Integrated Karyotyping of Woodland Strawberry (Fragaria vesca) with Oligopaint FISH Probes

2017 ◽  
Vol 153 (3) ◽  
pp. 158-164 ◽  
Author(s):  
Manman Qu ◽  
Kunpeng Li ◽  
Yanli Han ◽  
Lei Chen ◽  
Zongyun Li ◽  
...  
Keyword(s):  
Draft Genome ◽  
5S Rdna ◽  
Fish Analysis ◽  
Fragaria Vesca ◽  
Chromosome Identification ◽  
Multicolor Fish ◽  
Metaphase Chromosomes ◽  
Cross Species Chromosome Painting ◽  
Simultaneous Identification

Chromosome identification is critical for many aspects of cytogenetic research. However, for Fragaria vesca, definite identification of individual chromosomes is almost impossible because of their small size and high similarity. Here, we demonstrate that bulked oligonucleotide (oligo) probes can be used as chromosome-specific DNA markers for chromosome identification in F. vesca. Oligos specific to entire pseudochromosomes in the draft genome of F. vesca were identified and synthesized as libraries. In all, we synthesized 6 oligo libraries corresponding to 6 pseudochromosomes of F. vesca. These libraries were amplified and labeled as probes for fluorescence in situ hybridization (FISH). Two rounds of multicolor FISH analysis were sequentially conducted on the same metaphase cells with each round including 3 probe libraries, which permitted simultaneous identification of all chromosomes of F. vesca. Moreover, 45S and 5S rDNA were mapped to chromosomes 1, 2, and 7, respectively. A karyotype of metaphase chromosomes was constructed, representing the first FISH-based molecular cytogenetic karyotype of F. vesca. Our study can serve as a basis for future comparative cytogenetic research through cross-species chromosome painting using bulked oligo probes and will facilitate the application of breeding technologies that rely on the identification of chromosomes in the genus Fragaria.

FISH analysis of Zanthoxylum armatum based on oligonucleotides for 5S rDNA and (GAA)6

Genome ◽  
2018 ◽  
Vol 61 (9) ◽  
pp. 699-702 ◽  
Author(s):  
Xiaomei Luo ◽  
Juncheng Liu ◽  
Jingyan Wang ◽  
Wei Gong ◽  
Liang Chen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
5S Rdna ◽  
Fish Analysis ◽  
Oligonucleotide Probes ◽  
Mitotic Metaphase ◽  
Metaphase Chromosomes ◽  
Zanthoxylum Armatum ◽  
Rdna Loci ◽  
Cytogenetic Studies

Fluorescence in situ hybridization (FISH) using oligonucleotide probes for (GAA)6 (18 bp) and ribosomal DNA (rDNA) (5S rDNA, 41 bp) was applied to analyse Zanthoxylum armatum. (GAA)6 loci were detected on the pericentromeric regions of five chromosome pairs, and 5S rDNA loci were also detected on the pericentromeric regions of another two chromosome pairs. The densities and locations of (GAA)6 and 5S rDNA signals varied between individual chromosomes. High-intensity (GAA)6 signals were detected at the centromeres of two large and two smaller metacentric chromosomes. Relatively strong (GAA)6 signals were detected at the centromeres of two relatively small metacentric chromosomes, although strong 5S rDNA signals were detected at the centromeres of two additional smaller metacentric chromosomes. Weak (GAA)6 signals were detected at the centromeres of four large metacentric chromosomes, whereas weak 5S rDNA signals were detected at the centromeres of two smaller metacentric chromosomes. The remaining chromosomes exhibited no signals. Zanthoxylum armatum had 2n = ∼128. The lengths of the mitotic metaphase chromosomes ranged from 1.22 to 2.34 μm. Our results provide information that may be beneficial for future cytogenetic studies and could contribute to the physical assembly of the Zanthoxylum genome.

scholarly journals Repetitive DNA sequences accelerate molecular cytogenetic research in plants with small chromosomes

