We constructed chromosome-specific probes through microdissection of a single plant chromosome. With meticulous steps, the procedure is step-by-step summarized and, from now on, may be applied in other plants.

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). Different from the FISH for a specific DNA sequence, the probe for chromosome painting comprises a cocktail of numerous labeled DNA sequences from a specific chromosome obtained through microdissection or flow sorting (Yang et al., 2016). The DNA from the collected chromosome is then 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). Nonetheless, the specificity of probe hybridization is often hampered by the presence of ubiquitously distributed repetitive sequences. 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).

For humans and other mammals with relatively small genomes, such as rodents and primates (1C = ∼1.5 to 3.0 pg), chromosome painting is well established and has been widely used for clinical diagnosis of chromosome abnormalities (Langer et al., 2004). Besides, chromosome painting has been applied in cytotaxonomic studies to detect chromosomal rearrangements that occur during evolution (Wienberg et al., 1990), and to construct ancestral karyotypes through cross-species in situ hybridization (Müller et al., 1999; Rens et al., 2006). Contrastingly, the first painting of specific plant chromosomes was only achieved in 2001 for Arabidopsis thaliana (L.) Heynh. (Lysak et al., 2001) after approximately one decade of unsatisfactory results, which were probably due to the large amount of ubiquitous repetitive DNA sequences in plant genomes (Schubert et al., 2001). The successful painting of A. thaliana chromosome 4 was facilitated by its small genome size (1C = ∼0.16 pg; Bennet et al., 2003) and relatively low amount of repetitive DNA. In addition, the painting technique involved the application of a pool of BAC clones as probe, allowing to enrich the probe with low-copy sequences (Lysak et al., 2001, 2003). Nonetheless, chromosome painting using DNA probes from flow-sorted or microdissected chromosomes, even associated to the CISS hybridization, has still failed to yield satisfactory results in plant species (Hou et al., 2018). Thus, it is mainly restricted to B or Y chromosomes, most likely due to the presence of chromosome-specific repetitive sequences in these chromosomes, rather than low- or single-copy sequences (Houben et al., 2001). Oligo-FISH has showed to be an alternative for chromosome painting. This approach is based on the selection of thousands of oligonucleotides specific to a target chromosome. However, its application is restricted to species with sequenced genomes (Han et al., 2015; Braz et al., 2018).

Chromosome painting in plant species has another hindrance besides the unspecific signals due to repetitive sequences: the identification and collection of chromosomes. Flow karyotyping followed by chromosome sorting allows the purification of large amounts of a specific chromosome, as long as the chromosome of interest can be distinguished from the other chromosomes based on its optical properties (light scatter, fluorescence; Doležel et al., 2012). In humans the chromosomes are easily discriminated, while in plants the low metaphasic index, difficulties with chromosome release from the cell wall and homomorphic karyotypes are restraints for the successful application of the flow sorting technique (Doležel et al., 2012). On the other hand, probe construction through microdissection is performed by physically collecting multiple copies of a target chromosome or chromosome region (Christian et al., 1999). Usually, a relatively high number of copies (∼20–50) of the target chromosome must be collected, which is considered a limiting technical factor (Christian et al., 1999; Henning et al., 2008). Nonetheless, efforts have been made to adapt protocols in which a reduced amount of target DNA is enough to obtain the chromosome-specific probes (Christian et al., 1999; Henning et al., 2008; Passamani et al., 2018). In plants, the need to dissect more than one copy of the same chromosome is often hampered by the highly homomorphic karyotypes. Therefore, the construction of whole-chromosome probe through the dissection of a single chromosome and amplification through DOP-PCR, which to date has not yet been successfully performed in plants, would be profitable for the progress of plant chromosome painting.

In this work, we present an efficient and reproducible methodology for constructing plant chromosome-specific probes from a single dissected chromosome. Initially, the protocol was standardized in human chromosomes and then adapted and expanded for mitotic and meiotic chromosomes of two different plant species, Zea mays L. and Capsicum annuum L.

