Plant Cell Reports
Plant Cell Reports (1992) 11: 623- 626
9 Springer-Verlag1992
Isolation and molecular characterisation of a tuberisation-related cDNA clone from potato (Solanum tuberosum L.) M.A. Taylor 1, A. Kumar 2, L.A. George 1, and H.V. Davies 1 1 Department of Cellular and Environmental Physiology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland 2 Department of Cell and Molecular Genetics, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland Received May 4, 1992/Revised version received July 23, 1992 - Communicated by K. Hahlbrock
Summary. A e D N A clone of a gene which shows a large increase in transcript level in the stolon tip during the early stages of tuberisation in potato (Solanum tuberosum) has been isolated by differential screening. This gene is also expressed at low levels in other parts of the plant including leaves and stems. Sequence analysis and comparison did not reveal any significant similarity with other gene sequences in the E M B L database. D N A - b l o t analysis indicates that the gene is present as a single copy in the potato genome and a restriction fragment length polymorphism exists between wild type and cultivated potatoes. Introduction Tuberisation is a complex process that results in the differentiation of a modified stem, the stolon, into a specialised storage organ, the tuber. Histological studies have described in detail the changes in cell type, shape, size and growth pattern that occur during the early stages of tuberisation (reviewed by Peterson et al. 1985). The molecular mechanisms that underpin tuberisation, however, remain to be elucidated. Changes in gene expression have been shown to occur during the early stages of tuberisation by analysis of protein extracts from the tuberising tip and also by analysis of the products of in vitro translation of R N A extracted from the same stages (Hannapel 1991, Hendriks et al. 1991, Taylor et al. 1991). Until recently the only changes in gene expression to be identified were dramatic increases in two of the major tuber gene products, patatin (reviewed by Park 1990) and the protease inhibitor family ( K e i l e t al. 1990). The expression of both these major polypeptides has been shown in the absence of tuberisation (Paiva et al. 1983) and conversely, down-regulation ofpatatin expression has Correspondence to: M.A. Taylor
no obvious effect on tuber morphology (Hofgen and Willmitzer 1991). Our objective was to identify some of the other changes in gene expression that occur during tuberisation particularly those related to morphogenesis or involved in the regulation of tuberisation. In this study we describe the isolation and characterisation of a cDNA clone of a novel gene that is differentially expressed during tuberisation. In addition, the use of this single copy gene as a genetic marker is described.
Materials and Methods Plant material and growth conditions. Potato plants (Solarium
atberosumL.) cv. Recordwere grownha compostin a glasshouseunder conditions described in Taylor et aL 1991. Plants were harvested between21 and 60 days afterplantingto obtainmaterialat a varietyof differentgrowthstages. RNA was extractedtrom the apical5 mm of stolontipsor the entireswollenregion/smalltuber. On plantsharvested shortly after the emergence of green shoots there was a complete absenceof swellingstolons(stage 1) and so stolontips from this stage were designatedas being non-induced. On plants harvested 28-35 days after plantingthere was a mixtureof non-swollen (stage2) and swollen (stage 3) stolon tips. Small tubers (up to 1 g fresh weight) were harvestedafter approximately42 days (stage4) and25 g tubers (stage5) were harvested at 60 days after planting. Roots, intemodal green stem segmentsand leaveswere harvestedfrom plants in which therewere no visiblesigns of tuberisation(21 days afterplanting)and from plants which had started to tuberise (42 days after planting). Source of somatic hybrid plants and their parents. Somatic hybrid
plantswere producedbetween a dihaploidline, PDH 417 and a wild typeplant,Solanum brevidens by a protoplastfusionmethod described in detail by Cooper-Bland et aL (1990). RigA isolation and blot analysis. Total RNA was isolated using
Qiagen-pack100 cartridges(QiagenInc.,StudioCity,USA)following the manufacturer'sprotocol. Poly A(+) RIgAwas preparedby affinity chromatographyusing an oligo (dr) cellulosecolumn. RNA blotting was carried out using Hybond-N (Amersham) as directed by the manufacturer. Fifteen ~tgof totalRNA was loaded per track. A probe was prepared from the EcoRI insert of the TUBIO clone by gel purificationand was labelled with [~_3Zp] dCI~ to high specific activity (typicallylx109 cpmlg) using random primers(Feinburgand
624
Fig. 1, RNA -blot analysis of TUBIO expression. (a) Hybridisation of TUBIO to 15 Ixg total RNA extracted from stolon tips from stages 1-5, lane 1-5 respectively (stages as described in materials and methods). (b) Hybridisation of 25S rRNA probe to stripped filter identical to that used in Fig. la (lanes as in la).
