Next Article in Journal
The Immune Privilege of Cancer Stem Cells: A Key to Understanding Tumor Immune Escape and Therapy Failure
Next Article in Special Issue
Cytogenetic Evidence Clarifies the Phylogeny of the Family Rhynchocyclidae (Aves: Passeriformes)
Previous Article in Journal
Effects of High Dietary Carbohydrate and Lipid Intake on the Lifespan of C. elegans
Previous Article in Special Issue
Measurement of Chromosomal Arms and FISH Reveal Complex Genome Architecture and Standardized Karyotype of Model Fish, Genus Carassius
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Cells
Volume 10
Issue 9
10.3390/cells10092358
cells-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Subgenome Discrimination in Brassica and Raphanus Allopolyploids Using Microsatellites

by
Nicole Bon Campomayor
1,†,
Nomar Espinosa Waminal
1,†,
Byung Yong Kang
1,
Thi Hong Nguyen
1,
Soo-Seong Lee
2,
Jin Hoe Huh
3 and
Hyun Hee Kim
1,*
1
Department of Chemistry and Life Science, BioScience Institute, Sahmyook University, Seoul 01795, Korea
2
BioBreeding Institute, Ansung 17544, Korea
3
Department of Agriculture, Forestry and Bioresources, Plant Genomics and Breeding Institute, Seoul National University, Seoul 08826, Korea
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Cells 2021, 10(9), 2358; https://doi.org/10.3390/cells10092358
Submission received: 27 July 2021 / Revised: 1 September 2021 / Accepted: 3 September 2021 / Published: 8 September 2021
(This article belongs to the Collection Non-human Chromosome Analysis)
Browse Figures
Review Reports Versions Notes

Abstract

:
Intergeneric crosses between Brassica species and Raphanus sativus have produced crops with prominent shoot and root systems of Brassica and R. sativus, respectively. It is necessary to discriminate donor genomes when studying cytogenetic stability in distant crosses to identify homologous chromosome pairing, and microsatellite repeats have been used to discriminate subgenomes in allopolyploids. To identify genome-specific microsatellites, we explored the microsatellite content in three Brassica species (B. rapa, AA, B. oleracea, CC, and B. nigra, BB) and R. sativus (RR) genomes, and validated their genome specificity by fluorescence in situ hybridization. We identified three microsatellites showing A, C, and B/R genome specificity. ACBR_msat14 and ACBR_msat20 were detected in the A and C chromosomes, respectively, and ACBR_msat01 was detected in B and R genomes. However, we did not find a microsatellite that discriminated the B and R genomes. The localization of ACBR_msat20 in the 45S rDNA array in ×Brassicoraphanus 977 corroborated the association of the 45S rDNA array with genome rearrangement. Along with the rDNA and telomeric repeat probes, these microsatellites enabled the easy identification of homologous chromosomes. These data demonstrate the utility of microsatellites as probes in identifying subgenomes within closely related Brassica and Raphanus species for the analysis of genetic stability of new synthetic polyploids of these genomes.

1. Introduction

The genus Brassica (Brassicaceae) includes several economically valuable crops that are used as oilseeds, condiments, culinary vegetables [1,2], and sources of health-promoting phytochemicals [3,4]. Brassica is also a good model for studying polyploidization because of numerous intercrossing and morphological variants [5,6]. The genetic relationship of the six most economically important Brassica species was described in U’s triangle [7]. These include three diploids, B. rapa (AA genome, 2n = 2x = 20), B. nigra (BB, 2n = 2x = 16), and B. oleracea (CC, 2n = 2x = 18), and their allotetraploid hybrids, B. juncea (AABB, 2n = 4x = 36), B. napus (AACC, 2n = 4x = 38), and B. carinata (BBCC, 2n = 4x = 34).
Like many Brassica species, R. sativus L. (RR, 2n = 2x = 18) has also been cultivated worldwide [8,9]. R. sativus is valued more for its roots than Brassica species, whose shoots are the primary commodities. Breeders have attempted to synthesize hybrids with shoot and root features of Brassica crops and R. sativus, respectively, to maximize the use of plant parts for culinary purposes [10]. Therefore, several synthetic crops that involved crosses between the A, C, B, and R genomes have been generated [11,12,13,14,15]. One such example is the intergeneric allotetraploid ×Brassicoraphanus, commonly known as ‘Baemoochae’, which resulted from a cross between B. rapa ssp. pekinensis L. and R. sativus L. [10,16].
However, due to frequent genetic instability of the selected phenotypes, only a few stable ×Brassicoraphanus lines have been successfully registered as commercial cultivars to date. One of these is “BB#1′’ (2n = 38, AARR genome), which was stabilized through induced microspore mutation of the intergeneric hybrid between B. rapa cv. Weongyo#207 and R. sativus cv. Baekgyoung [10,17,18].
Several lines involving multiple genomes have also been developed from crosses of synthetic allotetraploids. One example is hybrid ×Brassicoraphanus 977 (Supplementary Figure S1), which possibly carries chromosome blocks from the A, C, and R genomes, considering its genomic background from a cross between two ×Brassicoraphanus allotetraploids, BB#50 (AARR, 2n = 4x = 38) and “Mooyangchae” (RRCC, 2n = 4x = 36), a fertile amphidiploid progeny between Raphanus sativus cv. HQ-04 (2n = 2x = 18, RR) and Brassica alboglabra Bailey (2n = 2x = 18, CC) [19]. At present, the karyotype and genome composition of ×Brassicoraphanus 977 have not yet been reported.
It is important to identify the donor genomes of all chromosomes in ×Brassicoraphanus 977 for efficient karyotyping. Genomic in situ hybridization (GISH) is a conventional technique used to analyze donor genomes in allopolyploids [20,21]. However, GISH is laborious and inefficient in discriminating the A and C Brassica genomes in B. napus [22,23,24]. However, GISH-like results have been achieved using conventional fluorescence in situ hybridization (FISH) with microsatellite repeats as probes instead of genomic DNA. This approach discriminated C from the A genome in B. napus [24,25] and the B genome in Triticum aestivum [26].
Microsatellites are often used in PCR-based genetic diversity studies because of their high rate of transferability to other closely and even distantly related taxa [27]. They have been used to identify subgenome alleles in some bryophytes [28]. Moreover, microsatellites are good cytogenomic markers because they are expected to be distributed evenly along chromosomes [29]. However, they have not yet been used to visually discriminate subgenomes in allopolyploid species, and there has not been an extensive comparative whole-genome microsatellite quantification and FISH analysis between the Brassica A, C, B, and R. sativus R genomes. Hence, we are advancing the use of microsatellites in analyzing their presence and absence in component chromosomes of subgenomes to identify homologous chromosomes in allopolyploids.
This study aimed to determine whether similar microsatellites, such as the C-genome-specific ones found in B. napus, are also found in the A, B, and R genomes. We quantified microsatellite repeats in the A, C, B, and R genomes and performed comparative FISH to identify potential genome-specific microsatellites that can be used as a cytogenomic resource in karyotyping polyploid hybrids between these four genomes. Finding similar microsatellites unique to the A, C, B, and R genomes would ease chromosome discrimination in ×Brassicoraphanus 977 and other natural and synthetic allopolyploids carrying these genomes.

2. Materials and Methods

2.1. Microsatellite Mining

To analyze genome-specific microsatellites, we downloaded 100-bp whole-genome sequence reads of the four diploid A, C, B, and R genomes from NCBI (Table 1). High-quality reads representing 0.04–0.06× of the A, C, B, and R genomes [30,31,32,33] were selected using the FastQC tool in the RepeatExplorer version 2 pipeline [34]. Following a script provided in RepeatExplorer2, a long single contig was generated by concatenating all reads from each species and inserting 50-bp Ns between reads.
Each contig from the A, C, B, and R genomes was scanned for tandem repeats of ≤ 20 bp using Tandem Repeats Finder version 4.09 [35], and the output was sorted and quantified using the TRAP program v1 [36]. The repeat units of each microsatellite were concatenated to approximately 200 bp. To filter out redundant sequences, we grouped similar sequences and generated consensus sequences using the Assembly Sequence features in the CLC Main Workbench (CLC Inc., Aarhus, Denmark). The microsatellites were named ACBR_msat# according to the closer relationship of the A/C and B/R genomes [37], msat for “microsatellite” and the number order according to decreasing cumulative abundance. For FISH validation, we selected microsatellites with > 6 kb cumulative total bases from the A, C, B, and R genomes. A summary of the mining and FISH validation workflow is presented in Supplementary Figure S2.

