Construction of a garlic BAC library and chromosomal assignment of BAC clones using the FISH technique

Genome ◽  
2003 ◽  
Vol 46 (3) ◽  
pp. 514-520 ◽  
Author(s):  
Hye-Ran Lee ◽  
Eun-Mi Eom ◽  
Yong-Pyo Lim ◽  
Jae-Wook Bang ◽  
Dong-Hee Lee
Keyword(s):  
Allium Sativum ◽  
Bac Library ◽  
Chromosomal Assignment ◽  
Fish Analysis ◽  
Average Insert Size ◽  
Insert Size ◽  
Bac Clone ◽  
Fish Technique ◽  
Bac Clones ◽  
Cytogenetic Studies

For molecular and cytogenetic studies, two partial bacterial artificial chromosome (BAC) libraries of the garlic cultivar Allium sativum L. 'Danyang' were constructed using high molecular weight (HMW) garlic DNA, the pBAC1–SACB1 vector, and the pIndigoBAC536 vector. The average insert size of the BAC library was about 90 kb. The sequence compositions of the BAC clones were characterized by Southern hybridization with garlic genomic DNA and a repetitive sequence clone of garlic. Two BAC clones with weak signals (thus implying mostly unique sequences), GBC2-5e and GBC2-4d, were selected for FISH analysis. FISH analysis localized the GBC2-5e (~100 kb) BAC clone on the long arm of garlic chromosome 7. The other BAC clone, GBC2-4d (~110 kb), gave rise to discrete FISH signals on a mid-size early metaphase chromosome. The FISH screening with BAC clones proved to be a useful resource for molecular cytogenetic studies of garlic, and will be useful for further mapping and sequencing studies of important genes of this plant.Key words: garlic, chromosomal assignment, Allium sativum L., FISH technique, BAC library.

scholarly journals Construction of a Populus tremuloides Michx. BAC library

2008 ◽  
Vol 57 (1-6) ◽  
pp. 65-69 ◽  
Author(s):  
M. Fladung ◽  
H. Kaufmann ◽  
T. Markussen ◽  
H. Hoenicka
Keyword(s):  
Genome Size ◽  
Populus Tremuloides ◽  
Random Sampling ◽  
Aflp Marker ◽  
Bac Library ◽  
Male Parent ◽  
Haploid Genome ◽  
Average Insert Size ◽  
Insert Size ◽  
Bac Clones

Abstract We have constructed an aspen (Populus tremuloides Michx., line Turesson141) BAC library containing 55,296 clones in total. A random sampling of 86 BAC clones indicated an average insert size of 76 kb with a range of 20 to 160 kb. Twelve percent of the BAC clones in the library have an insert size larger than 100 kb. Based on an estimated genome size for Populus of 500 Mbp, library coverage is about 8 haploid genome equivalents. This library will be screened using AFLP marker identified before co-segregating with gender in a P. tremula x P. tremuloides progeny, where Turesson141 was the male parent.

Construction of a Coix BAC library and isolation of the 22 kDa α-coixin gene cluster

Genome ◽  
2010 ◽  
Vol 53 (9) ◽  
pp. 667-674 ◽  
Author(s):  
Xiangzong Meng ◽  
Binbin Huang ◽  
Liangliang Zhou ◽  
Yunxia He ◽  
Qi Chen ◽  
...  
Keyword(s):  
Genetic Resource ◽  
Crop Improvement ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Gene Isolation ◽  
Close Relative ◽  
Average Insert Size ◽  
Insert Size ◽  
Bac Clones ◽  
Organellar Dna

Coix lacryma-jobi L. (Coix) is a close relative of maize and is considered a valuable genetic resource for crop improvement. Here we report the construction of the first Coix bacterial artificial chromosome (BAC) library using accession PI 324059. This BAC library contains about 230 400 clones with an average insert size of 113 kb, has low organellar DNA contamination, and provides 16.3-fold coverage of the genome. The library was stored in 12 × 96 pools that could be screened with a PCR protocol. Library screening was performed for the 22 kDa α-coixin gene family. A total of 57 positive pools were identified, and single clones were isolated from 19 of these pools. Based on DNA fingerprinting and Southern blot analysis, these 19 BAC clones form a single contig of about 340 kb in length, indicating that the 22 kDa α-coixin genes occur in a cluster. These results demonstrated the suitability of this BAC library for gene isolation and comparative genomics studies of the Coix genome.

