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.

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 Six promising selections from the Hungarian apple breeding program for multiple resistance

2005 ◽  
Vol 11 (3) ◽  
Author(s):  
M. Tóth
Keyword(s):  
Powdery Mildew ◽  
Fire Blight ◽  
Molecular Genetic ◽  
Apple Scab ◽  
Breeding Program ◽  
Scab Resistance ◽  
Multiple Resistance ◽  
Vf Gene ◽  
Apple Breeding ◽  
Malus Floribunda

An apple breeding program has been carried out at the Department of Fruit Science for more than a decade. Several apple selections have been released from the progenies of crosses in 1992 and 1993. Six candidates were submitted for national recognition out of the hybrids examined for more than a decade. The six selections are resistant against all the three most important apple diseases (apple scab, powdery mildew and fire blight). Scab resistance is controlled by the Vf gene originating from the species Malus floribunda 821 and transmitted by cultivar Prima. Heterozygote Vfvf genotype of the six cultivar candidates was proved by molecular genetic examinations of Dept. Genetics and Plant Breeding. Characteristics of these selections from 'Prima' progenies are shown on the base of our own observations.

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.

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.

Comparison of the chromosome maps around a resistance hot spot on chromosome 5 of potato and tomato using BAC-FISH painting

Genome ◽  
2010 ◽  
Vol 53 (2) ◽  
pp. 103-110 ◽  
Author(s):  
Ute C. Achenbach ◽  
Xiaomin Tang ◽  
Agim Ballvora ◽  
Hans de Jong ◽  
Christiane Gebhardt
Keyword(s):  
Hot Spot ◽  
Artificial Chromosome ◽  
Globodera Pallida ◽  
Chromosome 5 ◽  
Physical Size ◽  
Bac Clones ◽  
A Genome ◽  
Chromosome Maps ◽  
Bac Fish

Potato chromosome 5 harbours numerous genes for important qualitative and quantitative traits, such as resistance to the root cyst nematode Globodera pallida and the late blight fungus, Phytophthora infestans . The genes make up part of a “hot spot” for resistances to various pathogens covering a genetic map length of 3 cM between markers GP21 and GP179. We established the physical size and position of this region on chromosome 5 in potato and tomato using fluorescence in situ hybridization (FISH) on pachytene chromosomes. Five potato bacterial artificial chromosome (BAC) clones with the genetically anchored markers GP21, R1-contig (proximal end), CosA, GP179, and StPto were selected, labeled with different fluorophores, and hybridized in a five-colour FISH experiment. Our results showed the location of the BAC clones in the middle of the long arm of chromosome 5 in both potato and tomato. Based on chromosome measurements, we estimate the physical size of the GP21–GP179 interval at 0.85 Mb and 1.2 Mb in potato and tomato, respectively. The GP21–GP179 interval is part of a genome segment known to have inverted map positions between potato and tomato.

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.

scholarly journals Construction and Characterization of a Bacterial Artificial Chromosome Library for the Hexaploid Wheat Line 92R137

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Qingdong Zeng ◽  
Fengping Yuan ◽  
Xin Xu ◽  
Xue Shi ◽  
Xiaojun Nie ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Hexaploid Wheat ◽  
Positional Cloning ◽  
Bac Library ◽  
Restriction Enzymes ◽  
Artificial Chromosome ◽  
Stripe Rust Resistance ◽  
Wheat Line ◽  
Genomic Resource

For map-based cloning of genes conferring important traits in the hexaploid wheat line 92R137, a bacterial artificial chromosome (BAC) library, including two sublibraries, was constructed using the genomic DNA of 92R137 digested with restriction enzymesHindIII andBamHI. The BAC library was composed of total 765,696 clones, of which 390,144 were from theHindIII digestion and 375,552 from theBamHI digestion. Through pulsed-field gel electrophoresis (PFGE) analysis of 453 clones randomly selected from theHindIII sublibrary and 573 clones from theBamHI sublibrary, the average insert sizes were estimated as 129 and 113 kb, respectively. Thus, theHindIII sublibrary was estimated to have a 3.01-fold coverage and theBamHI sublibrary a 2.53-fold coverage based on the estimated hexaploid wheat genome size of 16,700 Mb. The 765,696 clones were arrayed in 1,994 384-well plates. All clones were also arranged into plate pools and further arranged into 5-dimensional (5D) pools. The probability of identifying a clone corresponding to any wheat DNA sequence (such as geneYr26for stripe rust resistance) from the library was estimated to be more than 99.6%. Through polymerase chain reaction screening the 5D pools withXwe173, a marker tightly linked toYr26, six BAC clones were successfully obtained. These results demonstrate that the BAC library is a valuable genomic resource for positional cloning ofYr26and other genes of interest.

Sign in / Sign up

Export Citation Format

Share Document