Construction and characterization of a BAC library for the molecular dissection of a single wild beet centromere and sugar beet (Beta vulgaris) genome analysis

Genome ◽  
2001 ◽  
Vol 44 (5) ◽  
pp. 846-855 ◽  
Author(s):  
Frank Gindullis ◽  
Daryna Dechyeva ◽  
Thomas Schmidt
Keyword(s):  
Sugar Beet ◽  
Beta Vulgaris ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Average Insert Size ◽  
Insert Size ◽  
Single Chromosome ◽  
Beta Procumbens ◽  
Wild Beet

We have constructed a sugar beet bacterial artificial chromosome (BAC) library of the chromosome mutant PRO1. This Beta vulgaris mutant carries a single chromosome fragment of 6-9 Mbp that is derived from the wild beet Beta procumbens and is transmitted efficiently in meiosis and mitosis. The library consists of 50 304 clones, with an average insert size of 125 kb. Filter hybridizations revealed that approximately 3.1% of the clones contain mitochondrial or chloroplast DNA. Based on a haploid genome size of 758 Mbp, the library represents eight genome equivalents. Thus, there is a greater than 99.96% probability that any sequence of the PRO1 genome can be found in the library. Approximately 0.2% of the clones hybridized with centromeric sequences of the PRO1 minichromosome. Using the identified BAC clones in fluorescence in situ hybridization experiments with PRO1 and B. procumbens chromosome spreads, their wild-beet origin and centromeric localization were demonstrated. Comparative Southern hybridization of pulsed-field separated PRO1 DNA and BAC inserts indicate that the centromeric region of the minichromosome is represented by overlapping clones in the library. Therefore, the PRO1 BAC library provides a useful tool for the characterization of a single plant centromere and is a valuable resource for sugar beet genome analysis.Key words: Beta vulgaris, BAC library, Beta procumbens minichromosome, centromere, FISH.

scholarly journals Construction of a BAC Library Covering a Common Region between Two Sugar Beet Translocation Lines with Two Cyst Nematode Resistance Genes

HortScience ◽  
2004 ◽  
Vol 39 (4) ◽  
pp. 822C-822
Author(s):  
Suren Samuelian*
Keyword(s):  
Sugar Beet ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Nematode Resistance ◽  
Cyst Nematode ◽  
Translocation Line ◽  
Average Insert Size ◽  
Insert Size ◽  
Specific Sequence ◽  
Wild Beet

Resistance against the beet cyst nematode (BCN) has been introduced into cultivated sugar beet from wild beet by conventional breeding. The first gene effective against the BCN, Hs1pro-1, was isolated from the sugar beet translocation line A906001. It is assumed that a second nematode resistance gene, Hs1pro-1, is present in the translocation line PRO3, which does not carry Hs1pro-1 but still imparts complete resistance against the nematode and resides in the overlapping region between the two lines. The overall goal of this study was to construct a bacterial artificial chromosome (BAC) library to facilitate the cloning of Hs1pro-1. A BAC library from PRO3 was constructed containing 45,041 clones with an average insert size of 108.36 kb. Screening of the library with organelle specific probes indicated less than 1% mitochondrial and 4% chloroplast DNA content. The library covers 6.17 genome equivalents which provides a 99.76% probability of recovering any specific sequence present in the genome.

Construction and characterization of the first bacterial artificial chromosome library for the cotton species Gossypium barbadense L.

