Average Insert Size Research Articles

Construction of a BAC library for Haplochromis chilotes, a cichlid fish from Lake Victoria.

Cichlid fishes in Lake Victoria are model organisms for studying rapid radiation and speciation. On the way to examine the molecular basis of how these cichlid fishes achieved such a remarkable morphological diversification, we constructed a bacterial artificial chromosome (BAC) library derived from a cichlid species, Haplochromis chilotes, from Lake Victoria. The library includes 157,056 clones with the average insert size of 128 kb, corresponding to a 10-fold coverage of the H. chilotes genome. Given that the cichlid fishes endemic to Lake Victoria are closely related to one another phylogenetically and their genomes are nearly identical, this BAC library can be utilized to isolate genes from the more than 200 Haplochromine cichlid species in Lake Victoria.

Ordered cosmid library of the Mesorhizobium loti MAFF303099 genome for systematic gene disruption and complementation analysis.

For effective exploitation of the genome sequence information of Lotus microsymbiont, Mesorhizobium loti MAFF303099, to discover gene functions, we have constructed an ordered and mutually overlapping cosmid library using an IncP broad host-range vector. The library consisted of 480 clones to cover approximately 99.6% of the genome with average insert size and overlap of 26.9 and 11.1 kbp, respectively. The genome of M. loti consists of a single chromosome and two plasmids. The chromosome (7,036,071 bp) was covered 99.68% by 445 clones with four gaps, although two clones were unstable in E. coli. The larger plasmid pMLa (351,911 bp) was completely covered by 23 clones, while the smaller pMLb (208,315 bp) was covered 98.85% by 12 clones with two gaps. We have also made ancillary plasmids to facilitate the construction of deletion mutants using derivatives of the library clones. As a pilot experiment to uncover regions which contain novel symbiotic genes, 13 deletion mutants were constructed to lack in total 180.5 kbp of the genome. All the mutants formed apparently normal nodules and supported symbiotic nitrogen fixation, however, one mutant that lacked a 5.3 kbp chromosomal region, 4,551,930-4,557,222, did not produce normal exopolysaccharides as judged by fluorescence on medium containing Calcofluor. The results supported the effectiveness of the approach to detect gene functions.

Construction and characterization of a bacterial artificial chromosome library of banana (Musa acuminata Colla).

A bacterial artificial chromosome (BAC) library for banana was constructed from leaves of the wild diploid 'Calcutta 4' clone (Musa acuminata subsp. Burmannicoides 2n = 2 x = 22). 'Calcutta 4' is widely used in breeding programs for its resistance to the current major disease of banana and is being used to build a genetic reference map of banana. As banana leaves are particularly rich in polyphenols and polysaccharides a protocol was adapted to isolate intact nuclei and high-molecular-weight (HMW) DNA. A total of 55,152 clones with an average insert size of 100 kb were picked. The frequency of BAC clones carrying inserts derived from chloroplast and mitochondrial DNA was estimated to be 1.5%. The coverage of the library is equivalent to 9.0-times the haploid genome. The BAC library was screened with 13 RFLP probes belonging to the 8 linkage groups of the consensus molecular map of banana. A total of 135 clones were identified giving an average of 10.38 clones for each locus. This BAC library will be a valuable starting tool for many of the goals of the recently emerged International Musa Genomic Consortium. One of our initial objectives will be to develop a banana physical map by BAC-FISH (fluorescent in situ hybridization) viewing the characterization of translocation break points.

Artificial chromosome libraries of Streptomyces coelicolor A3(2) and Planobispora rosea

Using an Escherichia coli–Streptomyces shuttle vector derived from a bacterial artificial chromosome (BAC), we developed methodologies for the construction of BAC libraries of filamentous actinomycetes. Libraries of Streptomyces coelicolor, the model actinomycete, and Planobispora rosea, a genetically intractable strain, were constructed. Both libraries have an average insert size of 60 kb, with maximal insert larger than 150 kb. The S. coelicolor library was evaluated by selected hybridisations to DraI fragments and by end sequencing of a few clones. Hybridisation of the P. rosea library to selected probes indicates a good representation of the P. rosea genome and that the library can be used to facilitate the genomic analysis of this actinomycete.

