scholarly journals A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps

2021 ◽  
Author(s):  
Natalya Kudryavtseva ◽  
Aleksey Ermolaev ◽  
Gennady Karlov ◽  
Ilya Kirov ◽  
Masayoshi Shigyo ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Assembly ◽  
Genetic Maps ◽  
Plant Genome ◽  
Probe Design ◽  
Fish Technique ◽  
Plant Chromosomes ◽  
Dual Color ◽  
Chromosome Level

In situ imaging of molecular markers on a physical chromosome is an indispensable tool for refin-ing of genetic maps and validation genome assembly at the chromosomal level. Despite tremen-dous progress in genome sequencing the plant genome assembly at chromosome level still remain a challenge. Recently developed optical and Hi-C mapping aim to assist in genome assembly. For high-confidence in the genome assembly at chromosome level more independent approaches will be required. The present study aimed to refined an ultrasensitive Tyr-FISH technique and to de-velop a reliable and easy method for in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH technique to find out the reasons for the failures of using the method. It has been shown that successful visualization of marker/gene depends significantly on method of chromosome slide preparation, probe design and labelling, high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.7Kb with a frequency of 51.6%. Based on our results, we developed a reliable and simple protocol for dual-color Tyr-FISH visualization of short unique DNA sequences on plant chromosomes. New protocol allows more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.

scholarly journals A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps

2021 ◽  
Vol 22 (11) ◽  
pp. 5860
Author(s):  
Natalya Kudryavtseva ◽  
Aleksey Ermolaev ◽  
Gennady Karlov ◽  
Ilya Kirov ◽  
Masayoshi Shigyo ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Assembly ◽  
Genetic Maps ◽  
Plant Genome ◽  
Probe Design ◽  
Simple Method ◽  
Plant Chromosomes ◽  
Dual Color ◽  
Chromosome Level

In situ imaging of molecular markers on a physical chromosome is an indispensable tool for refining genetic maps and validation genome assembly at the chromosomal level. Despite the tremendous progress in genome sequencing, the plant genome assembly at the chromosome level remains a challenge. Recently developed optical and Hi-C mapping are aimed at assistance in genome assembly. For high confidence in the genome assembly at chromosome level, more independent approaches are required. The present study is aimed at refining an ultrasensitive Tyr-FISH technique and developing a reliable and simple method of in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH to find out the reasons behind the flaws of this technique. The accurate visualization of markers/genes appeared to be significantly dependent on the means of chromosome slide preparation, probe design and labeling, and high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.6 Kb with a frequency of 51.6%. Based on our results, we developed a more reliable and simple protocol for dual-color Tyr-FISH visualization of unique short DNA sequences on plant chromosomes. This new protocol can allow for more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.

A combined PRINS-FISH technique for simultaneous localisation of DNA sequences on plant chromosomes

1998 ◽  
Vol 41 (2) ◽  
pp. 293-296 ◽  
Author(s):  
M. Kubalakova ◽  
M. Nouzova ◽  
M. Dolezelova ◽  
J. Macas ◽  
J. Dolezel
Keyword(s):  
Dna Sequences ◽  
Fish Technique ◽  
Plant Chromosomes

scholarly journals Chromosome-level genome assembly of the female western mosquitofish (Gambusia affinis)

GigaScience ◽  
2020 ◽  
Vol 9 (8) ◽  
Author(s):  
Feng Shao ◽  
Arne Ludwig ◽  
Yang Mao ◽  
Ni Liu ◽  
Zuogang Peng
Keyword(s):  
Dna Sequences ◽  
Genome Assembly ◽  
Gambusia Affinis ◽  
Comparative Genomic ◽  
Suitable Model ◽  
High Quality ◽  
Western Mosquitofish ◽  
Poeciliid Fish ◽  
Oxford Nanopore ◽  
Chromosome Level

Abstract Background The western mosquitofish (Gambusia affinis) is a sexually dimorphic poeciliid fish known for its worldwide biological invasion and therefore an important research model for studying invasion biology. This organism may also be used as a suitable model to explore sex chromosome evolution and reproductive development in terms of differentiation of ZW sex chromosomes, ovoviviparity, and specialization of reproductive organs. However, there is a lack of high-quality genomic data for the female G. affinis; hence, this study aimed to generate a chromosome-level genome assembly for it. Results The chromosome-level genome assembly was constructed using Oxford nanopore sequencing, BioNano, and Hi-C technology. G. affinis genomic DNA sequences containing 217 contigs with an N50 length of 12.9 Mb and 125 scaffolds with an N50 length of 26.5 Mb were obtained by Oxford nanopore and BioNano, respectively, and the 113 scaffolds (90.4% of scaffolds containing 97.9% nucleotide bases) were assembled into 24 chromosomes (pseudo-chromosomes) by Hi-C. The Z and W chromosomes of G. affinis were identified by comparative genomic analysis of female and male G. affinis, and the mechanism of differentiation of the Z and W chromosomes was explored. Combined with transcriptome data from 6 tissues, a total of 23,997 protein-coding genes were predicted and 23,737 (98.9%) genes were functionally annotated. Conclusions The high-quality female G. affinis reference genome provides a valuable omics resource for future studies of comparative genomics and functional genomics to explore the evolution of Z and W chromosomes and the reproductive developmental biology of G. affinis.

Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research

Genome ◽  
2006 ◽  
Vol 49 (9) ◽  
pp. 1057-1068 ◽  
Author(s):  
Jiming Jiang ◽  
Bikram S. Gill
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Molecular Cytogenetics ◽  
Plant Genome ◽  
Current Status ◽  
Sequence Information ◽  
Resolution Mapping

Fluorescence in situ hybridization (FISH), which allows direct mapping of DNA sequences on chromosomes, has become the most important technique in plant molecular cytogenetics research. Repetitive DNA sequence can generate unique FISH patterns on individual chromosomes for karyotyping and phylogenetic analysis. FISH on meiotic pachytene chromosomes coupled with digital imaging systems has become an efficient method to develop physical maps in plant species. FISH on extended DNA fibers provides a high-resolution mapping approach to analyze large DNA molecules and to characterize large genomic loci. FISH-based physical mapping provides a valuable complementary approach in genome sequencing and map-based cloning research. We expect that FISH will continue to play an important role in relating DNA sequence information to chromosome biology. FISH coupled with immunoassays will be increasingly used to study features of chromatin at the cytological level that control expression and regulation of genes.

Use of fluorescence in situ hybridization to study the arrangement of DNA sequences in normal and disease-related chromosomes

1995 ◽  
Vol 53 ◽  
pp. 748-749
Author(s):  
Barbara J. F. Trask ◽  
Hillary Massa ◽  
Cynthia Friedman ◽  
Richard Esposito ◽  
Ger van den Engh ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Dna Sequences ◽  
Single Copy ◽  
Two Dimensions ◽  
Genetic Maps ◽  
Epifluorescence Microscopy ◽  
Metaphase Chromosomes ◽  
Interphase Nuclei

The sites of specific DNA sequences can be fluorescently tagged by fluorescence in situ hybridization (FISH). Different sequences can be labeled with different fluorochromes so that their arrangement can be studied using epifluorescence microscopy. The distances between points on the same or different chromosomes can be determined easily in a large number of interphase nuclei or metaphase chromosomes. A variety of probe types, ranging from single-copy sequences to highly repeated sequences can be employed. Our work has focussed on the analysis of hybridization patterns in two dimensions using conventional fluorescence microscopy.We have used FISH to study various aspects of genome organization that are difficult to study using other techniques. Examples of these applications will be presented.FISH is now the method of choice for determining the chromosomal location of DNA sequences. DNA sequences can be positioned in the genome with <1:1000 accuracy (to a 3-Mbp region within a 3000-Mbp genome). Through FISH, the cytogenetic, physical and genetic maps of chromosomes can be linked.

Correlating the Genetic and Physical Map of Barley Chromosome 3H Revealed Limitations of the FISH-Based Mapping of Nearby Single-Copy Probes Caused by the Dynamic Structure of Metaphase Chromosomes

2017 ◽  
Vol 152 (2) ◽  
pp. 90-96 ◽  
Author(s):  
Fernanda O. Bustamante ◽  
Lala Aliyeva-Schnorr ◽  
Jörg Fuchs ◽  
Sebastian Beier ◽  
Andreas Houben
Keyword(s):  
Dna Sequences ◽  
Physical Map ◽  
Dynamic Structure ◽  
Single Copy ◽  
Physical Distance ◽  
Genetic Distances ◽  
Genetic Maps ◽  
Metaphase Chromosomes ◽  
Physical Maps

Genetic maps are based on the recombination frequency of molecular markers which often show different positions in comparison to the corresponding physical maps. To decipher the position and order of DNA sequences genetically mapped to terminal and interstitial regions of barley (Hordeum vulgare) chromosome 3H, fluorescence in situ hybridization (FISH) on mitotic metaphase chromosomes was performed with 16 genomic single-copy probes derived from fingerprinted BAC contigs. Long genetic distances at subterminal regions translated into short physical distances, confirming that recombination events occur more often at distal regions of chromosome 3H. Nonoverlapping FISH signals were frequently obtained for probes with a physical distance of at least 30-60 kb. Only 8% of the analyzed chromosomes showed a symmetric order of FISH signals on both sister chromatids. Due to the dynamic packing of metaphase chromatin, the order of 2 adjacent single-copy signals along the chromosome arms outside the (peri)centromeric region can only reliably be determined if the cytological distance is approximately 3%, corresponding to 21.6 Mb.

