Construction of a BAC library of pearl millet, Pennisetum glaucum

A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from pearl millet (Pennisetum glaucum), and used as a resource for the isolation of microsatellite sequences. The library contains a total of 159,100 clones with an average insert size of 90 kb, and corresponds to 5.8 haploid genome equivalents. The BAC library was pooled for screening by the polymerase chain reaction (PCR) as well as robotically gridded on high-density filters. PCR-based screening of a subset of the library (4.7 haploid genome equivalents) using five sequence-tagged site (STS) and six microsatellite markers identified between 2 and 11 positives superpools (5.4 on average). The frequency of BAC clones carrying inserts of chloroplast DNA was estimated to be less than 1% by hybridisation with a rice chloroplast probe. Received: 30 January 2000 / Accepted: 16 October 2000     

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.     

Generation of a soybean BAC library, and identification of DNA sequences tightly linked to the Rps1-k disease resistance gene

 A soybean bacterial artificial chromosome (BAC) library, comprising approximately 45 000 clones, was constructed from high-molecular-weight nuclear DNA of cultivar Williams 82, which carries the Rps1-k gene for resistance against Phytophthora sojae. The library is stored in 130 pools with about 350 clones per pool. Completeness of the library was evaluated for 21 random sequences including four markers linked to the Rps1 locus and 16 cDNAs. We identified pools containing BACs for all sequences except for one cDNA. Additionally, when screened for possible contaminating BAC clones carrying chloroplast genes, no sequences homologous to two barley chloroplast genes were found. The estimated average insert size of the BAC clones was about 105 kb. The library comprises about four genome equivalents of soybean DNA. Therefore, this gives a probability of 0.98 of finding a specific sequence from this library. This library should be a useful resource for the positional cloning of Rps1-k, and other soybean genes. We have also evaluated the feasibility of an RFLP-based screening procedure for the isolation of BAC clones specific for markers that are members of repetitive sequence families, and are linked to the Rps1-k gene. We show that BAC clones isolated for two genetically linked marker loci, Tgmr and TC1-2, are physically linked. Application of this method in expediting the map-based cloning of a gene, especially from an organism, such as soybean, maize and wheat, with a complex genome is discussed. Received: 12 May 1998/Accepted: 24 August 1998     

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 of a bacterial artificial chromosome library of Medicago truncatula and identification of clones containing ethylene-response genes

 To facilitate genome analysis and map-based cloning of symbiotic genes in the model legume Medicago truncatula, a bacterial artificial chromosome (BAC) library was constructed. The library consists of 30 720 clones with an average insert size of approximately 100 kb, representing approximately five haploid-genome equivalents. The frequency of BAC clones carrying inserts of chloroplast DNA was estimated to be 1.4%. Screening of the library with single- or low-copy genes as hybridization probes resulted in the detection of 1–12 clones per gene. Hybridization of the library with repeated sequences such as rDNA genes and transposon-like elements of M. truncatula revealed the presence of 60 and 374 BAC clones containing the two sequences, respectively. The BAC library was pooled for screening by polymerase chain reaction (PCR)-amplification. To demonstrate the utility of this system, we used primers designed from a conserved region of the ein3-like loci of Arabidopsis thaliana and isolated six unique BAC clones from the library. DNA gel-blot and sequence analyses showed that these ein3-like clones could be grouped into three classes, an observation consistent with the presence of multiple ein3-like loci in M. truncatula. These results indicate that the BAC library represents a central resource for the map-based cloning and physical mapping in M. truncatula and other legumes. Received: 27 July 1998 / Accepted: 5 August 1998     

Construction of a rice bacterial artificial chromosome library and identification of clones linked to the Xa-21 disease resistance locus

A bacterial artificial chromosome (BAC) library consisting of 11 000 clones with an average DNA insert size of 125 kb was constructed from rice nuclear DNA. The BAC clones were stable in E. coli after 100 generations of serial growth. Transformation of the BAC clones by electroporation into E. coli was highly efficient and increased with decreasing size of the DNA inserts. The library was evaluated for the presence of organellar, repeated, and telomeric sequences. A very low percentage (<0.3%) of the library consisted of chloroplast and mitochondrial clones. Eighteen BACs were identified that hybridized with an Arabidopsis telomere repeat. Sixteen BACs hybridized with the AA genome-specific repetitive sequence pOs48. Twelve clones were isolated that hybridized with three DNA markers linked to the Xa-21 disease resistance locus. The results indicate that the BAC system can be used to clone and manipulate large pieces of plant DNA efficiently.     

