Fluorescence in situ hybridization in plants: recent developments and future applications

  • Jiming JiangEmail author
Waldeyer-Flemming Special Collection


Fluorescence in situ hybridization (FISH) was developed more than 30 years ago and has been the most paradigm-changing technique in cytogenetic research. FISH has been used to answer questions related to structure, mutation, and evolution of not only individual chromosomes but also entire genomes. FISH has served as an important tool for chromosome identification in many plant species. This review intends to summarize and discuss key technical development and applications of FISH in plants since 2006. The most significant recent advance of FISH is the development and application of probes based on synthetic oligonucleotides (oligos). Oligos specific to a repetitive DNA sequence, to a specific chromosomal region, or to an entire chromosome can be computationally identified, synthesized in parallel, and fluorescently labeled. Oligo probes designed from conserved DNA sequences from one species can be used among genetically related species, allowing comparative cytogenetic mapping of these species. The advances with synthetic oligo probes will significantly expand the applications of FISH especially in non-model plant species. Recent achievements and future applications of FISH and oligo-FISH are discussed.


FISH Oligo-FISH Karyotype Chromosome Genome Evolution 



Bacterial artificial chromosome


Chromatin immunoprecipitation


Extrachromosomal circular DNA


Fluorescence in situ hybridization


Million years




Simple-sequence repeats


FISH has been the most important technique in plant cytogenetic research. DNA/RNA associated with a specific genomic region or an entire chromosome can be visualized by FISH on a range of cytological specimens. FISH was developed in the early 1980s (Langer-Safer et al. 1982) and became popular in the 1990s, owing to the introduction of CCD camera and imaging process of FISH signals. Searches using the keyword “fluorescence in situ hybridization” in the Web of Science database show a steady number of publications using FISH over the last 10 years (Fig. 1). Thus, FISH techniques will continue to play an important role in cytogenetic and genome research.
Fig. 1

The numbers of FISH publication since 1991 (65) in the Web of Science database. The keyword “fluorescence in situ hybridization” was used in search of the database. The highest publication number is 1430 in 2012

FISH was introduced in plants nearly 30 years ago (Leitch et al. 1991; Schwarzacher et al. 1989). Early development and applications of FISH in plants have been reviewed previously (Jiang and Gill 1994, 2006). In this review, I will discuss the advances in FISH since 2006 with a focus on recent developments using synthetic oligonucleotide (oligo) probes.

Recent development of the FISH techniques

Development of FISH probes based on synthetic oligos

Successful and efficient FISH experiments primarily rely on robust DNA probes. Repetitive DNA sequences and large-insert genomic DNA clones, such as bacterial artificial chromosome (BAC) clones, have been the most common FISH probes in plants (Jiang and Gill 1994, 2006). An important recent development is the application of FISH probes based on synthetic oligos. Oligo-based probes can be designed either from repetitive DNA elements or from single-copy DNA sequences, a paradigm shift from the traditional FISH probes using cloned DNA sequences.

Oligo probes designed from simple-sequence repeats or satellite repeats

Synthetic oligo-FISH probes were first developed to physically map simple-sequence repeats (SSRs) in plants. Oligos with repeated di-, tri-, or tetra-nucleotide motifs, such as (AG)12 or (AGG)5, were synthesized and used as FISH probes. These synthetic oligos can be end-labeled with biotin-dUTP or digoxigenin-dUTP (Cuadrado and Schwarzacher 1998; Schmidt and HeslopHarrison 1996), or are conjugated with a fluorochrome during synthesis (Danilova et al. 2012). The initial goals of FISH mapping of SSR-related oligos were to investigate the chromosomal organization of SSRs in plant genomes (Cuadrado and Schwarzacher 1998; Gortner et al. 1998; Schmidt and HeslopHarrison 1996). However, some of the SSR-related oligo probes produced distinct FISH signal patterns on individual chromosomes, which can be used for chromosome identification. For example, the FISH signals derived from the GAA repeats resemble the N-banding patterns on wheat and barley chromosomes (Danilova et al. 2012; Pedersen et al. 1996). Thus, SSR-related oligo probes have been used for chromosome identification in a number of plant species (Amosova et al. 2015; Carmona et al. 2013; Cuadrado and Jouve 2007; Danilova et al. 2012; Dou et al. 2009; Fuchs et al. 1998; Pedersen and Langridge 1997; Ruban and Badaeva 2018; Zheng et al. 2016).

