scholarly journals Development of a sequence-based reference physical map of pea (Pisum sativum L.)

2019 ◽  
Author(s):  
Krishna Kishore Gali ◽  
Bunyamin Tar’an ◽  
Mohammed-Amin Madoui ◽  
Edwin van der Vossen ◽  
Jan van Oeveren ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Positional Cloning ◽  
Physical Map ◽  
Repetitive Sequences ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Bac Clones ◽  
Pooled Dna ◽  
Mapping Technology ◽  
Generation Sequencing

AbstractWhole genome profiling (WGP) is a sequence-based physical mapping technology and uses sequence tags generated by next generation sequencing for construction of bacterial artificial chromosome (BAC) contigs of complex genomes. The physical map provides a framework for assembly of genome sequence and information for localization of genes that are difficult to find through positional cloning. To address the challenges of accurate assembly of the pea genome (~4.2 GB of which approximately 85% is repetitive sequences), we have adopted the WGP technology for assembly of a pea BAC library. Multi-dimensional pooling of 295,680 BAC clones and sequencing the ends of restriction fragments of pooled DNA generated 1,814 million high quality reads, of which 825 million were deconvolutable to 1.11 million unique WGP sequence tags. These WGP tags were used to assemble 220,013 BACs into contigs. Assembly of the BAC clones using the modified Fingerprinted Contigs (FPC) program has resulted in 13,040 contigs, consisting of 213,719 BACs, and 6,294 singleton BACs. The average contig size is 0.33 Mbp and the N50 contig size is 0.62 Mbp. WGPTM technology has proved to provide a robust physical map of the pea genome, which would have been difficult to assemble using traditional restriction digestion based methods. This sequence-based physical map will be useful to assemble the genome sequence of pea. Additionally, the 1.1 million WGP tags will support efficient assignment of sequence scaffolds to the BAC clones, and thus an efficient sequencing of BAC pools with targeted genome regions of interest.

scholarly journals DECONVOLUTING BAC–GENE RELATIONSHIPS USING A PHYSICAL MAP

2008 ◽  
Vol 06 (03) ◽  
pp. 603-622
Author(s):  
YONGHUI WU ◽  
LAN LIU ◽  
TIMOTHY J. CLOSE ◽  
STEFANO LONARDI
Keyword(s):  
Physical Map ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Hybridization Experiment ◽  
Pooling Design ◽  
Bac Clones ◽  
Hybridization Data ◽  
A Genome ◽  
The Right ◽  
Combinatorial Pooling

Deconvolution of relationships between bacterial artificial chromosome (BAC) clones and genes is a crucial step in the selective sequencing of regions of interest in a genome. It often includes combinatorial pooling of unique probes obtained from the genes (unigenes), and screening of the BAC library using the pools in a hybridization experiment. Since several probes can hybridize to the same BAC, in order for the deconvolution to be achievable the pooling design has to be able to handle a large number of positives. As a consequence, smaller pools need to be designed, which in turn increases the number of hybridization experiments, possibly making the entire protocol unfeasible. We propose a new algorithm that is capable of producing high-accuracy deconvolution even in the presence of a weak pooling design, i.e. when pools are rather large. The algorithm compensates for the decrease of information in the hybridization data by taking advantage of a physical map of the BAC clones. We show that the right combination of combinatorial pooling and our algorithm not only dramatically reduces the number of pools required, but also successfully deconvolutes the BAC–gene relationships with almost perfect accuracy. Software is available on request from the first author.

A bacterial artificial chromosome (BAC) library of Malus floribunda 821 and contig construction for positional cloning of the apple scab resistance gene Vf

Genome ◽  
2001 ◽  
Vol 44 (6) ◽  
pp. 1104-1113 ◽  
Author(s):  
Mingliang Xu ◽  
Junqi Song ◽  
Zhukuan Cheng ◽  
Jiming Jiang ◽  
Schuyler S Korban
Keyword(s):  
Positional Cloning ◽  
Apple Scab ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Scab Resistance ◽  
Vf Gene ◽  
Bac Clones ◽  
Malus Floribunda ◽  
Sequence Characterized Amplified Regions

