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.
Highlights
Genetic linkage maps that have been used for over 100 years aided greatly de novo assembly of plant genomes [1,2,3,4]
Linkage maps, which are most accurate in regions of the genome with high rates of recombination, can result in mistakes in arranging scaffolds in the region of suppressed recombination e.g., pericentromeric, knob, and subtelomeric heterochromatin [5,6,7]
The cytogenetic map can compensate for the discrepancy between the real position of the DNA sequence and its position on the genetic linkage map
Summary
Genetic linkage maps that have been used for over 100 years aided greatly de novo assembly of plant genomes [1,2,3,4]. Linkage maps based on recombination rate between markers can anchor de novo sequences and order small fragments into chromosome-scale sequences. The cytogenetic map can compensate for the discrepancy between the real position of the DNA sequence and its position on the genetic linkage map. Whole genomes can be quickly sequenced, which is not the case with genome assembly as this process goes slower due to its computational complexity and labor intensity. Whole-genome sequencing is much more informative when linked and oriented to chromosomes than unlinked and disordered scaffolds.
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