Satellite repeats, or tandem repeats, are popular FISH probes in plants because the FISH signals derived from such repeats can often be used for chromosome identification (Jiang and Gill 1994, 2006). If a short sequence motif unique to a satellite repeat can be identified, an oligo probe based on this motif can be synthesized for FISH (Danilova et al. 2012; Fu et al. 2015; Tang et al. 2016, 2014). Such short synthetic probes may allow to distinguish and visualize subfamilies of a satellite repeat, and offer several advantages compared to traditionally prepared probes from cloned satellite repeats, including consistent probe quality and reduction of time and cost for probe preparation. In addition, denaturation of the chromosomal DNA is not required in FISH with some of these probes (Fu et al. 2015; Tang et al. 2016), which can positively impact the quality of FISH results. Synthetic oligo probes can be designed directly from computationally identified satellite repeats from genomic sequence data (Lang et al. 2018; Waminal et al. 2018). Thus, a fully sequenced reference genome is not required to develop such probes.

Oligo probes designed from single-copy DNA sequences

Oligo probes can also be designed from single-copy DNA sequences. Although a large number of single-copy oligos may be required to visualize a specific chromosomal region (Beliveau et al. 2012; Boyle et al. 2011; Yamada et al. 2011), oligos specific to a chromosomal region or to an entire chromosome can be computationally identified and synthesized in parallel as a pool (Beliveau et al. 2012; Han et al. 2015). Each oligo in the pool can be added with sequence tags at both ends during synthesis, which allows PCR-based amplification of the entire pool (Beliveau et al. 2012; Han et al. 2015). Subsequently, FISH probes can be generated from the pool via amplification of oligos labeled directly with a fluorochrome or indirectly with biotin-dUTP or digoxigenin-dUTP (Albert et al. 2019; Beliveau et al. 2012; Han et al. 2015). Thus, each synthesized oligo pool can be used as an infinite probe resource since the synthesized DNA (< 500 ng) can be used for up to a million FISH applications (Han et al. 2015).

Fine-tuning of the FISH procedure

The basic procedure of FISH was developed in 1982 (Langer-Safer et al. 1982) and has essentially remained unchanged in modern FISH protocols. Nevertheless, plant labs have continued to fine-tune the procedure to improve the sensitivity of the technique for detecting small DNA probes. Early reports on FISH mapping of DNA probes as small as few kilobases (kb) were rare (Jiang and Gill 1994, 2006) and the results were sometimes controversial. However, several plant labs have recently demonstrated routine detection of small DNA probes, mostly in the range of few kilobases (Aliyeva-Schnorr et al. 2015a; Danilova et al. 2017; Danilova and Birchler 2008; Danilova et al. 2012; Khrustaleva et al. 2016; Kirov et al. 2014b; Lamb et al. 2007; Li et al. 2018b; Lou et al. 2014; Nani et al. 2018; Said et al. 2018; Tiwari et al. 2015; Yu et al. 2007; Zhao et al. 2017).

The increased sensitivity of the FISH techniques is at least partly due to the improvement of plant chromosome preparation. Several modified chromosome preparation techniques have been reported in different plant species (Aliyeva-Schnorr et al. 2015b; Dang et al. 2015; Kato et al. 2006; Kirov et al. 2014a; Kuo et al. 2016; Setiawan et al. 2018; Yu et al. 2007). Various modifications have been implemented in these new techniques, which although minor, can positively impact the digestion of plant cell walls, reduce cytoplasm background, and maintain chromosome morphology, which may ultimately enhance the sensitivity of FISH. Nevertheless, although FISH mapping of DNA probes < 1 kb was reported (Khrustaleva et al. 2016), these probes can usually only be detected in a low percentage of cells and are not robust markers for routine chromosome identification or cytogenetic studies.

Recent applications of FISH in plant cytogenetic and genome research

The most common application of FISH is mapping DNA probes to chromosomes, thus allowing establishment of a physical map of the probes. If the DNA probes are genetically mapped, FISH mapping of these probes would allow integration of genetic linkage maps with chromosomal maps. These common applications of FISH were reviewed previously (Jiang and Gill 1994, 2006). Thus, I will only discuss the new applications of FISH since 2006.