Fig. 2. RNA-biot analysis of TUBIO expression in root, leaf and stem. (a) Hybridisation of TUBIO to 15 Ixg total RNA extracted from root, leaf and stem (lanes 1-3 respectively) ~om plants harvested 21 days after planting (non-tuberising), and from plants harvested 42 days after planting (tuberising) (lanes 4-6 respectively). (b) Hybridisafion of 25S rRNA probe to skipped filter identical to that used in Fig. 2a (lanes as in 2a).
Vogelstein 1983). Following hybridisation (14 hours at 42~ in 50% formamide) filters were washed as described in Sambrook et aL (1989) and exposed to X-ray film at -70"C for 96 hours. The hybridisation signal was quantified by densitometry using a Quantimet 900 image analyser (Leica, Cambridge, UK). Equal loading of gels was verified by re-probing stripped filters with a 25S potato ribosomal RNA gene probe.
Sequenase version 2.0 (United States Biochemical Corporation). DNA sequence data were compiled and compared using the Genetics Computer Group programmes for the Vax (Devereux et aL 1984).
Results and Discussion
Differential gene expression during tuberisation
Library construction and differential screening. A cDNA library
was constructed from poly A(+) RNA extracted from swelling stolon tips (stage 3). cDNA was synthesised using a Pharmacia kit and was then ligated into the EcoRI site of the lambda Zap II vector (Stratagene) and packaged using an in vitro packaging kit (Amersham). The library contained approximately 1.5 x 106 pfu with a mean insert size of 1.2 kbp. Differential screening was carried out as described in Sambrook et aL (1989) using labelled probes from non-induced stolon tips (stage 1) and swelling stolon tips (stage 3). Seven recombinants that hybridised with the stage 3 probe but not the stage 1 probe were isolated from screening 40,000 initial plaques. DNA extraction and DNA blots. Plant genomic DNA was extracted from leaves as described by Draper et al. (1989), Five Fxg of DNA was digested with EcoRI and/or Hindlll, resolved by
electrophoresis on 0.8% agarose gels and transferred to Hybond-N (Amersham). The filter was hybridised with the labelled insert of pTUBIO prepared as above. Filters were washed at high stringency (Sambrook et al. 1989) and exposed to X-ray film for 48 hours at 70"C with intensifying screens. DNA sequence analysis. DNA sequence was obtained from both
strands of alkaline denatured plasmid by dideoxysequencing using
In order to isolate genes that are expressed in the early stages of tuberisation, a differential screen of a cDNA library from an early swelling stage of tuberisafion was carried out. TUBIO was one of the seven clones isolated from a screen of 40,000 plaques. RNA-blot analysis confirmed that no IUBIO specific transcript was detected in non-induced stolons from stage 1 (Fig. la, lane 1), whereas in non-sweUing stolon tips from induced plants (stage 2) there was a dramatic increase in the level of transcript (Fig. la, Iane 2). As tuber development progressed, the level of transcript declined to 11% of the maximal stage 2 level in 25 g tubers (Fig. la, lanes 3-5). Therefore the maximal level of the transcript appeared before any visible signs of tuberisation but at a stage when the plants were induced. This implies that the expression of IUBIO maybe required in order to prime the stolon tip for tuberisafion and once tuberisation is initiatedits transcript level rapidly declines. The size of the IUBIO specific transcript was approximately 800
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Fig. 3. Nucleotide and deduced amino acid sequence of the eDNA clone of TUBIO. The TAA terminationcodon is labelled *. A putative polyadenylation signal is underlined.