2.2. Designing Microsatellite Pre-Labeled Oligoprobes

We developed pre-labeled oligonucleotide probes (PLOPs) for FISH validation of candidate microsatellites (Supplementary Table S1). PLOPs were designed using the CLC Main Workbench (CLC, Inc.) and synthesized by Bio Basic Canada Inc. (Toronto, ON, Canada). In addition, we also used 5S rDNA, 45S rDNA, and telomeric repeat PLOPs for chromosome identification. The rDNA and telomeric PLOPs were designed and prepared following methods described in [38] and were purchased from Bioneer (Daejeon, Korea).

2.3. Plant Samples

Seeds of the four diploid and five allopolyploid species were purchased commercially and provided by the National Plant Germplasm System of the US Department of Agriculture, the National Academy of Agricultural Science of Korea, or the BioBreeding Institute, Ansung, Korea (Table 2). Seeds were germinated on filter paper and incubated at 25 °C. Harvested root tips were treated with 2 mM 8-hydroxyquinoline for 5 h at 18 °C, fixed with aceto-ethanol (1:3 v/v), and stored in 70% ethanol at 4 °C until use.

2.4. Chromosome Preparation

Somatic chromosome spreads were prepared following the technique described by Waminal and Kim [39]. Five meristematic tips (~2 mm) were digested with pectolytic enzyme solution (2% Cellulase R-10 (C224, Phytotechnology Laboratories, Lenexa, KS, USA) and 1% Pectolyase Y-23 (P8004.0001, Duchefa, Haarlem, The Netherlands) in 100 mM citrate buffer) at 37 °C for 90 min. Roots were transferred into a microtube with chilled Carnoy’s solution and vortexed for 30 s at room temperature. The supernatant was discarded, and the pellet was resuspended in an appropriate amount of aceto-ethanol (9:1 v/v). The cellular suspension was pipetted onto pre-cleaned slides and pre-warmed in a humid chamber. After air-drying, slides were fixed in 2% formaldehyde [39] for 5 min, quickly rinsed with distilled water, and dehydrated in a series of ethanol treatments (70%, 90%, and 100%).

2.5. FISH and Karyotyping

FISH was performed according to the modified procedure of Waminal et al. [40] and Lim et al. [41]. The FISH hybridization mixture was prepared with 100% formamide, 50% dextran sulfate, 20× SSC, 50 ng/μL of each DNA probe, and deionized water (Sigma-Aldrich, St. Louis, MO, USA). The mixture was pipetted onto each slide. Slides were incubated at 80 °C for 5 min and transferred to a humid chamber at 37 °C overnight. After overnight hybridization, incubated slides were washed in 2× SSC at room temperature (RT) for 10 min, 0.1× SSC at 42 °C for 25 min, and 2× SSC at RT for 5 min, and dehydrated in a series of alcohol (70%, 90%, 95%). Slides were air-dried and counterstained with 4′,6-diaminidino-2-phenylindole (DAPI) at a ratio of 1:100 DAPI (100 µg/mL stock) and Vectashield (Vector Laboratories, Burlingame, CA, USA). Five to 10 well-spread metaphase chromosomes were chosen and were observed under an Olympus BX53 fluorescence microscope with a built-in CCD camera (CoolSNAP ™ cf), using an oil lens (×100 magnification). One metaphase chromosome spread was selected as a representative for each genome for karyotype analyse. Karyograms were finalized using Adobe Photoshop CC version 25.3, whereas ideograms were generated using Adobe Illustrator CC version 22.4. Homologous chromosomes were identified based on their FISH signals, morphological characteristics, and lengths, considering previous karyotype data for B. rapa, B. oleracea, and B. napus [5], B. nigra and B. juncea [42], and R. sativus and ×Brassicoraphanus [43].

3. Results

3.1. Identification of Major Microsatellites in the A, C, B, and R Genomes

To capture microsatellites with higher-order organization, we loosen our definition of microsatellites from a stricter 2–5 bp [44] to an extended target length of ≤ 20 bp (Supplementary Figure S2). This configuration generated 467 microsatellites in both strand orientations, including redundant or imperfect motif sequences [45] as demonstrated by sequence clustering (Figure 1A). Among the 467 microsatellites, the A, C, and B genomes had 123 sequences each. However, the R genome had only 98. The total base count for all microsatellites in each genome represented up to 0.2–0.3% of each genome (Figure 1B). Clustering of the related sequences generated 184 unique consensus sequences. Most of these microsatellites were di- and trinucleotides (Figure 1C). Many of these sequences represented <6 kb of cumulative total bases from the A, C, B, and R genomes. We only selected microsatellites with >6 kb cumulative total bases for FISH validation because microsatellites with lower total bases may not generate detectable FISH signals. This filtering retained 22 microsatellites, of which ACBR_msat01 had the highest cumulative total bases of ~328 kb (Figure 1D), which was distributed proportionately among the four genomes (Figure 1E and Table 3). Moreover, the ranks of the 22 microsatellites varied in each genome. For example, although the ACBR_msat06 was ranked sixth based on cumulative abundance, it ranked third, ninth, ninth, and fourteenth in the C, A, R, and B genomes, respectively.
The lengths of the 22 microsatellites ranged from 2 to 17 bp. However, the most abundant microsatellites were di- and trinucleotides. Moreover, 7-bp microsatellites were also abundant, although significantly biased to the C genome (Figure 1C). The 7-bp microsatellites represented the canonical Arabidopsis-type plant telomere repeat, ACBR_msat06, and its variant, ACBR_msat20 (Table 3). The Arabidopsis-type telomeric repeat covered ~65 kb total bases in the C genome, whereas only <15 kb in the other genomes. The ACBR_msat20, which was also observed predominantly in the C genome, had the central “A” in the Arabidopsis-telomeric repeat sequence replaced with a “C”.
Furthermore, microsatellites comprising only T and A were predominantly abundant in the C genome (Table 3). Although this bias was observed in di-, tri-, tetra, and pentanucleotides, it was prominent in the TA dinucleotide microsatellite (ACBR_msat02), in which the C genome had a total base of ~180 kb, whereas the A and B genomes had only < 20 kb and the R genome was ~71 kb (Table 3). The biased abundance of several microsatellites toward the C genome explains the higher proportion of microsatellites in the C genome than in the other three genomes (Figure 1B).
Similarly, ACBR_msat14 showed a biased abundance in the A genome (Figure 1E and Table 3). ACBR_msat14 is a 17-bp higher-order organized Arabidopsis-type telomere repeat composed of two units and an insertion of a trinucleotide “AGG” in the second repeat unit, particularly between the second and third T nucleotides (Table 3).
Some microsatellites, such as ACBR_msat18 and ACBR_msat22, also showed biased abundance in the B and R genomes, respectively. However, whether these genome-specific biases in microsatellite abundance also reflect their usability as genome-specific cytogenomic markers should be verified by FISH.