Although chromosome-specific probe construction has been considered an extensive and challenging cytogenetic methodology, the standardization of each step allowed us to obtain chromosome-specific probes for human material as well as two plant species. Our research group has been concentrating efforts to improve the chromosome painting technique, aiming especially to reduce the amount of target DNA necessary for the first round of DOP-PCR. To this end, we combined an in situ PCR (PRINS) prior to microdissection with the DOP-PCR and successfully constructed a complex probe from ten copies of a small fragment of human chromosome X (Passamani et al., 2018). Advancing in our main goal, the most critical steps were resolved: the chromosome microdissection, the transferring of the chromosome to the microtube and the initial low stringency amplification. By combining the chromosome-specific probes obtained from a single microdissected chromosome with the stringency conditions applied in FISH, the dispersed repetitive sequences were efficiently blocked, and we were consequently able to obtain clear fluorescence painting of the entire target chromosomes. For the first time, chromosome specific probes were constructed from a single microdissected autosomal chromosome of the A complement in two plant species, allowing chromosomal in situ suppression (CISS) hybridization to be successfully performed in plants.

Whole chromosome-specific probes were generated for humans, Zea mays and Capsicum annuum through microdissection of a single chromosome and amplification with DOP-PCR (Figure 1). Although different strategies were used to obtain human and plant chromosomes, the efforts to standardize each step provided morphologically adequate and well-preserved mitotic and meiotic cells, containing individualized chromosomes and bivalents, with well-defined centromeres and telomeres, and with a low level of chromatin condensation. All slides were submitted to cellular dissociation, while air-drying techniques were only applied to the slides submitted to FISH, allowing the chromosomes to be well-spread and flattened on the slide. Although successfully used in plant cytogenetics, the squashing technique has some drawbacks, such as possible morphological deformation of chromosomes, poor spreading of chromosomes on the slide and the eventual loss of biological material when removing the coverslip (Carvalho and Saraiva, 1993). Therefore, we consider cell dissociation and air-drying techniques (Carvalho et al., 2010) fundamental for the successful results obtained through FISH, allowing proper access of the probe fragments to the target DNA immobilized on the slide.

The slides submitted to chromosome microdissection were prepared in the same way as those for FISH, except for the lack of air-drying. The quality of the microdissected DNA is fundamental for the success of chromosome-specific probe construction, being directly dependent on chromosome fixation and staining (Houben et al., 2001). In human cytogenetics, it is common to perform G-banding to correctly identify chromosomes during microdissection. Nonetheless, the staining process is a source of contamination itself and should be avoided in the process of constructing chromosome-specific probes (Christian et al., 1999; Passamani et al., 2018). The high morphological quality of the obtained human chromosomes dismissed the need of staining because chromosome 2 was easily identified. Furthermore, since the main goal of this work was to produce a specific whole-chromosome probe from a single chromosome, the identification of the dissected chromosome was not a requirement. This is especially advantageous for plant species with small and homomorphic chromosomes.

All fixation procedures adopted for the chromosomes to be microdissected should involve a minimal contact with acetic acid, as this substance promotes DNA depurination (Engelen et al., 1998; Houben et al., 2001). Here, the suspension cultures (human lymphocytes and C. annuum meiocytes) were fixed in methanol:acetic acid (3:1), but stored in absolute methanol, minimizing the exposure to acetic acid. On the other hand, the root meristems were fixed and stored in methanol:acetic acid (3:1). In our previous attempts, the storage in absolute methanol was not efficient for such biological material, as the cell dissociation was substantially hampered. Nevertheless, for any of the biological materials, the chromosomes submitted to microdissection were not exposed to acetic acid after slide preparation, and the microdissection procedure was performed immediately after obtaining the slides. In an attempt to reduce the contact of the root meristems with acetic acid, we fixed 10 min in 3:1 methanol:acetic acid, then transferred to ethanol 70% and prepared the slides with dH₂O, as proposed by Zhou et al. (1999). But this fixation condition also hampered cellular dissociation and generated chromosomes with a refringent (shining, vitreous) aspect when visualized in phase contrast, making it difficult to perform the chromosome microdissection.

