Construction of an arabidopsis BAC library and isolation of clones hybridizing with disease-resistance,gene-like sequences

A bacterial artificial chromosome (BAC) library consisting of 9,000 clones (representing a 4.5 genome equivalents) with an average DNA insert size of 60 kb was constructed from arabidopsis nuclear DNA. We have demonstrated the usefulness of this library by identifying one BAC clone that hybridizes with the arabidopsisPHYB gene and seven clones, representing four distinct classes, that hybridize to a putative disease-resistance gene. This library represents an additional resource for map-based cloning and physical mapping in arabidopsis.     

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.     

A novel manual pooling system for preparing three-dimensional pools of a deep coverage soybean bacterial artificial chromosome library

Compared with hybridization‐based techniques, polymerase chain reaction‐based screening of large insert libraries has been used widely as it is fast, easy and sensitive. However, various pooling strategies are needed to ensure efficient screening. It is time‐consuming and labourious to prepare three‐dimensional pools for a deep coverage bacterial artificial chromosome (BAC) library of soybean (1.12 × 10⁹ bp) in the absence of robotic facility. In the present study, we describe a novel manual pooling system for preparing three‐dimensional pools of a soybean BAC library. This simple technique enables a single researcher to construct three‐dimensional pools for a deep‐coverage (12 haploid genome equivalents) BAC library of soybean in less than 2 months without any robotic manipulation. When the prepared three‐dimensional pools were screened with 29 polymerase chain reaction‐based markers, an average of 9.2 clones per marker were identified. These identified clones will be useful either in quantitative trait loci gene isolation or in synteny study between soybean and other legumes including Lotus japonicus. This efficient pooling system could be applied to any other BAC libraries without the need for robotic manipulation.     

A fast and efficient method for isolation of the BAC end

As a new developmental vector system, the bacterial artificial chromosome (BAC) has been used widely in constructing genomic libraries and in generating transgenic animals. Isolation of the BAC insert end is useful to analyze the BAC clone. Here, we describe a fast and efficient method to obtain the BAC end by ligating the BAC fragments digested with Not I and another selected restriction enzyme into universal cloning vector, followed by determining the correct clones with HindIII digestion. Further DNA sequencing analysis verified the results mentioned above.     

A genome-wide, end-sequenced 129Sv BAC library resource for targeting vector construction

The majority of gene-targeting experiments in mice are performed in 129Sv-derived embryonic stem (ES) cell lines, which are generally considered to be more reliable at colonizing the germ line than ES cells derived from other strains. Gene targeting is reliant on homologous recombination of a targeting vector with the host ES cell genome. The efficiency of recombination is affected by many factors, including the isogenicity (H. te Riele et al., 1992, Proc. Natl. Acad. Sci. USA 89, 5128-5132) and the length of homologous sequence of the targeting vector and the location of the target locus. Here we describe the double-end sequencing and mapping of 84,507 bacterial artificial chromosomes (BACs) generated from AB2.2 ES cell DNA (129S7/SvEvBrd-Hprtb-m2). We have aligned these BACs against the mouse genome and displayed them on the Ensembl genome browser, DAS: 129S7/AB2.2. This library has an average insert size of 110.68 kb and average depth of genome coverage of 3.63- and 1.24-fold across the autosomes and sex chromosomes, respectively. Over 97% of the mouse genome and 99.1% of Ensembl genes are covered by clones from this library. This publicly available BAC resource can be used for the rapid construction of targeting vectors via recombineering. Furthermore, we show that targeting vectors containing DNA recombineered from this BAC library can be used to target genes efficiently in several 129-derived ES cell lines.     

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     

Isolation of HMW DNA from sunflower (Helianthus annuus L.) for BAC cloning

The cultivated sunflower (Helianthus annuus L.) is one of the most important oil crops in the world. The importance of sunflower oil in human nutrition and in the chemical industry makes the sunflower a major research interest. An essential element for genomic libraries is the preparation of high molecular weight (HMW) DNA. We developed 2 methods for isolating HMW sunflower DNA. We prepared the DNA from nuclei and from protoplasts isolated from mesophyll tissue with the enzymes cellulase RS and pectolyase Y23. The HMW DNA was digested with restriction endonucleases. The ethidium bromide-stained gel suggested the DNA to be completely digested. These results were confirmed by Southern analysis using a radiolabeled RFLP marker. Both methods made it possible to generate sufficient quantities of megabase-size sunflower DNA suitable for bacterial artificial chromosome (BAC) cloning.     

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.     

Isolation of megabase-size DNA from sorghum and applications for physical mapping and bacterial and yeast artificial chromosome library construction

A method was developed for the isolation of megabase-size DNA fromSorghum bicolor. Sorghum protoplasts were isolated from young leaf tissue, embedded in an agarose matrix as microbeads or plugs, followed by cell lysis and protein degradation. The DNA prepared by this method was larger than 1 Mb in size and readily digestible with restriction enzymes. The DNA was shown to be suitable for physical mapping, and was successfully used for the construction of BAC and YAC libraries.     