3.2. FISH Revealed Different Chromosome and Genome Distribution Patterns of the Microsatellites

To visualize the chromosomal distribution of the 22 microsatellites and analyze whether the microsatellites that showed putative genome specificity based on the in-silico analysis could also generate genome-specific FISH signals, we performed the FISH analysis in the four diploid A, C, B, and R genomes using PLOPs developed from the 22 microsatellite sequences (Supplementary Table S1).
Eleven of the 22 microsatellites did not show detectable FISH signals, indicating non-clustering of the microsatellite loci [46], making them undetectable by FISH (Supplementary Figure S3 and Table S1). Other microsatellites showed clustered loci that generated detectable FISH signals in either a few or all chromosomes in each genome. One microsatellite showed signals in the A, C, and B genomes, whereas five showed non-specific signals for all four genomes. Two microsatellites showed signals in both the C and R genomes, and one microsatellite showed specific signals to the A (ACBR_msat14) and C (ACBR_msat20) genomes (Figure 2 and Figure 3, Table 3). We did not observe any microsatellite that discriminated the B and R genomes. However, we identified one microsatellite that showed signals to both B and R genomes (ACBR_msat01) (Supplementary Figure S3, Figure 2 and Figure 3, Supplementary Table S1).
Genome-specific abundance from the in-silico analysis was partially supported by FISH data. The Arabidopsis-type telomeric repeat showed biased intense signals in the C genome, corroborating in silico data (Supplementary Figure S4 and Table S1). In addition, ACBR_msat14 and ACBR_msat20 showed A and C genome-specific signals, which also corroborated the in-silico data (Supplementary Figure S3, Figure 2 and Figure 3, Supplementary Table S1). However, ACBR_msat01 showed signals only in the B and R genomes as well as in one chromosome pair in the A genome. This did not coincide with the in-silico data, where relatively equal abundance in the four genomes was observed Figure 1E, Figure 2, and Figure 3, Table 3). These data suggest differences in the loci organization of this microsatellite in the four genomes: clustered in the B and R genomes as well as in chromosome 8 in the A genome and non-clustered in the C genome and other chromosomes in the A genome.
In summary, the FISH analysis revealed three microsatellites specific to the diploid species in the U’s triangle: ACBR_msat14 for the A genome, ACBR_msat20 for the C genome, and ACBR_msat01 for the B genome (Supplementary Figure S3). Additionally, ACBR_msat01 could also detect R-genome chromosomes, making it a candidate marker for identifying the R chromosomes in the intergeneric ×Brassicoraphanus hybrids.

3.3. Genome-Specific Microsatellites Discriminated Subgenomes in Allotetraploids

To verify whether the FISH signals of ACBR_msat01, ACBR_msat14, and ACBR_msat20 in the diploid Brassica genomes were genome-specific and could distinguish subgenomes in interspecific and intergeneric hybrids, we performed the FISH analysis in allotetraploids within the U’s triangle and ×Brassicoraphanus.
As expected, the 10, 9, and 8 homologous chromosomes of the A, C, and B genomes were clearly distinguished from each other in B. napus (AACC), B. juncea (AABB), and B. carinata (BBCC) using ACBR_msat14, ACBR_msat20, and ACBR_msat01, respectively (Figure 4, Figure 5 and Figure 6). The Arabidopsis-type telomeric repeat also enabled identification of the C-genome chromosomes in B. napus and B. carinata (Supplementary Figure S4), making this probe sufficient to distinguish the C-genome chromosomes among the allotetraploids in the U’s triangle.
Moreover, the A and R genomes were also clearly distinguished in ×Brassicoraphanus “BB#1” using ACBR_msat14 and ACBR_msat01, respectively (Figure 4 and Figure 5). However, although the R genome showed intense ACBR_msat01 signals, A-genome chromosomes also showed ACBR_msat01 signals in intercalary regions of all chromosomes with varied intensity, although weaker than those in the R genome, except for chromosome 8, which showed a more intense single locus at the proximal region of the long arm (Figure 5).
In summary, the three microsatellites proved to be efficient FISH probes to distinguish between the A, C, and B subgenomes of the allotetraploid species in the U’s triangle and between the A and R genomes in ×Brassicoraphanus (Figure 6).

3.4. FISH Revealed Genome Rearrangement and Chromatin Elimination in ×Brassicoraphanus 977

No cytogenetic analysis has been performed to investigate the chromosomal constitution of the putative tri-genome ×Brassicoraphanus 977. Therefore, we used the genome-specific microsatellites identified in this study to analyze the genomic composition of this plant. Line 977 was generated from a cross between two ×Brassicoraphanus allotetraploids, BB#50 (AARR, 2n = 4x = 38) and ‘‘Mooyangchae’’ (CCRR, 2n = 4x = 36). Therefore, we expected to see the complete set of the R chromosomes and a mixture of A and C chromosomes.
As expected, ×Brassicoraphanus 977 had 2n = 38 chromosomes, and ACBR_msat01 identified the complete R-genome chromosomes (Figure 5). ACBR_msat14 generated signals in the remaining 10 homologous pairs, indicating the A-genome origin of the other chromosomes. ACBR_msat20 generated FISH signals in three A-genome chromosomes, which we named A1C, A3C, and A6C, indicating introgression of C genome chromosome blocks into these chromosomes (Figure 5 and Figure 6). These data also suggest that the C genome chromatin is preferentially eliminated in ×Brassicoraphanus 977. The ACBR_msat20 signals were coincidentally localized in the 45S rDNA-bearing chromosomes and co-localized with the NOR signals. This observation indicates a role for 45S rDNA in the genome reshuffling of the A and C genomes and the homing of the ACBR_msat20 in the 45S rDNA array in ×Brassicoraphanus 977.
In summary, the FISH data of the genome-specific microsatellite probes in ×Brassicoraphanus 977 suggests the possibility that line 977 has an AARR genomic background with introgressed C-genome chromatin at the 45S rDNA loci, contrary to ARRC that was initially thought. This analysis also showed preferential elimination of the C genome in ×Brassicoraphanus 977.

3.5. Ribosomal DNA and Telomeric Repeat Distribution

We used 5S and 45S rDNA probes to identify known chromosomes and the Arabidopsis-type telomeric repeats to identify chromosome ends for karyotyping. All four diploid species revealed three 45S rDNA loci, whereas B. rapa, B. nigra, B. oleracea, and R. sativus had three, one, one, and two 5S rDNA loci, respectively, similar to those observed in previous studies [5,47,48,49]. In the allotetraploid species, B. juncea, B. napus, B. carinata, ×Brassicoraphanus ‘‘BB#1′’, and 977 had four, six, two, five, and six 5S rDNA loci and six, seven, six, five, and seven 45S rDNA loci. Arabidopsis-type telomeric repeat sequences were detected in all chromosomes of Brassica and Raphanus genome.

4. Discussion

4.1. Importance of Subgenome Discrimination in Allopolyploids

The ability to discriminate individual subgenomes or chromosomes in allopolyploids is invaluable for studying genome structure, dynamics, and stability [26,50]. This is particularly useful in analyzing the chromosomal composition and genetic stability of synthetic plants from intergeneric crosses, such as ×Brassicoraphanus, which generate sterile progenies from unstable meiotic chromosome paring [51].
Although GISH has been the conventional choice to discriminate subgenomes in allopolyploids [20,21], recent innovations, such as bulk oligo-FISH which uses single-copy oligomer probes, can allow easy discrimination of subgenomes and individual chromosomes [52]. However, this method requires high-quality genome assembly to design oligoprobe libraries, making it difficult to analyze species without a whole-genome assembly [52,53]. Moreover, the synthesis of bulk oligo libraries is costly relative to repeat-based pre-labeled oligomer probes (PLOPs) [38]. However, several bioinformatics tools (see Methods, for example) offer ways to analyze repeats and microsatellites from short next-generation sequences, enabling microsatellite analysis in species without genome assembly.
Microsatellites can have genome-specific distribution. Therefore, they can be useful in discriminating subgenomes in allopolyploids, as demonstrated in previous studies [25,26,29,54] and this study. Here, we identified 22 high-abundance microsatellites from the A, C, and B genomes of Brassica and the R genome of R. sativus using short next-generation sequencing reads. Three microsatellites, namely ACBR_14, ACBR_20, and ACBR_01, could discriminate the A, C, and B Brassica genomes and the R. sativus R genome, respectively, making them useful cytogenomic markers for natural and synthetic allopolyploids carrying the A, C, B, and R genomes.
Moreover, the FISH procedure using microsatellites is more straightforward and not as laborious and resource-intensive as GISH because PLOPs are used instead of genomic and blocking DNA. In addition, the use of rDNA and telomere repeat PLOPs and the new microsatellites enabled efficient homologous chromosome identification in each subgenome.

4.2. Organization of Microsatellite in the Chromosomes Determine FISH Detectability

The abundance of microsatellites in a genome cannot always predict detectability using FISH. Microsatellites are organized either in a clustered or non-clustered manner, making them detectable or undetectable by FISH, respectively [26,46,55,56,57]. Long microsatellite arrays or short arrays clustered together can allow amplified FISH signals to reach the fluorescence detection threshold [46].
This difference in locus organization was well demonstrated in ACBR_msat01. Intense ACBR_msat01 FISH signals were predominantly observed in the B and R genomes, despite being present in comparable abundance in all four genomes based on in silico quantification (Figure 1 and Table 3). This pattern suggests a more clustered organization of ACBR_msat01 in the B and R genomes compared to a predominantly non-clustered organization in the C and A genomes, except for one cluster in chromosome A8.