2019 ◽  
Vol 24 (2) ◽  
pp. 82
Author(s):  
Agus Budi Setiawan ◽  
Ari Wibowo ◽  
Chee How Teo ◽  
Shinji Kikuchi ◽  
Takato Koba
Keyword(s):  
Repetitive Dna ◽  
Dna Sequences ◽  
5S Rdna ◽  
Repetitive Dna Sequences ◽  
Type I ◽  
Chromosome Identification ◽  
Metaphase Chromosomes ◽  
Homologous Chromosomes ◽  
Centromeric Sequence ◽  
Subtelomeric Sequence

Repetitive DNA sequences are highly abundant in plant genomes and are favorable probes for chromosome identification in plants. However, it is difficult to conduct studies on the details of metaphase chromosome structures in plants with small chromosomes due to their highly condensed status. Therefore, identification of homologous chromosomes for karyotyping and analyzing chromosome structures is a challenging issue for cytogeneticists without specific probes and precise chromosome stages. In this study, five repetitive DNA probes, i.e., 5S and 45S ribosomal DNAs (rDNAs), melon centromeric sequence (Cmcent), cucumber subtelomeric sequence (Type I), and microsatellite (CT)10 repeats, were used to identify primary constrictions and homologous chromosomes for karyotyping. Four and two loci of 45S rDNA were respectively observed on metaphase and pachytene chromosomes of Abelia × grandiflora. Cmcent was detected on both primary constrictions of melon pachytene and metaphase chromosomes. Furthermore, one pair of 5S rDNA signals were hybridized on melon metaphase chromosomes. Eight and two loci of 45S and 5S rDNA were respectively detected on cucumber chromosomes. Type I and (CT)10 probes were specifically hybridized on subtelomeric and interstitial regions on the chromosomes, respectively. These results suggest that repetitive DNA sequences are versatile probes for chromosome identification in plants with small chromosomes, particularly for karyotyping analyses.

scholarly journals Fluorescence In Situ Hybridization (FISH) Analysis of the Locations of the Oligonucleotides 5S rDNA, (AGGGTTT)3, and (TTG)6 in Three Genera of Oleaceae and Their Phylogenetic Framework

Genes ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 375 ◽  
Author(s):  
Xiaomei Luo ◽  
Juncheng Liu
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
5S Rdna ◽  
Fish Analysis ◽  
Cytogenetic Map ◽  
Ligustrum Lucidum ◽  
Starting Point ◽  
Central Regions ◽  
Almost All

We report the cytogenetic map for a collection of species in the Oleaceae, and test similarities among the karyotypes relative to their known species phylogeny. The oligonucleotides 5S ribosomal DNA (rDNA), (AGGGTTT)3, and (TTG)6 were used as fluorescence in situ hybridization (FISH) probes to locate the corresponding chromosomes in three Oleaceae genera: Fraxinus pennsylvanica, Syringa oblata, Ligustrum lucidum, and Ligustrum × vicaryi. Forty-six small chromosomes were identified in four species. (AGGGTTT)3 signals were observed on almost all chromosome ends of four species, but (AGGGTTT)3 played no role in distinguishing the chromosomes but displayed intact chromosomes and could thus be used as a guide for finding chromosome counts. (TTG)6 and 5S rDNA signals discerned several chromosomes located at subterminal or central regions. Based on the similarity of the signal pattern (mainly in number and location and less in intensity) of the four species, the variations in the 5S rDNA and (TTG)6 distribution can be ordered as L. lucidum < L. × vicaryi < F. pennsylvanica < S. oblata. Variations have observed in the three genera. The molecular cytogenetic data presented here might serve as a starting point for further larger-scale elucidation of the structure of the Oleaceae genome, and comparison with the known phylogeny of Oleaceae family.

FISH landmarks for barley chromosomes (Hordeum vulgare L.)

