Abstract
Painting plant chromosomes through chromosomal in situ suppression (CISS) hybridization has long been considered impracticable. Seeking to build specific and complex probes from a single microdissected chromosome, we employed human chromosomes as models to standardize all the necessary steps for application in plants. Human metaphases were used to define the adequate conditions for microdissection, chromosome DNA amplification and labeling through degenerate oligonucleotide-primed PCR, and in situ hybridization stringency. Subsequently, these methodologies were applied in the plant species Zea mays (chromosome 1) and Capsicum annuum (chromosome 7 or 8). The high quality of human and plant cytogenetic preparations and the meticulous standardization of each step, especially the most critical ones – microdissection and first round of DNA amplification – were crucial to eliminate the signs of non-specific hybridization and for direct application in plants. By overcoming these challenges, we obtained chromosome-specific probes, which allowed to achieve a clear and uniform painting of the entire target chromosomes with little or no background, evidencing their complexity and specificity. Despite the high amount of ubiquitous repetitive sequences in plant genomes, the main drawback for chromosome painting, we successfully employed our methodology on two plant species. Both have more than 80% repetitive sequences, which is compared to the human genome (66–69%). This is the first time that plant chromosome-specific probes were successfully obtained from a single A mitotic or meiotic microdissected chromosome. Thereby, we assume that chromosome painting through microdissection and CISS hybridization can now be considered a reality in the field of plant cytogenetics.
Highlights
Chromosome painting is a molecular cytogenetic approach developed for chromosome classification and detection of chromosome aberrations (Pinkel et al, 1986)
This molecular cytogenetic approach is based on the painting of individual chromosomes or chromosome regions through fluorescence in situ hybridization (FISH)
In order to ensure the specific hybridization of the probe with the target chromosome or region, dispersed repetitive sequences must be suppressed, for instance by using an excess of unlabeled, whole genomic DNA, or DNA enriched with repetitive sequences
Summary
Chromosome painting is a molecular cytogenetic approach developed for chromosome classification and detection of chromosome aberrations (Pinkel et al, 1986). The DNA from the collected chromosome is amplified and labeled through polymerase chain reaction (PCR) using a degenerated primer in association with a low initial annealing temperature (DOP-PCR) In such conditions, multiple, evenly dispersed DNA sequences are expected to be amplified, ensuring that the target chromosome is almost completely represented in the probe cocktail (Telenius et al, 1992; Yang et al, 2016). In order to ensure the specific hybridization of the probe with the target chromosome or region, dispersed repetitive sequences must be suppressed, for instance by using an excess of unlabeled, whole genomic DNA, or DNA enriched with repetitive sequences (as Cot-1 DNA) For this reason, chromosome painting is often called chromosomal in situ suppression (CISS) hybridization (Lichter et al, 1988; Ried et al, 1998; Sharma and Sharma, 2001)
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