4.3. Genome-Specific Microsatellites for the A, C, and B Brassica Genomes

Our data corroborated the abundance of a telomeric repeat-like C-genome-specific microsatellite (ACBR_msat20) previously identified in B. napus using conventional molecular techniques [25]. However, although we only identified the consensus TTTCGGG sequence of ACBR_msat20, a previous study presented an intermittent organization of TTTCGGG, TTTGGGG, and a few imperfect derivative sequences [25], demonstrating that molecular analyses are relevant in defining actual sequences. However, for the FISH and genome discrimination, our results were comparable to previously reported data for discriminating the C-genome chromosomes from the A and B Brassica genomes.
In addition to the C genome-specific microsatellite, we identified ACBR_msat14, which is an A genome-specific microsatellite that efficiently discriminated A from the C and B genomes. Although it is challenging to precisely determine whether ACBR_msat14 is located in the telomeric or subtelomeric region just by looking at the FISH image, ACBR_msat14 is possibly localized at the subtelomeric region rather than the telomeres because (i) Arabidopsis-type telomeric signals were distinctly observed at chromosomal ends and (ii) subtelomeres are known to host different telomeric sequence variants, similar to the six different variants found in rice subtelomeres [58,59]. Subtelomeres are also prone to accumulation and rapid expansion of several tandem repeats, making subtelomere sites of frequent chromosomal rearrangements [60,61].
Similar to the C and A genomes, we also identified ACBR_msat01, which discriminated B from the A or C Brassica genomes in the U’s triangle. However, ACBR_msat01 failed to discriminate B from the R genome of R. sativus. In contrast, ACBR_msat01 can be an excellent cytogenetic marker between the species in the U’s triangle, it would not be useful to distinguish B and R chromosomes in allopolyploids comprising these two genomes. In this case, B. nigra and R. sativus centromeric satellites may be used to identify the B and R genomes, respectively [62,63,64]. However, no comparative analysis has been performed in allopolyploids with the B and R genomes that show genome specificity and a lack of cross-hybridization of these centromeric satellites.

4.4. Microsatellite Distribution Supports Phylogenetic Relationships of the Four Diploid Species

The distribution of the three genome-specific microsatellites reflected the phylogenetic relationships of the diploid species. Clustering of the ACBR_msat01 in the B and R genomes, but not in the A and C genomes, corroborates the observed closer relationship between B. nigra and R. sativus than B. nigra is to the B. rapa or B. oleracea [37]. Considering the high evolvability of microsatellites [65], the relatively recent divergence of the B. rapa and B. oleracea genomes from the more primitive B. nigra and R. sativus [5,37] may have abruptly de-clustered the ACBR_msat01 loci in the C genome and most of the A genome, except for that chromosome A8. This genome reshuffling may have also concomitantly spurred the differential evolution of telomere-like repeats between the A and C genomes.

4.5. Association of 45S rDNA in Genome Reshuffling

Several studies have implicated the involvement of the 45S rDNA array in plant genome rearrangements [60,66,67,68]. Similarly, we observed the participation of the 45S rDNA array in the rapid evolution of the ACBR_msat20 microsatellite in ×Brassicoraphanus 977. While the ACBR_msat20 microsatellite was dispersed in the C-genome chromosomes of the diploid and allopolyploid species in the U’s triangle, it was localized at the 45S rDNA loci in ×Brassicoraphanus 977. This observation further demonstrates the role of 45S rDNA in genome reshuffling. Although we have not characterized the sequence of the 45S rDNA in ×Brassicoraphanus 977, the ACBR_msat20 microsatellite must have been likely inserted into the 45S rDNA intergenic spacer, as this region is often considered a “logistics hub” of repeats in the genome, hosting different types of repeats into or out of the 45S rDNA intergenic spacer [68,69,70]. The 45S rDNA may also facilitate the rapid and efficient concerted expansion and contraction of genomic repeats, as observed in Senna [68] and the ACBR_msat20 microsatellite in ×Brassicoraphanus 977.
Although the exact mechanism by which 45S rDNA interacts with the genome and performs genome rearrangements is not yet fully understood, microhomology between sub-repeats in the 45S rDNA intergenic spacer and microsatellites may drive recombination, particularly given that microsatellites are known hotspots for recombination and chromosomal rearrangements [71].

5. Conclusions

We performed a comprehensive comparative microsatellite quantification and FISH analysis between the Brassica A, C, B, and R. sativus R genomes and identified three microsatellites that could discriminate the A, C, and B/R genomes. Along with the 45S and 5S rDNA and the Arabidopsis-type telomeric repeats, we efficiently identified the subgenomes and homologous chromosomes of the natural and synthetic allopolyploids between these four genomes. This study presents the first three-genome integrated karyotype of the ×Brassicoraphanus 977. The genome-specific microsatellites studied here could constitute an excellent cytogenomic marker to study several lines of synthetic ×Brassicoraphanus.
Although the ACBR_msat01 could not distinguish between the B and R genomes, further comparative analysis with longer satellite sequences may generate genome-specific probes, such as the centromeric repeats identified in the B and R genomes [62,63,64]. However, further FISH validation is needed to determine whether these centromeric repeats cross hybridize with each other, considering the close genetic distance between the B and R genomes.
We have also shown the rapid evolution of the ACBR_msat20 microsatellite and implied the involvement of 45S rDNA in genome reshuffling. Moreover, further sequence characterization of the complete 45S rDNA sequence, including the intergenic spacer in ×Brassicoraphanus 977, will explain the observed signals of the ACBR_msat20 in the 45S rDNA array and reveal its actual insertion site. This analysis will also help provide clues on the role and mechanism of 45S rDNA in genome rearrangements.

Supplementary Materials

The following information is available at https://www.mdpi.com/article/10.3390/cells10092358/s1. Figure S1: Breeding summary of ×Brassicoraphanus 977; Figure S2: Summary of the microsatellite mining and FISH validation pipeline; Figure S3: FISH screening of the 22 candidate microsatellites in the diploid B. rapa (AA), B. oleracea (CC), B. nigra (BB), and R. sativus (RR) genomes; Figure S4: Chromosomal distribution of Arabidopsis-type telomere repeats (ACBR_msat06) showing more intense FISH signals in 18 C genome chromosomes (yellow arrowheads) in B. napus (AACC) and B. carinata (BBCC). Table S1: Summary of PLOPs used for FISH.

Author Contributions

Conceptualization, N.E.W. and H.H.K.; investigation, N.B.C.; resources, S.-S.L. and J.H.H.; formal analysis, N.E.W.; writing—original draft preparation, N.B.C. and N.E.W.; writing—review and editing, N.E.W., N.B.C., T.H.N., B.Y.K., and H.H.K.; visualization, N.E.W. and N.B.C.; supervision, N.E.W. and H.H.K.; project administration, H.H.K.; funding acquisition, H.H.K., J.H.H., and S.-S.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by a grant from the Agricultural, Fisheries, and Food Technology Planning Evaluation Institute (Research No. 117045033SB020) and by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. NRF-2020R1G1A1102594).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data that support the findings of this study are presented in this article and the Supplementary Materials.