Genome ◽  
1999 ◽  
Vol 42 (2) ◽  
pp. 274-281 ◽  
Author(s):  
Susan E Brown ◽  
Janice L Stephens ◽  
Nora LV Lapitan ◽  
Dennis L Knudson
Keyword(s):  
Hordeum Vulgare ◽  
Digital Imaging ◽  
18S Rdna ◽  
Chromosomal Location ◽  
5S Rdna ◽  
Hordeum Vulgare L ◽  
Metaphase Chromosomes ◽  
Bac Clone ◽  
Rdna Probe

Barley metaphase chromosomes (2n = 14) can be identified by fluorescence in situ hybridization (FISH) and digital imaging microscopy using heterologous 18S rDNA and 5S rDNA probe sequences. When these sequences are used together, FISH landmark signals were seen so that all 7 chromosomes were uniquely identified and unambiguously oriented. The chromosomal location of the landmark signals was determined by FISH to a barley trisomic series using the 18S and 5S probes labeled with different fluorophores. The utility of these FISH landmarks for barley physical mapping was also demonstrated when an Amy-2 cDNA clone and a BAC clone were hybridized with the FISH landmark probes.Key words: Hordeum vulgare, barley, FISH, 5S, 18S, rDNA, landmarks, chromosome.

Chromosomal localization of two novel repetitive sequences isolated from the Chenopodium quinoa Willd. genome

Genome ◽  
2011 ◽  
Vol 54 (9) ◽  
pp. 710-717 ◽  
Author(s):  
B. Kolano ◽  
B.W. Gardunia ◽  
M. Michalska ◽  
A. Bonifacio ◽  
D. Fairbanks ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Repetitive Sequences ◽  
Chenopodium Quinoa ◽  
5S Rdna ◽  
Rrna Gene ◽  
Fish Analysis ◽  
Rdna Loci

The chromosomal organization of two novel repetitive DNA sequences isolated from the Chenopodium quinoa Willd. genome was analyzed across the genomes of selected Chenopodium species. Fluorescence in situ hybridization (FISH) analysis with the repetitive DNA clone 18–24J in the closely related allotetraploids C. quinoa and Chenopodium berlandieri Moq. (2n = 4x = 36) evidenced hybridization signals that were mainly present on 18 chromosomes; however, in the allohexaploid Chenopodium album L. (2n = 6x = 54), cross-hybridization was observed on all of the chromosomes. In situ hybridization with rRNA gene probes indicated that during the evolution of polyploidy, the chenopods lost some of their rDNA loci. Reprobing with rDNA indicated that in the subgenome labeled with 18–24J, one 35S rRNA locus and at least half of the 5S rDNA loci were present. A second analyzed sequence, 12–13P, localized exclusively in pericentromeric regions of each chromosome of C. quinoa and related species. The intensity of the FISH signals differed considerably among chromosomes. The pattern observed on C. quinoa chromosomes after FISH with 12–13P was very similar to GISH results, suggesting that the 12–13P sequence constitutes a major part of the repetitive DNA of C. quinoa.

scholarly journals Coexpression of Three Odorant-Binding Protein Genes in the Foreleg Gustatory Sensilla of Swallowtail Butterfly Visualized by Multicolor FISH Analysis

2021 ◽  
Vol 1 ◽  
Author(s):  
Atsushi Ugajin ◽  
Katsuhisa Ozaki
Keyword(s):  
Host Plants ◽  
Fish Analysis ◽  
Multicolor Fish ◽  
Plant Recognition ◽  
Lepidopteran Insects ◽  
Soluble Ligand ◽  
Multicolor Fluorescence ◽  
Odorant Binding ◽  
Gustatory Sensilla

Lepidopteran insects are mostly monophagous or oligophagous. Female butterflies distinguish their host plants by detecting a combination of specific phytochemicals through the gustatory sensilla densely distributed on their foreleg tarsi, thereby ensuring oviposition on appropriate host plants. In this study, to gain insight into the molecular mechanism underlying host plant recognition by the gustatory sensilla, using Asian swallowtail, Papilio xuthus, we focused on a family of small soluble ligand-binding molecules, odorant-binding proteins (OBPs), and found that three OBP genes showed enriched expression in the foreleg tarsus. Multicolor fluorescence in situ hybridization analyses demonstrated the coexpression of these three OBP genes at the bases of the foreleg gustatory sensilla. Further analyses on other appendages revealed that PxutOBP3 was exclusively expressed in the tissues which could have direct contact with the leaf surface, suggesting that this OBP gene specifically plays an important role in phytochemicals perception.