nucleotides by comparison with RNA size markers. Equal loading of the blot shown in Fig. la was demonstrated by re-probing the stripped filter with a 25S ribosomal RNA gene probe (Fig. lb). In order to interpret the data quantitatively the blots were scanned with a densitometer (not shown). The expression of TUBIO was also investigated in organs of the potato plant in addition to the tuberising stolon tip (Fig. 2). Prior to tuberisation the TUBIO specific transcript was not detectable in roots (Fig, 2a, lane 1) but was detectable at low levels in leaves and stems (Fig. 2a, lanes 2 and 3 respectively). Following tuberisation there was a large increase in the root transcript level (Fig. 2a, lane 4) but little change in the levels in leaves and stems (Fig. 2a, lanes 5 and 6 respectively). Figure 2b shows the stripped filter used in Fig. 2a reprobed with the 25S ribosomal RNA gene probe in order to confirm equal loading of lanes. A quantitative analysis of the blot in Fig. 2a was carried out using a densitometer (not shown). It is apparent that in addition to being induced in the stolon tip during tuberisation, TUBIO is also induced in roots. This may be a reflection of changes that occur throughout the whole plant on tuberisation although there is little change in the level in leaf and green stem. It also implies that there is a recruitment of genes that are expressed in other plant organs in the stolon tip during the early stages of tuberisation.
Sequence analysis of TUBIO The size of the insert inpTUBlO and 534 base pairs and contained an open reading frame from the nucleotide in position 2 to a TAA termination codon in position 426 (Fig. 3). The size of the TUBIO specific transcript was approximately 800 nucleotides (Fig. 2) and therefore if it is assumed that the transcript has a poly (A) tail of 200 nucleotides the clone is probably not full length but lacks approximately 100 base pairs at its 5' terminus. The ATG codon in position 6 is probably not the translation initiation point as it is not in the consensus context for initiation (Kozak 1986). The nucleotide and deduced amino acid sequences of this clone were used in a search of the EMBL database in order to identify any sequence similarities with known sequences and hence determine a role for this gene. No significant similarity was found with known sequences. Consequently, antisense experiments are being carried out in order to determine the effects of down-regulating gene expression. Use of TUBIO as a genetic marker to characterise interspecific somatic hybrid plants Genetic markers based on naturally existing polymorphisms between plants at the level of morphology, cytology, proteins and DNA have been very useful in plant genetic studies (reviewed by Paterson et a/. 1991). We have used the TUBIO clone as a genetic marker to characterise somatic hybrid plants. Somatic
626 and flower sizes and colours and tuber shape and sizes (Cooper-Bland et al. 1990). These morphological traits were found to be intermediate between the two parents. In order to conf'u'm their hybrid nature at the D N A l e v e l we used the TUBIO clone as a genetic marker. It was found that the TUBIO gene is present in a single copy in the genomes of the potato dihaploid line, PDH417 and the wild diploid Solanum brevidens, based on the D N A blot analyses using three different erm3ane combinations (EcoRI, HindlII and EcoRI/HindlII) and TUBIO as a probe (Fig. 4). Furthermore, different hybridisation fragment DNA sizes on the blot between PDH417 and S. brevidens were observed, making it an ideal genetic marker to use in the characterisation of hybrid plants between PDH417 and S. brevidens. Figure 4 shows the presence of both parental bands in the genome of a somatic hybrid plant, confirming its hybridity at the DNA level. W e are currently using the TUBIO probe as a genetic marker to characterise other sexual and somatic hybrid plants in the genus Solanum.
Acknowledganaffs. Hybridand parentalplantmaterialwere generously providedby Dr S. Cooper-Bland,Departmentof Cell and Molecular Genetics, SCRI. Figures were prepared with the help of the Visual Aids and Data Processing Departments, SCRI. We used the SERC computingfacilitiesat Daresbury. Thiswork was supportedfinancially by the Scottish Office Agriculture and Fisheries Department.
