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

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.

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

10.1101/518563 ◽

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.

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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 ◽

10.1139/g00-117 ◽

2001 ◽

Vol 44 (2) ◽

pp. 154-162 ◽

Cited By ~ 31

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.

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DECONVOLUTING BAC–GENE RELATIONSHIPS USING A PHYSICAL MAP

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720008003564 ◽

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.

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A Bacterial Artificial Chromosome Contig Spanning the Major Domestication Locus Q in Wheat and Identification of a Candidate Gene

Genetics ◽

10.1093/genetics/164.1.311 ◽

2003 ◽

Vol 164 (1) ◽

pp. 311-321 ◽

Cited By ~ 3

Author(s):

Justin D Faris ◽

John P Fellers ◽

Steven A Brooks ◽

Bikram S Gill

Keyword(s):

Physical Map ◽

Bac Library ◽

Wheat Chromosome ◽

Artificial Chromosome ◽

Physical Distance ◽

Bac Contig ◽

Complete Sequencing ◽

Bacterial Artificial Chromosome Contig ◽

Q Gene ◽

Similarity Searches

Abstract The Q locus played a major role in the domestication of wheat because it confers the free-threshing character and influences many other agronomically important traits. We constructed a physical contig spanning the Q locus using a Triticum monococcum BAC library. Three chromosome walking steps were performed by complete sequencing of BACs and identification of low-copy markers through similarity searches of database sequences. The BAC contig spans a physical distance of ∼300 kb corresponding to a genetic distance of 0.9 cM. The physical map of T. monococcum had perfect colinearity with the genetic map of wheat chromosome arm 5AL. Recombination data in conjunction with analysis of fast neutron deletions confirmed that the contig spanned the Q locus. The Q gene was narrowed to a 100-kb segment, which contains an APETALA2 (AP2)-like gene that cosegregates with Q. AP2 is known to play a major role in controlling floral homeotic gene expression and thus is an excellent candidate for Q.

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BAC contig development by fingerprint analysis in soybean

Genome ◽

10.1139/g97-056 ◽

1997 ◽

Vol 40 (4) ◽

pp. 420-427 ◽

Cited By ~ 52

Author(s):

L. Fredrick Marek ◽

R. C. Shoemaker

Keyword(s):

Disease Resistance ◽

Linkage Group ◽

Bacterial Artificial Chromosome ◽

Resistance Gene ◽

Bac Library ◽

Artificial Chromosome ◽

Disease Resistance Gene ◽

Average Insert Size ◽

Specific Sequence ◽

Class 1

We constructed a soybean bacterial artificial chromosome (BAC) library suitable for map-based cloning and physical mapping in soybean. This library consists of approximately 40 000 clones (4–5 genome equivalents) stored individually in 384-well microtiter dishes. A random sampling of 224 clones yielded an average insert size of 150 kb, giving a 98% probability of recovering any specific sequence. We screened the library for seven single or very low copy genie or genomic sequences using the polymerase chain reaction (PCR) and found between one and seven BACs for each of the seven sequences. When testing the library with a portion of the soybean psbA chloroplast gene, we found less than 1% chloroplast DNA representation. We also screened the library for eight different classes of disease resistance gene analogs (RGAs) and identified BACs containing all RGAs except class 8. We arranged nine of the class 1 RGA BACs and six of the class 3 RGA BACs into individual contigs based on fingerprint patterns observed after Southern probing of restriction digests of the member BACs with a class-specific sequence. This resulted in the partial localization of the different multigene family sequences without precise definition of their exact positions. Using PCR-based end rescue techniques and RFLP mapping of BAC ends, we mapped individual BACs of each contig onto linkage group J of the soybean public map. The class 1 contig mapped to the region on linkage group J that contains several disease resistance genes. The class 1 contig extended approximately 400 kb. The arrangement of the BACs within this contig has been confirmed using PCR. One end of the class 1 contig core BAC mapped to two positions on linkage group J and cosegregated with two class 1 RGA loci, suggesting that this segment is within an area of regional duplication.Key words: bacterial artificial chromosome, BAC library, soybean, contig, resistance gene analog.

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Construction of a bacterial artificial chromosome (BAC) library for potato molecular cytogenetics research

Genome ◽

10.1139/g99-099 ◽

2000 ◽

Vol 43 (1) ◽

pp. 199-204 ◽

Cited By ~ 32

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.

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Karyotype Evolution and Genomic Organization of Repetitive DNAs in the Saffron Finch, Sicalis flaveola (Passeriformes, Aves)

Animals ◽

10.3390/ani11051456 ◽

2021 ◽

Vol 11 (5) ◽

pp. 1456

Author(s):

Rafael Kretschmer ◽

Benilson Silva Rodrigues ◽

Suziane Alves Barcellos ◽

Alice Lemos Costa ◽

Marcelo de Bello Cioffi ◽

...

Keyword(s):

Biological Diversity ◽

Genomic Organization ◽

Repetitive Sequences ◽

Artificial Chromosome ◽

Sister Group ◽

Z Chromosome ◽

Domestic Species ◽

Dna Mapping ◽

Evolution Of Birds

The Saffron finch (Sicalis flaveola), a semi-domestic species, is tolerant of human proximity and nesting in roof spaces. Considering the importance of cytogenomic approaches in revealing different aspects of genomic organization and evolution, we provide detailed cytogenetic data for S. flaveola, including the standard Giemsa karyotype, C- and G-banding, repetitive DNA mapping, and bacterial artificial chromosome (BAC) FISH. We also compared our results with the sister groups, Passeriformes and Psittaciformes, bringing new insights into the chromosome and genome evolution of birds. The results revealed contrasting rates of intrachromosomal changes, highlighting the role of SSR (simple short repetition probes) accumulation in the karyotype reorganization. The SSRs showed scattered hybridization, but brighter signals were observed in the microchromosomes and the short arms of Z chromosome in S. flaveola. BACs probes showed conservation of ancestral syntenies of macrochromosomes (except GGA1), as well as the tested microchromosomes. The comparison of our results with previous studies indicates that the great biological diversity observed in Passeriformes was not likely accompanied by interchromosomal changes. In addition, although repetitive sequences often act as hotspots of genome rearrangements, Passeriformes species showed a higher number of signals when compared with the sister group Psittaciformes, indicating that these sequences were not involved in the extensive karyotype reorganization seen in the latter.

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

Genome ◽

10.1139/g99-076 ◽

1999 ◽

Vol 42 (6) ◽

pp. 1176-1182 ◽

Cited By ~ 66

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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