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Article

Exploration of B Chromosome Origin in Allotriploid Lily Associated with Anomalous Meiosis

by
Kongzhong Xiao
1,
Lijie Yang
1,
Hui Li
1,
Pengfei Du
1,
Rong Fu
1 and
Changlong Xiao
1,2,*
1
School of Soil and Water Conservation, Nanchang Institute of Technology, No. 289 of Tianxiang Avenue, Nanchang 330099, China
2
Key Laboratory of Horticultural Plant Genetic and Improvement of Jiangxi Province, Institute of Biological Resources, Jiangxi Academy of Sciences, No. 7777, Changdong Road, Gaoxin District, Nanchang 330096, China
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(3), 267; https://doi.org/10.3390/horticulturae11030267
Submission received: 4 January 2025 / Revised: 17 February 2025 / Accepted: 27 February 2025 / Published: 1 March 2025
(This article belongs to the Special Issue Genetic Innovation and Breeding in Ornamental Plants)
Browse Figures
Versions Notes

Abstract

:
Supernumerary (B) chromosomes are widespread in numerous plants, including the Lilium genus. However, their origin remains unclear. This study used traditional and modern cytogenetics to analyze the triploid lily cultivar ‘Eyeliner’ (LAA) to identify the microsporogenesis, fertility, and chromosome composition of its progeny and record a case of potential B chromosome formation. The results indicated anomalous meiosis of LAA in all processes. In microspores, different cells had different numbers of chromosomes and fragments. Moreover, the fluorescence in situ hybridization (FISH) results showed that some fragments contained telomere signals at both ends. The LAA × AA progenies were aneuploid, with one progeny containing a small aberrant chromosome (potential B chromosome). The genomic in situ hybridization (GISH) results showed that the aberrant chromosome originated from the L genome. In contrast, the FISH results showed that the aberrant chromosome contained two telomere signals. This suggests that the anomalous meiosis of the triploid lily forms chromosome bridges, fragments, and small aberrant chromosomes (isochromosome), which eventually form aneuploid gametes containing small aberrant chromosomes passed on to the progeny. This study provides a case in which the potential B chromosomes are derived from the A chromosomes.

1. Introduction

Lilies, (genus Lilium, family Liliaceae) are among the most important bulb flowers worldwide, with Lilium including 111 wild species classified into seven sections [1]. The modern lily groups Longiflorum (L), Asiatic (A), Oriental (O), and Trumpet (T) were bred from intra-section hybridization, while intergroup cultivars, such as LA, LO, OT, and OA, were obtained from inter-section hybridization [2]. Intra-sectional lilies are usually diploid (2n = 2x = 24), whereas most inter-sectional lilies are allotriploid (2n = 3x = 36), such as LAA, LOO, and OTO. Studies on the fertility of triploid lilies have identified anomalous meiosis during microsporogenesis, leading to male sterility; however, aneuploid females can also be produced to cross with suitable males [3,4,5,6].
B chromosomes (Bs) have been reported to occur in more than one thousand flowering plant species [7,8]. In the B chromosome database (http://www.bchrom.csic.es/, accessed on 10 November 2024; D’Ambrosio et al. [9]), 35 lily species were shown to contain B chromosomes. These B chromosomes vary in size, ranging from minute elements to normal-sized A chromosomes [10,11,12,13]. The number of reported B chromosomes varies among lily species, ranging from 1 in L. lancifolium [12] to 12 in L. medeoloides [14]. Although many studies have been conducted on the structure, occurrence, sequence, meiotic behavior, and transmission of B chromosomes [11,15,16], their origins remain unclear. The most likely explanation is that they originated from the basic A chromosomes as a by-product of chromosomal rearrangement or unbalanced separation and developed into the B chromosome from chromosome fragments or additional homologous chromosomes [17,18,19,20]. However, direct evidence for this is scant.
In this study, we aimed to investigate anomalous meiosis in allotriploid lilies and the chromosome composition of their hybrids. We detected small aberrant chromosomes that resemble potential Bs and analyzed their origin using genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH). Subsequently, the correlation between the origin of potential Bs and meiosis was discussed.

