Fluorescence in situ hybridization of potato somatohaploids and their somatic hybrid donors using two Solanum brevidens specific sequences

Two Solanum brevidens specific repetitive DNA clones (pSBI and pSB7) were used simultaneously as probes in fluorescence in situ hybridization (FISH) for cytological studies of somatohaploids and their somatic hybrid donors. pSBI was labelled with digoxigenin-11-dUTP and pSB7 was labelled with biotin-14-dATP and they were detected with reporter molecules conjugated to fluorescent dyes using digital imaging. The tandemly repeated sequences hybridized mostly near the telomeres of the chromosomes of S. brevidens. Using these two probes, it was possible to identify chromosomes containing repetitive DNA of S. brevidens both in the somatic hybrids between S. brevidens and S. tuberosum, and somatohaploids derived from the somatic hybrids. These cytological analyses showed that for the largest part genomes of the hexaploid somatic hybrids and their anther-derived triploid somatohaploids were composed of the genome of S. brevidens.

ntroduction Fluorescence in situ hybridization (FISH) is an important method in chromosome identification and physical mapping. Simultaneous localization of two or more probes (Leitch et al. 1991) or multiple colour FISH (Mukai and Nakahara 1993) and genomic in situ hybridization (GISH) (Le et al. 1989, Schwarzacher et al. 1989 can be used in cytological analyses of the organization of genomes in interspecific hybrids. We have previously been able to combine the genome ofSolanum brevidens, which has a broad virus resistance (Valkonen et al. 1992), with S. tuberosum, cultivated potato, through protoplast fusion (Rokka et al. 1994).These interspecific somatic hybrids have shown androgenic capacity (Rokka et al. 1995) and few somatohaploids using anther culture (Rokka et al. 1997) have been produced from somatic hybrids. Somatohaploids can be used in potato breeding as a step to combine valuable characters of wild species with good agronomic traits of cultivated potato. Somatohaploids also provide interesting material for genetic studies of interspecific somatic hybrids.
There have been few reports on cytogenetic analysis of tissue culture derived hybrids using in situ hybridization. Quantitative changes of repeated sequences, translocations and deletions in tissue culture regenerated sexual hybrids of wheat and rye have been documented (Lapitan et al. 1986(Lapitan et al. , 1988. However, there have been only a few reports using species-specific repetitive DNA probes or total genomic DNA for in situ hybridization to analyse somatic hybrids or their derivatives (e.g. Piastuch and Bates 1990, Tempelaaretal. 1991,Itohet al. 1991, Parokonny et al. 1992, Wolters et al. 1994, Jacobsen et al. 1995. In this paper we describe the cytological characterization of three somatohaploids of potato and two of their somatic hybrid donor plants with FISH using two species-specific DNA repeats, pSB 1 and pSB7, isolated from S. brevidens (Pehu et al. 1990) as probes. Both of these DNA clones contain highly repeated sequences and have previously been used as species-specific probes in dot blot analysis for the verification of somatic hybridity (Pehu et al. 1990) and in a survey of DNA sequence similarities between Solanum species (Malkamäki et al. 1996). The S. brevidens specific probes have also been determined for their chromosomal distribution in situ, and localized near the telomeres and in some centromeric and interstitial sites of S. brevidens chromosomes, but not in S. tuberosum (Rokka et al. 1998).
The probe mixture preparation and in situ hybridization were carried out as described by Rokka et al. (1998). The slides were incubated before the chromosome denaturation step in a solution of pepsin and rinsed in dH,O as described by Brown (1995). For hybridization the slides were incubated in a humidity chamber at 37°C for 15-16 hours. After hybridization, the slides were washed in 40% (v/v) formamide in 2xSSC at 42°C for 10 min, IxSSC at 37°C for 10min and O.IxSSC at room temperature for 10 min. The hybridized signals were simultaneous-ly detected with PN buffer (0.1 M Na 2 HP0 4 , 0.1 M NaH,P0 4 , pH 8.0, 0.5% (v/v) Nonidet P-40 (Sigma)) containing 2.5 pg/ml of anti-digoxigenin-rhodamine (Boehringer Mannheim) and 5 (lg/ml of fluorescein avidin DN (Vector Laboratories) (Rokka et al. 1998). DAPI stained chromosomes and the hybridized signals were captured with a lOOx Zeiss objective using a cooled array CCD (charge-coupled device) collector and digital imaging (Rokka et al. 1998). From one to three chromosome sets were analysed per genotype.

