Construction and characterization of two Citrus BAC libraries and identification of clones containing the phytoene synthase gene

Genome ◽  
2009 ◽  
Vol 52 (5) ◽  
pp. 484-489 ◽  
Author(s):  
M. N.R. Baig ◽  
An Yu ◽  
Wenwu Guo ◽  
Xiuxin Deng
Keyword(s):  
Dna Sequences ◽  
False Positive ◽  
Bac Library ◽  
Phytoene Synthase ◽  
Haploid Genome ◽  
Average Insert Size ◽  
Insert Size ◽  
Important Species ◽  
Dna Contamination ◽  
Bac Libraries

Two deep-coverage Bacterial Artificial Chromosome (BAC) libraries of Citrus sinensis (L.) Osbeck ‘Cara Cara’ navel orange and Citrus reticulata (L.) Blanco ‘Egan No. 1’ Ponkan mandarin, which belong to the two most important species of the Citrus genus, have been constructed and characterized to facilitate gene cloning and to analyze variety-specific genome composition. The C. sinensis BAC library consists of 36 000 clones with negligible false-positive clones and an estimated average insert size of 126 kb covering ~4.5 × 109 bp and thus providing an 11.8-fold coverage of haploid genome equivalents, whereas the C. reticulata library consists of 21 000 clones also with negligible false-positive clones and an estimated average of 120 kb covering ~2.5 × 109 bp representing a 6.6-fold coverage of haploid genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBAC536 vector, and organellar DNA sequences. Screening has been performed by Southern hybridization of BAC filters, which results in <0.5% chloroplast DNA contamination and no mitochondrial DNA contamination in both libraries. Eight and five positive clones harboring the gene encoding Phytoene synthase (Psy (EC 2.5.1.32)) were identified from the C. sinensis and C. reticulata libraries, respectively, using the filter hybridization procedure. These results suggest that the two BAC libraries are useful tools for the isolation of functional genes and advanced genomics research in the two important species C. sinensis and C. reticulata. Resources, high-density filters, individual clones, and whole libraries are available for public distribution and are accessible at the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University.

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.

scholarly journals Construction of Papaya Male and Female BAC Libraries and Application in Physical Mapping of the Sex Chromosomes

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Andrea R. Gschwend ◽  
Qingyi Yu ◽  
Paul Moore ◽  
Christopher Saski ◽  
Cuixia Chen ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Restriction Enzyme ◽  
Sex Chromosomes ◽  
Physical Map ◽  
Bac Library ◽  
Specific Region ◽  
Average Insert Size ◽  
Insert Size ◽  
Male And Female ◽  
Bac Libraries

Papaya is a major fruit crop in the tropics and has recently evolved sex chromosomes. Towards sequencing the papaya sex chromosomes, two bacterial artificial chromosome (BAC) libraries were constructed from papaya male and female genomic DNA. The female BAC library was constructed using restriction enzymeBstY I and consists of 36,864 clones with an average insert size of 104 kb, providing 10.3x genome equivalents. The male BAC library was constructed using restriction enzymeEcoR I and consists of 55,296 clones with an average insert size of 101 kb, providing 15.0x genome equivalents. The male BAC library was used in constructing the physical map of the male-specific region of the male Y chromosome (MSY) and in filling gaps and extending the physical map of the hermaphrodite-specific region of the Yhchromosome (HSY) and the X chromosome physical map. The female BAC library was used to extend the X physical map gap. The MSY, HSY, and X physical maps offer a unique opportunity to study chromosomal rearrangements, Y chromosome degeneration, and dosage compensation of the papaya nascent sex chromosomes.

BAC Library Development and Clone Characterization for Dormancy-Responsive DREB4A, DAM, and FT from Leafy Spurge (Euphorbia esula) Identifies Differential Splicing and Conserved Promoter Motifs

Weed Science ◽  
2013 ◽  
Vol 61 (2) ◽  
pp. 303-309 ◽  
Author(s):  
David P. Horvath ◽  
David Kudrna ◽  
Jayson Talag ◽  
James V. Anderson ◽  
Wun S. Chao ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Sequence Data ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Phylogenetic Footprinting ◽  
Leafy Spurge ◽  
Average Insert Size ◽  
Insert Size ◽  
Perennial Weed ◽  
Bac Libraries

We developed two leafy spurge bacterial artificial chromosome (BAC) libraries that together represent approximately 5× coverage of the leafy spurge genome. The BAC libraries have an average insert size of approximately 143 kb, and copies of the library and filters for hybridization-based screening are publicly available through the Arizona Genomics Institute. These libraries were used to clone full-length genomic copies of an AP2/ERF transcription factor of the A4 subfamily of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEINS (DREB) known to be differentially expressed in crown buds of leafy spurge during endodormancy, a DORMANCY ASSOCIATED MADS-BOX (DAM) gene, and several FLOWERING LOCUS T (FT) genes. Sequencing of these BAC clones revealed the presence of multiple FT genes in leafy spurge. Sequencing also provided evidence that two different DAM transcripts expressed in crown buds of leafy spurge during endo- and eco-dormancy result from alternate splicing of a single DAM gene. Sequence data from the FT promoters was used to identify several conserved elements previously recognized in Arabidopsis, as well as potential novel transcription factor binding sites that may regulate FT. These leafy spurge BAC libraries represent a new genomics-based tool that complements existing genomics resources for the study of plant growth and development in this model perennial weed. Furthermore, phylogenetic footprinting using genes identified with this resource demonstrate the usefulness of studying weedy species to further our general knowledge of agriculturally important genes.