2. Materials and Methods

2.1. Plant Material

The allotriploid lily cultivar ‘Eyeliner’ (2n = 3x = 36) and autodiploid Asiatic Lily ‘Black Magic’ (AA, 2n = 2x = 24) were assessed. The genomic DNA of the Longiflorum lily ‘White Heaven’ (LL) was extracted and used as a probe. All lilies were grown at the Eco-Science Park of Nanchang Institute of Technology.

2.2. Chromosome Preparation with Anthers

Chromosomes were prepared as previously described by Xiao et al. [21]. When the ‘Eyeliner’ flower buds were 26–30 mm, the anther was removed and placed in Carnot fixative (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 1 h. Then, the pollen mother cells in the anther were extracted on a slide and mixed with 20 μL of 45% acetic acid. A cover glass was placed over the slide and gently flattened using the thumb, and the slide was then frozen at −80 °C for approximately 1 h. Subsequently, a knife was then used to remove the cover glass, which was soaked in ice ethanol for 2–3 min and then air dried.

2.3. Mitotic Chromosome Preparation by Root Tips

Mitotic chromosomes were prepared as described by Xiao et al. [22]. When the roots were approximately 1 cm, they were cut off and incubated in 0.7 mM cycloheximide (Duchefa Biochemie, Haarlem, The Netherlands) at 25 °C for 4 h and then stored in Carnot fixative. The root tips were treated with 1% cellulase RS (Yukult, Tokyo, Japan) and pectinase Y23 (Yukult) mix at 37 °C for 1 h. The root tips were mixed with 20 μL of 45% acetic acid on a glass slide, covered with a cover glass, and squeezed. The method for removing the cover glass was outlined in Section 2.2.

2.4. Pollination and Embryo Rescue

Pollination and embryo rescue were performed as described by Xiao et al. [23]. When the ‘Eyeliner’ flowers were opening, their anthers were carefully removed. The stigmas were then pollinated with fresh ‘Black Magic’ pollen. After pollination, the stigmas were wrapped in aluminum. Approximately 50 days after pollination, the fruits were harvested for embryo rescue.

2.5. Telomere Repeats by Fluorescence In Situ Hybridization (FISH)

For the FISH experiments, the chromosomes were treated according to the procedures described by Xiao et al. [22]. The telomeric probes used were the Deoxyribonucleotide oligomer 5′-(TTTAGGG)4-3′, and they were synthesized with FAM (a green fluorescence dye) by Tsingke Company (Beijing, China). The experimental steps were referred to Cuadrado et al. [24] with minor modification. The hybridization mix (40 μL) consisted of 50% deionized formamide (Sigma-Aldrich (Shanghai) Trading Co., Ltd., Shanghai, China), 10% dextran sulfate (Amresco, New York, NY, USA), 2× SSC (0.3 M NaCl plus 30 mM sodium citrate, pH = 7.0), 0.25% SDS, and 50 ng probe DNA. The hybridization mix solution was added to the slide with the prepared chromosomes, and then the slide was quickly covered and incubated in a moist insulated box at 37 °C for 6 h. The slide was then washed at 42 °C with 2× SSC three times for 5 min each time and air dried. Subsequently, 10 μL of antifade solution (ProLong™ Diamond Antifade Mountant with DAPI, Thermo Fisher, Waltham, MA, USA) was added to each slide. The slides were observed under a fluorescence microscope (Nikon TI-S, Nikon Imaging Instruments Sales (China) Co., Ltd., Shanghai, China). For each slide, 5–20 cells were observed.

2.6. Genomic In Situ Hybridization (GISH) Analysis

The genomic DNA of ‘White Heaven’ (LL) was extracted based on the CTAB method and then labeled with biotin using a nick translation kit (Roche 11745824910, Basel, Switzerland) as a probe. Meanwhile, herring sperm (HS) DNA was used as a block for GISH.
The hybridization mix (40 μL) consisted of 50% deionized formamide, 10% dextran sulfate, 2× SSC, 0.25% SDS, 50 ng probe DNA, and 1 μg block DNA. In situ hybridization was performed as previously described by Xiao et al. [22]. Biotin signals were detected and amplified using Streptavidin-CY3 (Invitrogen, Camarillo, CA, USA) and Biotinylated anti-Streptavidin (Vector Laboratories, Burlingame, CA, USA). After mounting with VECTASHIELD (H-1200, Vector Laboratories, Burlingame, CA, USA), the slides were observed under a fluorescence microscope (Nikon TI-S). In addition, Adobe Photoshop 2018 was used for image processing.