One of the main features that set the adequate conditions for microdissection is the chromosome hydration. Chromosomes that are too dry become brittle and crumble when microdissected. In contrast, excessively hydrated chromosomes exhibited a gelatinous aspect and do not adhere to the glass microneedle. After washing the slides for 1 min in PBS prior to microdissection, the chromosomes exhibited an ideal hydration, while washing only in ultrapure water resulted in faster drying of the chromosomes. Moreover, the maintenance of the relative humidity of the room around 70% avoided the chromosomes to dry during the microdissection. These strategies facilitated the microdissection of whole chromosomes without loss of visible fragments and ensured the proper chromosome adherence to the glass microneedle and transference to the microtube (Supplementary Figure S1). Engelen et al. (1998) applied a 5 μL drop of ultrapure water over the target chromosomes for rehydration prior to microdissection. Nonetheless, in our experience, this process hampered chromosome visualization and removal due to the water layer. A different strategy was attempted by Christian et al. (1999) that washed the slide in 4x SSC prior to microdissection, but in this case, the excessive amount of salt also hampered chromosome visualization and microdissection.

Enzymatic treatment with 1.44 mg mL^–1 Proteinase-K for 2 h at 60°C was applied to degrade the chromatin proteins and to liberate the chromosomal DNA before the process of PCR amplification. Passamani et al. (2018) reported a treatment of 24 h with 0.5 mg mL^–1 proteinase K at 37°C. But despite also being effective to liberate the DNA, the 2 h treatment adapted from Yang et al. (2016) considerably reduced the total time required for probe construction. Each microdissected chromosome was directly transferred to a microtube containing 0,1 μL of the collection solution, minimizing the contamination with exogenous DNA. In approaches like microcloning or ligation mediated PCR, the multiple handling steps involving the microdissected DNA are time-consuming and increase the probability of contamination (Vega et al., 1994). The application of a DOP primer was a remarkable advance in chromosome painting for its simplicity, since there is no need to use adaptors or restriction enzymes, and no previous knowledge of the chromosome target sequences is necessary (Yang et al., 2016).

Fluorescence in situ hybridization results showed that the probe pool represented all target chromosome for the three species, even though some DNA sequences might be preferential amplified due to their representativeness in the genome (as repetitive sequences). The painting observed for the three chromosome-specific probes revealed that the DOP-PCR protocol was suitable to generate complex probes. One of the main concerns in chromosome painting is to produce a sufficiently complex pool of probe sequences, which results in a suitable painting along the entire length of the target chromosome (Christian et al., 1999; Yang et al., 2016). For a long time, chromosome painting through CISS hybridization with probes obtained by microdissection was not achievable in plant species (Fuchs et al., 1996; Lysak and Mandáková, 2013; Hou et al., 2018). The main constraint to the success in this technique was the inefficient blocking of dispersed repetitive sequences, which are characteristic of plant genomes (Fuchs et al., 1996). While humans possess around 66–69% repetitive sequences (Koning et al., 2011), the genomes of the plant species used in this work are composed of more than 80% repetitive sequences: ∼81% for C. annuum (Qin et al., 2014) and ∼85% for Z. mays (Schnable et al., 2009). Slight preferences in primer annealing in the initial low temperature PCR might produce asymmetries during amplification (Christian et al., 1999). Therefore, a high amount of non-chromosome-specific repetitive sequences, which are common in plant genomes (Shcherban, 2015), would favor the generation of asymmetric and poorly representative probes. In addition, the presence of contaminant DNA from other chromosomes due to insufficiently spread metaphases might also contribute to the generation of unspecific sequences in the probe pool (Christian et al., 1999; Guan, 2003). Based on these hindrances, our concern was to associate well-spread and preserved chromosomes with the proposed procedures of microdissection, DNA amplification and hybridization, which allowed us to overcome these issues by generating sufficiently complex and specific chromosome probes.