Construction of cDNA Libraries from Microdissected Benign and Malignant Thyroid Tissue

cDNA libraries were constructed from thyroid epithelial cells gained by laser capture microdissection for gene expression analysis of the progression of thyroid cancer. Six histologically diverse thyroid tissue specimens were used. A mean of 93 ng of total RNA was gained per tissue sample from a mean estimated number of 25,000 microdissected cells per sample. Analysis of randomly selected clones from six libraries showed an average insert size of 600 (range, 300–1500) bp. Preliminary sequencing of clones selected from the six libraries indicates a range of 46% to 62% known genes per library, 4% to 25% anonymous expressed sequence tags per library, and 15% to 43% novel expressed sequence tags per library. Thyroglobulin was found in normal thyroid epithelium and follicular thyroid adenoma, whereas calcitonin precursor transcripts were found in medullary thyroid carcinoma. We demonstrate production of high-quality cDNA libraries of microdissected tissue of the thyroid, which should prove useful for gene expression analysis of human thyroid tumors.

DNA LIBRARIES FOR SEQUENCING THE GENOME OF OSTREOCOCCUS TAURI (CHLOROPHYTA, PRASINOPHYCEAE): THE SMALLEST FREE‐LIVING EUKARYOTIC CELL1

Ostreococcus tauri is a marine photosynthetic picoeukaryote presenting a minimal cellular organization with one nucleus, one chloroplast, and one mitochondrion. It has the smallest genome described among free‐living eukaryotic cells, and we showed by pulsed‐field gel electrophoresis (PFGE) that it is divided between 15 bands ranging from 1.2 to 0.15 Mb, giving a total size of 9.7 Mb. A Bacterial Artificial Chromosome (BAC) library was prepared from genomic DNA extracted from a culture of O. tauri. A total of 2457 clones was obtained with an average insert size of around 70 kb, representing an 18‐fold coverage of the genome. The library was spotted on high density filters, and several probes of coding sequences were hybridized to both the high density BAC library filters and directly to the dried PFGE gels of the O. tauri genomic DNA. These hybridizations allowed a preliminary organization of the library and the localization of several markers on the chromosomes. Randomly selected fragments were also sequenced, representing 12% of the O. tauri genome. Many sequences showed significant similarities in data banks, mainly with plant and algae sequences. About 1000 coding sequences could be identified. These data confirmed the position of O. tauri in the green lineage and the hypothesis of a very compact organization of its genome.

A Blumeria graminisf.sp. hordei BAC library--contig building and microsynteny studies.

A bacterial artificial chromosome (BAC) library of Blumeria graminis f.sp. hordei, containing 12,000 clones with an average insert size of 41 kb, was constructed. The library represents about three genome equivalents and BAC-end sequencing showed a high content of repetitive sequences, making contig-building difficult. To identify overlapping clones, several strategies were used: colony hybridisation, PCR screening, fingerprinting techniques and the use of single-copy expressed sequence tags. The latter proved to be the most efficient method for identification of overlapping clones. Two contigs, at or close to avirulence loci, were constructed. Single nucleotide polymorphism (SNP) markers were developed from BAC-end sequences to link the contigs to the genetic maps. Two other BAC contigs were used to study microsynteny between B. graminis and two other ascomycetes, Neurospora crassa and Aspergillus fumigatus. The library provides an invaluable tool for the isolation of avirulence genes from B. graminis and for the study of gene synteny between this fungus and other fungi.

Resolving the aphid resistance locus Sd-1 on a BAC contig within a sub-telomeric region of Malus linkage group 7.