3. Results

3.1. Parental Chromosome Composition

To determine the chromosomal composition of allopolyploid lilies, the GISH method was applied to identify chromosomes originating from both the L genome and the A genomes. GISH signals showed that the allotriploid lily ‘Eyeliner’ contains 12 L chromosomes and 24 A chromosomes (2n = 3x = 12 L + 24 A; Figure 1a), and according to the telomere signal site, it does not contain a B chromosome (Figure 1b). ‘Black Magic’ is a diploid Asiatic lily (2n = 2x = 24 A; Figure 1c) that also does not have a B chromosome.

3.2. Anomalous Meiosis of ‘Eyeliner’ (LAA)

Telomere repeat sequences can be used as probes to mark the ends of chromosomes and be used to determine the number of association chromosomes in meiosis. The results showed that ‘Eyeliner’ (LAA) was anomalous at all phases of meiosis. (1) In metaphase I (Figure 2a–c), the chromosome association was relatively disordered; monovalent (I), bivalent (II), trivalent (III), and polyvalent associations were formed; and different pollen mother cells (PMCs) presented different results. (2) After early anaphase I, most of the univalent was separated, and the polyvalent can be observed to have a ring structure (Figure 2c white arrow). (3) At anaphase I, polyvalent separation was observed, and the univalent was also separated; some chromosomes formed chromosome bridges and fragments (or small aberrant chromosomes) due to the variation in chromosome structure, and the appearance of chromosome bridges was often accompanied by the loss of telomere signals (as shown by the red circle in Figure 2d–f). Moreover, smaller chromosomal fragments contained telomere signals at both ends (Figure 2d–f red arrow). Most of the small fragments appeared to be of the U-type, which will be further discussed in the Discussion.
The period from the second division of meiosis to the formation stage of pollen grains was characterized as follows. Anaphase II presented abnormalities such as lag chromosomes, free chromosomes, and chromosome bridges, and it had similar characteristics to anaphase I, with small fragments of chromosomes containing two telomere signals at both ends, chromosome bridges, and telomere deletion (short chromosome fragments shown by arrows in Figure 3a,b, and telomere signal deletion shown by red circles). At telophase II (Figure 3c–e), fragment chromosomes and micronuclei were quite common. Analysis of the chromosomal characteristics of the formed pollen grains (Figure 3f–h) revealed that different pollen grains contained variable chromosome numbers. Moreover, the broken small fragments formed by anaphase I and anaphase II were randomly distributed in different pollen grains. Telomere signals were absent in some chromosomes of the microspores, while telomere signals were observed at both ends of some small fragments (Figure 3d, red arrow). Chromosome fragments come not only from the L genome but also from the A genome. These results indicate that the allotriploid lily exhibited anomalous meiosis and formed chromosome fragments and small aberrant chromosomes.

3.3. Chromosomes Composition of the Progenies from LAA × AA and Identification of Potential B Chromosome

In total, 140 progenies were obtained by hybridization with LAA × AA. The chromosomal compositions of the 12 progenies were analyzed using GISH (Table 1), and Figure 4a,b show the representative GISH results. The chromosome number varied from 25 to 34, averaging 28.25. All progenies were aneuploid and contained variable chromosome numbers (25–34 ch) and L (1–4 ch) and LA (0–4 ch) recombinant chromosomes. One of the progenies (23103-12) was detected to contain a small aberrant chromosome (Figure 4c,d). The GISH results showed that it originated from the L genome (red arrow in Figure 4c). FISH using telomere repeat probes revealed that this chromosome contained telomere signals at both ends (red arrow in Figure 4d). Moreover, this small chromosome was present in all observed cells and could not complete the normal meiosis process; thus, it was a typical small aberrant chromosome. We hypothesize that this was a potential B chromosome.