Results and discussion
The chromosome composition of the somatic hybrids of the Solanaceae has not been extensively studied, because the potato chromosomes are small and morphologically similar. In the present work, two somatic hybrids and three anther-derived somatohaploids of S. brevidens and S. tuberosum were cytologically characterized using two S. brevidens specific repetitive DNA sequences simultaneously as probes for in situ hybridization.
The two S. brevidens specific repeated sequences, pSBI and pSB7, were previously shown to hybridize in situ to S. brevidens chromosomes, but not to chromosomes of S. tuberosum (Rokka et al. 1998). Using highly stringent washes (40% formamide), which allowed approximately 20% nucleotide mismatches, it was possible to distinguish S. brevidens chromosomes from the chromosomes of S. tuberosum. Under this stringency, pSBI and pSB7 were detected in chromosomal regions which are typically known to contain tandemly repeated sequences, such as telomeric areas and some centromeric and interstitial sites. pSB7 hybridized to all 24 chromosomes and pSBI hybridized to 17-18 chromosomes of S. brevidens (Rokka et al. 1998).
The somatohaploids, which were previously described (Rokka et al. 1997), are triploids (2n=3x=32-36) derived from hexaploid (2n=6x=6o-71) somatic hybrids between diploid S. brevidens (2n=2x=24) and dihaploid S. tuberosum (2n=2x=24) Based on counts of chromosomes showing FISH signals, when probed with pSBI and pSB7, two thirds (= 70%) of the genomes of the somatic hybrids (0502 and 0603) were derived from S. brevidens and one third (= 30%) from S. tuberosum (Table 1). This is probably a result of the electrofusion of two proto- Table I. Chromosome numbers of the somatic hybrids and somatohaploids between Solanum brevidens and Solanum tuberosum and the number of chromosomes containing DNA of S. brevidens based on FISH (fluorescence in situ hybridization) using two S. brevidens specific repetitive DNA sequences (pSB 1 and pSB7).  (Rokka et al. 1994). This observation is interesting, especially because the hybrids included in this study have shown androgenic capacity, although S. brevidens itself is recalcitrant in anther culture (Rokka et al. 1995). The exact determination of the number of donor species derived chromosomes in somatic hybrids was difficult, because the hybrids showed intragenomic variation in their chromosome numbers (Rokka et al. 1995). Secondly, chromosomal translocations within homoeologous chromosomes and origin of minichromosomes in somatic hybrids are common, as shown between Nicotiana plumbaginifolia and N. tabacum (Piastuch and Bates 1990), N. sylvestris and N. plumbaginifolia (Parokonny et al. 1992), Lycopersicon esculentum and S. tuberosum (Wolters et al. 1994). In the genomes of our somatic hybrids there were fewer than 24 chromosomes which were derived from S. tuberosum. Because of the translocations some chromosomes which may only have one arm of a S. brevidens chromosome may be distinguished as a chromosome of S. brevidens, when these two species-specific sequences are used in situ. Pijnacker et al. (1989) reported preferential elimination of S. phureja chromosomes in S. phureja (+) S. tuberosum somatic hybrids whereas Wolters et al. (1994) reported a random elimination of tomato chromosomes in potato (+) tomato somatic hybrids. In our hybrids, elimination of some S. tuberosum chromosomes and intergenomic chromosomal rearrangements may have occured, but the exact identification of donor genomes could be more accurately determined using GISH as described for tobacco hybrids by Parokonny et al. (1992) and for potato hybrids by Wolters et al. (1994). In GISH, labelled total DNA from one species is blocked with unlabelled DNA from the other species and used as a probe representing a broader proportion of the genome in in situ hybridization than cloned isolated probes of repetitive sequences (Anamthawat-Jonsson et al. 1990, Itoh et al. 1991. Rokka et al. (1995) found variation in the genome sizes of the somatic hybrids between S. brevidens and S. tuberosum. These aberrations may not only be due to changes in chromosome numbers, but also due to structural alterations in the karyotypes and differences in the replication of particular (mostly repetitive) DNA families.
Quantitative changes in repetitive sequences have been found in protoclones ofpotato (Landsmann and Uhrig 1985). These changes together with occasional translocations complicate the cytological analysis of the tissue culture derived regenerants.
Solanum brevidens specific DNA was present in 80% of the chromosomes of all the somatohaploids (Table 1). This is possible because some S. brevidens chromosomes can pair and recombine with S. tuberosum chromosomes as earlier reported by Williams et al. (1993) and McGrath et al. (1996). In meiosis, homoeologous pairing and crossing overs between tomato and potato chromosomes have also been shown in allotetraploid somatic hybrids (Wolters et al. 1994). Examples of in situ hybridizations of two somatohaploids (0502.1.1.1. and 0603.1.5.4.) are presented in Figures 1 and 2.