Construction and characterisation of a BAC library for genome analysis of the allotetraploid coffee species (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

In order to promote genome research on coffee trees, one of the most important tropical crops, a bacterial artificial chromosome (BAC) library of the coffee allotetraploid species, Coffea arabica, was constructed. The variety IAPAR 59, which is widely distributed in Latin America and exhibits a fair level of resistance to several pathogens, was chosen. High-efficiency BAC cloning of the high molecular weight genomic DNA partially digested by HindIII was achieved. In total, the library contains 88,813 clones with an average insert size of 130 kb, and represents approximately eight C. arabica dihaploid genome equivalents. One original feature of this library is that it can be divided into four sublibraries with mean insert sizes of 96, 130, 183 and 210 kb. Characterisation of the library showed that less than 4.5% of the clones contained organelle DNA. Furthermore, this library is representative and shows good genome coverage, as established by hybridisation screening of high-density filters using a number of nuclear probes distributed across the allotetraploid genome. This Arabica BAC library, the first large-insert DNA library so far constructed for the genus Coffea, is well-suited for many applications in genome research, including physical mapping, map-based cloning, functional and comparative genomics as well as polyploid genome analyses.Communicated by J.W. Snape     

Two-dimensional screening of the Wageningen chicken BAC library

We have constructed a Bacterial Artificial Chromosome (BAC) library that provides 5.5-fold redundant coverage of the chicken genome. The library was made by cloning partial HindIII-digested high-molecular-weight (HMW) DNA of a female White Leghorn chicken into the HindIII site of the vector pECBAC1. Several modifications of standard protocols were necessary to clone efficiently large partial HindIII DNA fragments. The library consists of 49,920 clones arranged in 130 384-well plates. An average insert size of 134 kb was estimated from the analysis of 152 randomly selected BAC clones. The average number of NotI restriction sites per clone was 0.77. After individual growth, DNA was isolated of the pooled clones of each 384-well plate, and subsequently DNA of each plate was isolated from the individual row and column pools. Screening of the Wageningen chicken BAC library was performed by two-dimensional PCR with 125 microsatellite markers. For 124 markers at least one BAC clone was obtained. FISH experiments of 108 BAC clones revealed chimerism in less than 1%. The number of different BAC clones per marker present in the BAC library was examined for 35 markers which resulted in a total of 167 different BAC clones. Per marker the number of BAC clones varied from 1 to 11, with an average of 4.77. The chicken BAC library constitutes an invaluable tool for positional cloning and for comparative mapping studies. Received: 26 October 1999 / Accepted: 6 January 2000     

Construction and characterisation of a yeast artificial chromosome library containing three haploid maize genome equivalents

We have constructed a yeast artificial chromosome (YAC) library using high-molecular-weight DNA prepared from agarose-embedded leaf protoplasts of the maize inbred line UE95. This library contains 79 000 clones with an average insert size of 145 kb and should therefore represent approximately three haploid genome equivalents. The library is organised as an ordered array in duplicate microtitre plates. Forty-one pools of DNA from 1920 individual clones have been prepared for rapid screening of the library by the polymerase chain reaction (PCR). Using this approach, together with conventional colony hybridisation, we have been able to identify between one and eight positive clones for every probe used.     

Construction of a BAC library and a physical map of a major QTL for CBB resistance of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

A major quantitative trait loci (QTL) conditioning common bacterial blight (CBB) resistance in common bean (Phaseolus vulgaris L.) lines HR45 and HR67 was derived from XAN159, a resistant line obtained from an interspecific cross between common bean lines and the tepary bean (P. acutifolius L.) line PI319443. This source of CBB resistance is widely used in bean breeding. Several other CBB resistance QTL have been identified but none of them have been physically mapped. Four molecular markers tightly linked to this QTL have been identified suitable for marker assisted selection and physical mapping of the resistance gene. A bacterial artificial chromosome (BAC) library was constructed from high molecular weight DNA of HR45 and is composed of 33,024 clones. The size of individual BAC clone inserts ranges from 30 kb to 280 kb with an average size of 107 kb. The library is estimated to represent approximately sixfold genome coverage. The BAC library was screened as BAC pools using four PCR-based molecular markers. Two to seven BAC clones were identified by each marker. Two clones were found to have both markers PV-tttc001 and STS183. One preliminary contig was assembled based on DNA finger printing of those positive BAC clones. The minimum tiling path of the contig contains 6 BAC clones spanning an estimated size of 750 kb covering the QTL region.     

Use of the IGF BAC library for physical mapping of the Arabidopsis thaliana genome

In order to generate a physical map of the Arabidopsis thaliana genome based on bacterial artificial chromosome clones (BACs), an iterative high throughput hybridisation strategy was applied and its efficiency was evaluated. Thus, probes generated from both ends of 500 BAC clones selected from the Arabidopsis –IGF–BAC library were hybridised to the entire library gridded on high density filters. The 1000 hybridisation reactions identified 4496 clones (41.8% of the complete library, or 50.3% if organellar, centromeric, and ribosomal DNA carrying clones are excluded) which were assembled into a minimum of 220 contigs. These results demonstrate the viability of the applied ‘double-end clone-limited/sampling without replacement’ hybridisation strategy for the generation of a high resolution physical map, and provide a highly useful resource for map-based gene cloning approaches and further genome analysis.     