4. Discussion

4.1. Mechanisms of Chromosome Bridge and Fragment Formation in Allotriploid Lilies

GISH and FISH are powerful cytogenetic tools that can be used to distinguish heterologous chromosomal behavior during the meiosis of pollen mother cells of distant hybrids, allotriploids, or odd-allotetraploids [14,23,25,26,27]. In this study, the GISH and FISH techniques were used to perform a detailed analysis of the meiotic process of allotriploid lilies, and many anomalies were found, including lagging chromosomes, chromosome bridges, unequal separation, and fragments. These results are consistent with previous studies [23,28,29,30].
Two primary mechanisms have been proposed to explain the origin of dicentric bridges and fragments during meiosis: paracentric inversion and U-type exchange [31,32,33,34]. To illustrate these processes, we provide schematic diagrams of U-type exchange (Figure 5a) and chromosome inversion (Figure 5b). During anomalous meiosis, the premature replication of univalent centromeres allows their separation during anaphase I (Figure 5a) [22,35]. When chromosomal breakage occurs in a univalent form, it can lead to anomalous fusion of the broken end, thus producing U-type exchange fragments [32]. The occurrence of a U-type exchange at the centromere region helps explain the origin of the potential B chromosome in the 2023-12 progeny. In addition, pachytene loops during meiosis provide direct evidence of chromosomal inversion [36]. The presence of chromosomal inversion leads to the appearance of broken fragments and bridge structures in anaphase I [13,29]; these phenomena are found in many plants, such as Lilium [13,37], Aesculus carnea [38], Fritillaria [39], Zea mays [40], Hordeum vulgare [41], Agave stricta [42], and many others. These findings demonstrate that the anomalous meiosis in LAA is characterized by chromosome inversion exchanges, resulting in the formation of chromosome bridges and fragments, along with U-type exchanges caused by abnormal fusion of broken ends between sister chromatids. These processes lead to reduced fertility, the production of aneuploid gametes, and the generation of gametes containing aberrant chromosomes.

4.2. Mechanisms of B Chromosome and Fertility in the Hybrid Between Allotriploid and Diploid Lilies

The present results showed that the allotriploid ‘Eyeliner’ exhibited high male sterility but could represent a female parent to hybridize with appropriate males and produce aneuploidy progenies. This finding is consistent with several previous reports (Lim et al. [3]; Khan et al. [4]; Xie et al. [43]; Zhou et al. [5,6]; Chung et al. [44], Zhou et al. [45], Xi et al. [46]). The mechanism underlying this phenomenon is associated with the fritillaria-type embryo sac characteristic of Lilium plants. During LAA × AA hybridization, the endosperm is euploid and can provide nutrients for embryo development [5,6,47]. However, there are few reports on the production of B chromosomes or potential B chromosomes through hybridization in lily for the following reasons: (1) The B chromosome is easily overlooked in aneuploid progeny because Lilium species have SAT-chromosomes, which are easily separated from the original chromosome during chromosome preparation (Figure 6). (2) The number of B chromosomes in the hybrid offspring was small because the probability of LAA meiosis forming functional aneuploid gametes and carrying aberrant chromosomes (potential Bs) was low, with only one such offspring observed among the 12 offspring analyzed in this study (Table 1). (3) The size of the B chromosome varies [11] because the hybrid offspring are all aneuploid. When the lengths of the Bs and basic A chromosomes are similar, it is difficult to distinguish them clearly.