Distribution of FISH oligo-5S rDNA and oligo-(AGGGTTT)3 in Hibiscus mutabilis L.

Genome ◽  
2021 ◽  
pp. 1-10
Author(s):  
Xiaomei Luo ◽  
Zhoujian He
Keyword(s):  
Chromosome Number ◽  
Physical Map ◽  
Molecular Cytogenetics ◽  
5S Rdna ◽  
Chromosome Length ◽  
Chromosome Identification ◽  
Karyotype Asymmetry ◽  
First Time ◽  
Distribution Of Fish

Hibiscus exhibits high variation in chromosome number both within and among species. The Hibiscus mutabilis L. karyotype was analyzed in detail using fluorescence in situ hybridization (FISH) with oligonucleotide probes for (AG3T3)3 and 5S rDNA, which were tested here for the first time. In total, 90 chromosomes were counted in prometaphase and metaphase, and all exhibited similarly intense (AG3T3)3 signals at both ends. (AG3T3)3 showed little variation and thus did not allow discrimination among H. mutabilis chromosomes, but its location at both ends confirmed the integrity of each chromosome, thus contributing to accurate counting of the numerous, small chromosomes. Oligo-5S rDNA marked the proximal/distal regions of six chromosomes: weak signals on chromosomes 7 and 8, slightly stronger signals on chromosomes 15 and 16, and very strong signals on chromosomes 17 and 18. Therefore, 5S rDNA could assist in chromosome identification in H. mutabilis. Metaphase chromosome lengths ranged from 3.00 to 1.18 μm, indicating small chromosomes. The ratios of longest to shortest chromosome length in prometaphase and metaphase were 2.58 and 2.54, respectively, indicating karyotype asymmetry in H. mutabilis. These results provide an exact chromosome number and a physical map, which will be useful for genome assembly and contribute to molecular cytogenetics in the genus Hibiscus.

scholarly journals Meiotic crossovers characterized by haplotype-specific chromosome painting in maize

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Lívia do Vale Martins ◽  
Fan Yu ◽  
Hainan Zhao ◽  
Tesia Dennison ◽  
Nick Lauter ◽  
...  
Keyword(s):  
Recombinant Inbred Lines ◽  
Critical Role ◽  
Fish Analysis ◽  
Recombinant Chromosome ◽  
Metaphase Chromosomes ◽  
Chromosome 10 ◽  
Homologous Chromosomes ◽  
Proximal Half ◽  
Maize Inbreds

Abstract Meiotic crossovers (COs) play a critical role in generating genetic variation and maintaining faithful segregation of homologous chromosomes during meiosis. We develop a haplotype-specific fluorescence in situ hybridization (FISH) technique that allows visualization of COs directly on metaphase chromosomes. Oligonucleotides (oligos) specific to chromosome 10 of maize inbreds B73 and Mo17, respectively, are synthesized and labeled as FISH probes. The parental and recombinant chromosome 10 in B73 x Mo17 F1 hybrids and F2 progenies can be unambiguously identified by haplotype-specific FISH. Analysis of 58 F2 plants reveals lack of COs in the entire proximal half of chromosome 10. However, we detect COs located in regions very close to the centromere in recombinant inbred lines from an intermated B73 x Mo17 population, suggesting effective accumulation of COs in recombination-suppressed chromosomal regions through intermating and the potential to generate favorable allelic combinations of genes residing in these regions.

scholarly journals Generation of microdissected DNA probes from metaphase chromosomes when chromosome identification by routine staining is impossible