Acknowledgments

We thank Remnyl Joyce Pellerin for training N.B.C. on FISH analysis.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Kiefer, M.; Schmickl, R.; German, D.A.; Mandáková, T.; Lysak, M.A.; Al-Shehbaz, I.A.; Franzke, A.; Mummenhoff, K.; Stamatakis, A.; Koch, M.A.J.P.; et al. BrassiBase: Introduction to a novel knowledge database on Brassicaceae evolution. J. Plant Cell Physiol. 2014, 55, e3. [Google Scholar] [CrossRef] [Green Version]
  2. Ebeed, H.T. Bioinformatics Studies on the Identification of New Players and Candidate Genes to Improve Brassica Response to Abiotic Stress, In The Plant Family Brassicaceae: Biology and Physiological Responses to Environmental Stresses; Hasanuzzaman, E., Ed.; Springer Nature: Singapore, 2020; pp. 483–496. [Google Scholar]
  3. Mandrich, L.; Caputo, E.J.N. Brassicaceae-derived anticancer agents: Towards a green approach to beat cancer. J. Nutr. 2020, 12, 868. [Google Scholar] [CrossRef] [Green Version]
  4. Pérez-Balibrea, S.; Moreno, D.A.; García-Viguera, C. Glucosinolates in broccoli sprouts (Brassica oleracea var. italica) as conditioned by sulphate supply during germination. J. Food Sci. 2010, 75, C673–C677. [Google Scholar]
  5. Waminal, N.E.; Perumal, S.; Lee, J.; Kim, H.H.; Yang, T.-J. Repeat evolution in Brassica rapa (AA), B. oleracea (CC), and B. napus (AACC) genomes. Plant Breed. Biotechnol. 2016, 4, 107–122. [Google Scholar] [CrossRef] [Green Version]
  6. Liu, S.; Liu, Y.; Yang, X.; Tong, C.; Edwards, D.; Parkin, I.A.; Zhao, M.; Ma, J.; Yu, J.; Huang, S. The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes. Nat. Commun. 2014, 5, 3930. [Google Scholar] [CrossRef] [PubMed]
  7. Nagaharu, U. Genome analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of fertilization. J. Jap. Bot. 1935, 7, 389–452. [Google Scholar]
  8. Khalid, M.; Ayayda, R.; Gheith, N.; Salah, Z.; Abu-Lafi, S.; Jaber, A.; Al-Rimawi, F.; Al-Mazaideh, G. Assessment of antimicrobial and anticancer activity of radish sprouts extracts. Jordan J. Biol. Sci. 2020, 13, 567–574. [Google Scholar]
  9. Chang, M.J.; Kim, B.; Lee, H.S.; Kim, W.K.; Cho, K.J.T.F.J. Anti-cancer effect of Korean radish (Raphanus sativus L.) leaf extract through induction of apoptosis and down-regulate Erb B2 signaling and Akt pathway in human breast MDA-MB-231 cell lines. FASEB J. 2010, 24, 720.4. [Google Scholar] [CrossRef]
  10. Lee, S.-S.; Choi, W.-J.; Woo, J.-G. Development of a new vegetable crop in xBrassicoraphanus by hybridization of Brassica campestris and Raphanus sativus. KHSH 2002, 43, 693–698. [Google Scholar]
  11. Prakash, S.; Bhat, S.; Quiros, C.; Kirti, P.; Chopra, V. Brassica and its close allies: Cytogenetics and evolution. Plant Breed. Rev. 2009, 31, 21–187. [Google Scholar]
  12. Bang, S.; Tsutsui, K.; Shim, S.; Kaneko, Y. Production and characterization of the novel CMS line of radish (Raphanus sativus) carrying Brassica maurorum cytoplasm. Plant Breed. 2011, 130, 410–412. [Google Scholar] [CrossRef]
  13. Wang, X.; Zeng, L.; Xu, L.; Chen, W.; Liu, F.; Yang, H.; Chi, P.; Ren, L.; Yan, R.; Lu, G. Clubroot resistance introgression in interspecific hybrids between Raphanus sativus and Brassica napus. OCS 2019, 4, 139–151. [Google Scholar]
  14. Gong, T.; Ray, Z.T.; Butcher, K.E.; Black, Z.E.; Zhao, X.; Brecht, J.K. A novel graft between Pac Choi (Brassica rapa var. chinensis) and Daikon Radish (Raphanus sativus var. longipinnatus). Agronomy 2020, 10, 1464. [Google Scholar]
  15. Zhan, Z.; Nwafor, C.C.; Hou, Z.; Gong, J.; Zhu, B.; Jiang, Y.; Zhou, Y.; Wu, J.; Piao, Z.; Tong, Y.J. Cytological and morphological analysis of hybrids between Brassicoraphanus, and Brassica napus for introgression of clubroot resistant trait into Brassica napus L. PLoS ONE 2017, 12, e0177470. [Google Scholar] [CrossRef]
  16. Lee, S.; Kim, H.; Oh, D.; Woo, J.K. Breeding a fertile intergeneric allotetraploid plant between heading Chinese cabbage and Korean radish. Korean J. Hortic. Sci. Technol. 1999, 17, 653. [Google Scholar]
  17. Lee, S.-S.; Lee, S.-A.; Yang, J.; Kim, J. Developing stable progenies of ×Brassicoraphanus, an intergeneric allopolyploid between Brassica rapa and Raphanus sativus, through induced mutation using microspore culture. Theor. Appl. Genet. 2011, 122, 885–891. [Google Scholar] [CrossRef] [PubMed]
  18. Lee, S.-S.; Son, C.Y.; Kim, J.; Park, J.E.; Yu, S.H.; Yi, G.; Huh, J.H. Properties of self-sterile but cross-fertile allopolyploids synthesized between Brassica rapa and Raphanus sativus. Korean J. Hortic. Sci. Technol. 2020, 61, 163–171. [Google Scholar] [CrossRef]
  19. Chen, H.; Wu, J. Characterization of fertile amphidiploid between Raphanus sativus and Brassica alboglabra and the crossability with Brassica species. Genet. Resour. Crop Evol. 2008, 55, 143–150. [Google Scholar] [CrossRef]
  20. Schwarzacher, T.; Leitch, A.; Bennett, M.D.; Heslop-Harrison, J. In situ localization of parental genomes in a wide hybrid. Ann. Bot. 1989, 64, 315–324. [Google Scholar] [CrossRef]
  21. Ramzan, F.; Younis, A.; Lim, K.-B. Application of genomic in situ hybridization in horticultural science. Int. J. Genomics 2017, 2017, 7561909. [Google Scholar] [CrossRef] [Green Version]
  22. Howell, E.C.; Kearsey, M.J.; Jones, G.H.; King, G.J.; Armstrong, S.J. A and C genome distinction and chromosome identification in Brassica napus by sequential fluorescence in situ hybridization and genomic in situ hybridization. Genetics 2008, 180, 1849–1857. [Google Scholar] [CrossRef] [Green Version]
  23. Snowdon, R.; Köhler, W.; Friedt, W.; Köhler, A. Genomic in situ hybridization in Brassica amphidiploids and interspecific hybrids. Theor. Appl. Genet. 1997, 95, 1320–1324. [Google Scholar] [CrossRef]
  24. Ohmido, N.; Ueda, K.; Fujii, K. Chromosome instability of allopolyploid resynthesized Brassica napus. Chromosome Sci. 2015, 18, 79–84. [Google Scholar]
  25. Dos Santos, K.G.B.; Becker, H.; Ecke, W.; Bellin, U. Molecular characterisation and chromosomal localisation of a telomere-like repetitive DNA sequence highly enriched in the C genome of Brassica. Cytogenet. Genome Res. 2007, 119, 147–153. [Google Scholar] [CrossRef]
  26. Cuadrado, A.; Cardoso, M.; Jouve, N. Physical organisation of simple sequence repeats (SSRs) in Triticeae: Structural, functional and evolutionary implications. Cytogenet. Genome Res. 2008, 120, 210–219. [Google Scholar] [CrossRef] [PubMed]
  27. Peakall, R.; Gilmore, S.; Keys, W.; Morgante, M.; Rafalski, A. Cross-species amplification of soybean (Glycine max) simple sequence repeats (SSRs) within the genus and other legume genera: Implications for the transferability of SSRs in plants. Mol. Biol. Evol. 1998, 15, 1275–1287. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Karlin, E.F.; Boles, S.B.; Seppelt, R.D.; Terracciano, S.; Shaw, A.J. The Peat Moss Sphagnum cuspidatum in Australia: Microsatellites Provide a Global Perspective. Syst. Bot. 2011, 36, 22–32. [Google Scholar] [CrossRef]