Bacterial artificial chromosome (BAC) library resource for positional cloning of pest and disease resistance genes in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Pest and disease problems are important constraints of cassava production and host plant resistance is the most efficient method of combating them. Breeding for host plant resistance is considerably slowed down by the crop’s biological constraints of a long growth cycle, high levels of heterozygosity and a large genetic load. More efficient methods such as gene cloning and transgenesis are required to deploy resistance genes. To facilitate the cloning of resistance genes, bacterial artificial chromosome (BAC) library resources have been developed for cassava. Two libraries were constructed from the cassava clones, TMS 30001, resistant to the cassava mosaic disease (CMD) and the cassava bacterial blight (CBB), and MECU72, resistant to cassava white fly. The TMS30001 library has 55 296 clones with an insert size range of 40–150 kb with an average of 80 kb, while the MECU72 library consists of 92 160 clones and an insert size range of 25–250 kb average of 93 kb. Based on a genome size of 772 Mb, the TMS30001 and MECU72 libraries have a 5 and 11.3 haploid genome equivalents and a 95 and 99 chance of finding any sequence, respectively. To demonstrate the potential of the libraries, the TMS30001 library was screened by southern hybridization using a cassava analog (CBB1) of the Xa21 gene from rice that maps to a region containing a QTL for resistance to CBB as probe. Five BAC clones that hybridized to CBB1 were isolated and a Hind III fingerprint revealed 2–3 copies of the gene in individual BAC clones. A larger scale analysis of resistance gene analogs (RGAs) in cassava has also been conducted in order to understand the number and organization of RGAs. To scan for gene and repeat DNA content in the libraries, end-sequencing was performed on 2301 clones from the MECU72 library. A total of 1705 unique sequences were obtained with an average size of 715 bp. Database homology searches using BLAST revealed that 458 sequences had significant homology with known proteins and 321 with transposable elements. The use of the library in positional cloning of pest and disease resistance genes is discussed.     

一种使用混合PCR筛选技术高效延伸水稻BAC—重叠群的方法

使用“克隆连克隆 (clonebyclone)”战略进行水稻基因组测序需要依赖于构建好的基因组物理图。工作着眼于水稻籼稻广陆矮 4号 (OryzasativaindicaGuangLuAi4)第四号染色体长臂上 5 6 .1～ 6 8cM的区域 ,采用PCR方法筛选BAC全库来延伸重叠群 ,构建物理图。通过参照特异遗传探针定位的BAC克隆 (seedBAC)末端序列设计了 14对引物 ,按特定规则分成 3组 ,分别以代表水稻BAC库 (共 2 2 36 8个BAC )的 2 33个BACpool为模板进行PCR反应 ,一共获得了 6 5个阳性BAC克隆 ,通过末端测序、酶切杂交等方法确定了其中 2 9个BAC克隆作为有效延伸的克隆 ,延伸了 8个重叠群。通过酶切杂交、末端测序等方法还获知阳性BAC的延伸方向、延伸长度以及与seedBAC之间的重叠长度。8个重叠群总的延伸长度达到5 10kb。与实验室原用于作物理图的其他方法如指纹图、点杂交等相比 ,该方法有高效率、高灵敏度、专一性好、可重复使用等优点。创新之处在于通过引物的合理分组和PCR实验条件的改进降低了假阳性和假阴性率     

A bacterial artificial chromosome (BAC) library for sunflower, and identification of clones containing genes for putative transmembrane receptors

Sunflower (Helianthus annuus L.) is an economically important oil seed crop with an estimated genome size of 3000 Mb. We have constructed a bacterial artificial chromosome (BAC) library for sunflower, which represents an estimated 4- to 5-fold coverage of the genome. Nuclei isolated from young leaves were used as a source of high-molecular-weight DNA and a partial restriction endonuclease digestion protocol was used to cleave the DNA. A random sample of 60 clones indicated an average insert size of 80 kb, implying a 95% probability of recovering any specific sequence of interest. The library was screened with chloroplast DNA probes. Only 0.1% of the clones were identified to be of chloroplast origin, indicating that contamination with organellar DNAs is very low. The utility of the library was evaluated by screening for the presence of genes for putative transmembrane receptors sharing epidermal growth factor (EGF) and integrin-like domains. First, a homologous sunflower EST (HaELP1) was obtained by degenerate RT-PCR cloning, using Arabidopsis thaliana genes (AtELP) as a source of consensus sequences. Three different BACs yielded positive hybridization signals when HaELP1 was used as a probe. BAC subcloning and sequencing demonstrated the presence of two different loci putatively homologous to genes for transmembrane proteins with EGF- and integrin-like domains from sunflower. This work demonstrates the suitability of the library for homology map-based cloning of sunflower genes and physical mapping of the sunflower genome.     

