequences were similar to DNA sequences in GenBank. Of these, 68% shared sequence with transposable elements and 25% with genes from other taxa. This result closely reflected the content of random sequences obtained from a small insert genomic library for L. tulipifera, indicating that the BAC library construction process was not biased. The first genomic DNA sequences for Liriodendron genes are also reported. All the Liriodendron genomic sequences described in this paper have been deposited in the GenBank data library. The end sequences from shotgun genomic clones and BAC clones are under accession DU169330–DU169684. Partial sequences of Gigantea, Frigida, LEAFY, cinnamyl alcohol dehydrogenase, 4-coumarate:CoA ligase, and phenylalanine ammonia-lyase genes are under accession DQ223429–DQ223434. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Sequencing,annotation and comparative analysis of nine BACs of giant panda (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>)

A 10-fold BAC library for giant panda was constructed and nine BACs were selected to generate finish sequences. These BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of giant panda newly generated by the Illumina GA sequencing technology. Complete sanger sequencing, assembly, annotation and comparative analysis were carried out on the selected BACs of a joint length 878 kb. Homologue search and de novo prediction methods were used to annotate genes and repeats. Twelve protein coding genes were predicted, seven of which could be functionally annotated. The seven genes have an average gene size of about 41 kb, an average coding size of about 1.2 kb and an average exon number of 6 per gene. Besides, seven tRNA genes were found. About 27 percent of the BAC sequence is composed of repeats. A phylogenetic tree was constructed using neighbor-join algorithm across five species, including giant panda, human, dog, cat and mouse, which reconfirms dog as the most related species to giant panda. Our results provide detailed sequence and structure information for new genes and repeats of giant panda, which will be helpful for further studies on the giant panda.     

水稻中一个NBS-LRR抗病同源基因家族的克隆和分析

利用克隆的抗病基因同源序列RS13作为探针,从水稻IR64的BAC文库中筛选到4个阳性克隆,其中一个克隆14E19能够覆盖其余3个克隆。对14E19进行全序列测定和分析,获得了73kb的全长DNA序列,基因预测显示其上有4个编码NBS-LRR结构域的基因(NL),分别命名为NL-A,B,C和D。对具有相同基因组背景的IRBB56同一染色体位置上跨度更大的BAC克隆106P13进行分析,发现其上有10个NL同源拷贝,其中4个同14E19上的NL一样。搜索日本晴、93—11、广陆矮4号的序列,发现三者有类似的同源序列。但与已知的NBS-LRR抗病基因同源性较低,说明NL是一个至少由10个成员(分别命名为NL-A至J)组成的新基因家族。对NL家族进行RT-PCR和cDNA库筛选分析,发现NL-B基因能够在抗白叶枯病品系IRBB4中表达,暗示该基因参与了抗病反应。     

BAC clones generated from sheared DNA

BAC libraries generated from restriction-digested genomic DNA display representational bias and lack some sequences. To facilitate completion of genome projects, procedures have been developed to create BACs from DNA physically sheared to create fragments extending up to 200 kb. The DNA fragments were repaired to create blunt ends and ligated to a new BAC vector. This approach has been tested by generating BAC libraries from Drosophila DNA with insert lengths between 50 and 150 kb. The libraries lack chimeric clone problems as determined by mapping paired BAC-end sequences to the assembled fly genome sequence. The utility of "sheared" libraries was demonstrated by closure of a previous clone gap and by isolation of clones from telomeric regions, which were notably absent from previous Drosophila BAC libraries.     

Construction and characterization of a papaya BAC library as a foundation for molecular dissection of a tree-fruit genome

A bacterial artificial chromosome (BAC) library was constructed from high-molecular-weight DNA isolated from young leaves of papaya (Carica papaya L.). This BAC library consists of 39168 clones from two separate ligation reactions. The average insert size of the library is 132 kb; 96.5% of the 18700 clones from the first ligation contained inserts that averaged 86 kb in size, 95.7% of the 20468 clones from the second ligation contained inserts that averaged 174 kb in size. Two sorghum chloroplast probes hybridized separately to the library and revealed a total of 504 chloroplast clones or 1.4% of the library. The entire BAC library was estimated to provide 13.7× papaya-genome equivalents, excluding the false-positive and chloroplast clones. High-density filters were made containing 94% or 36864 clones of the library with 12.7× papaya-genome equivalents. Eleven papaya-cDNA and ten Arabidopsis-cDNA probes detected an average of 22.8 BACs per probe in the library. Because of its relatively small genome (372 Mbp/1 C) and its ability to produce ripe fruit 9 to 15 months after planting, papaya shows promise as a model plant for studying genes that affect fruiting characters. A rapid approach to locating fruit-controlling genes will be to assemble a physical map based on BAC contigs to which ESTs have hybridized. A physical map of the papaya genome will significantly enhance our capacity to clone and manipulate genes of economic importance. Received: 11 April 2000 / Accepted: 28 July 2000     

Association and in Silico Assignment of Sequences from Turkey BACs

Bacterial artificial chromosomes (BACs) provide an important resource in genetic mapping. An initial set of BACs corresponding to microsatellite markers in the turkey (Meleagris gallopavo) was isolated from the CHORI-260 turkey BAC library. The selected markers were distributed on both macro- and microchromosomes and included a genetically unlinked marker. End sequences were obtained for a subset of the recovered BACs and compared to the chicken whole genome sequence. Close association of the turkey BAC-end sequences and original marker sequences was generally conserved in the chicken genome. Gene content of the turkey BACs is predicted from the comparative sequence alignments.