4.3. B Chromosome Origin

B chromosomes have been extensively studied over the past century; however, a scientific consensus has not been reached on their origin [48]. Although the origin of B chromosomes is commonly attributed to the standard A chromosome, evidence also points to their potential derivation from exogenous chromosomes introduced via hybridization with related species [13,49]. In this study, the small aberrant chromosome in 2023-12 was the result of interspecific hybridization derived from the A-chromosome (L genome), which is consistent with both previous views on the origin of the B chromosome [13,50,51,52,53]. Here, we provide some evidence for this result. First, the small supernumerary chromosomes in the LAA × AA hybrids originated independently because the parents did not have the same small aberrant chromosomes as the 23103-12 plants (Figure 1). Second, small abnormal chromosomes in the genotype originated from the L genome (GISH results, Figure 4c) and were the result of interspecific hybridization. Finally, many chromosome bridges and broken fragments were found during LAA meiosis. These fragments were still present in the formed microspores, which may have been of B chromosome origin. In addition, we found that the small B chromosomes in this study were morphologically similar to isochromosomes. There is evidence that in maize and wheat, the monovalent moves randomly to one pole during anaphase I separation and tends to misdivide at the centromere [54]. Centromere division results in centromere translocation, resulting in telocentrics and isochromosome [54,55]. The meiotic abnormalities observed in LAA in this study likely created conditions for the formation of isochromosomes (Bs).
As the origin of the B chromosome has long been deemed a “mystery”, it may be worthwhile to study the origin of these Bs observed in this study in more detail. The pattern of B chromosome origin was previously studied in Plantago lagopus, and it involves the formation of small chromosomes, amplification of 5S rDNA, stabilization of telomere repeats, and formation of isochromosomes [48,49]. Compared with this mode of origin, the formation of isochromosomes with short arms after centromeric breakage and division described in this study is a potential origin mechanism for B chromosomes. This study only analyzed the potential origin of the B chromosome at the chromosomal level. Nonetheless, whether the B chromosome has specific functions and whether its nucleotide sequences are highly homologous to that of the A chromosome are worthy of further consideration.

5. Conclusions

The anomalous meiosis of the triploid lily produced aneuploid gametes containing isochromosomes or small aberrant chromosomes (potential B chromosomes), which were eventually passed on to the progeny through LAA × AA hybridization. This provides direct evidence that the potential B chromosome of lilies originated from the A chromosome.

Author Contributions

Data curation, H.L.; Formal analysis, P.D.; Funding acquisition, C.X.; Investigation, L.Y.; Project administration, C.X.; Writing—original draft, R.F.; Writing—review and editing, K.X. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Major Science and Technology Research and Development Special Project of Jiangxi Province (20203ABC28W016); the Key Laboratory of Horticultural Plant Genetic and Improvement of Jiangxi Province (2021KFJJ004); the Jiangxi University Students Innovation and Entrepreneurship Training Program (S202311319002).