Construction of a bacterial artificial chromosome (BAC) library for potato molecular cytogenetics research

Genome ◽  
2000 ◽  
Vol 43 (1) ◽  
pp. 199-204 ◽  
Author(s):  
Junqi Song ◽  
Fenggao Dong ◽  
Jiming Jiang
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Physical Map ◽  
Molecular Cytogenetics ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Average Insert Size ◽  
Chromosome Identification ◽  
Insert Size ◽  
Potato Genome ◽  
Bac Clones

Lack of reliable techniques for chromosome identification is the major obstacle for cytogenetics research in plant species with large numbers of small chromosomes. To promote molecular cytogenetics research of potato (Solanum tuberosum, 2n = 4x = 48) we developed a bacterial artificial chromosome (BAC) library of a diploid potato species S. bulbocastanum. The library consists of 23 808 clones with an average insert size of 155 kb, and represents approximately 3.7 equivalents to the potato genome. The majority of the clones in the BAC library generated distinct signals on specific potato chromosomes using fluorescence in situ hybridization (FISH). The hybridization signals provide excellent cytological markers to tag individual potato chromosomes. We also demonstrated that the BAC clones can be mapped to specific positions on meiotic pachytene chromosomes. The excellent resolution of pachytene FISH can be used to construct a physical map of potato by mapping molecular marker-targeted BAC clones on pachytene chromosomes. Key words: potato, BAC library, chromosome identification, physical mapping, molecular cytogenetics.

scholarly journals 484 Constructing the BAC Contig for the Evergreen Gene Region in Peach [Prunus persica (L.) Batsch]

HortScience ◽  
2000 ◽  
Vol 35 (3) ◽  
pp. 477E-478
Author(s):  
Ying Wang ◽  
Laura L. George ◽  
Gregory L. Reighard ◽  
Ralph Scorza ◽  
Albert G. Abbott
Keyword(s):  
Prunus Persica ◽  
Physical Map ◽  
Single Gene ◽  
Bac Library ◽  
Gene Region ◽  
Average Insert Size ◽  
Insert Size ◽  
Bac Clone ◽  
Bac Contig ◽  
Peach Genome

Evergreen genotypes of peach [Prunus persica (L.) Batsch] have been identified in Mexico, where terminal growth on evergreen trees is continuous under favorable environmental conditions. This evergreen trait in peach is controlled by one single gene (evg), and this evergreen condition is homozygous recessive. Four dominant AFLP markers, EAT/MCAC, ETT/MCCA2, EAT/MCTA, and ETT/MACC, were found to be tightly linked to the evg locus at 1 cM, 4.6 cM, 5.8 cM, and 11 cM, respectively. All four markers were sequenced and identified. A peach BAC library was constructed by using the pBeloBAC11 vector for building the physical map for the evg gene. This library represents four times the coverage of the peach genome with the average insert size of 50 to 70 kb. The EAT/MCAC AFLP marker fragment was used for screening the peach BAC library. A single BAC clone, 18F12, was confirmed to contain this fragment. The final BAC contig for this evg gene region and the potential homology between peach and Arabidopsis thaliana will be presented and discussed.

Construction and characterization of bacterial artificial chromosome library of black-handed spider monkey (Ateles geoffroyi)

Genome ◽  
2004 ◽  
Vol 47 (2) ◽  
pp. 239-245 ◽  
Author(s):  
Yaping Qian ◽  
Li Jin ◽  
Bing Su
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Large Scale ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Spider Monkey ◽  
Ateles Geoffroyi ◽  
Comparative Genomic ◽  
Average Insert Size ◽  
Bac Clones ◽  
Genomic Study

The large-insert genomic DNA library is a critical resource for genome-wide genetic dissection of target species. We constructed a high-redundancy bacterial artificial chromosome (BAC) library of a New World monkey species, the black-handed spider monkey (Ateles geoffroyi). A total of 193 152 BAC clones were generated in this library. The average insert size of the BAC clones was estimated to be 184.6 kb with the small inserts (50-100 kb) accounting for less than 3% and the non-recombinant clones only 1.2%. Assuming a similar genome size with humans, the spider monkey BAC library has about 11× genome coverage. In addition, by end sequencing of randomly selected BAC clones, we generated 367 sequence tags for the library. When blasted against human genome, they showed a good correlation between the number of hit clones and the size of the chromosomes, an indication of unbiased chromosomal distribution of the library. This black-handed spider monkey BAC library would serve as a valuable resource in comparative genomic study and large-scale genome sequencing of nonhuman primates.Key words: black-handed spider monkeys, Ateles geoffroyi, BAC library.

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

Genome ◽  
2005 ◽  
Vol 48 (6) ◽  
pp. 1028-1036 ◽  
Author(s):  
P Ling ◽  
X M Chen
Keyword(s):  
Triticum Aestivum ◽  
Stripe Rust ◽  
Hexaploid Wheat ◽  
Rust Resistance ◽  
Puccinia Striiformis ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Stripe Rust Resistance ◽  
Insert Size ◽  
Bac Clones

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 3.58× 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.Key words: BAC library, BAC pools, hexaploid wheat, Puccinia striiformis f. sp. tritici, resistance gene, stripe rust, Triticum aestivum.

scholarly journals Construction, Characterization, and Preliminary BAC-End Sequence Analysis of a Bacterial Artificial Chromosome Library of the Tea Plant (Camellia sinensis)

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Jinke Lin ◽  
Dave Kudrna ◽  
Rod A. Wing
Keyword(s):  
Camellia Sinensis ◽  
Bac Library ◽  
Gc Content ◽  
Artificial Chromosome ◽  
Tea Plant ◽  
Average Insert Size ◽  
Plant Genomics ◽  
Insert Size ◽  
Sequence Class ◽  
End Sequences