Construction and characterization of a bacterial artificial chromosome library of peach

A peach [Prunus persica (L.) Batch] bacterial artificial chromosome (BAC) library of var. Jingyu was constructed. Jingyu is a traditional variety, that displays many of the important agronomic characters of stone fruits. Since peach leaves are rich in polysaccharides, high-molecular-weight (HMW) DNA was extracted from leaf nuclei using a protocol adapted to peach. The HMW DNA embedded in agarose plugs was partially digested by HindIII. After size-selection by pulsed field gel electrophoresis, the selected DNA fragments were ligated to pBeloBAC11 and transformed into E. coli DH10B cells by electroporation. In total 20,736 recombinant clones were obtained. The BAC library has an average insert size of 95 kb and represents approximately 6.7 peach haploid genome equivalents. The BAC clones were stable in E. coli cell after 100 generations. The lack of hybridization to chloroplast and mitochondrial genes demonstrated that the library is predominantly composed of nuclear DNA. The library was screened with two molecular markers, W4 and P20, that are linked to white flesh and nectarine genes of peach, respectively. Ten positive clones were detected. Their fingerprints will be used to determine clone relationships and assemble contigs. This library should be well-suited for the map-based cloning of peach genes and genome physical mapping. Received: 18 January 2000 / Accepted: 29 May 2000     

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     

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 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 for the Rongchang pig breed and its use for the identification of genes involved in intramuscular fat deposition

In a search for genes affecting intramuscular fat deposition, we constructed a bacterial artificial chromosome (BAC) library for the whole genome of Rongchang pig, a domestic Chinese swine breed. The library consisted of approximately 192,000 clones, with an averaged insert size of 116 kb. Frequency of non-insert clone of the BAC library was no higher than 1.8%, based on estimation of 220 BAC clones randomly selected. We estimated the coverage of the library to be more than seven porcine genome equivalents. Subsequent screening of the BAC library with a three-step PCR procedure resulted in identification of seven candidate genes that were potentially involved in intramuscular fat deposition. The number of positive BAC clones ranged from 2 to 4 for each of the seven genes. One positive clone, containing the lipin1 gene, was fully sequenced by shotgun method to generate 118,041 bp porcine genomic sequences. The BAC clone contained complete DNA sequence of porcine lipin1 gene including all the exons and introns. Our results indicate that this BAC library is a useful tool for gene identification and help to serve as an important resource for future porcine genomic study.     

Human BAC library: construction and rapid screening

We have constructed a human genomic bacterial artificial chromosome (BAC) library using high molecular weight DNA from a pre-pro-B cell line, FLEB14-14, with a normal male diploid karyotype. This BAC library consists of 96 000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. The library can be screened by three different methods. (1) Probe hybridization to 31 high-density replica (HDR) filters: each filter contains 3072 BAC clones which were gridded in a 6×6 pattern. (2) Probe hybridization to two Southern blot filters to which 31 HindIII digests of the pooled 3072 BAC clones were loaded. This identifies a particular HDR filter for which further probe hybridization is performed to identify a particular clone(s). (3) Two-step polymerase chain reaction (PCR). First, PCR is applied to DNA samples prepared from ten superpools of 9600 BAC clones each to identify a particular superpool and the second PCR is applied to 40 unique DNA samples prepared from the four-dimensionally assigned BAC clones of the particular superpool. We present typical examples of the library screening using these three methods. The two-step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The theoretical consideration of the advantage of this method is presented. Furthermore, we have adapted Vectorette method to our BAC library for the isolation of terminal sequences of the BAC DNA insert to facilitate contig formation by BAC walking.     

Construction of a bacterial artificial chromosome library containing large Eco RI and Hin dIII genomic fragments of lettuce

 Existing bacterial artificial chromosome (BAC) vectors were modified to have unique EcoRI cloning sites. This provided an additional site for generating representative libraries from genomic DNA digested with a variety of enzymes. A BAC library of lettuce was constructed following the partial digestion of genomic DNA with HindIII or EcoRI. Several experimental parameters were investigated and optimized. The BAC library of over 50,000 clones, representing one to two genome equivalents, was constructed from six ligations; average insert sizes for each ligation varied between 92.5 and 142 kb with a combined average insert size of 111 kb. The library was screened with markers linked to disease resistance genes; this identified 134 BAC clones from four regions containing resistance genes. Hybridization with low-copy genomic sequences linked to resistance genes detected fewer clones than expected from previous estimates of genome size. The lack of hybridization to chloroplast and mitochondrial sequences demonstrated that the library was predominantly composed of nuclear DNA. The unique EcoRI site in the BAC vector should allow the integration of BAC cloning with other technologies that utilize EcoRI digestion, such as AFLP^TM markers and RecA-assisted restriction endonuclease (RARE) cleavage, to clone specific large EcoRI fragments from genomic DNA. Received: 5 August 1996 / Accepted: 23 August 1996     

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.