Aphids cause serious physical and economic damage to most major crops throughout the world, and there is a pressing requirement to isolate genes conferring aphid resistance. The Sd-1 locus in Malus spp. (apple) confers resistance against the rosy leaf-curling aphid (Dysaphis devecta Wlk.), and was recently positioned within a 1.3-cM region on linkage group 7, flanked by molecular markers. These markers were used as a basis for development of a BAC contig spanning the locus, together with adapter-mediated amplification of flanking sequences to obtain BAC insert-end sequences, and fingerprinting of BAC clones. Approximately 800 kb of the Sd-1 genomic region was covered by 19 overlapping BACs, with an average insert size of 75-150 kb. The physical-genetic distance ratio was estimated at 460 kb/cM, although the distribution of recombination events was irregular with respect to estimated physical distance. Recombinant analysis and development of new markers allowed Sd-1 to be positioned within an interval of approximately 180 kb located on either of two overlapping BACs. From one of these, an insert end sequence showed a significant degree of similarity to nucleotide binding site-leucine rich repeat (NBS-LRR) resistance genes. Fluorescent in situ hybridization (FISH) of BAC clones within the contig enabled positioning and orientation of the locus within a euchromatic region, very close to the telomere of linkage group 7.

Cosmid library of the marine macroalga Bryopsis maxima (Bryopsidales, Ulvophyceae)

SUMMARY The purpose of this study was to construct a cosmid library from chromosomal DNA of a marine macroalga, Bryopsis maxima Okamura ex Segawa (Bryopsidales, Ulvophyceae), in a rapid, simple and inexpensive manner. In the DNA purification, polysaccharides were removed by covalently binding them to resin particles containing free boric acid groups. The DNA yield was 20 μg g−1 of B. maxima fresh weight. This DNA was 100–200 kb in length, and its A260/A280 and A230/A260 ratios were 1.8 and 0.4, respectively. It was of sufficient quality for molecular research. The cloning procedures were carried out in the following steps: controlled partial shearing of purified DNA through a microsyringe, optimal size separation of the DNA by biased sinusoidal field gel electrophoresis, ligation of the DNA to the cosmid vector in the gel, and in vitro packaging into the lambda phage. The library consisted of 2.0 × 103 independent clones with an average insert size of 40 kb. The fragment amplified by polymerase chain reaction in the library was hybridized with a DNA fragment (328 bp) encoding B. maxima glutamate dehydrogenase under high-stringency conditions by Southern blot analysis, thus demonstrating that the library contained B. maxima chromosomal DNA. This cosmid library is the first to be constructed for any species of marine macroalgae.

One large-insert plant-transformation-competent BIBAC library and three BAC libraries of Japonica rice for genome research in rice and other grasses.

We report one large-insert BIBAC library and three BAC libraries for japonica rice cv Nipponbare. The BIBAC library was constructed in the HindIII site of a plant-transformation-competent binary vector (pCLD04541) and the three BAC libraries were constructed in the BamHI, HindIII and EcoRI sites of a BAC vector (pECBAC1), respectively. Each library contains 23,040 clones, has an average insert size of 130 kb, 170 kb, 150 kb and 156 kb, and covers 6.7x, 8.7x, 7.7x and 8.0 x rice haploid genomes, respectively. The combined libraries contain 92,160 clones in total, covering 31.1 x rice haploid genomes. To demonstrate their utility, we screened the libraries with 55 DNA markers mapped to chromosome 8 of the rice genetic maps and analyzed a number of clones by the restriction fingerprinting and contig assembly method. The results indicate that the libraries completely cover the rice genome and, thus, are well-suited for genome research in rice and other gramineous crops. The BIBAC library represents the first plant-transformation-competent large-insert DNA library for rice, which will streamline map-based cloning, functional analysis of the rice genome sequence and molecular breeding in rice and other grass species. These libraries are being used in the development of a whole-genome, BAC/BIBAC-based, integrated physical, genetic and sequence map of rice and in the research of genome-wide comparative genomics of grass species.

