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

Abstract

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.

Integrating Genetic and Chromosome Maps of Allium Cepa: From Markers Visualization to Genome Assembly Verification

“…The stringency of hybridization and washing together define the percentages of hybridized nucleotides to keep the double helix hybrid stable [30]. Based on our previous work [17] and this study, a stringency of 80-82% was optimal for a unique target visualization with a genome-wide specific probe.…”

“…The most specific probe has none or the least amount of "dangerous" hits (see definition in Methods) in local alignment on genome assembly. In addition, high level (80-82%) of stringency washing was optimally applied for a unique target visualization with a genome-wide specific probe [17].…”

“…The design of genome-wide specific probes was performed as we described earlier [17] with several modifications. Markers from linkage group 2 (198 markers) and 6 (126 markers) of transcriptome genetic map of A. cepa reported by [29] were used.…”

“…Probe sequences containing masked regions were discarded. Genome-wide chromosome specificity of selected probes was validated using blastn 2.12.0+ with parameters -evalue 1e-5 and hits with identity >80% and length >100 bp were classified as "dangerous"the ones which can lead to non-specific hybridization because of the length and identity based on stringency of hybridization and washing selected for a dual-color Tyr-FISH [17]. For each genomic sequence probe with none or the least amount of "dangerous" hits was selected.…”

“…Direct visualization of molecular markers on physical chromosome is an indispensable tool for the chromosome-level genome assembly. We have improved the Tyr-FISH method for mapping of short unique DNA sequences on plant chromosomes and accurate determination of the physical distance between markers due to the simultaneous detection of two markers on the same chromosome with high detection frequency [17]. There are several molecular techniques that can generate physical genome maps such as optical mapping [18,19], long-read sequencing [20], chromosomal conformation capture (Hi-C) [4,21].…”