Vol 7 (1998): SI-38. Fluorescence in situ hybridization of potato somatohaploids and their somatic hybrid donors using two Solanum brevidens specific sequences Veli-Matti Rokka Agricultural Research Centre ofFinland, Plant Production Research, Crops and Soil, FIN-31600 Jokioinen, Finland, e-mail: veli-matti.rokka@mtl.fi Nora L.V. Lapitan Department ofSoil and Crop Sciences, Colorado State University, Fort Collins, CO 80523, USA Dennis L. Knudson Department ofBioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO 80523, USA Eija Pehu Department ofPlant Production, PO Box 27, FIN-00014 University ofHelsinki, Finland 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 con- taining repetitive DNA of S. brevidens both in the somatic hybrids between S. brevidens and S. tu- berosum, 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. Key words: chromosome, haploid, repetitive DNA, Solanum tuberosum, somatic hybrid, species-spe- cific sequences 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 organiza- tion of genomes in interspecific hybrids. © Agricultural and Food Science in Finland Manuscript received January 1998 31 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Rokka, V.-M. et al. Fluorescence in situ hybridization ofpotato somatohaploids 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 proto- plast fusion (Rokka et al. 1994).These interspe- cific somatic hybrids have shown androgenic ca- pacity (Rokka et al. 1995) and few somatohap- loids using anther culture (Rokka et al. 1997) have been produced from somatic hybrids. So- matohaploids 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 so- matic 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, 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, Tem- pelaaretal. 1991,Itohet al. 1991,Parokonny et al. 1992, Wolters et al. 1994, Jacobsen et al. 1995). In this paper we describe the cytological char- acterization of three somatohaploids of potato and two oftheir somatic hybrid donorplants 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 pre- viously 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. brevi- dens specific probes have also been determined for their chromosomal distribution in situ, and localized near the telomeres and in some cen- tromeric and interstitial sites of S. brevidens chromosomes, but not in S. tuberosum (Rokka et al. 1998). Material and methods Two hexaploid somatic hybrids (0502 and 0603) between S. brevidens and S. tuberosum (Rokka et al. 1994) and three of their triploid anther- derived somatohaploids (0502.1.1.1., 0507.1.2.1. and 0603.1.5.4.) (Rokka et al. 1997)were includ- ed in the study. Roots of the tissue cultured plant material (0502.1.1.1. and 0507.1.2.1.), grown on MS (Murashige and Skoog 1962) with 2% (w/v) su- crose (Merck) and 0.3 |iM (0.05 mg/1) NAA (a- naphthaleneacetic acid) (Sigma), were pretreat- ed overnight with 1.25 mM hydroxyurea (Sig- ma), at 23°C. After the pretreatment, the roots were washed three times with dH20 and trans- ferred to a Petri dish combining a wet filter pa- per. After 5-6 hours, theroot tips were collected and transferred to an icebath for 16-18 h. The tissue cultured roots of the genotypes 0502, 0603 and 0603.1.5.4. were not treated with hydroxy- urea but were directly transferred to an icebath at4°C for 18-21 h. After the icewater treatment, the root tips were fixed in ice cold methanoLglacial acetic acid (3:1). The met- aphase chromosomes were prepared as described by Rokka et al. (1998). Labelling the plasmids (pUCIB) containing the inserts of pSBI and pSB7 (Pehu et al. 1990) was done by random priming (oligolabelling). The probe pSB 1 was labelled with digoxigenin- -11 -dUTP (Boehringer Mannheim) and the probe pSB7 was labelled with biotin- 14-dATP (BRL) (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 de- scribed 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- 32 AGRICULTURAL AND FOOD SCIENCE IN FINLAND ly detected with PN buffer (0.1 M Na2 HP0 4, 0.1 M NaH,P04 , pH 8.0, 0.5% (v/v) Nonidet P-40 (Sigma)) containing 2.5 pg/ml of anti-digoxi- genin-rhodamine (Boehringer Mannheim) and 5 (lg/ml of fluorescein avidin DN (Vector Labora- tories) (Rokka et al. 1998). DAPI stained chro- mosomes and the hybridized signals were cap- tured 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 gen- otype. Results