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Comparative Molecular Cytogenetic Analysis of Ribosomal DNAs Distribution in Brassica Species
배추에서 Ribosomal DNAs 분포의 비교분자세포유전학적 분석
Korean J. Breed. Sci. 2021;53(3):206-216
Published online September 1, 2021
© 2021 Korean Society of Breeding Science.

Franklin H. Mancia1, Jung Sun Kim2, and Yoon-Jung Hwang3*
Franklin H. Mancia1⋅김정선2⋅황윤정3*

1Department of Environmental Horticulture, Sahmyook University, Seoul 01795, Republic of Korea
2Department of Agricultural Biotechnology, National Institute of Agricultural Science, Rural Development Administration, Jeonju 54874, Republic of Korea
3Department of Chemistry Life Science, Sahmyook University, Seoul 01795, Republic of Korea
1삼육대학교 대학원 환경원예학과, 2농촌진흥청 국립농업과학원, 3삼육대학교 화학생명과학과
Correspondence to: (E-mail: hyj@syu.ac.kr, Tel: +82-3399-1718)
Received April 28, 2021; Revised May 5, 2021; Accepted June 24, 2021.
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Three Brassica species, namely, Brassica rapa, B. nigra, and B. oleracea are considered economically important as they are grown for human consumption and biogas production. Like other crops facing agricultural constraints, selective crossing or hybridization in cruciferous vegetables has helped farmers to improve them. This study conducted a comparative evaluation across and within the species through cytogenetic analysis to provide fresh insights into their chromosome structures and evolutionary relationships. A new karyomorphological parameter confirmed symmetric karyotypes in all the accessions, thereby allowing for the depiction of the ancestral chromosome karyotype. Several lines of B. rapa, B. nigra, and B. oleracea were subjected to physical mapping using a fluorescence in situ hybridization technique to elucidate the chromosomal distribution of the two types of rDNAs. The signal number and distribution of 18S rDNA across the metaphase chromosomes of B. rapa accessions did not vary as compared to 5S rDNA, which was also observed in several lines of B. nigra. In contrast, the number and distribution of 5S rDNA loci across the chromosomes in several lines of B. oleracea were found to be more conserved than those corresponding to the 18S rDNA. Overall, this study revealed the evolutionary dynamics of rDNA, which may play an important role in shaping the chromosome karyotypes of Brassica species.
Keywords : FISH, interspecies, karyotype evolution, polymorphism
Introduction

The genus Brassica in the mustard family comprises 38 species, which include economically important crops (Kim et al. 2014). Of these crops, many are valuable sources of vegetables (Masarirambi et al. 2020), oils (Sun 2017), feed and forage (Westwood and Mulcock 2012), and medicines (Saeidnia and Gohari 2012). Owing to their high market demand, several genera of Brassicaceae have been subjected to plant breeding, and considerable research, including the identification of agronomic traits, diversity analyses, and genetic mapping (Nishio 2014, Sharma et al. 2014, Velasco et al. 2017). Moreover, three diploid species, that is, Brassica rapa (2n = 2× = 20), Brassica nigra (2n = 2× = 16), and Brassica oleracea (2n = 2× = 18) are denoted as A, B and C genomes, respectively, in the triangle of U (U 1935). Based on their close relationships, interspecific hybridization has been effectively conducted among the Brassica diploids for the production of new species and the introgression of useful adaptive traits for targeted crop improvement (Dhaliwal et al. 2017, Elvis et al. 2020, Navabi et al. 2010). Furthermore, the progress in understanding the genome structures of Brassica species has been greatly underpinned by molecular cytogenetic studies (Chèvre et al. 2018, Hong et al. 2008).

Fluorescence in situ hybridization (FISH) is a widely used technique in plant molecular cytogenetic research that has been proved promising in relating DNA sequence information to chromosome biology, thereby allowing a comprehensive view of the evolution of plant species (Bačovský et al. 2018, Jiang & Gill 2006, Zheng et al. 2016). A unique FISH pattern of repetitive DNA sequences on individual chromosomes can help discriminate plant genomes and analyze the somatic pairing of homologous chromosomes (Hasterok et al. 2005b, Hwang et al. 2009, Iourov et al. 2005). Moreover, this technique aids in the identification of species using taxonomic and phylogenetic studies, validation of karyotypic and chromosomal evolution, analysis of chromatin features associated with gene expression, and provides a valuable complementary approach to genome sequencing and map-based cloning research strategies (Braz et al. 2018, Deng et al. 2019, Jiang & Gill 2006).