Identification and validation of satellite repeats in plant genomes

Repetitive DNA elements can be cataloged as dispersed repeats, which are distributed throughout the genome, or satellite repeats, which are organized as tandem arrays and located in distinct region(s) on one or multiple chromosome(s). Satellite repeats are often the main DNA components in the centromeric and subtelomeric regions in plant genomes (Sharma and Raina 2005) and are the most popular probe resource for chromosome identification and cytogenetic studies in plants (Jiang and Gill 1994, 2006). The traditional approach of cloning and characterizing satellite repeats is tedious and time-consuming. RepeatExplorer (, a graph-based sequence clustering program, was developed for de novo identification of various types of repetitive DNA elements (Novak et al. 2010, 2013). A set of random shotgun genomic sequences from a target species is the only required resource for RepeatExplorer. Putative satellite repeats can be predicted based on their unique graphic characteristics. However, the predicted repeats need to be confirmed by FISH analysis. A combination of RepeatExplorer and FISH has become a popular methodology to identify and characterize major satellite repeats in many plant species (Belyayev et al. 2018; Camacho et al. 2015; Dluhosova et al. 2018; He et al. 2015; Heitkam et al. 2015; Iwata-Otsubo et al. 2016; Kirov et al. 2017; Macas et al. 2011; Perumal et al. 2017; Puterova et al. 2017; Ribeiro et al. 2017; Robledillo et al. 2018; Ruiz-Ruano et al. 2016; Torres et al. 2011; Yang et al. 2017).

One important application of RepeatExplorer and FISH is to uncover repeats associated with the centromeres of plant chromosomes. Centromeres in higher eukaryotes are marked with a centromere-specific histone H3 variant, CENH3, and are often composed of long arrays of repetitive DNA elements (Henikoff et al. 2001; Jiang et al. 2003). Centromeric DNA can be identified by chromatin immunoprecipitation (ChIP) using anti-CENH3 antibodies followed by sequencing of the immunoprecipitated DNA (ChIP-seq) (Yan et al. 2008). Repetitive DNA elements within the ChIP-seq data can be identified using RepeatExplorer. In addition, the relative enrichment of each computationally identified repeat in the ChIPed DNA versus a random genomic DNA sequence dataset would predict if each repeat is enriched in centromeres. The predicted centromere-specific repeats can then be confirmed by FISH (Gong et al. 2012; Neumann et al. 2012). This methodology allows identification of all centromeric repeats in a single experiment and has been successfully demonstrated in a number of plant species (Han et al. 2016; Kowar et al. 2016; Li et al. 2018a; Marques et al. 2015; Nagaki et al. 2015; Robledillo et al. 2018; Yang et al. 2018; Zhang et al. 2014; Zhang et al. 2017).

FISH-based assays of chromosome synteny and evolution

Comparative genetic linkage mapping with a common set of DNA markers was the traditional methodology to study the synteny and evolution of homoeologous chromosomes from different species (Paterson et al. 2000). This methodology, however, is time-consuming and relies on established mapping populations. Comparative FISH mapping of a common set of DNA probes provides an alternative method to reveal the synteny of homoeologous chromosomes in different species. The FISH-based approach is highly complementary to linkage mapping since it does not require a population and can be accomplished in a relatively short time.

Comparative FISH mapping of BAC clones developed in potato (Solanum tuberosum) or tomato (Solanum lycopersicum) was conducted in a number of Solanum species (Gaiero et al. 2017; Iovene et al. 2008; Lou et al. 2010; Szinay et al. 2012; Tang et al. 2008). Many of the potato and tomato BACs generated distinct FISH signals on distantly related Solanum species, including eggplant (Solanum melongena), which diverged from potato/tomato nearly 12 million years (Mys) ago (Doganlar et al. 2002). Mapping of a common set of BACs on meiotic pachytene chromosomes in different species was demonstrated to be a highly efficient approach to reveal the evolution of individual Solanum chromosomes in the last 12 million years (Lou et al. 2010; Szinay et al. 2012). Similarly, BAC-based comparative FISH mapping has been reported in a number of other plant species (Betekhtin et al. 2014; Han et al. 2009; Idziak et al. 2014; Iovene et al. 2011; Lysak et al. 2006, 2005; Vasconcelos et al. 2015).