The apple scab resistance gene Vf, originating from the wild species Malus floribunda 821, has been incorporated into a wide variety of apple cultivars through a classical breeding program. With the aim of isolating the Vf gene, a bacterial artificial chromosome (BAC) library consisting of 31 584 clones has been constructed from M. floribunda 821. From the analysis of 88 randomly selected BAC clones, the average insert size is estimated at 125 kb. If it is assumed that the genome size of M. floribunda 821 is 769 Mb/haploid, the library represents about 5× haploid genome equivalents. This provides a 99% probability of finding any specific sequence from this library. PCR-based screening of the library has been carried out using eight random genomic sequence-characterized amplified regions (SCARs), chloroplast- and mitochondria-specific SCARs, and 13 high-density Vf-linked SCAR markers. An average of five positive BAC clones per random SCAR has been obtained, whereas less than 1% of BAC clones are derived from the chloroplast or mitochondrial genomes. Most BAC clones identified with Vf-linked SCAR markers are physically linked. Three BAC contigs along the Vf region have been obtained by assembling physically linked BAC clones based on their fingerprints. The overlapping relatedness of BAC clones has been further confirmed by cytogenetic mapping using fiber fluorescence in situ hybridization (fiber-FISH). The M. floribunda 821 BAC library provides a valuable genetic resource not only for map-based cloning of the Vf gene, but also for finding many other important genes for improving the cultivated apple.Key words: apple, resistance Vf gene, BAC library, sequence-characterized amplified regions (SCARs), fiber fluorescence in situ hybridization (fiber-FISH), positional cloning.

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.

Genomic organization of the 260 kb surrounding the waxy locus in a Japonica rice

Genome ◽  
1999 ◽  
Vol 42 (6) ◽  
pp. 1121-1126 ◽  
Author(s):  
Hironori Nagano ◽  
Lihua Wu ◽  
Shinji Kawasaki ◽  
Yuji Kishima ◽  
Yoshio Sano
Keyword(s):  
Genomic Organization ◽  
Physical Map ◽  
Repetitive Sequences ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Molecular Organization ◽  
Japonica Rice ◽  
Class 1 ◽  
Average Size ◽  
Waxy Locus

The present study was carried out to characterize the molecular organization in the vicinity of the waxy locus in rice. To determine the structural organization of the region surrounding waxy, contiguous clones covering a total of 260 kb were constructed using a bacterial artificial chromosome (BAC) library from the Shimokita variety of Japonica rice. This map also contains 200 overlapping subclones, which allowed construction of a fine physical map with a total of 64 HindIII sites. During the course of constructing the map, we noticed the presence of some repeated regions which might be related to transposable elements. We divided the 260-kb region into 60 segments (average size of 5.7 kb) to use as probes to determine their genomic organization. Hybridization patterns obtained by probing with these segments were classified into four types: class 1, a single or a few bands without a smeared background; class 2, a single or a few bands with a smeared background; class 3, multiple discrete bands without a smeared background; and class 4, only a smeared background. These classes constituted 6.5%, 20.9%, 3.7%, and 68.9% of the 260-kb region, respectively. The distribution of each class revealed that repetitive sequences are a major component in this region, as expected, and that unique sequence regions were mostly no longer than 6 kb due to interruption by repetitive sequences. We discuss how the map constructed here might be a powerful tool for characterization and comparison of the genome structures and the genes around the waxy locus in the Oryza species.Key words: BAC library, genomic organization, physical map, rice (Oryza sativa), the waxy locus.

scholarly journals A4Mb High Resolution BAC Contig on Bovine Chromosome1q12and Comparative Analysis With Human Chromosome21q22

2005 ◽  
Vol 6 (4) ◽  
pp. 194-203 ◽  
Author(s):  
Cord Drögemüller ◽  
Anne Wöhlke ◽  
Tosso Leeb ◽  
Ottmar Distl
Keyword(s):  
Positional Cloning ◽  
Physical Map ◽  
Dominant Gene ◽  
Bac Library ◽  
Exact Order ◽  
Bac Contig ◽  
Nucleotide Sequence Data ◽  
New Genes ◽  
Human Genes ◽  
First Time

The bovine RPCI-42 BAC library was screened to construct a sequence-ready ~4 Mb single contig of 92 BAC clones on BTA 1q12. The contig covers the region between the genesKRTAP8P1andCLIC6. This genomic segment in cattle is of special interest as it contains the dominant gene responsible for the hornless or polled phenotype in cattle. The construction of the BAC contig was initiated by screening the bovine BAC library with heterologous cDNA probes derived from 12 human genes of the syntenic region on HSA 21q22. Contig building was facilitated by BAC end sequencing and chromosome walking. During the construction of the contig, 165 BAC end sequences and 109 single-copy STS markers were generated. For comparative mapping of 25 HSA 21q22 genes, genomic PCR primers were designed from bovine EST sequences and the gene-associated STSs mapped on the contig. Furthermore, bovine BAC end sequence comparisons against the human genome sequence revealed significant matches to HSA 21q22 and allowed thein silicomapping of two new genes in cattle. In total, 31 orthologues of human genes located on HSA 21q22 were directly mapped within the bovine BAC contig, of which 16 genes have been cloned and mapped for the first time in cattle. In contrast to the existing comparative bovine–human RH maps of this region, these results provide a better alignment and reveal a completely conserved gene order in this 4 Mb segment between cattle, human and mouse. The mapping of known polled linked BTA 1q12 microsatellite markers allowed the integration of the physical contig map with existing linkage maps of this region and also determined the exact order of these markers for the first time. Our physical map and transcript map may be useful for positional cloning of the putative polled gene in cattle. The nucleotide sequence data reported in this paper have been submitted to EMBL and have been assigned Accession Numbers AJ698510–AJ698674.