Important applications of molecular cytogenetic analysis have been reported for Brassica crops, which include the following: information about genome organization in diploid and polyploid species leading to the establishment of a common ancestral genome and description of the three differentially fractionated Brassica ancestral sub-genomes, and an enhanced understanding of intergenic relationships and structural rearrangements in interspecific Brassica hybrids (Chèvre et al. 2018). Previously, FISH-mapping has been conducted in several Brassica crops to evaluate their karyotypic structures, genome compositions, and chromosomal distribution of molecular cytogenetic markers such as ribosomal DNAs (Kamiński et al. 2020, Koo et al. 2004, Hwang et al. 2009, Perumal et al. 2017, Waminal et al. 2016, Wang et al. 2007, Xiong & Pires 2011, Zheng et al. 2014). Likewise, the distribution and number of ribosomal DNA (rDNA) sites have been evaluated in several genotypes of B. rapa, B. nigra, and B. oleracea, which have revealed polymorphism in 5S rDNA and 35S rDNA (Hasterok et al. 2006, Zheng et al. 2015).

Despite the abundance of molecular cytogenetic maps generated for rDNA in the three Brassica diploids, FISH-based mitotic karyotypes need to be studied for these genotypes. Such analyses are important to make the genus Brassica a model for conducting extensive studies on complex chromosomal evolution in plants. Therefore, we conducted the present study to evaluate the cytogenetic diversity of Brassica genotypes that might prompt biologists to delve comprehensively into research regarding species diversification. The data from this study would enhance our current understanding of the genetic diversity that could facilitate breeding in Brassica species.

Materials and Methods

Chromosome slide preparation

Seeds used in this research were provided by National Agrobiodiversity Center (http://genebank.rda.go.kr), Korea. The seeds were stored inside a properly sealed container and placed at 4℃. A petri dish with moist filter paper was used to germinate the seeds at 24ºC in dark conditions. The root tips were harvested after they attained a length of 1-2 cm. Then, these were soaked in 8-hydroxyquinoline at 18ºC for 5 hrs. A mixture of ethanol and acetic acid in a ratio of 3:1 (v/v) was used to fix the pre-treated roots overnight at room temperature. The fixative substance was decanted and replaced by 70% ethanol and stored at 4ºC. The pre-treated young roots were enzymatically digested to prepare a chromosome slide. It was carried out by following the protocol of Zheng et al. (2014) with some minor modifications. Root tips were treated with 2% cellulase R-10 (Sigma, USA), cytohelicase (Sigma, USA), and pectolyase γ-23 (Duchefa, Germany) at 37ºC for 80 min to digest the cellular components excluding DNA materials. Carefully, the enzyme solution was discarded and replaced with distilled water. Next, water was eliminated without disturbing the delicate roots and homogenized with Carnoy’s solution. The steam-drop method by Kirov et al. (2014) was followed to prepare well-distributed and morphologically intact chromosomes on a slide.

Polymerase chain reaction and DNA probes

The total genomic DNA from B. rapa L. (50 ng⋅µl-1) was extracted using the CTAB method of Devi et al. (2013). A pair of primers (forward, 5'-GGA TGG GTG ACC TCC CGG GAA GTCC-3'; reverse, 5'-CGC TTA ACT GCG GAG TTC TGA TGGG-3') were used to amplify the 5S rDNA (Yang et al. 1998). PCR conditions involved an initial denaturation at 94℃ for 3 min, 35 cycles of denaturation at 94ºC for 1 min, annealing at 55℃ for 45 sec, extension at 72℃ for 1 min, and a final extension at 72℃ for 3 min. The primers used to amplify the 18S rDNA are as follows: forward, 5′-AAC CTG GTT GAT CCT GCC AGT-3′; reverse, 5′-TGA TCC TTC TGC AGG TTC ACC TAC-3′ (Matsuda et al. 2011). The amplification was conducted with an initial denaturation at 95℃ for 5 min, 35 cycles of denaturation at 94℃ for 30 sec, annealing at 60℃ for 30 sec, extension at 72℃ for 1 min, and a final extension at 72℃ at 10 min. The amplified DNA products were labeled with dig 11-dUTP or biotin 16-dUTP through nick translation (Roche, Germany). The reaction mixture constituted 2% TaKaRa Ex Taq, 10% 10× exTaq buffer, 10% dNTP mix, 1% template DNA (< 500 ng), 20% primer mix (2 µm), and 54% DNase/RNase-Free Water (Takara Bio, Japan).

In situ hybridization and imaging

The FISH protocol by Lim et al. (2001) was performed with some modifications. The DNA probe (50-100 ng⋅µl-1) was added to the hybridization mixture (50% dextran sulfate, 10% SDS, 20× SSC, 100% formamide, 5 ng⋅µL-1 salmon sperm DNA). Next, the mixture was denatured at 90℃ for 10 min and transferred on ice for 5 min before adding onto the slide. The somatic metaphase chromosomes on a slide were denatured at 90℃ for 5 min. The slide was kept inside a moistened container at 37℃ for 16-18 hrs. Stringent washing was performed by immersing the slide in a copulin jar containing 20% deionized formamide in 0.1× SSC at 42℃ for 10 min (Hasterok et al. 2005a). The probes labeled with digoxigenin-11- dUTP and biotin 16-dUTP were detected with anti- digoxigenin- rhodamine and streptavidin-cy3 (Sigma-Aldrich, USA). Finally, the preparations were mounted in 100 ug⋅µl-1 DAPI (Roche, USA) in Vectashield (Vector Laboratories, USA). Visualization was conducted using a model BX63 microscope (Olympus, Japan) equipped with a CoolSNAP cf camera.

Chromosome evaluation

The metaphase chromosomes from several accessions of the three Brassica species were analyzed using the computer application developed by Altinordu et al. (2016). The karyotype formula, chromosome size variation, and total haploid chromo-some lengths were used to characterize the chromosome complements. Karyotype asymmetry was assessed using indices for interchromosomal level, which illustrate the heterogeneity of chromosome sizes within a complement. Likewise, indices were used for the intrachromosomal asymmetry, marking the predominance of certain chromosomes with terminal/subterminal or median centromeres. Interchromosomal indices include: 1) A2 (0 indicates ‘no variation’ and values > 0 indicate ‘increasing variation’), and 2) CVCL (0 indicates ‘no variation’ and 100 indicates ‘very heterogenous’); intrachromosomal indices include: 1) S/AI (1 indicates ‘full symmetric’ and 4 indicates ‘full asymmetric’), 2) TF% (increasing values indicate ‘symmetric’ and decreasing values indicate ‘asymmetric’), 3) A (0 indicates ‘symmetric’ and values ≥ 1 indicate ‘asymmetric’), and 4) MCA (0 indicates ‘symmetric’ and 100 indicates ‘asymmetric’).

Results

FISH-based karyotype analysis

Conventional chromosome analysis coupled with FISH mapping using two types of rDNAs provided information on diverse cytological characters such as chromosome number, ploidy level, chromosome size variation, karyotype formula, and haploid chromosome length (Table 1). Further, insights into chromosomal and genome evolution were provided based on the generated cytogenetic maps in several Brassica genotypes (Figs. 1-3). Moreover, karyotype asymmetry was quantified in the different accessions of Brassica diploids with intrachromosomal and interchromosomal indices.

Table 1

Karyotypic characteristics and karyotype asymmetry assessment for different accessions of B. rapa, B. nigra, and B. oleracea.

Taxa IT
No.z
Place of origin 2n KFy CSVx (µm) hclw (µm) CSv Inter- and Intrachromosomal indices
A2u CVCLt S/AIs TF%r Aq MCAp
B. rapa subsp. pekinensis 032747 Korea 20 12m+8sm+2st 5.4-11.2 81 2 0.24 23.6 1.5 36.9 0.28 28.5
B. campestris var. rapifera 108867 Korea 20 12m+6sm+2st 4.3-8.9 60.8 2 0.20 20.29 1.5 38.2 0.25 28.9
B. rapa var. perviridis 112272 Japan 20 12m+6sm+2st 4.4-9.4 65.7 3 0.16 16.23 1.5 35.8 0.29 29.24
B. rapa subsp. rapa 175952 Korea 20 12m+6sm+2st 4.7-10 70.2 2 0.15 14.7 1.5 36.7 0.28 27.7
B. rapa var. chinensis 185735 Korea 20 12m+6sm+2st 4.6-11.2 64.3 3 0.21 20.9 1.5 37.2 0.26 27.4
B. rapa subsp. narinosa 221794 Japan 20 12m+6sm+2st 5.3-11.1 64.9 4 0.18 17.9 1.5 37 0.27 27.2
B. rapa subsp. oleifera 223924 Italy 20 12m+6sm+2st 6-11.8 81.4 4 0.17 17.3 1.5 36 0.30 29.8
B. rapa var. narinosa 293391 China 20 12m+6sm+2st 5.1-11.8 78 3 0.21 21.1 1.5 35.6 0.31 31
Mean 0.19 19 1.5 36.7 0.28 28.7
B. nigra 305656 Germany 16 10m+6sm 3.7-5.8 36.5 0 0.08 7.88 1.4 37.6 0.23 22.8
B. nigra 305086 Germany 16 10m+6sm 3.3-4.7 32.2 2 0.10 10.14 1.4 39 0.22 22.4
B. nigra 279264 UK 16 10m+6sm 4.2-5.7 39 0 0.06 6.34 1.4 38.9 0.22 22.4
B. nigra 279261 Italy 16 10m+6sm 3-4.8 30 0 0.10 10.2 1.4 38.3 0.23 23.1
B. nigra 279259 Pakistan 16 10m+6sm 4.2-5.9 40.3 0 0.07 7.21 1.4 38.2 0.23 23.4
B. nigra 279258 India 16 10m+6sm 3.8-5.2 40 1 0.09 9.2 1.4 38.2 0.23 23.5
B. nigra 279253 Ethiopia 16 10m+6sm 2.8-4.5 28 2 0.11 10.5 1.4 39.4 0.21 21
B. nigra 279251 Pakistan 16 10m+6sm 2.8-4.1 26 2 0.09 8.51 1.4 38 0.24 24
Mean 0.09 8.75 1.4 38.5 0.22 22.8
B. oleracea var. capitata 136508 India 18 10m+8sm 4.2-7.8 48.3 0 0.11 10.9 1.4 40 0.21 21
B. oleracea var. capitata 180722 Egypt 18 10m+8sm 3-4 32 0 0.07 7.28 1.4 39 0.23 23
B. oleracea var. capitata 188172 Tanzania 18 10m+8sm 3-4.6 32 0 0.09 9.08 1.4 38.9 0.22 22
B. oleracea var. capitata 206707 Uzbekistan 18 10m+8sm 3.3-4.5 34 0 0.27 7.37 1.4 37 0.27 27
B. oleracea var. gemmifera 267606 UK 18 10m+8sm 4.2-6.6 46.1 0 0.09 8.70 1.4 37 0.26 26
B. oleracea var. gongylodes 257512 Japan 18 10m+8sm 3.6-5.3 39 1 0.09 8.8 1.4 37.5 0.25 25
B. oleracea var. alboglabra 210060 Thailand 18 10m+8sm 3-4.9 32 1 0.11 10.6 1.4 37.1 0.25 25
B. oleracea var. alboglabra 278477 Netherlands 18 10m+8sm 3-4.1 31 0 0.08 7.8 1.4 39 0.23 23
B. oleracea var. alboglabra 278479 Netherlands 18 10m+8sm 3-5.4 36.1 0 0.12 12.3 1.4 37 0.25 28
Mean 0.11 9.2 1.4 38 0.24 24.4

zThe seeds and IT no. used in this study were obtained from the National Agrobiodiversity Center, Korea., yKF: Karyotype Formula, xCSV: Chromosome Size Variation, whcl: total sum of haploid chromosome length, vCS: Colocalized rDNA Site pair, uA2 index (Zarco 1986), tCVCL (Lavania & Srivastava 1992, Paszko 2006, Watanabe et al. 1999), sS/A1 (Eroğlu 2015), rTF% (Huziwara 1962), qA (Watanabe et al. 1999), pMCA (Peruzzi & Eroğlu 2013).



Fig. 1. FISH-based mapping of 18S (red) and 5S rDNAs (green) in eight B. rapa accessions. A) Karyotypes of B. rapa subsp. pekinensis (a), B. campestris var. rapifera (b), B. rapa var. perviridis (c), B. rapa subsp. rapa (d), B. rapa var. chinensis (e), B. rapa subsp. narinosa (f), B. rapa subsp. oleifera (g), B. rapa var. narinosa (h). B) Haploid ideograms of 8 genotypes showing the conserved distribution and locus number of 18S rDNA. *polymorphic sites in chromosome-bearing 5S rDNA, +hemizygous sites in chromosome-bearing 5S rDNA (Scale bar = 10 µm).

Fig. 2. Chromosomal localization of 18S (red) and 5S rDNAs (green) in eight B. nigra accessions. A) Karyotypes of B. nigra (IT no. 305656) (a), B. nigra (IT no. 305086) (b), B. nigra (IT no. 279264) (c), B. nigra (IT no. 279261) (d), B. nigra (IT no. 279259) (e), B. nigra (IT no. 279258) (f), B. nigra (IT no. 279253) (g), B. nigra (IT no. 279251) (h). B.) Haploid ideograms of eight genotypes depicting the conserved distribution and locus number of 18S rDNA. *polymorphic sites in chromosome-bearing 5S rDNA (Scale bar = 10 µm).

Fig. 3. Detection of rDNAs in metaphase chromosomes of B. oleracea accessions. A) Karyotypes of B. oleracea var. capitata from India (a), B. oleracea var. capitata from Egypt (b), B. oleracea var. capitata from Tanzania (c), B. oleracea var. capitata from Uzbekistan (d), B. oleracea var. gemmifera from the UK (e), B. oleracea var. gongylodes from Japan (f), B. oleracea var. alboglabra from Thailand (g), B. oleracea var. alboglabra from the Netherlands (h), B. oleracea var. alboglabra from the Netherlands (i). B) Haploid ideograms of 10 genotypes showing the conserved distribution and locus number of 5S rDNA (green). +hemizygous sites in chromosome-bearing 18S rDNA (red) (Scale bar = 10 µm).

B. rapa chromosome evaluation

The number and distribution of 5S and 18S rDNA loci across the somatic metaphase chromosomes were determined, and the latter being highly conserved in all eight genotypes of B. rapa (Fig. 1). Six rDNA-bearing chromosomes were primarily painted with FISH signals on the pericentromeric regions, while the rest were on the terminal regions. Co-localization of 5S and 18S rDNA loci on two pairs of chromosomes (Chrs. #2 and #3) was observed in all the genotypes. Moreover, an extra locus and hemizygous site of 5S rDNA were observed in B. rapa var. perviridis (Figs. 1A-c and B-c, Chr. #1), B. rapa var. chinensis (Figs. 1A-e and B-e, Chr. #1), B. rapa subsp. narinosa (Figs. 1A-f and B-f, Chr. #1 and #5), B. rapa subsp. oleifera (Figs. 1A-g and B-g, Chrs. #1 and #5), and B. rapa var. narinosa (Figs. 1A-h and B-h, Chr. #5).

The average values for A2 and CVCL (Coefficient of Variation of Chromosome Length) indices across eight B. rapa genotypes are 0.19 and 19, respectively. Additionally, four indices were measured to determine the intrachromosomal asymmetry of the karyotypes. The average values corresponding to each index were observed as follows: S/AI (1.5), TF % (36.7), A (0.28), and MCA (Mean Centromeric Asymmetry) (28.7) (Table 1).

B. nigra chromosome evaluation

Two types of rDNAs were detected along the somatic metaphase chromosomes corresponding to the eight B. nigra genotypes (Fig. 2A). Three pairs of 18S rDNA were continuously observed across all genotypes (Chrs. #4, #6, and #7). Similar to our observations for the B. rapa genotypes, 5S rDNA exhibited three different loci patterns in B. nigra (Fig. 2B). In four genotypes of B. nigra (IT no. 305656, 279264, 279261, and 279259), only one major 5S rDNA was found in the short arm of Chr. #3 (Fig. 2B-a, c-e). In the other genotype (IT no. 279258), an extra locus of 5S rDNA was observed in the telomeric region of Chr. #4 (Fig. 2B-f). Interestingly, three other genotypes (IT no. 305086, 279253, and 279251) carried additional site of 5S rDNA in Chr. #6 (Fig. 2B-b, g, and h).

Interchromosomal asymmetry was measured for all eight genotypes (Table 1). The average values of A2 and CVCL indices were observed as 0.09 and 8.75, respectively. Similarly, intrachromosomal asymmetry was measured, and the average values of S/AI, TF %, A, and MCA were found as 1.4, 38.5, 0.22, and 22.8, respectively.

B. oleracea chromosome evaluation

In contrast to B. rapa and B. nigra, the number and distribution of 5S rDNA loci across nine B. oleracea genotypes were found to be conserved (Fig. 3A). Seven genotypes possessed two loci of 18S rDNA in Chr. #3 and #7 (Fig. 3B). However, two genotypes namely, B. oleracea var. gongylodes (Fig. 3B-g) and B. oleracea var. alboglabra (Fig. 3B-f), contained a hemizygous site of 18S rDNA juxtaposed to 5S rDNA in Chr. #2. The homogeneity of chromosome sizes and predominance of chromosomes with terminal/sub terminal centromeres were evaluated in each chromosome complements. The average values of A2 and CVCL indices were observed as 0.11 and 9.2, respectively (Table 1). Additionally, the average values for other indices such as S/AI, TF%, A, and MCA were 1.4, 38, 0.24, and 24.4, respectively.

Discussion

Homogeneous karyotypes were observed for the morphotypes of the three Brassica diploids (Table 1). Based on our results, the three Brassica diploids species exhibited high intrachromosomal symmetry with more chromosomes having centromeres positioned in the median and submedian regions. However, marginally higher interchromosomal asymmetry was observed across the accessions of B. rapa than those of B. nigra and B. oleracea (Table 1). Further, the results obtained upon assessment of karyotype asymmetry have suggested that the Brassica species (being examined in this study) did not undergo consequential and extreme genomic makeover resulting from genome evolution (i.e., genome triplication and diploidization events) (Tang and Lyons 2012). The genomes remain to an ancestral type with more metacentric and submetacentric chromosomes (Baltisberger and Hörandl 2016). However, Peruzzi and Eroğlu (2013) have highlighted that a symmetric karyotype does not necessarily imply ‘primitivity’. Overall the three species did not vary drastically in the karyotypic symmetry and chromosomal size homogeneity.

Interspecific variation of rDNA locus has been observed among several lines of B. rapa, B. nigra, and B. oleracea. Although, Hasterok et al. (2005a) have reported similar findings, the inclusion of new accessions for this specific analysis would ascertain the earlier outcome. Moreover, the interspecific differences of 5S rDNA chromosomal locations observed in several accessions of B. rapa and B. nigra (Figs. 1B and 2B), have also been observed in Arabidopsis thaliana and Cucumis spp. (Tutois et al. 2002, Zhang et al. 2016). Further, variation of rDNA loci has been documented to occur in a population of the same plant species (Breda et al. 2012). Cytogenetic changes and inferred dynamism have been reported to be triggered by genomic rearrangement, which results from contemporary hybridization and rDNA region instability due to deleterious recombination of repeats (Rosato et al. 2017, Kobayashi 2014). The interspecific variation of 5S rDNA locus has demonstrated how a specific gene locus can reorganize through interchromosomal translocation, which can further substantially impact genome function (Simon et al. 2018). However, the conjecture about the propensity of 5S rDNA gene to integrate into different genomic locations in Brassica is still unclear.

Nevertheless, the insertion of a transposable element in the 5S rDNA unit of Triticum aestivum showed differences in the distribution and intensity of signals on the chromosomes (Sergeeva et al. 2017), suggesting a similar phenomenon to occur in Brassica species. In addition, the RNA interference (RNAi) pathway has been determined to play a role in the dispersal of rDNA repeats from their wild-type loci to other chromosomal regions (Begnis et al. 2018). This circumstance might explain the unequal number of rDNA locus and its differential distribution within the species. The heterogeneity of 5S rDNA concerning to its copy number and sequence has been determined to exist across and within species (Cloix et al. 2002). Heterochromatic repeats in the centromere and rDNA regions are predisposed to rapid divergence leading to genetic variations (Koo et al. 2011, Mehrotra and Goyal 2014). Moreover, the expression of 5S rDNA is moderated by two silencing pathways, methylation-dependent (RNAi) and methylation-independent (MOM pathway), resulting in only a fraction of genes being expressed in A. thaliana (Douet and Tourmente 2007). These variations might indicate differential usage of 5S rDNA loci in distinct ecotypes (Simon et al. 2018). On the contrary, a more homogenous distribution of 5S rDNA among the accessions of B. oleracea was observed (Fig. 3B), consistent with the results reported by Hasterok et al. (2006).

Conserved locus number and chromosomal distribution of 18S rDNA can be observed among B. rapa and B. nigra accessions (Figs. 1B and 2B). Similarly, a conserved number of rDNA sites were reported in varieties and subspecies of Cucumis spp. (Zhang et al. 2016). Ribosomal DNA homogeneity has been observed across the major clades of early land plants, which suggests an evolutionary rDNA stasis during land colonization and diversification (Rosato et al. 2016). On the contrary, B. oleracea accessions exhibited polymorphism in 18S rDNA for the site number and location (Fig. 3B). One accession of B oleracea was reported to contain three loci of 45S rDNA (Armstrong et al. 1998), as opposed to two loci found in 80% of the genotypes studied in this paper. Several genotypes of Lolium multiflorum Lam, Brachypodium pinnatum, and Brachypodium sylvaticum have also shown variability in 45S rDNA loci that might be associated with their fragile sites (Bustamante et al. 2014, Breda et al. 2012). Differential characteristics such as the variability of rDNA site distribution or location and signal intensities would indicate structural changes in the nuclear genome at the chromosome level involved in speciation and diversification (Bustamante et al. 2014, Storme and Mason 2014).

The polymorphisms in site number and distribution of 5S and 18S rDNA observed within each species strongly suggest an intraspecific genome size variation. A recent study has shown that more rDNA and Gypsy elements are present in B. rapa ‘Z1’ compared to B. rapa ‘Chiifu’, which greatly impacted genome size intraspecific variability (Boutte et al. 2020). The three diploid Brassica species have multiple 5S and 18S rDNA repeat clusters across all accessions, but B. oleracea has only one locus of 5S rDNA. In addition, the studied species manifest evident interspecific genome size variation. Recent reports indicate the smallest genome size was found in B. rapa with 442.9 Mb, followed by the other two diploids (B. nigra and B. oleracea) with 570-608 Mb and 630 Mb, respectively (Zhang et al. 2018, Perumal et al. 2020, Liu et al. 2014). A positive relationship between rDNA copy number and genome size for 162 species of plants and animals was reported (Prokopowich et al. 2003). This relationship is unquestionably important in facilitating evolutionary processes and maintaining genome stability (Kobayashi 2011). Genome sizes of two subspecies of Japonica were observed to vary due to differences in the copy number of the repetitive sequences (Haiyan et al. 2012, Rubio-Piña et al. 2016). Nuclear DNA content variation among plant genomes, mediated by the loss and gain of repeated DNA sequences, is connected to their adaptation to different environments (Cavallini & Natali 1991).

Overall, the obtained molecular cytogenetic data from mostly unreported Brassica genotypes have exhibited complex evolutionary dynamics of rDNA loci within and across the Brassica species, which have mounted evidence about the generative roles of repetitive sequences in shaping the chromosome karyotypes in plants (Li et al. 2017, Volkov et al. 2017).

Acknowledgements

This work was carried out with the support of the Cooperative Research Program for National Agricultural Genome Program (Project No. PJ01338202), Rural Development Administration, Republic of Korea.

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