BACs are valuable probe resources for comparative FISH mapping. However, BACs from plant species with large complex genomes, such as wheat, often contain a high percentage of repetitive DNA sequences and cannot be used for FISH mapping (Janda et al. 2006; Zhang et al. 2004). Instead, comparative FISH mapping can be performed using single-copy DNA probes in these plants (Aliyeva-Schnorr et al. 2016; Danilova et al. 2012, 2014). For example, a large number of single-copy cDNA probes from wheat were used for FISH mapping in various related diploid and polyploid species (Danilova et al. 2017, 2012, 2014; Said et al. 2018). The comparative FISH mapping results revealed the evolution of several known translocations within the A genome chromosomes of wheat (Danilova et al. 2012) and detected several new chromosome rearrangements in the wild species (Danilova et al. 2017, 2014; Said et al. 2018).

FISH-based assays on gene duplication and amplification

Gene duplication is a common molecular mechanism for adaptation to a changing environment (Kondrashov 2012). Tandem duplication resulting from unequal crossover is the most common type of gene duplication and can be readily analyzed using traditional molecular methods. However, it is often difficult to characterize complex or massive duplication/amplification of a gene or a gene cluster. FISH can be a powerful tool to analyze such duplication/amplification events.

Overexpression of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene can render crops resistant to the herbicide glyphosate. Unfortunately, many weeds have developed resistance to glyphosate due to extensive application of this herbicide. Duplication or amplification of the EPSPS gene is a common and naturally occurring mechanism for the acquired resistance in glyphosate-tolerant weeds (Ngo et al. 2018; Powles 2010; Salas et al. 2012; Zhang et al. 2015). Interestingly, the duplication mode of the EPSPS gene is different among glyphosate-resistant weeds. For example, the copy number of the EPSPS gene in glyphosate-resistant Amaranthus tuberculatus and Kochia scoparia plants ranged from a few copies up to 16 copies, and the duplications were likely derived from the classical “unequal crossover” mechanism based on the FISH signal patterns of the EPSPS gene on the target chromosomes as well as on DNA fibers (Dillon et al. 2017; Jugulam et al. 2014). FISH mapping also revealed that the EPSPS gene escaped to an extra chromosome in A. tuberculatus (Dillon et al. 2017). This extra chromosome was likely derived from the original EPSPS-carrying chromosome via rearrangements (Koo et al. 2018a). The copy number of the EPSPS gene in glyphosate-resistant Amaranthus palmeri plants can reach up to 160 (Gaines et al. 2010). FISH mapping revealed that the amplified EPSPS genes were dispersed along all chromosomes, indicating that the amplification was not derived from unequal crossovers (Gaines et al. 2010). Strikingly, a recent FISH-based study revealed that the amplification of the EPSPS gene in A. palmeri was based on a 297-kb extrachromosomal circular DNA (eccDNA) molecule. The eccDNAs are transmitted through mitosis and meiosis by an unknown mechanism of tethering to chromosomes (Koo et al. 2018b).

The niche applications of fiber-FISH

FISH can be applied to DNA molecules spread on glass slides (Fransz et al. 1996; Jackson et al. 1999, 1998). Fiber-FISH were mostly used to analyze the structure and organization of repetitive DNA sequences and to measure the length of long DNA molecules (Jiang and Gill 2006). Another unique value of fiber-FISH is the analysis of tandem duplications associated with large genomic duplicons. An early example was the fiber-FISH characterization of a 620-kb mitochondrial DNA (mtDNA) fragment inserted in chromosome 2 of Arabidopsis thaliana (Stupar et al. 2001). This large mtDNA insert was derived from complex duplication and deletion events (Stupar et al. 2001), which could not be characterized using traditional methods, such as PCR or Southern blot hybridization.

Fiber-FISH has continued to be a valuable technique to characterize large complex DNA loci. For example, the soybean cyst nematode (SCN) resistance locus Rhg1 was mapped to a 31-kb region spanning several different genes (Cook et al. 2012). This 31-kb DNA segment occurs as a single copy in SCN-susceptible lines, but is present with multiple copies in SCN-resistant lines. The copy numbers of this 31-kb duplicon were unambiguously visualized by fiber-FISH (Cook et al. 2014, 2012). Fiber-FISH can also be used to visualize circular DNA molecules (Koo et al. 2018b) as well as artificially assembled long linear DNA molecules (Lin et al. 2011). In addition, fiber-FISH can be combined with immunodetection of methylated cytosine on DNA fibers. Combining these two techniques allows mapping DNA methylation associated with repetitive DNA sequences (Koo et al. 2011; Lough et al. 2015), which cannot be accurately assessed by DNA sequencing–based techniques.

