Euchromatic Supernumerary Chromosomal Segments—Remnants of Ongoing Karyotype Restructuring in the Prospero autumnale Complex?

Abstract

Supernumerary chromosomal segments (SCSs) represent additional chromosomal material that, unlike B chromosomes, is attached to the standard chromosome complement. The Prospero autumnale complex (Hyacinthaceae) is polymorphic for euchromatic large terminal SCSs located on the short arm of chromosome 1 in diploid cytotypes AA and B⁷B⁷, and tetraploid AAB⁷B⁷ and B⁶B⁶B⁷B⁷, in addition to on the short arm of chromosome 4 in polyploid B⁷B⁷B⁷B⁷ and B⁷B⁷B⁷B⁷B⁷B⁷ cytotypes. The genomic composition and evolutionary relationships among these SCSs have been assessed using fluorescence in situ hybridisation (FISH) with 5S and 35S ribosomal DNAs (rDNAs), satellite DNA PaB6, and a vertebrate-type telomeric repeat TTAGGG. Neither of the rDNA repeats were detected in SCSs, but most contained PaB6 and telomeric repeats, although these never spanned whole SCSs. Genomic in situ hybridisation (GISH) using A, B⁶, and B⁷ diploid genomic parental DNAs as probes revealed the consistently higher genomic affinity of SCSs in diploid hybrid B⁶B⁷ and allopolyploids AAB⁷B⁷ and B⁶B⁶B⁷B⁷ to genomic DNA of the B⁷ diploid cytotype. GISH results suggest a possible early origin of SCSs, especially that on chromosome 1, as by-products of the extensive genome restructuring within a putative ancestral P. autumnale B⁷ genome, predating the complex diversification at the diploid level and perhaps linked to B-chromosome evolution.

1. Introduction

Karyotypes of plants and animals sometimes carry supernumerary genetic material, either in the form of B-chromosomes (Bs) or physically integrated into the standard chromosome complement as supernumerary chromosomal segments (SCSs) [1,2]. SCSs in plants are widespread [3], but are most frequently found in monocotyledons [4,5,6,7,8,9,10]. They are usually heterochromatic, but euchromatic segments also occur [11,12]. SCSs are frequently located terminally, and are thus seen at meiosis as heteromorphic bivalents resulting from chiasma formation proximal to SCSs [3,5,7,13]. SCSs usually behave as selfish genetic elements [9], but their inheritance can sometimes be Mendelian [14], and both of these patterns may be found within a single species [3].

A model system for investigating SCSs is provided by the Prospero autumnale complex (L.) Speta (Hyacinthaceae) [6,12]. This species complex has four evolutionarily and phylogenetically well-characterised and genomically distinct diploid cytotypes AA, B⁵B⁵, B⁶B⁶, and B⁷B⁷, each with unique combinations of genome size, base chromosome number (x = 5, 6 and 7), and repetitive DNA amounts and distribution [15]. Such chromosome number variation results from the relatively high rate of genome rearrangements in Prospero, involving translocations and inversions [6,15]. Hybridisation and polyploidisation of three of these diploid cytotypes (AA, B⁶B⁶, and B⁷B⁷) have given rise to further polyploid cytotypes, including autopolyploids of genome B⁷ (x = 7) [16,17,18,19,20] and two classes of allopolyploids, of A/B⁷ (both x = 7) and of B⁶/B⁷ genome composition [20,21,22,23]. Both B chromosomes (Bs) and SCSs have been reported in the three diploid cytotypes AA, B⁶B⁶, and B⁷B⁷ [15], and in various polyploids [6,17,21,24]. SCSs in P. autumnale are preferentially located terminally on the short arms of chromosomes 1 and 4 in the A and B⁷ genomes and are frequently quite large. Thus, the SCS on chromosome 1 may be nearly double the length of the chromosome. Remarkably, no phenotypic effects have been ascribed to the presence of these massive elements [16,23], although SCSs are geographically widespread and reach polymorphic proportions in many populations [6].

The origins of SCSs, particularly of the euchromatic ones, are obscure [2,3]. They may result from amplification of part of the genome, such as tandem repeats, especially satellite DNAs [25], or may represent inessential chromosome blocks generated by karyotype rearrangements and retained during chromosomal evolution [11,26,27]. In this paper, the structure, repeat composition, and origins of SCSs have been addressed in the P. autumnale complex using molecular cytological tools based on FISH (fluorescence in situ hybridisation) and GISH (genomic in situ hybridisation). In particular, the distribution patterns of tandem repeats (rRNA genes, satellite DNA, telomeric sequences) within SCSs have been established in a range of diploid and polyploid cytotypes to assess whether SCSs reflect single or multiple origins and what their relationships to specific genomes or chromosomal locations might be.

