A dual-color tyr-FISH method for visualizing genes/markers on plant chromosomes to create integrated genetic and cytogenetic maps

Natalya Kudryavtseva, Aleksey Ermolaev, Gennady Karlov, Ilya Kirov, Masayoshi Shigyo, Shusei Sato, Ludmila Khrustaleva

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Article number5860
JournalInternational journal of molecular sciences
Volume22
Issue number11
DOIs
Publication statusPublished - 2021 Jun 1

Keywords

  • Allium cepa
  • Genome assembly
  • Plant chromosome preparation
  • Recombinant and cytogenetic maps
  • Transcript-based markers
  • Tyr-FISH

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Spectroscopy
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

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