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

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. Recen...

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Autores principales: Kudryavtseva, Natalya, Ermolaev, Aleksey, Karlov, Gennady, Kirov, Ilya, Shigyo, Masayoshi, Sato, Shusei, Khrustaleva, Ludmila
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8215642/
https://www.ncbi.nlm.nih.gov/pubmed/34070753
http://dx.doi.org/10.3390/ijms22115860
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author Kudryavtseva, Natalya
Ermolaev, Aleksey
Karlov, Gennady
Kirov, Ilya
Shigyo, Masayoshi
Sato, Shusei
Khrustaleva, Ludmila
author_facet Kudryavtseva, Natalya
Ermolaev, Aleksey
Karlov, Gennady
Kirov, Ilya
Shigyo, Masayoshi
Sato, Shusei
Khrustaleva, Ludmila
author_sort Kudryavtseva, Natalya
collection PubMed
description 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.
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spelling pubmed-82156422021-06-22 A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps Kudryavtseva, Natalya Ermolaev, Aleksey Karlov, Gennady Kirov, Ilya Shigyo, Masayoshi Sato, Shusei Khrustaleva, Ludmila Int J Mol Sci Article 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. MDPI 2021-05-30 /pmc/articles/PMC8215642/ /pubmed/34070753 http://dx.doi.org/10.3390/ijms22115860 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Kudryavtseva, Natalya
Ermolaev, Aleksey
Karlov, Gennady
Kirov, Ilya
Shigyo, Masayoshi
Sato, Shusei
Khrustaleva, Ludmila
A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps
title A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps
title_full A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps
title_fullStr A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps
title_full_unstemmed A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps
title_short A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps
title_sort dual-color tyr-fish method for visualizing genes/markers on plant chromosomes to create integrated genetic and cytogenetic maps
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8215642/
https://www.ncbi.nlm.nih.gov/pubmed/34070753
http://dx.doi.org/10.3390/ijms22115860
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