Future applications of oligo-FISH

Application of the FISH techniques has mainly been limited by the lack of robust DNA probes in most plant species, especially in non-model plants. Cloned DNA probes, such as BACs, are either not available or not useful in most plants. Thus, the recent development of FISH using synthetic oligo probes designed from single-copy DNA sequences will dramatically expand the applications of FISH in many plant species.

Development of oligo-FISH probes

Oligo-FISH probes can be designed from any plant species with a sequenced genome. Oligos specific to a specific region(s) of a chromosome or to an entire chromosome can be selected using the Chorus software ( Several questions rise in designing probes for different plant species:
  1. 1.

    How to design a probe for plants without a reference genome? Since chromosome-specific oligos are selected from single-copy sequences, including genes, oligo probes designed from one species are likely useful in other related species. Indeed, the first oligo probes developed from cucumber (Cucumis sativus) produced excellent signals on chromosomes of melon (Cucumis melo) and other Cucumis species that diverged from cucumber up to 12 Mys ago (Han et al. 2015). Similarly, oligo probes designed from potato (Solanum tuberosum) showed high-quality FISH signals on chromosomes of tomato (S. lycopersicum) (Fig. 2A), which diverged from potato approximately 5–8 Mys ago (Braz et al. 2018). Nevertheless, the quality of FISH signals from heterologous probes will reduce as the genetic distance of the two species increases. Although potato oligo probes generated signals on eggplant (S. melongena) chromosomes, which diverged from a common ancestor of potato/tomato 15.5 Mys ago, the signals on eggplant chromosomes were weak and not as punctuated as those on potato chromosomes (Braz et al. 2018). To avoid the quality problem associated with heterologous probes, shotgun genomic sequences can be generated from a plant species and mapped to the reference genome of a related model species. Oligos can then be designed based on the sequence reads from the target species, rather than from a heterologous reference genome.

  2. 2.

    How to design a chromosome-specific probe in a polyploid species? Many plants are polyploids containing multiple genomes with various levels of homology. Thus, a probe designed from one chromosome will cross-hybridize with other homologous or homoeologous chromosome(s). In autopolyploids where multiple genomes are identical, such as potato (2n=4x=48), an oligo probe will generate identical FISH signals on all homologous chromosomes (Braz et al. 2018) (Fig. 2C). In allopolyploids, however, the level of cross-hybridization is correlated with the level of sequence similarity between the homoeologous chromosomes. We have recently tested the possibility to develop chromosome-specific probes in switchgrass (Panicum virgatum, 2n=4x=36), an allotetraploid species. We first designed a chromosome painting probe for switchgrass chromosome 8a. All 27,000 oligos were designed based on the sequence of chromosome 8a. Any oligos mapped to a second location (> 75% homology) were discarded to minimize the hybridization of the probe to chromosome 8b. This probe generated strong signals on chromosome 8a (Fig. 2D1), but very weak signals on chromosome 8b (Fig. 2D2). We then designed a chromosome painting probe for the short arms of both chromosomes 4a and 4b in switchgrass (Fig. 2E1). All 27,000 oligos were designed based on the sequence of chromosome 4a. Only the oligos that show > 90% sequence similarity to the sequences of chromosome 4b were selected to maximize the hybridization of the probe to both 4a and 4b. The FISH signals on chromosomes 4a and 4b showed a similar intensity (Fig. 2E2). Alternatively, each oligo can be designed to have a similar sequence similarity against all homoeologous chromosomes in an allopolyploid species. Such an oligo probe would hybridize with all homoeologous chromosomes with a similar level of signal intensity.

  3. 3.