  29. Santos, J.; Serra, L.; Solé, E.; Pascual, M. FISH mapping of microsatellite loci from Drosophila subobscura and its comparison to related species. Chromosome Res. 2010, 18, 213–226. [Google Scholar] [CrossRef]
  30. Zhang, L.; Cai, X.; Wu, J.; Liu, M.; Grob, S.; Cheng, F.; Liang, J.; Cai, C.; Liu, Z.; Liu, B. Improved Brassica rapa reference genome by single-molecule sequencing and chromosome conformation capture technologies. Hortic. Res. 2018, 5, 50. [Google Scholar] [CrossRef] [Green Version]
  31. Paritosh, K.; Pradhan, A.K.; Pental, D. A highly contiguous genome assembly of Brassica nigra (BB) and revised nomenclature for the pseudochromosomes. BMC Genomics 2020, 21, 887. [Google Scholar] [CrossRef] [PubMed]
  32. Parkin, I.A.; Koh, C.; Tang, H.; Robinson, S.J.; Kagale, S.; Clarke, W.E.; Town, C.D.; Nixon, J.; Krishnakumar, V.; Bidwell, S.L. Transcriptome and methylome profiling reveals relics of genome dominance in the mesopolyploid Brassica oleracea. Genome Biol. 2014, 15, R77. [Google Scholar] [CrossRef] [Green Version]
  33. Shirasawa, K.; Hirakawa, H.; Fukino, N.; Kitashiba, H.; Isobe, S. Genome sequence and analysis of a Japanese radish (Raphanus sativus) cultivar named ‘Sakurajima Daikon’possessing giant root. DNA Res. 2020, 27, dsaa010. [Google Scholar] [CrossRef]
  34. Novák, P.; Neumann, P.; Macas, J. Global analysis of repetitive DNA from unassembled sequence reads using RepeatExplorer2. Nat. Protoc. 2020, 15, 3745–3776. [Google Scholar] [CrossRef]
  35. Benson, G. Tandem repeats finder: A program to analyze DNA sequences. Nucleic Acids Res. 1999, 27, 573–580. [Google Scholar] [CrossRef] [Green Version]
  36. Sobreira, T.J.P.; Durham, A.M.; Gruber, A. TRAP: Automated classification, quantification and annotation of tandemly repeated sequences. Bioinformatics 2006, 22, 361–362. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Kim, C.-K.; Seol, Y.-J.; Perumal, S.; Lee, J.; Waminal, N.E.; Jayakodi, M.; Lee, S.-C.; Jin, S.; Choi, B.-S.; Yu, Y. Re-exploration of U’s Triangle Brassica species based on chloroplast genomes and 45S nrDNA sequences. Sci. Rep. 2018, 8, 7353. [Google Scholar] [CrossRef]
  38. Waminal, N.E.; Pellerin, R.J.; Kim, N.-S.; Jayakodi, M.; Park, J.Y.; Yang, T.-J.; Kim, H.H. Rapid and efficient FISH using pre-labeled oligomer probes. Sci. Rep. 2018, 8, 8224. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  39. Waminal, N.E.; Kim, H.H. Dual-color FISH karyotype and rDNA distribution analyses on four Cucurbitaceae species. Hortic. Environ. Biotechnol. 2012, 53, 49–56. [Google Scholar] [CrossRef]
  40. Vrána, J.; Šimková, H.; Kubaláková, M.; Číhalíková, J.; Doležel, J. Flow cytometric chromosome sorting in plants: The next generation. Methods 2012, 57, 331–337. [Google Scholar] [CrossRef] [PubMed]
  41. Lim, K.B.; Yang, T.J.; Hwang, Y.J.; Kim, J.S.; Park, J.Y.; Kwon, S.J.; Kim, J.; Choi, B.S.; Lim, M.H.; Jin, M.; et al. Characterization of the centromere and peri-centromere retrotransposons in Brassica rapa and their distribution in related Brassica species. Plant J. 2007, 49, 173–183. [Google Scholar] [CrossRef] [PubMed]
  42. Kulak, S.; Hasterok, R.; Maluszynska, J. Karyotyping of Brassica amphidiploids using 5S and 25S rDNA as chromosome markers. Hereditas 2002, 136, 144–150. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Belandres, H.R.; Waminal, N.E.; Hwang, Y.-J.; Park, B.-S.; Lee, S.-S.; Huh, J.H.; Kim, H.H. FISH karyotype and GISH meiotic pairing analyses of a stable intergeneric hybrid xBrassicoraphanus line BB# 5. Hortic. Sci. Technol. 2015, 33, 83–92. [Google Scholar]
  44. Mehrotra, S.; Goyal, V. Repetitive sequences in plant nuclear DNA: Types, distribution, evolution and function. GPB 2014, 12, 164–171. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Vieira, M.L.C.; Santini, L.; Diniz, A.L.; de Freitas Munhoz, C. Microsatellite markers: What they mean and why they are so useful. Genet. Mol. Biol. 2016, 39, 312–328. [Google Scholar] [CrossRef] [PubMed]
  46. Ruiz-Ruano, F.J.; López-León, M.D.; Cabrero, J.; Camacho, J.P.M. High-throughput analysis of the satellitome illuminates satellite DNA evolution. Sci. Rep. 2016, 6, 28333. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Hwang, Y.-J.; Yu, H.-J.; Mun, J.-H.; Bok, K.; Park, B.-S.; Lim, K.-B. Centromere repeat DNA originated from Brassica rapa is detected in the centromere region of Raphanus sativus chromosomes. Hortic. Sci. Technol. 2012, 30, 751–756. [Google Scholar] [CrossRef]
  48. Perumal, S.; Waminal, N.E.; Lee, J.; Lee, J.; Choi, B.-S.; Kim, H.H.; Grandbastien, M.-A.; Yang, T.-J. Elucidating the major hidden genomic components of the A, C, and AC genomes and their influence on Brassica evolution. Sci. Rep. 2017, 7, 17986. [Google Scholar] [CrossRef]
  49. Xiong, Z.; Pires, J.C. Karyotype and identification of all homoeologous chromosomes of allopolyploid Brassica napus and its diploid progenitors. Genetics 2011, 187, 37–49. [Google Scholar] [CrossRef] [Green Version]
  50. Chester, M.; Gallagher, J.P.; Symonds, V.V.; da Silva, A.V.C.; Mavrodiev, E.V.; Leitch, A.R.; Soltis, P.S.; Soltis, D.E. Extensive chromosomal variation in a recently formed natural allopolyploid species, Tragopogon miscellus (Asteraceae). PNAS 2012, 109, 1176–1181. [Google Scholar] [CrossRef] [Green Version]
  51. Belandres, H.R.; Zhou, H.C.; Waminal, N.E.; Lee, S.-S.; Huh, J.H.; Kim, H.H. Cytogenetic analyses revealed different genome rearrangement footprints in four ×Brassicoraphanus lines with different fertility rates. Plant Breed. Biotechnol. 2019, 7, 95–105. [Google Scholar] [CrossRef]
  52. Han, Y.; Zhang, T.; Thammapichai, P.; Weng, Y.; Jiang, J. Chromosome-specific painting in Cucumis species using bulked oligonucleotides. Genetics 2015, 200, 771–779. [Google Scholar] [CrossRef] [Green Version]
  53. Jiang, J. Fluorescence in situ hybridization in plants: Recent developments and future applications. Chromosome Res. 2019, 27, 153–165. [Google Scholar] [CrossRef]
  54. Cuadrado, A.; Schwarzacher, T.; Jouve, N. Identification of different chromatin classes in wheat using in situ hybridization with simple sequence repeat oligonucleotides. Theor. Appl. Genet. 2000, 101, 711–717. [Google Scholar] [CrossRef]
  55. Zattera, M.L.; Gazolla, C.B.; de Araújo Soares, A.; Gazoni, T.; Pollet, N.; Recco-Pimentel, S.M.; Bruschi, D.P. Evolutionary dynamics of the repetitive DNA in the karyotypes of Pipa carvalhoi and Xenopus tropicalis (Anura, Pipidae). Front. Genet. 2020, 11, 637. [Google Scholar] [CrossRef] [PubMed]
  56. Utsunomia, R.; Melo, S.; Scacchetti, P.C.; Oliveira, C.; de Andrade Machado, M.; Pieczarka, J.C.; Nagamachi, C.Y.; Foresti, F. Particular chromosomal distribution of microsatellites in five species of the genus Gymnotus (Teleostei, Gymnotiformes). Zebrafish 2018, 15, 398–403. [Google Scholar] [CrossRef]
  57. Megyeri, M.; Mikó, P.; Farkas, A.; Molnár-Láng, M.; Molnár, I. Cytomolecular discrimination of the A chromosomes of Triticum monococcum and the A chromosomes of Triticum aestivum using microsatellite DNA repeats. J. Appl. Genet. 2017, 58, 67–70. [Google Scholar] [CrossRef] [Green Version]