2. Materials and Methods

2.1. Plant Materials

Plants for cytogenetic analysis were collected from natural populations across the Mediterranean basin [15,20] and grown in the Botanical Garden of the University of Vienna. Ten diploid and nine polyploid (4x and 6x) plants carrying SCSs were studied and their collection details are listed in

Table 1. List of plant material of the Prospero autumnale complex studied with voucher information and chromosome numbers. The chromosomal location, genomic affinity (as established using GISH), and length of supernumerary chromosomal segments (SCSs) are indicated.

Cytotype (Accession Number)	Locality; Collector	2n	Genomic Location/Genomic Affinity of SCSs	SCS Length (µm)	Proportion of SCS (%) in the Chromosome †	Figure No.
Diploids with SCSs
AA (H541)	Spain, Huelva; J. Parker	14	A 1/genome B7	2.67	28.8	Figure 1, Figure 2e,f, Figure 3a and Figure S1a
B7B7 (H1)	Greece, Rhodos; F. Speta	14	B7 1	2.02	28.8	Figure 1 and Figure 2a,b
B7B7 (H247)	Greece, Crete; F. Speta	14	B7 1	2.02	24.5	Figure 1
B7B7 (H423)	Montenegro; F. Speta	14	B7 1	2.07	31.3	Figure 1
B7B7 (H500)	Greece, Crete; F. Speta	14	B7 1	2.02	30.6	Figure 1
B7B7 (H502)	Greece, Crete; F. Speta	14	B7 1	2.07	33.3	Figure 1
B7B7 (H614)	Israel, HaCarmel Park; J. Parker	14	B7 3	2.29	36.4	Figure 1 and Figure 2c,d
B7B7 (H641)	Spain; J. Parker	14	B7 1	1.98	32.0	Figure 1, Figure 3d and Figure S1d
B7B7 (H642)	Spain; J. Parker	14	B7 1	2.07	27.8	Figure 1
B6B7 (H258)	Greece, Crete; F. Speta	13	B6 1	2.11	23.4	Figure 1, Figure 2g,h,e and Figure S1e
Polyploids with SCSs
AAB7B7 (H110–1)	Portugal, Cheleiros; F. Speta	28	A 1 *	3.16	21.4	Figure 1, Figure 3b and Figure S1b
AAB7B7 (H110–2)	Portugal, Cheleiros; F. Speta	28	A 1	2.63	33.3	Figure 1, Figure 2i,j, Figure 3c and Figure S1c
AAB7B7 (H606)	Portugal, Castro Marin; J. Parker	28	A 1	2.89	26.2	Figure 1
B6B6B7B7 (H574–1)	Greece, Naxos; F. Speta	28	B6 1	3.85	27.3	Figure 1, Figure 2m,n, Figure 3f and Figure S1f
B6B6B7B7 (H574–2)	Greece, Naxos; F. Speta	28	B6 1	3.21	31.3	Figure 1
B7B7B7B7 (H360)	Greece, Kos; F. Speta	28	B7 4	1.94	35.3	Figure 1 and Figure 2k,l
B7B7B7B7B7B7 (H31)	No information; F. Speta	42	B7 4 * and (5 or 6) §	1.92 §	33.3 §	Figure 1 and Figure 2o,p
B7B7B7B7B7B7 (H308)	Croatia, Solta; F. Speta	42	B7 4	2.88	34.6	Figure 1
B7B7B7B7B7B7 (H453)	Croatia, Mosor; F. Speta	42 + 1B	B7 4 *	2.56	33.3	Figure 1

*: Homozygous SCS; ^†: Proportion of SCSs within the chromosomes carrying them, ^§: Only SCSs of chromosomes 4 were measured because the other SCSs could not be unambiguously assigned to chromosome 5 or 6. GISH: Genomic in situ hybridization.

.

For cytological investigations, root meristems were pretreated with a solution of 0.05% colchicine (Sigma Aldrich, Vienna, Austria) for 4.5 h at room temperature, fixed in ethanol: acetic acid (3:1) for at least 3 h at room temperature, and stored at −20 °C until use.