Nonisotopic in situ hybridization and plant genome mapping: the first 10 years

Genome ◽  
1994 ◽  
Vol 37 (5) ◽  
pp. 717-725 ◽  
Author(s):  
Jiming Jiang ◽  
Bikram S. Gill
Keyword(s):  
In Situ Hybridization ◽  
Physical Mapping ◽  
Dna Sequences ◽  
Genome Mapping ◽  
Molecular Cytogenetics ◽  
Gene Families ◽  
Single Copy ◽  
Plant Genome ◽  
Metaphase Chromosomes

Nonisotopic in situ hybridization (ISH) was introduced in plants in 1985. Since then the technique has been widely used in various areas of plant genome mapping. ISH has become a routine method for physical mapping of repetitive DNA sequences and multicopy gene families. ISH patterns on somatic metaphase chromosomes using tandemly repeated sequences provide excellent physical markers for chromosome identification. Detection of low or single copy sequences were also reported. Genomic in situ hybridization (GISH) was successfully used to analyze the chromosome structure and evolution of allopolyploid species. GISH also provides a powerful technique for monitoring chromatin introgession during interspecific hybridization. A sequential chromosome banding and ISH technique was developed. The sequential technique is very useful for more precise and efficient mapping as well as cytogenetic determination of genomic affinities of individual chromosomes in allopolyploid species. A critical review is made on the present resolution of the ISH technique and the future outlook of ISH research is discussed.Key words: in situ hybridization, physical mapping, genome mapping, molecular cytogenetics.

Physical mapping of wheat-Aegilops longissima breakpoints in mildew-resistant recombinant lines using FISH with highly repeated and low-copy DNA probes

Genome ◽  
1999 ◽  
Vol 42 (5) ◽  
pp. 1013-1019 ◽  
Author(s):  
Marco Biagetti ◽  
Francesca Vitellozzi ◽  
Carla Ceoloni
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Common Wheat ◽  
Dna Sequences ◽  
Dna Probes ◽  
Genetic Maps ◽  
Chromosome Engineering ◽  
Aegilops Longissima ◽  
Copy Dna

Fluorescence in situ hybridization (FISH) with multiple probes, consisting of highly repeated DNA sequences (pSc119.2 and pAs1) and of a low-copy, 3BS-specific RFLP sequence (PSR907), enabled determination of the physical position of the wheat-alien breakpoints (BPs) along the 3BS and 3DS arms of common wheat recombinant lines. These lines harbour 3SlS Aegilops longissima segments containing the powdery mildew resistance gene Pm13. In all 3B recombinants, the wheat-Aegilops longissima physical BPs lie within the interval separating the two most distal of the three pSc119.2 3BS sites. In all such recombinants a telomeric segment, containing the most distal of the pSc119.2 3BS sites, was in fact replaced by a homoeologous Ae. longissima segment, marked by characteristic pSc119.2 hybridization sites. Employment of the PSR907 RFLP probe as a FISH marker allowed to resolve further the critical region in the various 3B recombinant lines. Three of them, like the control common wheat, exhibited between the two most distal pSc119.2 sites a single PSR907 FISH site, which was missing in a fourth recombinant line. The amount of alien chromatin can thus be estimated to represent around 20% of the recombinant arm in the three former lines and a maximum of 27% in the latter. A similar physical length was calculated for the alien segment contained in three 3D recombinants, all characterized by the presence of the Ae. longissima pSc119.2 sites distal to the nearly telomeric pAs1 sites of normal 3DS. Comparison between the FISH-based maps and previously developed RFLP maps of the 3BS-3SlS and 3DS-3SlS arms revealed substantial differences between physical and genetic map positions of the wheat-alien BPs and of molecular markers associated with the critical chromosomal portions.Key words: wheat-alien recombinants, chromosome engineering, fluorescence in situ hybridization, highly repeated and low-copy DNA probes, physical versus genetic maps.

DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 496-497
Author(s):  
Barbara Trask ◽  
Susan Allen ◽  
Anne Bergmann ◽  
Mari Christensen ◽  
Anne Fertitta ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Dual Band ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

A Chromosome-Level Genome Assembly Identifies the Origin of Neo-Y Chromosome to the Unique X 1X 2Y System

2019 ◽  
Author(s):  
Yongshuang Xiao ◽  
Zhizhong Xiao ◽  
Daoyuan Ma ◽  
Chenxi Zhao ◽  
Lin Liu ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Genome Assembly ◽  
Chromosome Level
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