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.

scholarly journals Use of a Mycobacterium tuberculosisH37Rv Bacterial Artificial Chromosome Library for Genome Mapping, Sequencing, and Comparative Genomics

1998 ◽  
Vol 66 (5) ◽  
pp. 2221-2229 ◽  
Author(s):  
Roland Brosch ◽  
Stephen V. Gordon ◽  
Alain Billault ◽  
Thierry Garnier ◽  
Karin Eiglmeier ◽  
...  
Keyword(s):  
Comparative Genomics ◽  
Bacterial Artificial Chromosome ◽  
Genome Mapping ◽  
Sequence Data ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Excellent Correlation ◽  
Sequencing Project ◽  
Polysaccharide Biosynthesis ◽  
Bac Clones

ABSTRACT The bacterial artificial chromosome (BAC) cloning system is capable of stably propagating large, complex DNA inserts in Escherichia coli. As part of the Mycobacterium tuberculosis H37Rv genome sequencing project, a BAC library was constructed in the pBeloBAC11 vector and used for genome mapping, confirmation of sequence assembly, and sequencing. The library contains about 5,000 BAC clones, with inserts ranging in size from 25 to 104 kb, representing theoretically a 70-fold coverage of the M. tuberculosisgenome (4.4 Mb). A total of 840 sequences from the T7 and SP6 termini of 420 BACs were determined and compared to those of a partial genomic database. These sequences showed excellent correlation between the estimated sizes and positions of the BAC clones and the sizes and positions of previously sequenced cosmids and the resulting contigs. Many BAC clones represent linking clones between sequenced cosmids, allowing full coverage of the H37Rv chromosome, and they are now being shotgun sequenced in the framework of the H37Rv sequencing project. Also, no chimeric, deleted, or rearranged BAC clones were detected, which was of major importance for the correct mapping and assembly of the H37Rv sequence. The minimal overlapping set contains 68 unique BAC clones and spans the whole H37Rv chromosome with the exception of a single gap of ∼150 kb. As a postgenomic application, the canonical BAC set was used in a comparative study to reveal chromosomal polymorphisms between M. tuberculosis, M. bovis, and M. bovis BCG Pasteur, and a novel 12.7-kb segment present in M. tuberculosis but absent from M. bovis and M. bovis BCG was characterized. This region contains a set of genes whose products show low similarity to proteins involved in polysaccharide biosynthesis. The H37Rv BAC library therefore provides us with a powerful tool both for the generation and confirmation of sequence data as well as for comparative genomics and other postgenomic applications. It represents a major resource for present and future M. tuberculosis research projects.

Genetic and contig map of a 2200-kb region encompassing 5.5 cM on chromosome 1 of Arabidopsis thaliana

Genome ◽  
1996 ◽  
Vol 39 (6) ◽  
pp. 1086-1092 ◽  
Author(s):  
Christian S. Hardtke ◽  
Thomas Berleth
Keyword(s):  
Arabidopsis Thaliana ◽  
Positional Cloning ◽  
Yeast Artificial Chromosome ◽  
Physical Map ◽  
Primary Root ◽  
Artificial Chromosome ◽  
Chromosome 1 ◽  
Rflp Markers ◽  
Caps Markers ◽  
Yac Contig

In the course of the isolation of the MONOPTEROS (MP) gene, required for primary root formation in Arabidopsis thaliana, a yeast artificial chromosome (YAC) contig encompassing approximately 2200 kilobases corresponding to 5.5 cM on the top arm of chromosome 1 was established. Forty-six YAC clones were characterized and 12 new restriction fragment length polymorphism (RFLP) markers are presented. Three new codominant amplified polymorphic sequence (CAPS) markers were generated that enabled high resolution genetic mapping and correlation of physical and genetic distances along the contig. The map contributes to the completion of a physical map of the Arabidopsis genome and should facilitate positional cloning of other genes in the region as well as studies on genome organization. We also present another set of 11 physically linked probes, as well as mapping data for additional RFLP markers within a broader interval of 10.4 cM. Key words : Arabidopsis, CAPS markers, MONOPTEROS gene, physical map, RFLP markers, YAC contig.

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.

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.

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