2.2. Karyotyping and FISH (Fluorescence In Situ Hybridisation with 5S & 35S Ribosomal DNAs, Vertebrate-Type Telomeric Repeats, and Satellite DNA PaB6)

Chromosome numbers and karyotypes were analysed as described by Jang et al. [15,20] using standard Feulgen staining. Chromosomal spreads for FISH and GISH were prepared by enzymatic digestion and squashing, as described in Jang et al. [15,20].

Probes used for FISH were: satellite DNA PaB6 isolated from the B⁶ genome in plasmid pGEM-T easy [28], 35S rDNA (18S/25S rDNA) from Arabidopsis thaliana in plasmid pSK+, and 5S rDNA from Melampodium montanum (Asteraceae) in plasmid pGEM-T easy, labeled with biotin or digoxygenin (Roche, Vienna, Austria). Probes were labeled either directly by PCR (5S rDNA and satellite DNA PaB6) or using a nick translation kit (35S rDNA; Roche). A commercially available, directly Cy3-labelled PNA (peptide nucleic acid) probe to vertebrate telomeric sequences (CCCTAA)₃ (Dako, Glostrup, Denmark) was used as described in [10]. FISH was performed as described in Jang et al. [15,20].

2.3. GISH (Genomic In Situ Hybridisation)

GISH has been performed in one diploid hybrid individual B⁶B⁷ (H258) and four allopolyploid individuals, two of AAB⁷B⁷ (H110–1 & 2) and two of B⁶B⁶B⁷B⁷ (H574–1 & 2) composition, using labelled parental diploid genomic DNA as probes [24,29]. Total genomic DNA from diploid cytotypes AA, B⁶B⁶, and B⁷B⁷ was isolated using the CTAB (Cetyltrimethylammonium Bromide) method [29] and sheared at 98 °C for 5 min. Approximately 1 μg of genomic DNA of each cytotype was labeled using either digoxigenin or a biotin nick translation kit (Roche). GISH was carried out following the method of [29].

All preparations after FISH and GISH were analysed with an AxioImager M2 epifluorescent microscope (Carl Zeiss, Vienna, Austria), and images were captured with a CCD camera and processed using AxioVision ver. 4.8 (Carl Zeiss) with only those functions that apply to all pixels of the image equally.

3. Results and Discussion

3.1. Karyotype Structure and Localisation of Supernumerary Chromosomal Segments

Nineteen SCS-carrying plants of the P. autumnale complex have been analysed (Table 1; Figure 1): ten diploids (one AA, eight B⁷B⁷, and one diploid hybrid B⁶B⁷; Figure 1) and nine polyploids (two B⁶/B⁷ allotetraploids, three A/B⁷ allotetraploids, and four B⁷ autopolyploids, one tetra- and three hexaploids; Figure 1). All SCSs are located distally, most frequently on chromosomes 1 and 4, and occasionally on chromosome 3 or either 5 or 6 (it is not possible to unambiguously assign this SCS to a specific chromosome; Figure 1). Chromosomes 1, 3, and 4 carry SCSs on their short arms and are thus are more symmetrical than their standard counterparts, while chromosome 5/6 carries an SCS on the long arm and is thus more asymmetric (Figure 1). The SCSs are massive blocks of chromatin and their length varies from 1.92 µm to 3.85 µm, representing a 21.4% to 36.4% increase in chromosome length, as found previously (Table 1) [6,16,21]. The SCS on chromosome 1 in the allotetraploid B⁶B⁶B⁷B⁷ is reported here for the first time (Figure 1).

The SCSs of chromosome 1 are remarkably constant in size across the distribution range of P. autumnale and also across ploidy levels. Thus, the SCS in B⁷B⁷ diploids has a length of about 2 μm in plants from Greece, Montenegro, and Spain, while that in the A genome is about 2.75 μm in length in AA diploids and in A/B⁷ allotetraploids (Table 1). The same constancy is not found in SCSs attached to other chromosomes.

3.2. Tandem Repeats in Supernumerary Chromosomal Segments

Four types of tandem repeats–35S rDNA, 5S rDNA, satellite DNA PaB6, and telomeric repeats–have been used as FISH probes on standard and SCS-carrying chromosomes of P. autumnale cytotypes (Figure 2). No signals for 35 or 5S rDNAs were detected within SCSs (Figure 2).