Data Availability Statement

All data are presented in the article; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. GISH and FISH results for the parental mitotic chromosome metaphase. (a): ‘Eyeliner’ GISH results (Ach is blue and Lch is red); (b): ‘Eyeliner’ telomeric FISH results; (c): ‘Black magic’ telomeric FISH results.
Figure 1. GISH and FISH results for the parental mitotic chromosome metaphase. (a): ‘Eyeliner’ GISH results (Ach is blue and Lch is red); (b): ‘Eyeliner’ telomeric FISH results; (c): ‘Black magic’ telomeric FISH results.
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Figure 2. FISH analysis of allotriploid lily chromosomes during metaphase I and anaphase I. (a,b) Metaphase I; (c) after metaphase I bias; and (d–f) anaphase I. Bars are 20 μm. Note: Telomere signals are green. ‘I’, ‘II’, ‘III’, and ‘IV’ mean monovalent, bivalent, trivalent, and tetravalent associations of different synapses; white arrows point to the chromosome ring; red arrows point to broken chromosome fragments; and the red ‘circle’ indicates chromosomes that lack telomeres.
Figure 2. FISH analysis of allotriploid lily chromosomes during metaphase I and anaphase I. (a,b) Metaphase I; (c) after metaphase I bias; and (d–f) anaphase I. Bars are 20 μm. Note: Telomere signals are green. ‘I’, ‘II’, ‘III’, and ‘IV’ mean monovalent, bivalent, trivalent, and tetravalent associations of different synapses; white arrows point to the chromosome ring; red arrows point to broken chromosome fragments; and the red ‘circle’ indicates chromosomes that lack telomeres.
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Horticulturae 11 00267 g003
Figure 3. Chromosome characteristics of ‘Eyeliner’ (LAA) meiosis anaphase II and microspores. (a,b): FISH results of anaphase II; red arrows indicate chromosome segments that contain telomere signals at both ends, and red circles indicate chromosome bridges that lack telomeres. (c–e): Telophase II (tetrad). (f): Microspore chromosomes were stained conventionally and contained three distinct chromosome segments; (g): FISH analysis of microspore chromosome telomeres revealed that certain chromosomes are missing telomere signals, as shown in red circles, and fragments of chromosomes may contain telomere signals; (h): GISH analysis of microspore chromosomes showing fragments of chromosomes from the L genome (pink) and A genome (blue), shown by red arrows. Bars are 20 μm.
Figure 3. Chromosome characteristics of ‘Eyeliner’ (LAA) meiosis anaphase II and microspores. (a,b): FISH results of anaphase II; red arrows indicate chromosome segments that contain telomere signals at both ends, and red circles indicate chromosome bridges that lack telomeres. (c–e): Telophase II (tetrad). (f): Microspore chromosomes were stained conventionally and contained three distinct chromosome segments; (g): FISH analysis of microspore chromosome telomeres revealed that certain chromosomes are missing telomere signals, as shown in red circles, and fragments of chromosomes may contain telomere signals; (h): GISH analysis of microspore chromosomes showing fragments of chromosomes from the L genome (pink) and A genome (blue), shown by red arrows. Bars are 20 μm.
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Figure 4. Representative GISH and FISH results for the metaphase chromosomes of root tips of seedlings obtained from LAA × AA. (a): 23103-01, (b): 20103-07, (c): 20103-12. (d): Telomere labeling with telomere repeat sequences as a probe in 23103-12. The white arrow indicates the location of the chromosome recombination, and the red arrow indicates the B chromosome. Bar = 20 μm.
Figure 4. Representative GISH and FISH results for the metaphase chromosomes of root tips of seedlings obtained from LAA × AA. (a): 23103-01, (b): 20103-07, (c): 20103-12. (d): Telomere labeling with telomere repeat sequences as a probe in 23103-12. The white arrow indicates the location of the chromosome recombination, and the red arrow indicates the B chromosome. Bar = 20 μm.
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Figure 5. Illustration of chromosome fragment formation. (a): U-type exchange; (b): chromosome inversion, in which meiosis forms chromosome rings and subsequently forms bridges and fragments in the crossover. The a–g and A–G represent alleles corresponding to chromosomes.
Figure 5. Illustration of chromosome fragment formation. (a): U-type exchange; (b): chromosome inversion, in which meiosis forms chromosome rings and subsequently forms bridges and fragments in the crossover. The a–g and A–G represent alleles corresponding to chromosomes.
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Figure 6. Comparison of the B chromosome and satellite chromosome. (a): Comparison of autosomes and B chromosomes; (b): Metaphase mitotic plate, comparing B chromosome and satellite.
Figure 6. Comparison of the B chromosome and satellite chromosome. (a): Comparison of autosomes and B chromosomes; (b): Metaphase mitotic plate, comparing B chromosome and satellite.
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Table 1. Genome composition of 12 progenies based on the GISH analysis.
Table 1. Genome composition of 12 progenies based on the GISH analysis.
CodeChromosomeLchAchL/AchPollenEgg
23103-012712331215
23103-023022531218
23103-032522211213
23103-042722141215
23103-062822601216
23103-072512401213
23103-082822511216
23103-092712511215
23103-102932331217
23103-113052321218
23103-1234 + 1B32921222
23103-133342721221
Average28.252.1724.331.7512.0016.25
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Xiao, K.; Yang, L.; Li, H.; Du, P.; Fu, R.; Xiao, C. Exploration of B Chromosome Origin in Allotriploid Lily Associated with Anomalous Meiosis. Horticulturae 2025, 11, 267. https://doi.org/10.3390/horticulturae11030267

AMA Style

Xiao K, Yang L, Li H, Du P, Fu R, Xiao C. Exploration of B Chromosome Origin in Allotriploid Lily Associated with Anomalous Meiosis. Horticulturae. 2025; 11(3):267. https://doi.org/10.3390/horticulturae11030267

Chicago/Turabian Style

Xiao, Kongzhong, Lijie Yang, Hui Li, Pengfei Du, Rong Fu, and Changlong Xiao. 2025. "Exploration of B Chromosome Origin in Allotriploid Lily Associated with Anomalous Meiosis" Horticulturae 11, no. 3: 267. https://doi.org/10.3390/horticulturae11030267

APA Style

Xiao, K., Yang, L., Li, H., Du, P., Fu, R., & Xiao, C. (2025). Exploration of B Chromosome Origin in Allotriploid Lily Associated with Anomalous Meiosis. Horticulturae, 11(3), 267. https://doi.org/10.3390/horticulturae11030267

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