  58. Mizuno, H.; Wu, J.; Matsumoto, T. Characterization of chromosomal ends on the basis of chromosome-specific telomere variants and subtelomeric repeats in rice (Oryza sativa L.). In Subtelomeres; Springer: Berlin/Heidelberg, Germany, 2014; pp. 187–194. [Google Scholar]
  59. Mizuno, H.; Matsumoto, T.; Wu, J. Composition and structure of rice centromeres and telomeres. In Rice Genomics, Genetics and Breeding; Springer: Berlin/Heidelberg, Germany, 2018; pp. 37–52. [Google Scholar]
  60. Waminal, N.E.; Pellerin, R.J.; Kang, S.-H.; Kim, H.H. Chromosomal mapping of tandem repeats revealed massive chromosomal rearrangements and insights into Senna tora dysploidy. Front. Plant Sci. 2021, 12, 154. [Google Scholar] [CrossRef]
  61. Vershinin, A.V.; Evtushenko, E.V. What is the Specificity of Plant Subtelomeres? In Subtelomeres; Louis, E.J., Becker, M.M., Eds.; Springer: Berlin/Heidelberg, Germany, 2014; pp. 195–209. [Google Scholar]
  62. Schelfhout, C.; Snowdon, R.; Cowling, W.; Wroth, J.M.; Genetics, A. A PCR based B-genome-specific marker in Brassica species. Theor. Appl. Genet. 2004, 109, 917–921. [Google Scholar] [CrossRef] [PubMed]
  63. Schelfhout, C.; Snowdon, R.; Cowling, W.; Wroth, J.M. Tracing B-genome chromatin in Brassica napus × B. juncea interspecific progeny. Genome 2006, 49, 1490–1497. [Google Scholar] [CrossRef]
  64. He, Q.; Cai, Z.; Hu, T.; Liu, H.; Bao, C.; Mao, W.; Jin, W. Repetitive sequence analysis and karyotyping reveals centromere-associated DNA sequences in radish (Raphanus sativus L.). BMC Plant Biol. 2015, 15, 105. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  65. Flynn, J.M.; Long, M.; Wing, R.A.; Clark, A.G. Evolutionary dynamics of abundant 7-bp satellites in the genome of Drosophila virilis. Mol. Biol. 2020, 37, 1362–1375. [Google Scholar] [CrossRef]
  66. Havlová, K.; Dvořáčková, M.; Peiro, R.; Abia, D.; Mozgová, I.; Vansáčová, L.; Gutierrez, C.; Fajkus, J. Variation of 45S rDNA intergenic spacers in Arabidopsis thaliana. Plant Mol. Biol. 2016, 92, 457–471. [Google Scholar] [CrossRef]
  67. Garcia, S.; Kovařík, A.J.H. Dancing together and separate again: Gymnosperms exhibit frequent changes of fundamental 5S and 35S rRNA gene (rDNA) organisation. Heredity 2013, 111, 23–33. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  68. Ta, T.D.; Waminal, N.E.; Nguyen, T.H.; Pellerin, R.J.; Kim, H.H. Comparative FISH analysis of Senna tora tandem repeats revealed insights into the chromosome dynamics in Senna. Genes Genom. 2021, 43, 237–249. [Google Scholar] [CrossRef] [PubMed]
  69. Almeida, C.; Fonsêca, A.; dos Santos, K.G.B.; Mosiolek, M.; Pedrosa-Harand, A.J.G. Contrasting evolution of a satellite DNA and its ancestral IGS rDNA in Phaseolus (Fabaceae). Genome 2012, 55, 683–689. [Google Scholar] [CrossRef] [PubMed]
  70. Falquet, J.; Creusot, F.; Dron, M. Molecular analysis of Phaseolus vulgaris rDNA unit and characterization of a satellite DNA homologous to IGS subrepeats. Plant Physiol. 1997, 35, 611–622. [Google Scholar]
  71. Dos Santos, J.M.; Diniz, D.; Rodrigues, T.A.S.; Cioffi, M.D.B.; Waldschmidt, A.M. Heterochromatin distribution and chromosomal mapping of microsatellite repeats in the genome of Frieseomelitta stingless bees (Hymenoptera: Apidae: Meliponini). Fla. Entomol. 2018, 101, 33–39. [Google Scholar] [CrossRef] [Green Version]
Cells 10 02358 g001 550
Figure 1. Quantification of microsatellites in the A, C, B, and R genomes. (A) Clustering of 467 microsatellites that were ≤ 20 bp in the A, B, C, and R genomes. The three microsatellites that showed genome-specific distribution are shown in circles. (B) Distribution of the 467 microsatellites by genome in terms of total counts and genome proportions (GP). (C) Abundance of microsatellites according to monomer length. (D) Cumulative base count of the 22 microsatellites that had a cumulative base count of ≥ 6 kb. The numbers in the x-axis correspond to the microsatellite names listed in Table 3. (E) Cumulative proportion of each microsatellite in the A, C, B, and R genomes. Note the predominant abundance of ACBR_msat14 and ACBR_msat20 in the A and C genomes, respectively. ACBR_msat01 showed relative equal abundance in the four genomes. Bar graph colors in C–E are same as those in B.
Figure 1. Quantification of microsatellites in the A, C, B, and R genomes. (A) Clustering of 467 microsatellites that were ≤ 20 bp in the A, B, C, and R genomes. The three microsatellites that showed genome-specific distribution are shown in circles. (B) Distribution of the 467 microsatellites by genome in terms of total counts and genome proportions (GP). (C) Abundance of microsatellites according to monomer length. (D) Cumulative base count of the 22 microsatellites that had a cumulative base count of ≥ 6 kb. The numbers in the x-axis correspond to the microsatellite names listed in Table 3. (E) Cumulative proportion of each microsatellite in the A, C, B, and R genomes. Note the predominant abundance of ACBR_msat14 and ACBR_msat20 in the A and C genomes, respectively. ACBR_msat01 showed relative equal abundance in the four genomes. Bar graph colors in C–E are same as those in B.
Cells 10 02358 g001
Cells 10 02358 g002 550
Figure 2. FISH analysis of ACBR_msat01, ACBR_msat14, and ACBR_msat20 in the four diploid genomes. ACBR_msat14, and ACBR_msat20 were exclusively detected in B. rapa and B. oleracea genomes, respectively. ACBR_msat01 was detected in both B. nigra and R. sativus genomes including one pair of B. rapa chromosomes. Scale bar = 10 µm.
Figure 2. FISH analysis of ACBR_msat01, ACBR_msat14, and ACBR_msat20 in the four diploid genomes. ACBR_msat14, and ACBR_msat20 were exclusively detected in B. rapa and B. oleracea genomes, respectively. ACBR_msat01 was detected in both B. nigra and R. sativus genomes including one pair of B. rapa chromosomes. Scale bar = 10 µm.
Cells 10 02358 g002
Cells 10 02358 g003 550
Figure 3. Karyogram of B. rapa, B. oleracea, B. nigra, and R. sativus chromosomes from Figure 2. The chromosomal distribution of ACBR_msat01, ACBR_msat14, ACBR_msat20, 5S rDNA, 45S rDNA and Arabidopsis-type telomere repeats as well as the merged images of the six probes are shown. Note the hybridization of ACBR_msat01 in chromosome A8. Also note the disperse distribution of ACBR_msat20 signal in the C genome. Scale bar = 10 µm.
Figure 3. Karyogram of B. rapa, B. oleracea, B. nigra, and R. sativus chromosomes from Figure 2. The chromosomal distribution of ACBR_msat01, ACBR_msat14, ACBR_msat20, 5S rDNA, 45S rDNA and Arabidopsis-type telomere repeats as well as the merged images of the six probes are shown. Note the hybridization of ACBR_msat01 in chromosome A8. Also note the disperse distribution of ACBR_msat20 signal in the C genome. Scale bar = 10 µm.
Cells 10 02358 g003
Cells 10 02358 g004 550