2020 ◽  
Vol 24 (5) ◽  
pp. 519-524 ◽  
Author(s):  
K. S. Zadesenets ◽  
N. B. Rubtsov
Keyword(s):  
Metaphase Plate ◽  
Single Copy ◽  
Dna Probes ◽  
Dna Probe ◽  
Karyotypic Evolution ◽  
Chromosome Identification ◽  
Metaphase Chromosomes ◽  
Cell Line Establishment ◽  
Dna Libraries

Application of microdissected DNA libraries and DNA probes in numerous and various modern molecular cytogenetic studies showed them as an efficient and reliable tool in the analysis of chromosome reorganization during karyotypic evolution and in the diagnosis of human chromosome pathology. An important advantage of DNA probe generation by metaphase chromosome microdissection followed by sequence-independent polymerase chain reaction in comparison with the method of DNA probe generation using chromosome sorting is the possibility of DNA probe preparation from chromosomes of an individual sample without cell line establishment for the production of a large number of metaphase chromosomes. One of the main requirements for successful application of this technique is a possibility for identification of the chromosome of interest during its dissection and collection of its material from metaphase plates spread on the coverslip. In the present study, we developed and applied a technique for generation of microdissected DNA probes in the case when chromosome identification during microdissection appeared to be impossible. The technique was used for generation of two sets of Whole Chromosome Paints (WCPs) from all chromosomes of two species of free-living flatworms in the genus Macrostomum, M. mirumnovem and M. cliftonensis. The single-copy chromosome technique including separate collection of all chromosomes from one metaphase plate allowed us to generate WCPs that painted specifically the original chromosome by Chromosome In Situ Suppression Hybridization (CISS-Hybridization). CISS-Hybridization allowed identifying the original chromosome(s) used for DNA probe generation. Pooled WCPs derived from homologous chromosomes increased the intensity and specificity of chromosome painting provided by CISS-Hybridization. In the result, the obtained DNA probes appeared to be good enough for application in our studies devoted to analysis of karyotypic evolution in the genus Macrostomum and for analysis of chromosome rearrangements among the worms of laboratory cultures of M. mirumnovem.

scholarly journals Development of new cytogenetic markers for Thinopyrum ponticum (Podp.) Z.-W. Liu & R.-C. Wang

2019 ◽  
Vol 13 (3) ◽  
pp. 231-243 ◽  
Author(s):  
Pavel Yu. Kroupin ◽  
Victoria M. Kuznetsova ◽  
Ekaterina A. Nikitina ◽  
Yury Ts. Martirosyan ◽  
Gennady I. Karlov ◽  
...  
Keyword(s):  
Aegilops Tauschii ◽  
Whole Genome Sequence ◽  
Chromosome Identification ◽  
Multicolor Fish ◽  
Thinopyrum Ponticum ◽  
Real Time Quantitative Pcr ◽  
Lower Quantity ◽  
Cytogenetic Characterization

Thinopyrum ponticum (Podpěra, 1902) Z.-W. Liu &amp; R.-C.Wang, 1993 is an important polyploid wild perennial Triticeae species that is widely used as a source of valuable genes for wheat but its genomic constitution has long been debated. For its chromosome identification, only a limited set of FISH probes has been used. The development of new cytogenetic markers for Th. ponticum chromosomes is of great importance both for cytogenetic characterization of wheat-wheatgrass hybrids and for fundamental comparative studies of phylogenetic relationships between species. Here, we report on the development of five cytogenetic markers for Th. ponticum based on repetitive satellite DNA of which sequences were selected from the whole genome sequence of Aegilops tauschii Cosson, 1849. Using real-time quantitative PCR we estimated the abundance of the found repeats: P720 and P427 had the highest abundance and P132, P332 and P170 had lower quantity in Th. ponticum genome. Using fluorescence in situ hybridization (FISH) we localized five repeats to different regions of the chromosomes of Th. ponticum. Using reprobing multicolor FISH we colocalized the probes between each other. The distribution of these found repeats in the Triticeae genomes and its usability as cytogenetic markers for chromosomes of Th. ponticum are discussed.

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