Construction and Characterization of a Deep‐Coverage Bacterial Artificial Chromosome Library for Maize

Modern cultivated maize (Zea mays L.)is one of the primary agronomic crops in the USA with an estimated genome size of 2500 megabases (Mb). To develop the resources for positional cloning and structural genomics in maize, we constructed a bacterial artificial chromosome (BAC) library for the inbred line B73 using the cloning enzyme Hind III. The library contains 247 680 clones (645 384‐well plates). A random sampling of 697 clones indicated an average insert size of 136 kilobase (kb) (range = 42 to 379 kb) and 0.4% empty vectors. Screening the colony filters for chloroplast DNA content indicated an exceptionally low 0.18% contamination with chloroplast DNA. Thus, the library provides 13.5 haploid genome equivalents allowing >99% probability of recovering any specific sequence of interest. High‐density filters were gridded robotically using a Genetix Q‐BOT (Hampshire, UK) in a 4 by 4 double‐spotted array on 22.5‐cm2 filters. Partial screening (6× coverage) of the library with 20 single copy probes identified an average 7.1 positive signals per probe, with a range of 3 to 15 positive signals per probe. To evaluate the utility of the library for sequence tagged connector (STC) analysis, 768 BAC clones were end sequenced in both forward and reverse directions giving a total of 1415 successful reads. End sequences were queried against SWISS‐PROT, Genbank NR, MIPS Arabidopsis, maize genomic sequence dbGSS, and maize cDNA database dbEST. Results in spreadsheet format from these searches is publicly available at the CUGI website (www.genome.clemson.edu/projects/stc/maize/ZMMBBb/).

Construction of a cDNA library of Bemisia tabaci for use in the ‘yeast two‐hybrid screen’ method

The molecular mechanisms involved in the circulative, non‐propagative transmission pathway of TYLCV through its vector the whitefly Bemisia tabaci have hardly been studied. Points requiring investigation include the specific adhesion of virus coat protein to insect structures, the proteins involved in membrane passage in the insect and the possibility of replication of the virus in the vector. To isolate the insect proteins which are involved in transmission by interaction with viral proteins, we propose to use the ‘yeast two‐hybrid screen’ genetic method. For this method, it is indispensable to have a ‘cDNA library’ of the organism concerned, cloned in plasmids, and our first step has been to develop this. A new method was developed for isolating whitefly mRNA. From this mRNA, cDNA was synthesized, ligated in the plasmid pGADT7 (Clontech) and transformed in bacteria to amplify the plasmid DNA. The number of independent clones and average insert size of the plasmids were determined.

Distribution and characterization of microsatellites in the emu (Dromaius novaehollandiae) genome.

This study generates data concerning the genome of a flightless species of bird, the emu. We examined and ultimately rejected the following hypotheses: (1) Microsatellites are randomly distributed throughout the emu genome. (2) The relative order of abundance of dinucleotides will be constant across genomes. (3) Interspersion distances for a given dinucleotide will be equal across vertebrate genomes. (4) In all genomes, a dinucleotide will be more frequent than any trinucleotide. (5) The percentage of single-copy DNA will remain the same in emus as in other volant birds. A cosmid library representing 4.48% of the emu genome was probed with 23 microsatellites. Hybridizations were scored on a scale of 0-3. The average insert size, approximately 40 kb, was used to determine frequency and interspersion. The cosmid library was probed with genomic DNA to determine the percent single copy. Co-occurrence frequencies and confidence intervals were compared to expected using chi-squared. The genome is estimated to contain a microsatellite repeat every 48 kb. Of 1632 clones probed for single-copy DNA, 643 displayed maximal hybridization, 220 displayed moderate hybridization, and 202 had minimal hybridization. After 3 days, 567 showed no hybridization.

Construction of BAC libraries from two apomictic grasses to study the microcolinearity of their apospory-specific genomic regions.