We describe the construction and characterization of a publicly available BAC library for the tea plant,Camellia sinensis. Using modified methods, the library was constructed with the aim of developing public molecular resources to advance tea plant genomics research. The library consists of a total of 401,280 clones with an average insert size of 135 kb, providing an approximate coverage of 13.5 haploid genome equivalents. No empty vector clones were observed in a random sampling of 576 BAC clones. Further analysis of 182 BAC-end sequences from randomly selected clones revealed a GC content of 40.35% and low chloroplast and mitochondrial contamination. Repetitive sequence analyses indicated that LTR retrotransposons were the most predominant sequence class (86.93%–87.24%), followed by DNA retrotransposons (11.16%–11.69%). Additionally, we found 25 simple sequence repeats (SSRs) that could potentially be used as genetic markers.

scholarly journals Construction and Preliminary Characterization Analysis of Wuzhishan Miniature Pig Bacterial Artificial Chromosome Library with Approximately 8-Fold Genome Equivalent Coverage

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Changqing Liu ◽  
Yuo Guo ◽  
Taofeng Lu ◽  
Hongmei Wu ◽  
Risu Na ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Blood Cells ◽  
Large Scale ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Genetic Dissection ◽  
Average Insert Size ◽  
Miniature Pig ◽  
Insert Size ◽  
Statistical Probability

Bacterial artificial chromosome (BAC) libraries have been invaluable tools for the genome-wide genetic dissection of complex organisms. Here, we report the construction and characterization of a high-redundancy BAC library from a very valuable pig breed in China, Wuzhishan miniature pig (Sus scrofa), using its blood cells and fibroblasts, respectively. The library contains approximately 153,600 clones ordered in 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 152.3 kb, representing approximately 7.68 genome equivalents of the porcine haploid genome and a 99.93% statistical probability of obtaining at least one clone containing a unique DNA sequence in the library. 19 pairs of microsatellite marker primers covering porcine chromosomes were used for screening the BAC library, which showed that each of these markers was positive in the library; the positive clone number was 2 to 9, and the average number was 7.89, which was consistent with 7.68-fold coverage of the porcine genome. And there were no significant differences of genomic BAC library from blood cells and fibroblast cells. Therefore, we identified 19 microsatellite markers that could potentially be used as genetic markers. As a result, this BAC library will serve as a valuable resource for gene identification, physical mapping, and comparative genomics and large-scale genome sequencing in the porcine.

scholarly journals A Census of rRNA Genes and Linked Genomic Sequences within a Soil Metagenomic Library

2003 ◽  
Vol 69 (5) ◽  
pp. 2684-2691 ◽  
Author(s):  
Mark R. Liles ◽  
Brian F. Manske ◽  
Scott B. Bintrim ◽  
Jo Handelsman ◽  
Robert M. Goodman
Keyword(s):  
16S Rrna ◽  
Bac Library ◽  
Metagenomic Library ◽  
Pcr Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Acid Uptake ◽  
Direct Pcr ◽  
Bac Clone ◽  
Bac Clones

ABSTRACT We have analyzed the diversity of microbial genomes represented in a library of metagenomic DNA from soil. A total of 24,400 bacterial artificial chromosome (BAC) clones were screened for 16S rRNA genes. The sequences obtained from BAC clones were compared with a collection generated by direct PCR amplification and cloning of 16S rRNA genes from the same soil. The results indicated that the BAC library had substantially lower representation of bacteria among the Bacillus, α-Proteobacteria, and CFB groups; greater representation among the β- and γ-Proteobacteria, and OP10 divisions; and no rRNA genes from the domains Eukaryota and Archaea. In addition to rRNA genes recovered from the bacterial divisions Proteobacteria, Verrucomicrobia, Firmicutes, Cytophagales, and OP11, we identified many rRNA genes from the BAC library affiliated with the bacterial division Acidobacterium; all of these sequences were affiliated with subdivisions that lack cultured representatives. The complete sequence of one BAC clone derived from a member of the Acidobacterium division revealed a complete rRNA operon and 20 other open reading frames, including predicted gene products involved in cell division, cell cycling, folic acid biosynthesis, substrate metabolism, amino acid uptake, DNA repair, and transcriptional regulation. This study is the first step in using genomics to reveal the physiology of as-yet-uncultured members of the Acidobacterium division.

scholarly journals A Bacterial Artificial Chromosome Library of Lotus japonicus Constructed in an Agrobacterium tumefaciens-Transformable Vector

2001 ◽  
Vol 14 (3) ◽  
pp. 422-425 ◽  
Author(s):  
Artem E. Men ◽  
Khalid Meksem ◽  
My Abdelmajid Kassem ◽  
Dasharath Lohar ◽  
Jiri Stiller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Agrobacterium Tumefaciens ◽  
Plant Transformation ◽  
Lotus Japonicus ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Average Insert Size ◽  
Insert Size ◽  
Model Legume ◽  
Genome Coverage

We constructed a BAC library of the model legume Lotus japonicus with a 6-to 7-fold genome coverage. We used vector PCLD04541, which allows direct plant transformation by BACs. The average insert size is 94 kb. Clones were stable in Escherichia coli and Agrobacterium tumefaciens.

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