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.

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.

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.

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.

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.

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.

Bacterial artificial chromosome–based physical map of Gibberella zeae (Fusarium graminearum)

Genome ◽  
2007 ◽  
Vol 50 (10) ◽  
pp. 954-962 ◽  
Author(s):  
Yueh-Long Chang ◽  
Seungho Cho ◽  
H. Corby Kistler ◽  
Chun-Sheng Hsieh ◽  
Gary J. Muehlbauer
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Fusarium Graminearum ◽  
Genetic Map ◽  
Genome Sequence ◽  
Restriction Enzyme ◽  
Physical Map ◽  
Artificial Chromosome ◽  
Gibberella Zeae ◽  
Restriction Enzyme Site ◽  
Insert Size

Fusarium graminearum is the primary causal pathogen of Fusarium head blight of wheat and barley. To accelerate genomic analysis of F. graminearum, we developed a bacterial artificial chromosome (BAC)–based physical map and integrated it with the genome sequence and genetic map. One BAC library, developed in the HindIII restriction enzyme site, consists of 4608 clones with an insert size of approximately 107 kb and covers about 13.5 genome equivalents. The other library, developed in the BamHI restriction enzyme site, consists of 3072 clones with an insert size of approximately 95 kb and covers about 8.0 genome equivalents. We fingerprinted 2688 clones from the HindIII library and 1536 clones from the BamHI library and developed a physical map of F. graminearum consisting of 26 contigs covering 39.2 Mb. Comparison of our map with the F. graminearum genome sequence showed that the size of our physical map is equivalent to the 36.1 Mb of the genome sequence. We used 31 sequence-based genetic markers, randomly spaced throughout the genome, to integrate the physical map with the genetic map. We also end-sequenced 17 BamHI BAC clones and identified 4 clones that spanned gaps in the genome sequence. Our new integrated map is highly reliable and useful for a variety of genomics studies.

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 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 Integrative Physical and Genetic Mapping of the Chickpea Genome for Fine Mapping and Analysis of Agronomic Traits

2010 ◽  
Author(s):  
Hongbin Zhang ◽  
Shahal Abbo ◽  
Weidong Chen ◽  
Amir Sherman ◽  
Dani Shtienberg ◽  
...  
Keyword(s):  
Ssr Markers ◽  
Dna Markers ◽  
Agronomic Traits ◽  
Physical Map ◽  
Ascochyta Blight ◽  
Genetic Maps ◽  
Average Insert Size ◽  
Marker Assisted Breeding ◽  
Insert Size ◽  
A Genome

Chickpea is the third most important pulse crop in the world and ranks first in the Middle East; however, it has been subjected to only limited research in modern genomics. In the first period of this project (US-3034-98R) we constructed two large-insert BAC and BIBAC libraries, developed 325 SSR markers and mapped QTLs controlling ascochyta blight resistance (ABR) and days to first flower (DTF). Nevertheless, the utilities of these tools and results in gene discovery and marker-assisted breeding are limited due to the absence of an essential platform. The goals of this period of the project were to use the resources and tools developed in the first period of the project to develop a BAC/BIBAC physical map for chickpea and using it to identify BAC/BIBACcontigs containing agronomic genes of interest, with an emphasis on ABR and DTF, and develop DNA markers suitable for marker-assisted breeding. Toward these goals, we proposed: 1) Fingerprint ~50,000 (10x) BACs from the BAC and BIBAC libraries, assemble the clones into a genome-wide BAC/BIBAC physical map, and integrate the BAC/BIBAC map with the existing chickpea genetic maps (Zhang, USA); 2) fine-map ABR and DTFQTLs and enhance molecular tools for chickpea genetics and breeding (Shahal, Sherman and DaniShtienberg, Israel; Chen and Muehlbauer; USA); and 3) integrate the BAC/BIBAC map with the existing chickpea genetic maps (Sherman, Israel; Zhang and Chen, USA). For these objectives, a total of $460,000 was requested originally, but a total of $300,000 was awarded to the project. We first developed two new BAC and BIBAC libraries, Chickpea-CME and Chickpea- CHV. The chickpea-CMEBAC library contains 22,272 clones, with an average insert size of 130 kb and equivalent to 4.0 fold of the chickpea genome. The chickpea-CHVBIBAC library contains 38,400 clones, with an average insert size of 140 kb and equivalent to 7.5 fold of the chickpea genome. The two new libraries (11.5 x), along with the two BAC (Chickpea-CHI) and BIBAC (Chickpea-CBV) libraries (7.1 x) constructed in the first period of the project, provide libraries essential for chickpea genome physical mapping and many other genomics researches. Using these four libraries we then developed the proposed BAC/BIBAC physical map of chickpea. A total of 67,584 clones were fingerprinted, and 64,211 (~11.6 x) of the fingerprints validated and used in the physical map assembly. The physical map consists of 1,945 BAC/BIBACcontigs, with each containing an average of 39.2 clones and having an average physical length of 559 kb. The contigs collectively span ~1,088 Mb, being 1.49 fold of the 740- Mb chickpea genome. Third, we integrated the physical map with the two existing chickpea genetic maps using a total of 172 (124 + 48) SSR markers. Fourth, we identified tightly linked markers for ABR-QTL1, increased marker density at ABR-QTL2 and studied the genetic basis of resistance to pod abortion, a major problem in the east Mediterranean, caused by heat stress. Finally, we, using the integrated map, isolated the BAC/BIBACcontigs containing or closely linked to QTL4.1, QTL4.2 and QTL8 for ABR and QTL8 for DTF. The integrated BAC/BIBAC map resulted from the project will provide a powerful platform and tools essential for many aspects of advanced genomics and genetics research of this crop and related species. These includes, but are not limited to, targeted development of SNP, InDel and SSR markers, high-resolution mapping of the chickpea genome and its agronomic genes and QTLs, sequencing and decoding of all genes of the genome using the next-generation sequencing technology, and comparative genome analysis of chickpea versus other legumes. The DNA markers and BAC/BIBACcontigs containing or closely linked to ABR and DTF provide essential tools to develop SSR and SNP markers well-suited for marker-assisted breeding of the traits and clone their corresponding genes. The development of the tools and knowledge will thus promote enhanced and substantial genetic improvement of the crop and related legumes.

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