We have constructed bacterial artificial chromosome (BAC) libraries from two grass species that reproduce by apospory, a form of gametophytic apomixis. The library of an apomictic polyhaploid genotype (line MS228-20, with a 2C genome size of approximately 4,500 Mbp) derived from a cross between the obligate apomict, Pennisetum squamulatum, and pearl millet ( P. glaucum) comprises 118,272 clones with an average insert size of 82 kb. The library of buffelgrass ( Cenchrus ciliaris, apomictic line B-12-9, with a 2C genome size of approximately 3,000 Mbp) contains 68,736 clones with an average insert size of 109 kb. Based on the genome sizes of these two lines and correcting for the number for false-positive and organellar clones, library coverages were found to be 3.7 and 4.8 haploid genome equivalents for MS 228-20 and B12-9, respectively. Both libraries were screened by hybridization with six SCARs (sequence-characterized amplified regions), whose tight linkage in a single apospory-specific genomic region had been previously demonstrated in both species. Analysis of these BAC clones indicated that some of the SCAR markers are actually amplifying duplicated regions linked in coupling in both genomes and that restriction enzyme mapping will be necessary to sort out the duplications.

Molecular diversity of CMS sources and fertility restoration in the genus: Helianthus

Molecular characterization of 28 CMS sources and the fertile cytoplasm of H. annuus in sunflower demonstrated that cytoplasms different by their origin show a considerable similarity that cannot be expected from their pedigree. Cluster analysis using the UPGMA method allowed differentiating 10 mitochondrial (mt) types based on RFLP data. Characteristic proteins specific for one or more CMS sources could be identified which correspond to the mt types. In addition, the data obtained on fertility restoration for a subset of nine new CMS sources support the classification of the cytoplasms made on mtDNA level as demonstrated in the UPGMA dendrogram. A map-based cloning approach is followed for the isolation of the restorer gene Rf1, which restores pollen fertility in hybrids based on PET1. Markers closely linked to the restorer gene could be identified using AFLP and RAPD technologies. A bacterial artificial chromosome (BAC) library was constructed using the sunflower restorer line RHA325 and pBeloBAC11 as vector. The first sunflower BAC library corresponds to 1.7 haploid genome equivalents (104,736 clones average insert size approx. 50 kb). Use of STS markers, which had been developed from RAPD markers, allowed to identify three BAC clones by colony hybridization against high density filters. DNA fingerprinting using HindIII allowed to build a contig for the restorer locus Rf1.

Structural analysis of a Lotus japonicus genome. II. Sequence features and mapping of sixty-five TAC clones which cover the 6.5-mb regions of the genome.

Sixty-five TAC (transformation-competent artificial chromosomes) clones were selected from a genomic library of Lotus japonicus accession MG-20 based on the sequence information of expressed sequences tags (ESTs), cDNA and gene information, and their nucleotide sequences were determined. The average insert size of the TAC clone was approximately 100 kb, and the total length of the sequenced regions in this study is 6,556,100 bp. Together with the nucleotide sequences of 56 TAC clones previously reported, the regions sequenced so far total 12,029,295 bp. By comparison with the sequences in protein and EST databases and by analysis with computer programs for gene modeling, a total of 711 potential protein-encoding genes with known or predicted functions, 239 gene segments and 90 pseudogenes were identified in the newly sequenced regions. The average gene density assigned so far was 1 gene/9140 bp. The average length of the assigned genes was 2.6 kb, which is considerably larger than that assigned in the Arabidopsis thaliana genome (1.9 kb for 6451 genes). Introns were identified in approximately 73% of the potential genes, and the average number and length of the introns per gene were 3.4 and 377 bp, respectively. Simple sequence repeat length polymorphism (SSLP) or derived cleaved amplified polymorphic sequence (dCAPS) markers were generated based on the nucleotide sequences of the genomic clones obtained, and each clone was mapped onto the linkage map using the F2 mapping population derived from a cross of two accessions of L. japonicus, Gifu B-129 and Miyakojima MG-20. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.

Physical mapping, BAC-end sequence analysis, and marker tagging of the soilborne nematicidal bacterium, Pseudomonas synxantha BG33R.