Melon bacterial artificial chromosome (BAC) library construction using improved methods and identification of clones linked to the locus conferring resistance to melon Fusarium wilt (Fom-2)

Genome ◽  
2001 ◽  
Vol 44 (2) ◽  
pp. 154-162 ◽  
Author(s):  
Meizhong Luo ◽  
Yi-Hong Wang ◽  
David Frisch ◽  
Tarek Joobeur ◽  
Rod A Wing ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Fusarium Wilt ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Microtiter Plates ◽  
Bac Libraries ◽  
Average Size ◽  
Improved Methods

Utilizing improved methods, two bacterial artificial chromosome (BAC) libraries were constructed for the multidisease-resistant line of melon MR-1. The HindIII library consists of 177 microtiter plates in a 384-well format, while the EcoRI library consists of 222 microtiter plates. Approximately 95.6% of the HindIII library clones contain nuclear DNA inserts with an average size of 118 kb, providing a coverage of 15.4 genome equivalents. Similarly, 96% of the EcoRI library clones contain nuclear DNA inserts with an average size of 114 kb, providing a coverage of 18.7 genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBac536 vector, and organellar DNA sequences. High-density filters were screened with two genetic markers FM and AM that co-segregate with Fom-2, a gene conferring resistance to races 0 and 1 of Fusarium wilt. Fourteen and 18 candidate BAC clones were identified for the FM and AM probes, respectively, from the HindIII library, while 34 were identified for the AM probe from filters A, B, and C of the EcoRI library.Key words: bacterial artificial chromosome (BAC) library, Fusarium wilt, melon, pCUGIBAC1, resistant gene.

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 two BAC libraries from Brachypodium distachyon, a new model for grass genomics

Genome ◽  
2006 ◽  
Vol 49 (9) ◽  
pp. 1099-1108 ◽  
Author(s):  
Naxin Huo ◽  
Yong Q. Gu ◽  
Gerard R. Lazo ◽  
John P. Vogel ◽  
Devin Coleman-Derr ◽  
...  
Keyword(s):  
Genomic Sequence ◽  
Brachypodium Distachyon ◽  
Repetitive Sequences ◽  
Average Insert Size ◽  
Insert Size ◽  
Genome Coverage ◽  
Wheat Ests ◽  
A Genome ◽  
Bac Libraries ◽  
Brachypodium Genome

Brachypodium is well suited as a model system for temperate grasses because of its compact genome and a range of biological features. In an effort to develop resources for genome research in this emerging model species, we constructed 2 bacterial artificial chromosome (BAC) libraries from an inbred diploid Brachypodium distachyon line, Bd21, using restriction enzymes HindIII and BamHI. A total of 73 728 clones (36 864 per BAC library) were picked and arrayed in 192 384-well plates. The average insert size for the BamHI and HindIII libraries is estimated to be 100 and 105 kb, respectively, and inserts of chloroplast origin account for 4.4% and 2.4%, respectively. The libraries individually represent 9.4- and 9.9-fold haploid genome equivalents with combined 19.3-fold genome coverage, based on a genome size of 355 Mb reported for the diploid Brachypodium, implying a 99.99% probability that any given specific sequence will be present in each library. Hybridization of the libraries with 8 starch biosynthesis genes was used to empirically evaluate this theoretical genome coverage; the frequency at which these genes were present in the library clones gave an estimated coverage of 11.6- and 19.6-fold genome equivalents. To obtain a first view of the sequence composition of the Brachypodium genome, 2185 BAC end sequences (BES) representing 1.3 Mb of random genomic sequence were compared with the NCBI GenBank database and the GIRI repeat database. Using a cutoff expectation value of E  < 10−10, only 3.3% of the BESs showed similarity to repetitive sequences in the existing database, whereas 40.0% had matches to the sequences in the EST database, suggesting that a considerable portion of the Brachypodium genome is likely transcribed. When the BESs were compared with individual EST databases, more matches hit wheat than maize, although their EST collections are of a similar size, further supporting the close relationship between Brachypodium and the Triticeae. Moreover, 122 BESs have significant matches to wheat ESTs mapped to individual chromosome bin positions. These BACs represent colinear regions containing the mapped wheat ESTs and would be useful in identifying additional markers for specific wheat chromosome regions.

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 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.

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.

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