Figure 4. FISH analysis of ACBR_msat01, ACBR_msat14, and ACBR_msat20 in B. napus, B. juncea, B. carinata, ×Brassicoraphanus ‘BB#1′, and ×Brassicoraphanus 977. ACBR_msat14, and ACBR_msat20 clearly discriminated the A and C genomes in the allopolyploids, respectively. ACBR_msat01 was useful in discriminating the B-genome in the interspecific AABB and BBCC allopolyploids, the R genome in the intergeneric AARR genome, and chromosome A8 (yellow arrowheads). Note the six chromosomes with ACBR_msat20 in ×Brassicoraphanus 977 indicating C-genome chromosome blocks (white arrowheads). Scale bar = 10 µm.
Figure 4. FISH analysis of ACBR_msat01, ACBR_msat14, and ACBR_msat20 in B. napus, B. juncea, B. carinata, ×Brassicoraphanus ‘BB#1′, and ×Brassicoraphanus 977. ACBR_msat14, and ACBR_msat20 clearly discriminated the A and C genomes in the allopolyploids, respectively. ACBR_msat01 was useful in discriminating the B-genome in the interspecific AABB and BBCC allopolyploids, the R genome in the intergeneric AARR genome, and chromosome A8 (yellow arrowheads). Note the six chromosomes with ACBR_msat20 in ×Brassicoraphanus 977 indicating C-genome chromosome blocks (white arrowheads). Scale bar = 10 µm.
Cells 10 02358 g004
Cells 10 02358 g005 550
Figure 5. Karyogram of natural and synthetic Brassica and Raphanus allotetraploids. The chromosomal distribution of ACBR_msat01, ACBR_msat14, ACBR_msat20, 5S rDNA, 45S rDNA and Arabidopsis-type telomere repeats are shown. ACBR_msat14 localized at the subterminal with some in the interstitial regions in the A-genome chromosomes (A1–A10). ACBR_msat20 localized at the interstitial regions of C-genome chromosomes (C1–C9). ACBR_msat01 localized at the interstitial regions of B-genome chromosomes (B1–B8) and extra signals at the paracentric region of chromosome A8 (yellow arrowheads). A-genome chromosomes with ACBR_msat20 signals are shown in white arrowheads. Scale bar = 10 μm.
Figure 5. Karyogram of natural and synthetic Brassica and Raphanus allotetraploids. The chromosomal distribution of ACBR_msat01, ACBR_msat14, ACBR_msat20, 5S rDNA, 45S rDNA and Arabidopsis-type telomere repeats are shown. ACBR_msat14 localized at the subterminal with some in the interstitial regions in the A-genome chromosomes (A1–A10). ACBR_msat20 localized at the interstitial regions of C-genome chromosomes (C1–C9). ACBR_msat01 localized at the interstitial regions of B-genome chromosomes (B1–B8) and extra signals at the paracentric region of chromosome A8 (yellow arrowheads). A-genome chromosomes with ACBR_msat20 signals are shown in white arrowheads. Scale bar = 10 μm.
Cells 10 02358 g005
Cells 10 02358 g006 550
Figure 6. Karyotype ideograms and genome relationships between the four diploids and their allotetraploid hybrids. Green arrows show the genome relationships among species within the U’s Triangle, and blue arrows for those involving the R genome. The A, C, B, and R genomes are distinguished by chromosome color. The chromosome numbers of ×Brassicoraphanus 977 with ACBR_msat20 signals are highlighted red.
Figure 6. Karyotype ideograms and genome relationships between the four diploids and their allotetraploid hybrids. Green arrows show the genome relationships among species within the U’s Triangle, and blue arrows for those involving the R genome. The A, C, B, and R genomes are distinguished by chromosome color. The chromosome numbers of ×Brassicoraphanus 977 with ACBR_msat20 signals are highlighted red.
Cells 10 02358 g006
Table
Table 1. Description of Brassica and Raphanus next-generation sequencing reads used to generate the microsatellites and summary of the coverage.
Table 1. Description of Brassica and Raphanus next-generation sequencing reads used to generate the microsatellites and summary of the coverage.
SpeciesCultivarGenomeGenome Size (Mbp)Accession NumberReadsCoverage (×)
B. rapaChiifu-401-42AA353GCA_000309985.3199,6620.06
B. oleraceaTO1000CC522GCA_000695525.1200,8800.04
B. nigrainbred line YZ12151BB468GCA_001682895.1201,7980.04
R. sativusXYB36-2RR414GCA_002197605.1201,2100.05
Table
Table 2. List of plant materials with their corresponding haploid chromosome number, genome type, seed source, and accession number.
Table 2. List of plant materials with their corresponding haploid chromosome number, genome type, seed source, and accession number.
Species2nGenome TypeSourceAccession Number
B. rapa20AANAASaIT 032730
B. oleracea18CCNPGSbPI 24501510GI
B. nigra16BBNPGSPI 649154
R. sativus18RRdanong.co.kr (accessed on 31 April 2019)N/A
B. napus38AACCNPGSTI 031006
B. juncea36AABBNPGSPI 633077
B. carinata34BBCCNPGSAmes 2779
×Brassicoraphanus ‘BB#1′38AARRBioBreeding Institute (Ansung, Korea)PRJNA353741
×Brassicoraphanus 97738ARRCBioBreeding InstituteN/A
a National Academy of Agricultural Sciences, (Jeollabuk-do, Korea); b National Plant Germplasm System, (US Department of Agriculture, USA).
Table
Table 3. Summary of top 22 microsatellites identified in the A, C, B, and R genomes.
Table 3. Summary of top 22 microsatellites identified in the A, C, B, and R genomes.
NameSequenceLengthTotal Bases
ACBRGrand Total
ACBR_msat01AG280,00465,64587,52395,545328,717
ACBR_msat02TA217,429180,55018,85271,783288,614
ACBR_msat03AAG362,10150,47463,61161,099237,285
ACBR_msat04TGA331,24323,21023,24628,005105,704
ACBR_msat05TAA3854151,85111,96828,179100,539
ACBR_msat06TTTAGGG714,62964,754443810,47194,292
ACBR_msat07TTG333,03214,32315,29222,71385,360
ACBR_msat08TG229,81515,74521,19816,03382,791
ACBR_msat09GAG318,862888012,87014,82355,435
ACBR_msat10AAAT4372027,736439010,22846,074
ACBR_msat11ACC3849440847375625426,207
ACBR_msat12CTTT4536151636096713723,757
ACBR_msat13TGC3696727104611572620,014
ACBR_msat14TTTAGGGTTAGGTAGGG1718,9032970019,200
ACBR_msat15AAAC4393344034519481317,668
ACBR_msat16TTCGG5350570881709184314,145
ACBR_msat17ACT3348440862441355213,563
ACBR_msat18ATAG485027017258168412,493
ACBR_msat19ATTTT55094749866419110,315
ACBR_msat20TTTCGGG7088458352709950
ACBR_msat21AATT4722492579216618100
ACBR_msat22TGAACAGTGTTTCGA150097351266099
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Campomayor, N.B.; Waminal, N.E.; Kang, B.Y.; Nguyen, T.H.; Lee, S.-S.; Huh, J.H.; Kim, H.H. Subgenome Discrimination in Brassica and Raphanus Allopolyploids Using Microsatellites. Cells 2021, 10, 2358. https://doi.org/10.3390/cells10092358

AMA Style

Campomayor NB, Waminal NE, Kang BY, Nguyen TH, Lee S-S, Huh JH, Kim HH. Subgenome Discrimination in Brassica and Raphanus Allopolyploids Using Microsatellites. Cells. 2021; 10(9):2358. https://doi.org/10.3390/cells10092358

Chicago/Turabian Style

Campomayor, Nicole Bon, Nomar Espinosa Waminal, Byung Yong Kang, Thi Hong Nguyen, Soo-Seong Lee, Jin Hoe Huh, and Hyun Hee Kim. 2021. "Subgenome Discrimination in Brassica and Raphanus Allopolyploids Using Microsatellites" Cells 10, no. 9: 2358. https://doi.org/10.3390/cells10092358

APA Style

Campomayor, N. B., Waminal, N. E., Kang, B. Y., Nguyen, T. H., Lee, S. -S., Huh, J. H., & Kim, H. H. (2021). Subgenome Discrimination in Brassica and Raphanus Allopolyploids Using Microsatellites. Cells, 10(9), 2358. https://doi.org/10.3390/cells10092358

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop