Plant Breed. Biotech. 2019 (June) 7(2):95~105 Online ISSN: 2287-9366 https://doi.org/10.9787/PBB.2019.7.2.95 Print ISSN: 2287-9358 RESEARCH ARTICLE

Cytogenetic Analyses Revealed Different Genome Rearrangement Footprints in Four ×Brassicoraphanus Lines with Different Fertility Rates

Hadassah Roa Belandres1, Hui Chao Zhou1, Nomar Espinosa Waminal1, Soo-Seong Lee2, Jin Hoe Huh3, Hyun Hee Kim1* 1Chromosome Research Institute, Department of Life Science, Sahmyook University, Seoul 01795, Korea 2BioBreeding Institute, Ansung 17544, Korea 3Department of Science, Plant Genomics and Breeding Institute and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea

ABSTRACT ×Brassicoraphanus (AARR, 2n = 38) is a synthetic intergeneric allopolyploid between rapa L. ssp. pekinensis (AA, 2n = 20) and sativus L. var. rafiphera (RR, 2n = 18). Abnormalities in meiosis are main causes for infertility, especially in recent intergeneric allopolyploids. Several ×Brassicoraphanus lines showing varied fertility rates were produced previously, but no cytogenetic data specifying the reasons for infertility have been reported. In this study, we performed cytogenetic analyses in BB4, BB6, BB12, and BB50 lines to evaluate their chromosomal composition and behavior during meiosis. The four lines had relatively small , ranging in length from 1.82 to 5.72 μm. BB6, BB12, and BB50 have euploid number of 2n = 38, whereas BB4 is an aneuploid with 2n ‒ 1 = 37. Fluorescent in situ hybridization karyotype analysis by using 5S/45S rDNA revealed 5/7, 6/7, 5/5 and 5/5 pairs in BB4, BB6, BB12 and BB50, respectively. Genomic in situ hybridization analysis on cells in prophase I revealed varying frequencies of tetravalent pairing and sticky, ring, rod, and laggard chromosomes across the lines, which were more abundant in BB4 and BB6. Unlike BB4 and BB6, both BB12 and BB50 are known to have relatively higher seed fertility and uniform plant morphology. The varied degrees of chromosomal pairing stability during meiosis could explain the different fertility rates among the four ×Brassicoraphanus lines in this study. These data might facilitate breeding programs of ×Brassicoraphanus and further cyto- genomic analyses. Keywords Intergeneric , ×Brassicoraphanus, Cytogenetic study

INTRODUCTION foods like kimchi (Kim et al. 2000). While either shoot or root are utilized in either B. rapa and R. sativus, breeding In recent years, breeding of Brassica and Raphanus for a with combined shoot and root features of both species has received considerable attention (Namai et al. species would optimize utility of plant parts. Thus, previ- 1980; Prakash et al. 2009) because of their agricultural ous breeding efforts have initiated this program (Lee et al. importance worldwide as major crops as fodders, edible 2002). vegetables, and functional compounds sources (Lee et al. Morphological and cytological methods have been used 2017). Brassica rapa (Chinese cabbage, 2n = 20) and Raphanus to select synthetic hybrid species between the two genomes sativus (big root radish, 2n = 18) produce economically of Brassica and Raphanus (Dolstra 1982; Kato and Tokumasu important shoot and root parts, respectively, especially in 1983). A newly synthesized intergeneric allotetraploid, eastern Asia, where they are the main ingredients of staple ×Brassicoraphanus, was developed by crossing B. rapa L.

Received March 14, 2019; Revised May 7, 2019; Accepted May 8, 2019; Published June 1, 2019 *Corresponding author Hyun Hee Kim, [email protected], Tel: +82-2-3399-1715, Fax: +82-2-3399-1729

Copyright ⓒ 2019 by the Korean Society of Breeding Science This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 96 ∙ Plant Breed. Biotech. 2019 (June) 7(2):95~105

ssp. pekinensis and R. sativus (Lee et al. 1999; Lee et al. constructed a dual-color FISH karyotype with both mar- 2002; Lee et al. 2017); however it was found to be unstable kers to identify the chromosome complement of each in- with low seed fertility and uniformity. The progenies from bred line. Moreover, meiotic chromosome pairing patterns these unstable materials were stabilized using microspore were analyzed to discriminate both parental genomes, mutagenesis by using N-nitroso-N-methyl urethane (NMU) Brassica and Raphanus, by using GISH method. (Lee et al. 2011; Lee et al. 2017). The same procedure involving the use of NMU was applied to develop another allotetraploid hybrid between B. rapa L. ssp. pekinensis MATERIALS AND METHODS and R. sativus L. var. rafiphera, and the F1 hybrid was crossed with a mutagen-induced stabilized line to produce Plant materials different lines of ×Brassicoraphanus. The floral buds and seeds of the ×Brassicoraphanus Before the development of fluorescence in situ hybridi- lines BB4, BB6, BB12, and BB50 were kindly provided by zation (FISH), chromosome-based cytogenetic studies per- Soo-Seong Lee who developed the intergeneric hybrids in formed using conventional staining method were applied to the Bio-Breeding Institute, Ansung, Korea. The floral buds Brassica species; however, the technique limited the eval- were collected from the adult of each line and the uation among the homologous chromosomes with similar seeds were sent for this experiment after harvesting from size (Olin-Fatih and Heneen 1992; Olin-Fatih 1994; Cheng each line. The seeds were germinated in Petri dishes with et al. 1995; Hwang et al. 2009). Chromosome characteri- moist filter paper at 25℃ for 48 hours. Root tips (length, 2 zation and genome identification have been advanced sig- cm) were harvested and pretreated with 2 mM 8-hydroxy- nificantly with the development of molecular cytogenetics quinoline at 18℃ for 5 hours, fixed in aceto-ethanol (1:3 involving the use of FISH and genomic in situ hybridi- v/v) solution for 2 to 24 hours, and then stored in 70% zation (GISH) techniques (Fukui 2005; Capdeville et al. ethanol until use. Floral buds were fixed in the same 2008; Park et al. 2010; Hwang et al. 2012; Pendinen et al. solution for 24 hours and stored in 70% ethanol at ‒20℃. 2012). These methods have been used to investigate al- lopolyploid species (Yang et al. 1999; Cao 2003; Devi et Chromosome and probe preparation for FISH and GISH al. 2005), recombinant breeding lines (Lou et al. 2010; Mitotic and meiotic chromosomes, rDNA probes, FISH Vasconcelos et al. 2010; Hwang et al. 2012), and genome and GISH procedures were done following the same pro- structure and inter-genomic relationships of hybrid plants cedures of previous study (Belandres et al. 2015). (Kenton et al. 1993; Jellen et al. 1994). Homologous chromosomes were identified based on the Tandemly repeated short sequences or multigene fa- FISH karyotype of ×Brassicoraphanus line BB5 (Belandres milies, including 5S and 45S ribosomal DNAs (rDNAs), et al. 2015). Meiotic pairing configurations were analyzed have primarily been used as cytogenetic markers in FISH in late prophase I. The varying degree of meiotic abnor- (Kato et al. 2004; Lim et al. 2005; Lim et al. 2012). malities were examined in 500 pollen mother cells (PMCs). Maluszynska and Heslop-Harrison (1993) first reported the number of 45S rDNA loci in diploids and allotetraploids of Brassica. RESULTS Recently, a FISH karyotype analysis of ×Brassicoraphanus line BB#5 (Belandres et al. 2015) and a conventional rDNA repeats Giemsa-staining karyotype analysis of line BB4 were con- The fluorescence in situ hybridization (FISH) karyotype ducted (Lim et al. 2012), however no cytogenetic data on of ×Brassicoraphanus lines with 5S/45S rDNA revealed the developed hybrid lines is available. In this study, we 5/7, 6/7, 5/5, and 5/5 pairs in BB4, BB6, BB12, and BB50, analyzed the chromosomal distribution and localization of respectively (Figs. 1 and 2, Table 1). The 5S rDNA loci 5S and 45S rDNA on four synthetic hybrid lines and were observed in the para-centromeric region of the long Cytogenetic Analyses in ×Brassicoraphanus ∙ 97

Fig. 1. Dual color FISH metaphase of the four inbred lines of ×Brassicoraphanus. Three mitotic metaphase spreads of lines BB6 (B, F), BB12 (C, G), BB50 (D, H) showing chromosome number 2n = 38, and BB4 (A, E, arrow), 2n ‒ 1 = 37. The 5S and 45S rDNA loci are shown as green and red signals, respectively. FISH metaphase chromosomes were arranged in decreasing order according to the length and morphology of each chromosome (E-H). Line BB4 has an incomplete chromosome number indicated by an arrow (E). Scale bar, 5 μm.

Fig. 2. FISH karyotypic idiograms showing chromosome number, length, morphology, and 5s and 45s rDNAs signals in ×Brassicoraphanus inbred lines, BB4 (2n ‒ 1 = 37, A) with missing chromosomes indicated by an arrow, BB6 (2n = 38, B), BB12 (2n = 38, C), and BB50 (2n = 38, D). The 5S and 45S rDNA loci are shown as green and red signals, respectively. The chromosomes were arranged according to the length, morphology, and rDNAs distri- bution described by Belandres et al. (2015). arms of chromosomes 2 and 7, terminal part of the short arm of chromosome 16. However, in BB6, another pair of arms of chromosome 13 and 14 being juxtaposed to the 5S rDNA loci was observed in the para-centromeric region satellite chromosome, and on the terminal part of the short of the long arm of chromosome 9. The 45S rDNA loci 98 ∙ Plant Breed. Biotech. 2019 (June) 7(2):95~105

Table 1. Summary of FISH karyotype analyses of intergeneric hybrid ×Brassicoraphanus lines BB4, BB6, BB12, and BB50. rDNA Inbred Chr. no. Chr. length (μm) signals Karyotype formula (2n) lines (2n) Shortest Longest Total 5S 45S BB4 37 1.82 3.45 49.69 5 7 2m + 2m + 2sm + 2m + 2m + 2m + 2m + 2m + 2m + 2sm + 2m + 2m + 2smz) + 2mz) + 2m + 2sm + 2m + 2m + 2m BB6 38 2.31 4.43 61.39 6 7 2m + 2m + 2m + 2m + 2m + 2m + 2m + 2m + 2m + 2m + 2sm + 2sm + 2smz) + 2mz) + 2sm + 2m + 2m + 2m + 2m BB12 38 2.58 4.78 68.02 5 5 2m + 2m + 2m + 2m + 2m + 2m + 2m + 2m + 2m + 2m + 2m + 2m + 2smz) + 2mz) + 2m + 2m + 2m + 2m + 2m BB50 38 2.40 5.72 74.34 5 5 2m + 2m + 2m + 2m + 2sm + 2m + 2m + 2m + 2m + 2m + 2m + 2m + 2smz) + 2mz) + 2sm + 2sm + 2sm + 2sm + 2sm z)satellite chromosomes. m: metacentric, sm: submetacentric. pattern of BB12 and BB50 was identical, whereas two lengths ranging from 2.31 to 4.43 μm and a total length of more pairs were observed for BB4 and BB6 (Figs. 1 and 2, 61.39 μm (Table 1). The chromosome complement com- Table 1). The 45S rDNA loci were observed at the para- prised fifteen metacentric and four submetacentric chromo- centromeric region of the long arms of chromosomes 4, 6, somes. BB12 somatic chromosome number was 2n = 38, and 7 and on the nuclear organizer region (NOR) regions of (Fig. 1C and G), with chromosome lengths ranging from chromosome 13 and 14. However, two additional pairs of 2.58 to 4.78 μm and a total length of 68.02 μm (Table 1). 45S rDNA loci were observed in the para-centromeric The chromosome complement comprised eighteen meta- region of the long arms of chromosome 5 and 9 in BB6 and centric and one submetacentric chromosomes. BB50 chro- the para-centromeric region of the long arms of chromo- mosome number was 2n = 38 (Fig. 1D and H), with lengths some 5 and near the short arm of chromosome 11 in BB4. ranging from 2.40 to 5.72 μm and a total length of 74.34 μm When compared with the FISH karyotype of the stable (Table 1). The chromosome complement comprised twelve line BB5, some of the 45S rDNA loci were absent, parti- metacentric and seven submetacentric chromosomes (Fig. 2). cularly in chromosome 8 of BB4, chromosomes 8 and 11 of BB6, and chromosomes 5, 8, and 11 of BB12 and BB50. Meiotic chromosome behavior However, the five pairs of 5S rDNA loci were all present in GISH analysis was done to identify chromosomes from the four lines. In addition, a distinct pair of co-localized 5S each parent, B. rapa (Fig. 3B-K, raw) and R. sativus (Fig. and 45S rDNA loci was observed only in chromosome 9 of 3A-J, DAPI-blue) on the PMCs at prophase I of meiosis in BB6, which was even absent in BB5. the four lines (Fig. 3). In meiotic pairing, nineteen bivalent chromosome pairs were observed in BB50 (Fig. 3J-L), FISH karyotype consisting of ten and nine bivalent chromosome pairs of B. BB4 was aneuploidy with a somatic chromosome num- rapa (Fig. 3J, red arrowheads, K-raw) and R. sativus, res- ber of 2n ‒ 1 = 37 (Fig. 1A, E), with lengths ranging from pectively (Fig. 3J, white arrowheads), whereas the rest of 1.82 to 3.45 μm and a total length of 49.64 μm (Table 1). the lines BB4, BB6, and BB12 consisted of combined The chromosome complement comprised fifteen meta- bivalent and tetravalent chromosome pairs from both the centric and four submetacentric chromosomes. Chromo- genomes (Fig. 3A-I). For instance, BB4 has six bivalent some 12 of BB4 has a missing copy of its homolog, (Fig. 3A, red arrowheads) and two tetravalent chromosome resulting in a low total chromosome length compared to pairs of B. rapa (Fig. 3A, red asterisk, B-raw), and seven that for the other lines (Fig. 1E, white arrows). BB6 somatic bivalent (Fig. 3A, white arrowheads) and one tetravalent chromosome number was 2n = 38 (Fig. 1B and F), with chromosome pairs from R. sativus (Fig. 3A, white asterisk); Cytogenetic Analyses in ×Brassicoraphanus ∙ 99

Fig. 3. Meiotic chromosome pairing in prophase I of ×Brassicoraphanus inbred lines BB4 (A-C), BB6 (D-F), BB12 (G-I), and BB50 (J-L). Nineteen complete bivalents were observed in BB50, as shown in (J), 10 of which are of B. rapa (green fluorescence, K- raw) and nine are of R. sativus chromosomes (DAPI, blue), whereas the remaining lines show multivalent and univalent chromosome pairing. In the panels above, white arrowheads and white asterisks indicate R. sativus chromosomes; red arrowheads, red asterisks, and the raw images in the middle panels are of B. rapa; yellow arrowheads indicate univalent chromosomes; and asterisks (red and white) indicate tetravalent chromosomes. The bottom panels are raw DAPI images of the four lines. Scale bar, 5 μm.

BB6 has nine bivalent (Fig. 3D, red arrowheads) and two nuclei (Fig. 4H, red arrowheads) were identified in telo- univalent chromosome pairs of B. rapa (Fig. 3D, yellow phase II due to acentric chromosome fragments or lagging arrowheads, E-raw) and five bivalent (Fig. 3D, white chromosomes that were not incorporated into one of the arrowheads) and two tetravalent chromosome pairs from R. daughter nuclei during cell division. The unstable lines sativus (Fig. 3D, white asterisk); BB12 has eight bivalent BB4 and BB6 showed high frequencies of meiotic abnor- (Fig. 3G, red arrowheads) and one tetravalent chromosome malities, which accounted for 57% (287) and 34% (169), pairs from B. rapa (Fig. 3G, red asterisk, H-raw) and nine respectively, compared to 12% (60) and below in the stable bivalent chromosome pairs of R. sativus (Fig. 3G, white lines BB50 and BB12. High degree of laggard chromo- arrowheads). somes in metaphase I was observed, which accounted for Meiotic abnormalities were noted in all lines from 31% (154), followed by bridge fragmentation in anaphase different stages of meiosis, although all the lines were not I for 10% (49) in BB4, whereas laggards in metaphase I and equally affected. The irregularities observed include chro- II accounted for 13% (65) and 7% (36) in BB6, examined mosome stickiness (Fig. 4A), rings (Fig. 4B, yellow arrow- using 500 PMCs (Table 2). heads), rods (Fig. 4B, red arrowheads), laggard chromo- somes (Fig. 4C-G, red arrowheads), and bridge fragment- ation (Fig. 4E, yellow arrowheads). In addition, micro- 100 ∙ Plant Breed. Biotech. 2019 (June) 7(2):95~105

Fig. 4. Meiotic abnormalities observed in the four lines of ×Brassicoraphanus. (A-H) DAPI raw images of abnormal cells observed including sticky chromosomes (A), ring (yellow arrows) and rod (red arrows) (B), lagging chromosomes in metaphase I/II and anaphase I/II (C-G, red arrows), bridge fragmentation in anaphase I and telophase II (E, H, yellow arrows) with two micronuclei (H, red arrow), all of which contributes to the characteristic increase in aneuploidy and sterility.

Table 2. The frequency of meiotic abnormalities in different meiotic stages of the four inbred lines of ×Brassicoraphanus. No. of abnormal PMCs Stages BB4 BB6 BB12 BB50 Prophase I (diakinesis) Sticky chromosomes 35 28 4 6 Rod/ring chromosomes18780 Laggard chromosomes 22 5 12 5 Metaphase I Laggard chromosomes 154 65 15 17 Anaphase I Bridge fragmentation 49 14 5 27 Metaphase II Laggard chromosomes 8 36 6 5 Anaphase II Bridge fragmentation1900 Total meiotic abnormality (%) 287 (57%) 169 (34%) 50 (10%) 60 (12%) (per 500 PMCs)

