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.

Integrated karyotyping of sorghum by in situ hybridization of landed BACs

Genome ◽  
2002 ◽  
Vol 45 (2) ◽  
pp. 402-412 ◽  
Author(s):  
Jeong-Soon Kim ◽  
Kevin L Childs ◽  
M Nurul Islam-Faridi ◽  
Monica A Menz ◽  
Robert R Klein ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Genetic Manipulation ◽  
Artificial Chromosome ◽  
Linkage Groups ◽  
Cytogenetic Map ◽  
Structural Genomic ◽  
Bac Clones ◽  
Flow Sorting ◽  
Fish Signal

The reliability of genome analysis and proficiency of genetic manipulation are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, and orientation with respect to telomeres. We have endeavored to establish means to enable these steps in sorghum (Sorghum bicolor (L.) Moench), the genome of which contains ca. 780 Mbp spread across n = 10 chromosomes. Our approach relies on fluorescence in situ hybridization (FISH) and integrated structural genomic resources, including large-insert genomic clones in bacterial artificial chromosome (BAC) libraries. To develop robust FISH probes, we selected sorghum BACs by association with molecular markers that map near the ends of linkage groups, in regions inferred to be high in recombination. Overall, we selected 22 BACs that encompass the 10 linkage groups. As a prelude to development of a multiprobe FISH cocktail, we evaluated BAC-derived probes individually and in small groups. Biotin- and digoxygenin-labeled probes were made directly from the BAC clones and hybridized in situ to chromosomes without using suppressive unlabelled C0t-1 DNA. Based on FISH-signal strength and the relative degree of background signal, we judged 19 BAC-derived probes to be satisfactory. Based on their relative position, and collective association with all 10 linkage groups, we chose 17 of the 19 BACs to develop a 17-locus probe cocktail for dual-color detection. FISH of the cocktail allowed simultaneous identification of all 10 chromosomes. The results indicate that linkage and physical maps of sorghum allow facile selection of BAC clones according to position and FISH-signal quality. This capability will enable development of a high-quality molecular cytogenetic map and an integrated genomics system for sorghum, without need of chromosome flow sorting or microdissection. Moreover, transgeneric FISH experiments suggest that the sorghum system might be applicable to other Gramineae.Key words: integrated karyotyping, FISH, sorghum, BAC.

Karyotyping of Brachypodium pinnatum (2n = 18) chromosomes using cross-species BAC–FISH

Genome ◽  
2013 ◽  
Vol 56 (4) ◽  
pp. 239-243 ◽  
Author(s):  
Elzbieta Wolny ◽  
Wojciech Fidyk ◽  
Robert Hasterok
Keyword(s):  
Brachypodium Distachyon ◽  
Genome Structure ◽  
In Situ Hybridisation ◽  
Artificial Chromosome ◽  
Fluorescence In Situ Hybridisation ◽  
Morphometric Features ◽  
Sequential Experiments ◽  
Brachypodium Pinnatum ◽  
Bac Fish

Identification of individual chromosomes in a complement is usually a difficult task in the case of most plant species, especially for those with small, numerous, and morphologically uniform chromosomes. In this paper, we demonstrate that the landmarks produced by cross-species fluorescence in situ hybridisation (FISH) of Brachypodium distachyon derived bacterial artificial chromosome (BAC) clones can be used for discrimination of Brachypodium pinnatum (2n = 18) chromosomes. Selected sets of clones were hybridised in several sequential experiments performed on exactly the same chromosome spreads, using reprobing of cytological preparations. Analysis of the morphometric features of B. pinnatum chromosomes was performed to establish their total length, the position of centromeres, and the position of BAC-based landmarks in relation to the centromere, thereby enabling their effective karyotyping, which is a prerequisite for more complex study of the grass genome structure and evolution at the cytomolecular level.

scholarly journals Chromosome Identification and Cytogenetic Map Construction of Zhikong Scallop (Chlamys farreri) Based on Fluorescence in situ Hybridization

2021 ◽  
Vol 8 ◽  
Author(s):  
Liping Hu ◽  
Liming Jiang ◽  
Qiang Xing ◽  
Zujing Yang ◽  
Qiang Zhao ◽  
...  
Keyword(s):  
Molecular Breeding ◽  
Repetitive Sequences ◽  
Genomic Analysis ◽  
Artificial Chromosome ◽  
Chlamys Farreri ◽  
Cytogenetic Map ◽  
Zhikong Scallop ◽  
Bac Clones ◽  
Map Construction

Zhikong scallop (Chlamys farreri) is a bivalve species with broad economic and biological value, and an essential species of aquaculture in North China. Recently, efforts have been made to improve knowledge of genome, genetics, and cytogenetics, which is devoted to develop the molecular breeding project for the scallop. In this study, we constructed a cytogenetic map and identified all chromosomes of C. farreri using fluorescence in situ hybridization (FISH). A total of 100 Bacterial Artificial Chromosome (BAC) clones and 27 fosmid clones, including 58 microsatellite marker-anchored BAC clones, 4 genes-anchored BAC clones, 38 random BAC clones, 22 repetitive sequences-anchored fosmid clones, and 5 gene-anchored fosmid clones, were tested as probes, and 69 of them produced specific and stable signal on one pair of chromosomes. Then, multiple co-hybridizations were conducted to distinguish all the submetacentric and subtelocentric chromosomes with similar morphology by the abovementioned chromosome-specific markers. On this basis, a cytogenetic map of C. farreri containing 69 clones was constructed by co-hybridization and karyotype analysis. The markers covered all 19 pairs of chromosomes, and the average number of markers on each chromosome was 3.6. The cytogenetic map provides a platform for genetic and genomic analysis of C. farreri, which facilitates the molecular breeding project of C. farreri and promotes the comparative studies of chromosome evolution in scallops and even bivalves.