    What is the appropriate oligo number/density of a probe? The number of oligos of an oligo-FISH probe is critical for the quality (intensity) of the FISH signals. In general, a probe with more oligos (or a higher density) will cost more but generate brighter signals (Fig. 2C). Our experience gained in several different plant species indicates that a density of 0.1–0.5 oligo/kb is sufficient to generate high-quality whole-chromosome painting signals on condensed metaphase chromosomes. Pachytene chromosomes can be extended 10–20 times longer than somatic metaphase chromosomes. Thus, a higher oligo density is recommended if the probe will be used for painting meiotic pachytene chromosomes. A rice chromosome 9 probe with a density of 2/kb showed excellent signals on pachytene chromosomes (Hou et al. 2018). A potato chromosome 11 probe with a density of 0.6/kb was sufficient to visualize the entire pachytene chromosomes (Fig. 2F). However, if the oligo probe is designed to visualize a small genomic region (< 10 kb), then the probe should include as many oligos as possible. Overlapping oligos and oligos targeting both strands of the corresponding DNA sequence can be incorporated to maximize the FISH signal strength of the probe.

Fig. 2

FISH using synthetic oligo probes. (A) Upper panel: a two-color oligo-FISH barcode designed from 12 potato chromosomes (Braz et al. 2018). Lower panel: a somatic metaphase cell of tomato was hybridized to the two potato oligo-FISH barcode probes. Individual tomato chromosomes are identified based on the barcode. (B) A somatic metaphase cell of Solanum demissum (2n=6x=72) was hybridized to the two potato oligo-FISH barcode probes. The 6 copies of chromosomes 1 and 12 are labeled. S. demissum chromosomes show identical barcode patterns as potato chromosomes (Braz et al. 2018). (C) Oligo-based painting of potato chromosomes 4 (green) and 11 (red), respectively. Chromosome 4 (72 Mb) is significantly larger than chromosome 11 (45 Mb). Since the two painting probes contain the same number of oligos (27,392), the FISH signal on chromosome 11 is more intense and uniform than that on chromosome 4. (D1) Oligo-based painting of switchgrass chromosome 8a. A total of 27,000 oligos were designed based on sequences of chromosome 8a. Oligos that showed > 75% homology to sequences of chromosome 8b were not included. (D2) The FISH signals were digitally separated from D1. Arrows indicate the cross-hybridization signals associated with chromosome 8b. (E1) Oligo-based painting of the short arms of chromosomes 4a and 4b in switchgrass. A total of 27,000 oligos were designed based on the sequences of chromosome 4a. All selected oligos showed > 90% sequence similarity to the sequences of chromosome 4b. (E2) The FISH signals were digitally separated from E1. Arrows point to the FISH signals with a similar level intensity on the short arms of all four chromosomes. (F) Painting of potato chromosome 11 at the pachytene stage in meiosis. The four homologous copies of potato chromosome 11 paired as cross-shaped quadrivalent and are traced by white lines. Bars are 5 μm in (D1) and (E1), 10 μm in (A), (B), (C), and (F)

Chromosome identification and karyotyping

An oligo-FISH barcode system was recently developed as a new tool for chromosome identification in potato and related Solanum species (Braz et al. 2018). In this system, two oligo probes were designed from one or multiple regions, or “spots,” on every chromosome. Thus, the FISH signals derived from the two probes resemble a chromosome banding pattern, or a “barcode” that uniquely labels each of the 12 potato chromosomes (Braz et al. 2018) (Fig. 2A). This FISH barcode allowed identification of every chromosome in cultivated potato (2n=4x=48) as well as in Solanum demissum (2n=6x=72), a hexaploid wild species (Fig. 2B). Remarkably, the same barcode can be used to identify the 12 homoeologous chromosomes among distantly related Solanum species, including tomato and eggplant (Braz et al. 2018).

For most plant species, identification of all chromosomes in a single metaphase cell has not been possible. Although FISH-based chromosome identification systems using BAC or repetitive DNA probes were developed in several plant species (Jiang and Gill 2006), these systems are time-consuming to develop and/or cannot be applied to other plant species, especially to plants with smaller chromosomes or species with a large number of chromosomes. The oligo-FISH barcode system has the potential to become a universal system for plant chromosome identification. Nevertheless, we anticipate a few technical challenges for some plant species. (1) Plants with a large number of small chromosomes. The sizes of potato chromosomes range from 45 to 89 Mb. Since the distance between two “spots” needs to be separated by 5–10 Mb to ensure consistent separation of the FISH signals, only a single spot may be designed on some of the short potato chromosomal arms (Fig. 2A). If a plant species has smaller but more chromosomes than potato, it may not be possible to develop a similar two-color FISH barcode and additional probes (colors) may be required to cover all chromosomes. (2) Plants with very large genomes. To ensure strong and punctuated signals, each “spot” should include ~ 1000 oligos that are restricted within a relatively small chromosomal region (these regions span 184–707 kb in potato (Braz et al. 2018)). Such chromosomal regions may not be common in plants with very large genomes, such as wheat, which contains > 90% repetitive DNA sequences.