References Fig. 4.
DNA-blot analysis of genomic DNA isolated from Solanum brevidens (.panel 1), PDH417 (panel 3) and a somatic hybrid of X brevidens and PDH417 (pane 2). DNA (10 ixg per track) was digested with EcoRl (E), HindlII (H) or both (F/H). Filters were probed with the labelled insert of pTUBIO. For the reference EcoRI digest of S. brevidens DNA gave a 6 kb band in a DNA-blot.
hybridisation by protoplast fusion provides a novel means of combining genomes of two parents even if they are sexually incompatible and thereby increases the range of genetic variability available for manipulation in a plant breeding programme (Davey and K u m a r 1982). For instance we have used somatic hybridisation methodology to introduce disease resistance genes from a sexually incompatible wild type, Solanum brevidens (a non-tuberising plant which has a potato leaf roll virus resistance gene), into the potato dihaploid line, PDH417 (a potato line derived from a tetraploid potato cultivar with good tuber shape and size properties). The somatic hybrid plants between S. brevidens and PDH417 have been characterised for their hybridity based on several easily identifiable morphological markers such as leaf
Cooper-BlandS, BairdE, Kumar A, De,MaineM, PowellW (1990) Annual Report of the Scottish Crop Research Institute, Dundee, Scotland: 31-33. Davey MR, KumarA (1982) Plant Protoplasts: IntemationalReview of Cytology Supp. 16: 219-263. Academic Press, New York. DevereuxJ, HaeberliP, SmithiesO (1984) NucleicAcids Research12: 387-395. Draper I, Scott RI, Armitage P, Walden R (ed) (1989) Plant genetic transformation and gene expression: A laboratory manual. Blackwell Scientific Publications. Feinberg AP, Vogelstein B (1983) Anal. Biochem. 132: 6-13. Hannapel DJ (1991) Physiol. Plant. 83: 568-573. Hendriks T, VreugdenhilD, Stiekema I (1991) Plant Mol. Biol. 17: 385-394. Hofgen R, WillmitzerL (1991) In: 2ridInternationalPotatoMolecular Biology Symposium, St Andrews, UK. Abstracts. KeflM, Sanchez-SerannoII, SchellJ-,WillmitzerL (1990) Plant Cell 2: 61-70. Paiva E, Lister RM, Park WD (1983) Plant Physiol 71: 161-168. ParkWD (1990) In: VaydaME, Park WD (eds)Molecularand cellular biology of the potato. CAB International, Oxford, UK: 43-56. PatersonAH, TanksleySD, SorreRsME (1991) Advancesin Agronomy 46: 39-90. Peterson RL, Barker WG, Howarth MI (1985) In: Li PH (ed) Potato Physiology. Academic Press, Orlando FI: 123-152. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratorymanual(secondedition).ColdSpringHarborLaboratory, NY. TaylorMA, DaviesI-IV,ScobieLA (1991) Physiol.Plant.81: 244-269. Added in proof: Kozak M (1986) Cell 44:283-292
Plant Cell Reports (1992) 11: 623- 626
9 Springer-Verlag1992
Isolation and molecular characterisation of a tuberisation-related cDNA clone from potato (Solanum tuberosum L.) M.A. Taylor 1, A. Kumar 2, L.A. George 1, and H.V. Davies 1 1 Department of Cellular and Environmental Physiology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland 2 Department of Cell and Molecular Genetics, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland Received May 4, 1992/Revised version received July 23, 1992 - Communicated by K. Hahlbrock
Summary. A e D N A clone of a gene which shows a large increase in transcript level in the stolon tip during the early stages of tuberisation in potato (Solanum tuberosum) has been isolated by differential screening. This gene is also expressed at low levels in other parts of the plant including leaves and stems. Sequence analysis and comparison did not reveal any significant similarity with other gene sequences in the E M B L database. D N A - b l o t analysis indicates that the gene is present as a single copy in the potato genome and a restriction fragment length polymorphism exists between wild type and cultivated potatoes. Introduction