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 Construction and Characterization of a Bacterial Artificial Chromosome Library for the A-Genome of Cotton (G.arboreumL.)

2011 ◽  
Vol 2011 ◽  
pp. 1-5 ◽  
Author(s):  
Yan Hu ◽  
Yamin Lu ◽  
Dan Ma ◽  
Wangzhen Guo ◽  
Tianzhen Zhang
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Gene Isolation ◽  
Comparative Genome Analysis ◽  
Bac Clones ◽  
A Genome ◽  
Reference Map ◽  
Insert Length

A bacterial artificial chromosome (BAC) library for the A-genome of cotton has been constructed from the leaves ofG. arboreumL cv. Jianglinzhongmian. It is used as elite A-genome germplasm resources in the present cotton breeding program and has been used to build a genetic reference map of cotton. The BAC library consists of 123,648 clones stored in 322 384-well plates. Statistical analysis of a set of 103 randomly selected BAC clones indicated that each clone has an average insert length of 100.2 kb per plasmid, with a range of 30 to 190 kb. Theoretically, this represents 7.2 haploid genome equivalents based on an A-genome size of 1697 Mb. The BAC library has been arranged in column pools and superpools allowing screening with various PCR-based markers. In the future, the A-genome cotton BAC library will serve as both a giant gene resource and a valuable tool for map-based gene isolation, physical mapping and comparative genome analysis.

scholarly journals The Puzzling Fate of a Lupin Chromosome Revealed by Reciprocal Oligo-FISH and BAC-FISH Mapping

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1489
Author(s):  
Wojciech Bielski ◽  
Michał Książkiewicz ◽  
Denisa Šimoníková ◽  
Eva Hřibová ◽  
Karolina Susek ◽  
...  
Keyword(s):  
Artificial Chromosome ◽  
Oligonucleotide Probes ◽  
Genome Reorganization ◽  
Depth Study ◽  
Genome Assemblies ◽  
Polyploidization Event ◽  
Lupinus Pilosus ◽  
Genome Triplication ◽  
Bac Fish

Old World lupins constitute an interesting model for evolutionary research due to diversity in genome size and chromosome number, indicating evolutionary genome reorganization. It has been hypothesized that the polyploidization event which occurred in the common ancestor of the Fabaceae family was followed by a lineage-specific whole genome triplication (WGT) in the lupin clade, driving chromosome rearrangements. In this study, chromosome-specific markers were used as probes for heterologous fluorescence in situ hybridization (FISH) to identify and characterize structural chromosome changes among the smooth-seeded (Lupinus angustifolius L., Lupinus cryptanthus Shuttlew., Lupinus micranthus Guss.) and rough-seeded (Lupinus cosentinii Guss. and Lupinus pilosus Murr.) lupin species. Comparative cytogenetic mapping was done using FISH with oligonucleotide probes and previously published chromosome-specific bacterial artificial chromosome (BAC) clones. Oligonucleotide probes were designed to cover both arms of chromosome Lang06 of the L. angustifolius reference genome separately. The chromosome was chosen for the in-depth study due to observed structural variability among wild lupin species revealed by BAC-FISH and supplemented by in silico mapping of recently released lupin genome assemblies. The results highlighted changes in synteny within the Lang06 region between the lupin species, including putative translocations, inversions, and/or non-allelic homologous recombination, which would have accompanied the evolution and speciation.

Simultaneous painting of three genomes in hexaploid wheat by BAC-FISH

Genome ◽  
2004 ◽  
Vol 47 (5) ◽  
pp. 979-987 ◽  
Author(s):  
Peng Zhang ◽  
Wanlong Li ◽  
Bernd Friebe ◽  
Bikram S Gill
Keyword(s):  
Triticum Aestivum ◽  
In Situ Hybridization ◽  
Transposable Elements ◽  
Fluorescence In Situ Hybridization ◽  
Hexaploid Wheat ◽  
D Genome ◽  
Bac Clones ◽  
Dispersed Repeat ◽  
Bac Fish

Fluorescence in situ hybridization (FISH) is widely used in the physical mapping of genes and chromosome landmarks in plants and animals. Bacterial artificial chromosomes (BACs) contain large inserts, making them amenable for FISH mapping. In our BAC-FISH experiments, we selected 56 restriction fragment length polymorphism (RFLP)-locus-specific BAC clones from the libraries of Triticum monococcum and Aegilops tauschii, which are the A- and D-genome donors of wheat (Triticum aestivum, 2n = 6x = 42), respectively. The BAC clone 676D4 from the T. monococcum library contains a dispersed repeat that preferentially hybridizes to A-genome chromosomes, and two BAC clones, 9I10 and 9M13, from the Ae. tauschii library contain a dispersed repeat that preferentially hybridizes to the D-genome chromosomes. These repeats are useful in simultaneously discriminating the three different genomes in hexaploid wheat, and in identifying intergenomic translocations in wheat or between wheat and alien chromosomes. Sequencing results show that both of these repeats are transposable elements, indicating the importance of transposable elements, especially retrotransposons, in the genome evolution of wheat.Key words: bacterial artificial chromosome (BAC), fluorescence in situ hybridization (FISH), transposable elements (TEs), wheat, Triticum aestivum.

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