Although karyotypes have been developed in many plant species, individual chromosomes were not identified in most reported karyotypes. Therefore, these karyotypes are expected to be error-prone due to chromosome misidentification and are not comparable to those of related species. The oligo-FISH barcode system allows unambiguous development of karyotypes based on individually identified chromosomes, which can be used for comparative analysis (Braz et al. 2018). Modifications to the FISH barcode among related species would indicate putative chromosomal rearrangement and evolution. Two reciprocal chromosomal translocations were discovered in S. etuberosum and S. caripense, respectively, based on comparative karyotyping (Braz et al. 2018). The oligo-FISH barcode system will allow karyotyping of a large number of accessions or ecotypes within a species and studies of chromosome-scale genetic adaptation and evolution.

Applications of chromosome painting in different plant species

Painting of individual chromosomes has been impossible for most plant species due to the lack of methods to develop chromosome-specific DNA probes (Schubert et al. 2001). Chromosome painting has only been accomplished in A. thaliana and Brachypodium distachyon, both with very small genomes, by pooling a large number of repeat-free BACs derived from a specific chromosome as a FISH probe (Idziak et al. 2011; Lysak et al. 2001). The oligo-based chromosome painting technique should be applicable to any plant species with a sequenced genome (Han et al. 2015). This technique has already been successfully applied to a number of plant species, including cucumber (Han et al. 2015), strawberry (Qu et al. 2017), Aquilegia coerulea (Filiault et al. 2018), potato (Braz et al. 2018; He et al. 2018), rice (Hou et al. 2018), poplar (Xin et al. 2018), sugarcane (Meng et al. 2018), and maize (Albert et al. 2019). The maize genome contains a high percentage of repetitive DNA sequences derived from various types of complete and decayed transposable elements. Chromosome painting probes on all 10 maize chromosomes showed high specificity (Albert et al. 2019), indicating effective elimination of repeats during oligo selection using the Chorus software.

Chromosome painting can be applied to various cytogenetic and genome research in plants, including chromosomal evolution based on cross-species painting (Braz et al. 2018; Filiault et al. 2018; Han et al. 2015), characterization of cytogenetic stocks (Albert et al. 2019; Hou et al. 2018), monitoring chromosome pairing and transmission in meiosis (He et al. 2018) (Fig. 2E), and examining the quality of genomic sequence assembly (Xin et al. 2018). We expect an increasing number of new applications of this technique in different plant species.

Conclusions and future directions

FISH has been the most important technique in plant cytogenetic research since the 1990s. Few techniques have dominated a research field for more than 30 years. Unfortunately, the application of FISH techniques has been hindered by the lack of robust DNA probes in many plant species, especially non-model plant species with no or limited genomic resources. This obstacle, however, is significantly alleviated by application of synthetic oligo probes. Thus, we expect a significant expansion of FISH applications in plants. Several exciting new developments of FISH have recently been reported in model animal species. For example, CAS-FISH used a fluorescently labeled nuclease-deficient Cas9 (dCas9) proteins to label and detect genomic regions in mammalian cells without using a DNA-denaturing step that would disturb the nuclear genomic organization of fixed or living cells (Deng et al. 2015). Single-molecule RNA-FISH (smRNA-FISH) techniques have been developed to measure transcription of multiple genes or non-coding RNAs within single cells (Cabili et al. 2015; Lubeck and Cai 2012). We expect adaption and application of some of these new FISH techniques in plants (Dreissig et al. 2017; Duncan et al. 2016; Fujimoto et al. 2016). Nevertheless, chromosome-based FISH experiments will continue to be the primary application in plants and oligo-based synthetic probes will become a cornerstone methodology in the future.



FISH images in Fig. 2 were developed by Guilherme Braz, Li He, Tao Zhang, and Pingdong Zhang.

Funding information

Cytogenetic research in the author’s lab has been supported by National Science Foundation (NSF) grants IOS-1444514 and MCB-1412948.


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© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Plant Biology, Department of HorticultureMichigan State UniversityEast LansingUSA

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