Tuberisation is a complex process that results in the differentiation of a modified stem, the stolon, into a specialised storage organ, the tuber. Histological studies have described in detail the changes in cell type, shape, size and growth pattern that occur during the early stages of tuberisation (reviewed by Peterson et al. 1985). The molecular mechanisms that underpin tuberisation, however, remain to be elucidated. Changes in gene expression have been shown to occur during the early stages of tuberisation by analysis of protein extracts from the tuberising tip and also by analysis of the products of in vitro translation of R N A extracted from the same stages (Hannapel 1991, Hendriks et al. 1991, Taylor et al. 1991). Until recently the only changes in gene expression to be identified were dramatic increases in two of the major tuber gene products, patatin (reviewed by Park 1990) and the protease inhibitor family ( K e i l e t al. 1990). The expression of both these major polypeptides has been shown in the absence of tuberisation (Paiva et al. 1983) and conversely, down-regulation ofpatatin expression has Correspondence to: M.A. Taylor
no obvious effect on tuber morphology (Hofgen and Willmitzer 1991). Our objective was to identify some of the other changes in gene expression that occur during tuberisation particularly those related to morphogenesis or involved in the regulation of tuberisation. In this study we describe the isolation and characterisation of a cDNA clone of a novel gene that is differentially expressed during tuberisation. In addition, the use of this single copy gene as a genetic marker is described.
Materials and Methods Plant material and growth conditions. Potato plants (Solarium
atberosumL.) cv. Recordwere grownha compostin a glasshouseunder conditions described in Taylor et aL 1991. Plants were harvested between21 and 60 days afterplantingto obtainmaterialat a varietyof differentgrowthstages. RNA was extractedtrom the apical5 mm of stolontipsor the entireswollenregion/smalltuber. On plantsharvested shortly after the emergence of green shoots there was a complete absenceof swellingstolons(stage 1) and so stolontips from this stage were designatedas being non-induced. On plants harvested 28-35 days after plantingthere was a mixtureof non-swollen (stage2) and swollen (stage 3) stolon tips. Small tubers (up to 1 g fresh weight) were harvestedafter approximately42 days (stage4) and25 g tubers (stage5) were harvested at 60 days after planting. Roots, intemodal green stem segmentsand leaveswere harvestedfrom plants in which therewere no visiblesigns of tuberisation(21 days afterplanting)and from plants which had started to tuberise (42 days after planting). Source of somatic hybrid plants and their parents. Somatic hybrid
plantswere producedbetween a dihaploidline, PDH 417 and a wild typeplant,Solanum brevidens by a protoplastfusionmethod described in detail by Cooper-Bland et aL (1990). RigA isolation and blot analysis. Total RNA was isolated using
Qiagen-pack100 cartridges(QiagenInc.,StudioCity,USA)following the manufacturer'sprotocol. Poly A(+) RIgAwas preparedby affinity chromatographyusing an oligo (dr) cellulosecolumn. RNA blotting was carried out using Hybond-N (Amersham) as directed by the manufacturer. Fifteen ~tgof totalRNA was loaded per track. A probe was prepared from the EcoRI insert of the TUBIO clone by gel purificationand was labelled with [~_3Zp] dCI~ to high specific activity (typicallylx109 cpmlg) using random primers(Feinburgand
624
Fig. 1, RNA -blot analysis of TUBIO expression. (a) Hybridisation of TUBIO to 15 Ixg total RNA extracted from stolon tips from stages 1-5, lane 1-5 respectively (stages as described in materials and methods). (b) Hybridisation of 25S rRNA probe to stripped filter identical to that used in Fig. la (lanes as in la).