and discussion The chromosome composition of the somatic hybrids of the Solanaceae has not been exten- sively studied, because the potato chromosomes are small and morphologically similar. In the present work, two somatic hybrids and three an- ther-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 se- quences, 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 dis- tinguish S. brevidens chromosomes from the chromosomes of S. tuberosum. Under this strin- gency, pSBI and pSB7 were detected in chro- mosomal regions which are typically known to contain tandemly repeated sequences, such as telomeric areas and some centromeric and inter- stitial sites. pSB7 hybridized to all 24 chromo- somes and pSBI hybridized to 17-18 chromo- somes 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. tubero- sum (2n=2x=24) Based on counts of chromo- somes showing FISH signals, when probed with pSBI and pSB7, two thirds (= 70%) of the ge- nomes of the somatic hybrids (0502 and 0603) were derived from S. brevidens and one third (= 30%) from S. tuberosum (Table 1). This is prob- ably 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 ofS. brevidens based on FISH (fluorescence in situ hybridiza- tion) using two S. brevidens specific repetitive DNA sequences (pSB 1 and pSB7). Plant total no. of no. of chromosomes total no. of chromosomes chromosomes showing signals of: containingDNA of S. brevidens, (ploidy level) pSB 1 pSB7 both probes simultaneously' 1 Hybrids: Pito dh.45/4(+)5. brevidens 0502 68±4 (6x) 45/70 39/70 50/70 -70% Pito dh.45/4(+)S. brevidens 0603 65±5 (6x) nd.770 nd.770 nd.770 ~70%c Somatohaploids: Pito dhA5/4(+)S. brevidens 0502.1.1.1. 35±1 (3x) 18/36 23/36 29/36 -80% Pito dh.45/4(+)S. brevidens 0507.1.2.1. 33±1 (3x) 21/32 17/32 25/32 -80% Pito dh.45/4(+)S. brevidens 0603.1.5.4. 35±1 (3x) 16/33'' 26-27/33 J 26-27/33 J -80% ■ analysed from combined captured images h exact number of chromosomes containing repetitive DNA of 5. brevidens is not determined ' approximative result '' one chromosome missing from the set analysed 33 Vol. 7 (1998): 31-38. AGRICULTURAL AND FOOD SCIENCE IN FINLAND Rokka, V.-M. et al. Fluorescence in situ hybridization ofpotato somatohaploids plasts of S. brevidens with one protoplast of di- haploid S. tuberosum line (Rokka et al. 1994). This observation is interesting, especially be- cause the hybrids included in this study have shown androgenic capacity, although S. brevi- dens itself is recalcitrant in anther culture (Rok- ka 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 chromo- some numbers (Rokka et al. 1995). Secondly, chromosomal translocations within homoeolo- gous chromosomes and origin of minichromo- somes in somatic hybrids are common, as shown between Nicotiana plumbaginifolia and N. tab- acum (Piastuch and Bates 1990), N. sylvestris and N. plumbaginifolia (Parokonny et al. 1992), Lycopersicon esculentumand S. tuberosum (Wol- ters 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 chro- mosome 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 so- matic hybrids whereas Wolters et al. (1994) re- ported a random elimination of tomato chromo- somes 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 broad- er proportion of the genome in in situ hybridiza- tion 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 structuralalterations 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 (Lands- mann 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 somato- haploids (Table 1). This is possible because some S. brevidens chromosomes can pair and recom- bine 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 allotetra- ploid somatic hybrids (Wolters et al. 1994). Ex- amples of in situ hybridizations of two somato- haploids (0502.1.1.1. and 0603.1.5.4.) are pre- sented in Figures 1 and 2. In this work the potential of using species- specific tandemly repeated sequences in cytolog- ical characterization of interspecific potato hy- brids and their derivatives was shown. Using in situ hybridization with S. brevidens specific DNA clones, hexaploid somatic hybrids and so- matohaploids derived from them via anther cul- ture were shown to have the largest part of their genomes derivedfrom S. brevidens, which itself is an androgenically recalcitrant species. Acknowledgements. The authors thankThe Finnish Minis- try