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The ability to directly look into genome sequences has opened great opportunities in plant breeding. Yet, the assembly of full-length chromosomes remains one of the most difficult problems in modern genomics. Genetic maps are commonly used in de novo genome assembly and are constructed on the basis of a statistical analysis of the number of recombinations. This may affect the accuracy of the ordering and orientation of scaffolds within the chromosome, especially in the region of recombination suppression. Moreover, it is impossible to assign contigs lacking DNA markers. Here, we report the use of Tyr-FISH to determine the position of the short DNA sequence of markers and non-mapped unique copy sequence on the physical chromosomes of a large-genome onion (Allium cepa L.). In order to minimize potential background masking of the target signal, we improved our earlier developed pipeline for probe design. A total of 23 markers were located on physical chromosomes 2 and 6. The order of markers was corrected by the integration of genetic, pseudochromosome maps and cytogenetic maps. Additionally, the position of the mlh1 gene, which was not on the genetic map, was defined on physical chromosome 2. Tyr-FISH mapping showed that the order of 23.1% (chromosome 2) and 27.3% (chromosome 6) of the tested genes differed between physical chromosomes and pseudochromosomes. The results can be used for the improvement of pseudochromosome 2 and 6 assembly. The present study aims to demonstrate the value of the in situ visualization of DNA sequences in chromosome-scaffold genome assembly. Full article