The three enzymes used for the initial DOP-PCR (Thermo Sequenase DNA Polymerase, Platinum Taq DNA Polymerase High Fidelity and USB Sequenase Version 2.0 DNA Polymerase) amplified the DNA from a single microdissected chromosome. While both Taq produced fragments that ranged from 100 to 900 bp, the fragments generated by Sequenase varied from 100 to 600 pb (Supplementary Figure S2). Nonetheless, some Taq polymerases are not able to amplify the DNA from a chromosome template. Besides, all basic components must be optimized for a successful reaction, mainly the pH of the PCR buffer and the Mg²⁺ concentration (Yang et al., 2016). The initiation is the most critical step of the PCR when a microdissected chromosome is used as target DNA. As the initial annealing temperature is low (30°C), to allow a broad annealing throughout the entire chromosome, thermostable enzymes with optimal temperature around 72°C were initially considered unsuitable for PCR initiation, in accordance with Guan (2003). In our experiments, both Taq polymerases successfully initiated the amplification and provided a larger final concentration of amplified fragments when compared with USB Sequenase Version 2.0 DNA Polymerase (Supplementary Figure S2). Six of the ten different enzymes tested for DOP-PCR labeling successfully incorporated the labeled uridine nucleotides Tetramethyl-rhodamine 5-dUTP or ChromaTide Alexa Fluor 488-5-dUTP. The practicality is the main advantage of this methodology, since the same conditions of the second DOP-PCR amplification step can also be used for labeling (Telenius et al., 1992). Moreover, labeling through PCR allows to restrict probe length to 200–600 pb, which is considered the optimum length for in situ hybridization (Morrison et al., 2003).

The probe obtained for human chromosome 2 exhibited a strong hybridization signal with low background (Figures 1A,B) and a high reproducibility (95%). The labeling of Z. mays chromosome 2 was consistent with the microdissection of a large chromosome, which was only identified after karyotype assembly (Figures 1C,D). Similarly, the probe obtained for C. annuum also showed specific labeling of a single chromosome pair (Figures 1E,F). However, the highly homomorphic karyotype did not allow us to distinguish between chromosomes 7 and 8. Based on these FISH results obtained for the three species, the produced probes were sufficiently complex to allow a uniform painting across the entire chromosome without individual spots. The hybridization efficiency for both plant species was 65–80%, an impressive value considering the well-known difficulties for obtaining suitable preparations in plant cytogenetics. In plants, each species requires a specific and refined adjustment to obtain chromosomes. Consequently, the slides are more heterogeneous regarding the number of prometaphases and/or metaphases and the level of chromatin condensation, which might have contributed to the lower efficiency in hybridization.

Surpassing the assumptions that CISS hybridization is not feasible in plants, mainly in species with a large number of repetitive sequences, we can here show a pathway (Supplementary Figure S3) starting with the microdissection of a single mitotic or meiotic chromosome. With this, plant chromosome-specific probes from microdissection of a single chromosome have become a reality, representing a new method for painting besides the artificial chromosome clones (BACs/YACs) and amplification of a large number of single-copy sequences. The methodology described here has the potential to be applied in any plant species, with the pre-requisite of high quality cytogenetic preparations to supply chromosomal DNA of similarly high quality. We hope that the application of the methodology contributes to a breakthrough in the field of plant cytogenetics, aiding the development of taxonomic and evolutive studies.

All datasets generated for this study are included in the article/Supplementary Material.

FS, CC, MS, and WC designed the research. FS and MS performed the molecular cytogenetic experiments. DP, PP, and JS performed the classical cytogenetic experiments. All authors analyzed the data and wrote the manuscript, equally contributed to the manuscript edition and revision, and approved the final manuscript version for submission.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.