Fig. 2. RNA-biot analysis of TUBIO expression in root, leaf and stem. (a) Hybridisation of TUBIO to 15 Ixg total RNA extracted from root, leaf and stem (lanes 1-3 respectively) ~om plants harvested 21 days after planting (non-tuberising), and from plants harvested 42 days after planting (tuberising) (lanes 4-6 respectively). (b) Hybridisafion of 25S rRNA probe to skipped filter identical to that used in Fig. 2a (lanes as in 2a).
Vogelstein 1983). Following hybridisation (14 hours at 42~ in 50% formamide) filters were washed as described in Sambrook et aL (1989) and exposed to X-ray film at -70"C for 96 hours. The hybridisation signal was quantified by densitometry using a Quantimet 900 image analyser (Leica, Cambridge, UK). Equal loading of gels was verified by re-probing stripped filters with a 25S potato ribosomal RNA gene probe.
Sequenase version 2.0 (United States Biochemical Corporation). DNA sequence data were compiled and compared using the Genetics Computer Group programmes for the Vax (Devereux et aL 1984).
Results and Discussion
Differential gene expression during tuberisation
Library construction and differential screening. A cDNA library
was constructed from poly A(+) RNA extracted from swelling stolon tips (stage 3). cDNA was synthesised using a Pharmacia kit and was then ligated into the EcoRI site of the lambda Zap II vector (Stratagene) and packaged using an in vitro packaging kit (Amersham). The library contained approximately 1.5 x 106 pfu with a mean insert size of 1.2 kbp. Differential screening was carried out as described in Sambrook et aL (1989) using labelled probes from non-induced stolon tips (stage 1) and swelling stolon tips (stage 3). Seven recombinants that hybridised with the stage 3 probe but not the stage 1 probe were isolated from screening 40,000 initial plaques. DNA extraction and DNA blots. Plant genomic DNA was extracted from leaves as described by Draper et al. (1989), Five Fxg of DNA was digested with EcoRI and/or Hindlll, resolved by
electrophoresis on 0.8% agarose gels and transferred to Hybond-N (Amersham). The filter was hybridised with the labelled insert of pTUBIO prepared as above. Filters were washed at high stringency (Sambrook et al. 1989) and exposed to X-ray film for 48 hours at 70"C with intensifying screens. DNA sequence analysis. DNA sequence was obtained from both
strands of alkaline denatured plasmid by dideoxysequencing using
In order to isolate genes that are expressed in the early stages of tuberisation, a differential screen of a cDNA library from an early swelling stage of tuberisafion was carried out. TUBIO was one of the seven clones isolated from a screen of 40,000 plaques. RNA-blot analysis confirmed that no IUBIO specific transcript was detected in non-induced stolons from stage 1 (Fig. la, lane 1), whereas in non-sweUing stolon tips from induced plants (stage 2) there was a dramatic increase in the level of transcript (Fig. la, Iane 2). As tuber development progressed, the level of transcript declined to 11% of the maximal stage 2 level in 25 g tubers (Fig. la, lanes 3-5). Therefore the maximal level of the transcript appeared before any visible signs of tuberisation but at a stage when the plants were induced. This implies that the expression of IUBIO maybe required in order to prime the stolon tip for tuberisafion and once tuberisation is initiatedits transcript level rapidly declines. The size of the IUBIO specific transcript was approximately 800
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Fig. 3. Nucleotide and deduced amino acid sequence of the eDNA clone of TUBIO. The TAA terminationcodon is labelled *. A putative polyadenylation signal is underlined.