Sugar beet BAC library construction and assembly of a contig spanning <Emphasis Type="Italic">Rf1</Emphasis>, a restorer-of-fertility gene for Owen cytoplasmic male sterility

Rf1 is a nuclear gene that controls fertility restoration in cases of cytoplasmic male sterility caused by the Owen cytoplasm in sugar beet. In order to isolate the gene by positional cloning, a BAC library was constructed from a restorer line, NK198, with the genotype Rf1Rf1. The library contained 32,180 clones with an average insert size of 97.8 kb, providing 3.4 genome equivalents. Five AFLP markers closely linked to Rf1 were used to screen the library. As a result, we identified eight different BAC clones that were clustered into two contigs. The gap between the two contigs was filled by chromosome walking. To map the Rf1 region in more detail, we developed five cleaved amplified polymorphic sequence (CAPS) markers from the BAC DNAs identified, and carried out genotyping of 509 plants in the mapping population with the Rf1-flanking AFLP and CAPS markers. Thirteen plants in which recombination events had occurred in the vicinity of the Rf1 locus were identified and used to map the molecular markers relative to each other and to Rf1. In this way, we were able to restrict the possible location of the Rf1 gene to a minimum of six BAC clones spanning an interval of approximately 250 kb. The first two authors contributed equally to this work.     

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.     

一种快速、简便、高效cDNA合成及克隆策略

以鱼呼肠孤病毒双链ＲＮＡ为模板,建立一种快速、简便、高效的ｃＤＮＡ合成及克隆策略。在一定量模板条件下,采用随机六聚体为引物合成ｃＤＮＡ第一链。以退火方式形成双链ｃＤＮＡ,直接通过琼脂糖凝胶电泳检测其ｃＤＮＡ合成产量。双链ｃＤＮＡ经两端补齐后,平头连接于具有阳性选择标记的载体中,以高效电转化的方式进行快速克隆。     

A vector system with temperature-sensitive replication for gene disruption and mutational cloning in streptomycetes

Summary Replication of the Streptomyces ghanaensis plasmid pSG5 was shown to be temperature sensitive. The pSG5 replicon is stably inherited at temperatures below 34° C, but is lost at incubation temperatures above this. A family of cloning vectors was constructed using the pSG5 minimal replicon and different marker genes. The vectors obtained are small in size, have an intermediate copy number, possess a broad host range and are compatible with some other streptomycete vector systems. By increasing the incubation temperature, these vectors can be eliminated from their host cells very efficiently. The suitability of the pSG5 vector family for mutating chromosomal genes by gene disruption was demonstrated: pBN10, a pSG5 derivative containing an internal fragment of the phosphinothricyl-alanyl-alanine (PTT) resistance gene pat, was integrated into the chromosomal pat gene of the PTT-producer Streptomyces viridochromogenes thus inactivating PTT resistance. The integrated pBN10 plasmid was rescued from the chromosome, together with an adjacent fragment carrying DNA of the PTT biosynthetic cluster.     

Anopheles gambiae collagen IV genes: cloning,phylogeny and midgut expression associated with blood feeding and Plasmodium infection

A prerequisite for understanding the role that mosquito midgut extracellular matrix molecules play in malaria parasite development is proper isolation and characterisation of the genes coding for components of the basal lamina. Here we have identified genes coding for alpha1 and alpha2 chains of collagen IV from the major malaria vector, Anopheles gambiae. Conserved sequences in the terminal NC1 domain were used to obtain partial gene sequences of this functional region, and full sequence was isolated from a pupal cDNA library. In a DNA-derived phylogeny, the alpha1 and alpha2 chains cluster with dipteran orthologs, and the alpha2 is ancestral. The expression of collagen alpha1(IV) peaked during the pupal stage of mosquito development, and was expressed continuously in the adult female following a blood meal with a further rise detected in older mosquitoes. Collagen alpha1(IV) is also upregulated when the early oocyst of Plasmodium yoelii was developing within the mosquito midgut and may contribute to a larger wound healing response. A model describing the expression of basal lamina proteins during oocyst development is presented, and we hypothesise that the development of new basal lamina between the oocyst and midgut epithelium is akin to a wound healing process.     

A simple and reliable method for SSU rRNA gene DNA extraction, amplification, and cloning from single AM fungal spores

 We describe a method that allows quick and easy PCR amplification and cloning of nearly complete SSU rRNA genes from arbuscular mycorrhizal fungi. The procedure tested on spores from 37 different glomalean isolates was based on magnetic separation with Dynabeads, followed by nested PCR with two primer pairs. All trials led to visible amplification products of the expected size. Thereafter, the PCR fragments could be quickly and efficiently cloned by means of a topoisomerase-activated vector (pCR2.1-TOPO). The technique is rapid, uncomplicated and comparatively inexpensive. The use of single spores for DNA extraction has some advantages over multispore-preparations, e.g. it is less susceptible to contamination with other organisms present in the cultures. The method can be used for the quick and reliable preparation of a large number of samples and is highly reproducible. It could also be used for genes other than the SSU rRNA gene. Accepted: 25 October 2000