A bacterial artificial chromosome (BAC) library was constructed for the genome of the rhizosphere-inhabiting fluorescent pseudomonad Pseudomonas synxantha BG33R. Three thousand BAC clones with an average insert size of 140 kbp and representing a 70-fold genomic coverage were generated and arrayed onto nylon membranes. EcoRI fingerprint analysis of 986 BAC clones generated 23 contigs and 75 singletons. Hybridization analysis allowed us to order the 23 contigs and condense them into a single contig, yielding an estimated genome size of 5.1 Mb for P. synxantha BG33R. A minimum-tile path of 47 BACs was generated and end-sequenced. The genetic loci involved in ring nematode egg-kill factor production in BG33R Tn5 mutants, 246 (vgrG homolog), 1122 (sensor kinase homolog), 1233 (UDP-galactose epimerase homolog), 1397 (ferrisiderophore receptor homolog), and 1917 (ribosomal subunit protein homolog), have been mapped onto the minimum-tile BAC library. Two of the genetic regions that flank Tn5 insertions in BG33R egg-kill-negative mutants 1233 and 1397 are separated by a single BAC clone. Fragments isolated by ligation-mediated PCR of the Tn5 mutagenized regions of 29 randomly selected, non-egg-kill-related, insertion mutants have been anchored onto the ordered physical map of P. synxantha.

Repeats of an oleate desaturase region cause silencing of the normal gene explaining the high oleic pervenets sunflower mutant

SUMMARY This work aims to clone the genomic region of the sunflower genome that may carry the Pervenets mutation causing high oleic content of seed oil. We previously reported that the oleHOS allele at the oleate-desaturase locus (oleHL) carries or is genetically closely linked to Pervenets mutation. We pointed out that the fragments that border the 5.8 kb EcoRI fragments carry the Pervenets mutation. The oleHOS reference physical map was constructed. It displays a common part made of the 5.8 kb EcoRI fragment, also present in oleLOR allele, and a specific region with the 7.9 kb EcoRI fragment, also carrying oleate-desaturase sequences. A genomic library was constructed in λfixII with an average insert size of 15 kb. Two millions clones were screened enabling to isolate clones from Group I, which were entirely sequenced and revealed carrying a gene for an oleate probably located in the RE. This corresponds to the invariant part of the oleHL locus. Two clones of Group II (11.1, 27.1) are overlapping but 11.1 is characterized by new specific restriction fragments and instability leading to a smear when probed with the oleate cDNA. Consequently, 11.1 sequence probably carries oleate repeated sequences that cause instability of the clone. The clone 11.1 is a good candidate to carry a part of the specific oleHOS allele, but due to its organization in repeats it is not yet sequenced. This organization leads to speculate on the mechanisms that could disturb oleate function in Pervenets [HOAC] genotypes.

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.

Development of new transformation-competent artificial chromosome vectors and rice genomic libraries for efficient gene cloning

The transformation-competent artificial chromosome vector (TAC) system has been shown to be very useful for efficient gene isolation in Arabidopsisthaliana (Proc. Natl. Acad. Sci. USA 96 (1998) 6535). To adapt the vector system for gene isolation in crops, two new TAC vectors and rice genomic libraries were developed. The new vectors pYLTAC17 and pYLTAC27 use the Bar gene and Hpt gene driven by the rice Act1 promoter as the plant selectable markers, respectively, and are suitable for transformation of rice and other grasses. Two representative genomic libraries (I and II) of an Indica rice variety Minghui63, a fertility restorer line for hybrid rice, were constructed with pYLTAC17 using different size classes of partially digested DNA fragments. Library I and library II consisted of 34560 and 1.2×105 clones, with average insert sizes of approximately 77 and 39 kb, respectively. The genome coverage of the libraries I and II was estimated to be about 5 and 11 haploid genome equivalents, respectively. Clones of the library I were stored individually in ninety 384-well plates, and those of the library II were collected as bulked pools each containing 30–50 clones and stored in eight 384-well plates. A number of probes were used to hybridize high-density colony filters of the library I prepared by an improved replicating method and each detected 2–9 positive clones. A method for rapid screening of the library II by pooled colony hybridization was developed. A TAC clone having an 80 kb rice DNA insert was successfully transferred into rice genome via Agrobacterium-mediated transformation. The new vectors and the genomic libraries should be useful for gene cloning and genetic engineering in rice and other crops.