In standard karyotypes of diploids of P. autumnale, satellite DNA PaB6 is located in pericentromeric regions of at least one chromosome (AA) and up to all chromosomes (B⁶B⁶, B⁷B⁷) [28]. By contrast, in SCSs, it is located terminally on chromosome 1 of B⁷B⁷ (2 of 7 plants), chromosome 1A of AAB⁷B⁷, chromosome 3 of B⁷B⁷, chromosomes 4 of B⁷B⁷B⁷B⁷ and B⁷B⁷B⁷B⁷B⁷B⁷, and chromosome 5/6 of B⁷B⁷B⁷B⁷B⁷B⁷ individuals (Figure 1 and Figure 2). No PaB6 signals were detected on the SCSs of the AA diploid, of the B⁶B⁷ diploid hybrid, and of the five remaining B⁷B⁷ plants (Figure 2).

In the P. autumnale complex, vertebrate-type telomeric signals are found at the termini of both arms of all standard chromosomes (Figure S1) [28], and also in pericentromeric regions, coinciding with satellite DNA PaB6 as its monomers contain a few full repeats of telomeric sequence TTAGGG (Figure S1b,c,e,f). Telomeric repeat signals in telomeric positions in standard chromosomes (i.e., lacking SCSs) were often very weak. In SCSs, slight amplification of telomeric repeats was detected in SCSs on chromosomes 1 of some B⁷ diploids (Figure S1d) and AAB⁷B⁷ allotetraploids (Figure S1b,c), always subterminally and coincident with amplification of satellite DNA PaB6.

3.3. Genomic DNA Affinities of the Supernumerary Chromosomal Segments (Genomic In Situ Hybridisation)

The relationships of SCSs to parental genomes can be established using formamide-free GISH [20,29] in the diploid hybrid B⁶B⁷ (Figure 3e), and in the allotetraploids B⁶B⁶B⁷B⁷ (Figure 3f) and AAB⁷B⁷ (Figure 3b,c). In all cases, the SCSs have a higher affinity for the B⁷ genomic probe than for either of the other two parental genomic probes (A or B⁶ genomes; Figure 3b,c,e,f).

Remarkably, the SCS of chromosome 1 of the AA diploid shows hybridisation with B⁷ genomic DNA (Figure 3a). As expected, no discrimination is shown by the chromosome 1 SCS in B⁷B⁷ diploid plants used as the control (Figure 3d). A possible explanation for this is hybridisation at the diploid level between AA and B⁷B⁷ plants. Recombination between the SCSs-carrying B⁷1 chromosome and an A1 chromosome may have occurred in the diploid background, allowing the transfer of this SCS from B⁷1 to A1 (A/B chiasma formation has been seen in AB⁷B⁷ triploids) [30]. Subsequent recurrent backcrossing to AA would restore the AA complement, but with the addition of an SCS derived from B⁷1.

The current study by means of FISH and GISH indicates that, in the complex evolutionary system of P. autumnale, the origin of at least some SCSs can be traced back to the B⁷ genome (Figure 4). Previously, it was hypothesized that the ancestral karyotype of the complex closely resembled that of B⁷B⁷ [15,28]. From this diploid complement, both B⁶B⁶ and B⁵B⁵ cytotypes have been derived by one and two independent fusions, respectively [15]. The generation of the SCSs, so remarkably prevalent in the complex, may thus be an outcome of chromosomal rearrangements associated with the generation of new cytotypes with new base chromosome numbers and karyotype structures. At least some of these SCSs might be quite old, their origin preceding the diversification of the extant diploid cytotypes. Extensive chromosomal rearrangements in P. autumnale have also been proposed to be the most likely reason for its extraordinary variability (both structural and genomic) and the ongoing origin of B chromosomes [24]. It is possible that the putative ancestral SCS of chromosome 1 has originated from a B chromosome that translocated to chromosome 1 early in the diversification of the P. autumnale complex. Other SCSs may be of a different age and their formation might also be ongoing.

We may postulate, then, that all of the SCSs of chromosomes 1 found in P. autumnale derived from a single event, and the presence of a B⁷-like SCS on the short arm of chromosome 1 of the A-genome supports this contention. It is clear, however, that the SCSs now differ in their molecular structure between chromosomes and between cytotypes, as we have demonstrated with repetitive DNA probes. This may simply reflect the profound genomic changes that have swept through the complex since its origin [15,28]. This common descent of SCSs, however, should be further explored through GISH studies and molecular analyses of many more plants, making use of the extraordinary levels of chromosomal polymorphism found in natural populations of P. autumnale [16,20,21,23].