of Agriculture and Forestry, The Academy of Finland, and Agronomiliitto (Finnish Association of Academic Agronomists), for the financial support to Veli-Matti Rok- ka. 34 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 7 (1998): 31-38. Fig. I. Fluorescence in situ hybridization (FISH) of two Solanum brevidens specificDNA repeats (pSB I and pSB7) on to the chromosomes of the somatohaploid 0603.1.5.4. Root tips were treated for 20 h in icewater before fixation and squashing, a) Spread of the 34 chromosomes counter- stained with DAPI. b) The same spread with a S. brevidens specific probe pSB7 hybridized in situ, c) The same spread with a S. brevidens specific probe pSB 1 hybridized in situ. Visible signals are seen on 26 out of the 34 chromosomes, i.e. those chromosomes (~ 76%) contain DNA derived from S. brevidens. (Photos: Veli-Matti Rokka). 35 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Rokka , V.-M. et al. Fluorescence in situ hybridization ofpotato somatohaploids Fig. 2. Localization of two Solanum brevidens specific DNA repeats (pSBI and pSB7) on to the chromosomes of the somatohaploid 0502.1.1.1. Root tips were treated with hy- droxyurea overnight and placed in icewater for 16 h before fixation and squashing, a) The spread of the 36 chromo- somes counterstained with DAPI. b) The same spread with a 5. brevidens specific probe pSB7 hybridized in situ, c) The same spread with a S. brevidens specific probe pSBI hybridized in situ. Visible signals are seen on 29 out of the 36 chromosomes, i.e. those chromosomes (~ 78%) contain DNA derived from S. brevidens. (Photos: Veli-Matti Rok- ka), 36 AGRICULTURAL AND FOOD SCIENCE IN FINLAND References Anamthawat-Jönsson, K., Schwarzacher, T, Leitch, A.R., Bennett, M.D. & Heslop-Harrison, J.S. 1990. Discrim- ination between closely related Triticeae species using genomic DNA as a probe. Theoretical and Ap- plied Genetics 79: 721-728. 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SELOSTUS Perunan somaattisten hybridien ja niiden somatohaploidien fluoresenssi in situ -hybridisaatio Solanum brevidens -lajin spesifisten sekvenssien avulla Veli-Matti Rokka, Nora L.V. Lapitan, Dennis L. Knudson ja Eija Pehu Maatalouden tutkimuskeskus, Colorado State University ja Helsingin yliopisto Somaattisia hybridejä, jotka on tuotettu villin peru- nalajin (Solanum brevidens ) ja viljellyn perunan (S. tuberosum) soluja fuusioimalla, on käytetty pon- siviljelyssä somatohaploidien tuottamiseksi. Somato- haploidit ovat mielenkiintoisia kasvinjalostustutki- muksessa, koska niiden avulla voidaan tutkia hybri- dien ominaisuuksien periytymistä meioosin jälkeen. Lisäksi somatohaploideja voidaan hyödyntää perunan lajikejalostuksessa risteyttämällä niitä haploidien pe- runalinjojen kanssa protoplastifuusioiden avulla. Täl- löin on kyseessä suvullisen takaisinristeytyksen vaih- toehtoinen jalostusmenetelmä, ja villien perunala- jien haluttuja ominaisuuksia voidaan siirtää todennä- köisesti tehokkaammin agronomisilta ominaisuuksil- taan hyviin perunalinjoihin. Tässä työssä tutkittiin kahden S. brevidens -lajil- ta eristetyn toistuvajaksoisen DNA-koettimen (pSBI ja pSB7) avulla somaattisten hybridien ja niistä tuo- tettujen somatohaploidien kromosomistoja. Tutki- muksessa käytettiin fluoresenssi in situ -hybridisaa- liota, jonka avulla yksijuosteinen toistuvajaksoinen DNA voitiin paikallistaa denaturoituihin kromo- someihin, jotka sisälsivät S. brevidens -lajin vastaa- vaa komplementaarista DNA:ta. Fluoresenssi in situ -hybridisaatiota varten pSBI-koetin leimattiin digok- sigeniini-1 l-dUTP:IIa ja pSB7-koetin biotiini-14- dATPdIa. Kromosomeihin hybridisoituneet leimatut DNA-koettimet paikallistettiin niihin konjugoitunei- den fluoresenssiväriaineiden avulla. Molemmat koet- timet olivat peräkkäisjaksoista DNA:ta ja hybridisoi- tuivat S. brevidens -lajin kromosomien telomeerisiin päihin. Kyseisten koettimien avulla voitiin erottaa somaattisten hybridien ja niiden somatohaploidien ne kromosomit, jotka sisälsivät S. brevidens -lajin tois- tuvajaksoista DNA:ta. Fluoresenssi in situ -hybri- disaation avulla todettiin, että heksaploidit somaatti- set hybridit ja niistä tuotetut triploidit somatohap- loidit sisälsivät enemmän S. brevidens -lajin genomia kuin S. tuberosum -lajilta peräisin olevaa genomia. 38 AGRICULTURAL AND FOOD SCIENCE IN FINLAND