Genome ◽  
2006 ◽  
Vol 49 (11) ◽  
pp. 1393-1398 ◽  
Author(s):  
X.F. Wang ◽  
J. Ma ◽  
W.S. Wang ◽  
Y.M. Zheng ◽  
G.Y. Zhang ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Gossypium Barbadense ◽  
Average Insert Size ◽  
Insert Size ◽  
Breeding Programs ◽  
Fiber Properties ◽  
Cotton Species

As the second most widely cultivated cotton, Gossypium barbadense is well known for its superior fiber properties and its high levels of resistance to Fusarium and Verticillium wilts. To enhance our ability to exploit these properties in breeding programs, we constructed the first bacterial artificial chromosome (BAC) library for this species. The library contains 167 424 clones (49 920 BamHI and 117 504 HindIII clones), with an estimated average insert size of 130 kb. About 94.0% of the clones had inserts over 100 kb, and the empty clones accounted for less than 4.0%. Contamination of the library with chloroplast clones was very low (0.2%). Screening the library with locus-specific probes showed that BAC clones represent 6.5-fold genome equivalents. This high-quality library provides an additional asset with which to exploit genetic variation for cotton improvement.

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.

Construction and characterization of a sugar beet (Beta vulgaris) fosmid library

Genome ◽  
2008 ◽  
Vol 51 (11) ◽  
pp. 948-951 ◽  
Author(s):  
Cornelia Lange ◽  
Daniela Holtgräwe ◽  
Britta Schulz ◽  
Bernd Weisshaar ◽  
Heinz Himmelbauer
Keyword(s):  
Sugar Beet ◽  
Beta Vulgaris ◽  
Fosmid Library ◽  
Average Insert Size ◽  
Size Estimation ◽  
Insert Size ◽  
Pcr Screening ◽  
Organellar Dna ◽  
Fosmid End Sequences ◽  
End Sequences

A sugar beet ( Beta vulgaris ) fosmid library from the doubled haploid accession KWS2320 encompassing 115 200 independent clones was constructed and characterized. The average insert size of the fosmid library was determined by pulsed field gel electrophoresis to be 39 kbp on average, thus representing 5.9-fold coverage of the sugar beet genome (758 Mbp). PCR screening of plate pools with primer pairs against nine sugar beet genes supported the insert size estimation. BLAST searches with 2951 fosmid end-sequences originating from 1510 clones (1536 clones attempted) revealed little contamination with organellar DNA (2.1% chloroplast DNA, 0.3% mitochondrial DNA). The sugar beet fosmid library will be integrated in the presently ongoing efforts to determine the sequence of the sugar beet genome. Fosmids will be publicly available in the format of plate pools and individual clones.

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 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.

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 Construction and characterization of a bacterial artificial chromosome (BAC) library for the A genome of wheat

Genome ◽  
1999 ◽  
Vol 42 (6) ◽  
pp. 1176-1182 ◽  
Author(s):  
D Lijavetzky ◽  
G Muzzi ◽  
T Wicker ◽  
B Keller ◽  
R Wing ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Repetitive Sequences ◽  
Bac Library ◽  
Artificial Chromosome ◽  
High Density ◽  
Triticum Monococcum ◽  
Average Insert Size ◽  
Insert Size ◽  
High Molecular Weight Dna ◽  
A Genome

A genomic bacterial artificial chromosome (BAC) library of the A genome of wheat has been constructed. Triticum monococcum accession DV92 was selected for this purpose because it is a cultivated diploid wheat and one of the parental lines used in the construction of a saturated genetic map. Leaves from this accession were used to isolate high-molecular-weight DNA from nuclei. This DNA was partially digested with restriction enzyme Hind III, subjected to double size selection, electroeluted and cloned into the pINDIGO451 BAC vector. The library consists of 276 480 clones with an average insert size of 115 kb. Excluding the 1.33% of empty clones and 0.14% of clones with chloroplast DNA, the coverage of this library is 5.6 genome equivalents. With this genome coverage the probability of having any DNA sequence represented in this library is higher than 99.6%. Clones were sorted in 720 384-well plates and blotted onto 15 high-density filters. High-density filters were screened with several single or low-copy clones and five positive BAC clones were selected for further analysis. Since most of the T. monococcum BAC ends included repetitive sequences, a modification was introduced into the classical end-isolation procedure to select low copy sequences for chromosome walking.Key words: bacterial artificial chromosome, BAC library, Triticum monococcum, wheat.

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