DISCUSSION by using microspore culture, which became a commercial vegetable crop. In this study, we assessed the somatic me- Intergeneric allopolyploids between Brassica and taphase chromosome karyotype by fluorescence in situ Raphanus have remained unstable despite numerous at- hybridization (FISH), meiotic pairing behavior by genomic tempts to improve their chromosomal pairing stability. in situ hybridization (GISH) and meiotic abnormalities in However, Lee et al. (2011) successfully developed stable four lines of ×Brassicoraphanus. progenies, ×Brassicoraphanus, through induced mutation Various studies have been conducted on cytogenetics of Cytogenetic Analyses in ×Brassicoraphanus ∙ 101

natural and synthetic allopolyploids (Bennett et al. 1992; rearrangement, gene dosage effects, unbalanced parental Yao et al. 2010; Fujii and Ohmido 2011). FISH karyo- genome contributions, existence of different genomes in typing has been found to be a useful method for identi- the nucleus, and variations in chromosome number (Leitch fication of each chromosome homolog and providing de- and Leitch 2008). In a synthetic hybrid, several chromo- tailed chromosomal information of a genome. The two somal variations and abnormal meiosis were observed, rDNA families, the non-NOR-forming 5S rDNA and implying that the genetic instability of the hybrid polyploid NOR-forming 45S rDNA (Singh et al. 2009), which are was due to abnormal chromosome number and structure, composed of many repeat units, have been primarily used which were attributed to aberrant meiosis (Fujii and Ohmido as FISH probes (Hasterok et al. 2006; Lim et al. 2007; Fujii 2011). In the present study, the unstable line BB4 was and Ohmido 2011). These two rDNAs have been used in aneuploid with 2n ‒ 1 = 37, showing a high degree of meiotic phylogenetic studies in which the varying distribution irregularities and nondisjunction and anaphase chromo- patterns among species and population have been exploited some lagging could be the two mechanisms responsible for (Drouin and de Sá 1995). Reports on FISH karyotype by chromosome loss or aneuploidy. The nuclear envelope was using 5S and 45S rDNA probes showed three 5S and five formed with a laggard might form a micronucleus. 45S rDNA pairs in Brassica rapa (Lim et al. 2005; Hwang interspecific hybrid subgenomes were suc- et al. 2009; Koo et al. 2011; Xiong and Pires 2011; Belandres cessfully identified using GISH method (Wang et al. 2006; et al. 2015), whereas two 5S and three 45S rDNA pairs in Yao et al. 2010; Howell and Armstrong 2013). Moreover, Raphanus sativus (Hwang et al. 2012; Belandres et al. the parental subgenomes, Brassica and Raphanus, were 2015). A recent study showed that both Brassica- and clearly discriminated in somatic metaphase (Lim et al. Raphanus genomes-derived 5S and 45S rDNA distribution 2012) and meiotic chromosomes (Belandres et al. 2015) of patterns showing eight and five pairs of 5S and 45S rDNA the hybrid ×Brassicoraphanus. In this study, we discrimi- loci, respectively, were identified in a stable allopolyploid nated ten and nine pairs of Brassica and Raphanus bivalent hybrid ×Brassicoraphanus line, BB5 (Belandres et al. 2015). chromosomes in BB50, and the remaining lines BB4, BB6, In this study, we observed varying numbers of 5S and and BB12 consisted of the combined univalent, bivalent, 45S rDNA loci within and among the several lines of and tetravalent chromosome pairs of both parental sub- ×Brassicoraphanus. However, two pairs of nucleolar or- genomes. Genomic reports showed that a high degree of ganizers of Brassica and Raphanus were present in all synteny is shared between B. rapa and R. sativus genomes lines. These factors might have contributed to the concerted (Kitashiba et al. 2014; Moghe and Shiu 2014). This might changes in multiple tandem repeats (Eickbush and Eickbush imply that multivalent chromosome pairing between B. 2007), in which the common mechanisms involve unequal rapa and R. sativus chromosomes might be attributed to the crossing-over and gene conversion (Dover 1989; Shaked et possible pairing of nonhomologous chromosomes to the al. 2001). In addition, these changes in rDNA distribution homeologous region at the early stage of meiosis. In add- can be attributed to genomic rearrangements that include ition, multivalent formation can be generated because of rDNA loci exchanges, fragment loss, and homoeologous chromosomal changes due to abnormal segregation in exchange, as reported previously (Pontes et al. 2004; Gaeta meiosis (Comai 2005; Leitch and Leitch 2008; Fujii and et al. 2007; Lim et al. 2008). Major karyotypic changes Ohmido 2011), which could increase the chance of chromo- such as aneuploidy can result in alterations and irregu- somal rearrangement such as translocation and deletion larities in somatic and meiotic cell division. 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