nucleotides by comparison with RNA size markers. Equal loading of the blot shown in Fig. la was demonstrated by re-probing the stripped filter with a 25S ribosomal RNA gene probe (Fig. lb). In order to interpret the data quantitatively the blots were scanned with a densitometer (not shown). The expression of TUBIO was also investigated in organs of the potato plant in addition to the tuberising stolon tip (Fig. 2). Prior to tuberisation the TUBIO specific transcript was not detectable in roots (Fig, 2a, lane 1) but was detectable at low levels in leaves and stems (Fig. 2a, lanes 2 and 3 respectively). Following tuberisation there was a large increase in the root transcript level (Fig. 2a, lane 4) but little change in the levels in leaves and stems (Fig. 2a, lanes 5 and 6 respectively). Figure 2b shows the stripped filter used in Fig. 2a reprobed with the 25S ribosomal RNA gene probe in order to confirm equal loading of lanes. A quantitative analysis of the blot in Fig. 2a was carried out using a densitometer (not shown). It is apparent that in addition to being induced in the stolon tip during tuberisation, TUBIO is also induced in roots. This may be a reflection of changes that occur throughout the whole plant on tuberisation although there is little change in the level in leaf and green stem. It also implies that there is a recruitment of genes that are expressed in other plant organs in the stolon tip during the early stages of tuberisation.
Sequence analysis of TUBIO The size of the insert inpTUBlO and 534 base pairs and contained an open reading frame from the nucleotide in position 2 to a TAA termination codon in position 426 (Fig. 3). The size of the TUBIO specific transcript was approximately 800 nucleotides (Fig. 2) and therefore if it is assumed that the transcript has a poly (A) tail of 200 nucleotides the clone is probably not full length but lacks approximately 100 base pairs at its 5' terminus. The ATG codon in position 6 is probably not the translation initiation point as it is not in the consensus context for initiation (Kozak 1986). The nucleotide and deduced amino acid sequences of this clone were used in a search of the EMBL database in order to identify any sequence similarities with known sequences and hence determine a role for this gene. No significant similarity was found with known sequences. Consequently, antisense experiments are being carried out in order to determine the effects of down-regulating gene expression. Use of TUBIO as a genetic marker to characterise interspecific somatic hybrid plants Genetic markers based on naturally existing polymorphisms between plants at the level of morphology, cytology, proteins and DNA have been very useful in plant genetic studies (reviewed by Paterson et a/. 1991). We have used the TUBIO clone as a genetic marker to characterise somatic hybrid plants. Somatic
626 and flower sizes and colours and tuber shape and sizes (Cooper-Bland et al. 1990). These morphological traits were found to be intermediate between the two parents. In order to conf'u'm their hybrid nature at the D N A l e v e l we used the TUBIO clone as a genetic marker. It was found that the TUBIO gene is present in a single copy in the genomes of the potato dihaploid line, PDH417 and the wild diploid Solanum brevidens, based on the D N A blot analyses using three different erm3ane combinations (EcoRI, HindlII and EcoRI/HindlII) and TUBIO as a probe (Fig. 4). Furthermore, different hybridisation fragment DNA sizes on the blot between PDH417 and S. brevidens were observed, making it an ideal genetic marker to use in the characterisation of hybrid plants between PDH417 and S. brevidens. Figure 4 shows the presence of both parental bands in the genome of a somatic hybrid plant, confirming its hybridity at the DNA level. W e are currently using the TUBIO probe as a genetic marker to characterise other sexual and somatic hybrid plants in the genus Solanum.
Acknowledganaffs. Hybridand parentalplantmaterialwere generously providedby Dr S. Cooper-Bland,Departmentof Cell and Molecular Genetics, SCRI. Figures were prepared with the help of the Visual Aids and Data Processing Departments, SCRI. We used the SERC computingfacilitiesat Daresbury. Thiswork was supportedfinancially by the Scottish Office Agriculture and Fisheries Department.
References Fig. 4.
DNA-blot analysis of genomic DNA isolated from Solanum brevidens (.panel 1), PDH417 (panel 3) and a somatic hybrid of X brevidens and PDH417 (pane 2). DNA (10 ixg per track) was digested with EcoRl (E), HindlII (H) or both (F/H). Filters were probed with the labelled insert of pTUBIO. For the reference EcoRI digest of S. brevidens DNA gave a 6 kb band in a DNA-blot.
hybridisation by protoplast fusion provides a novel means of combining genomes of two parents even if they are sexually incompatible and thereby increases the range of genetic variability available for manipulation in a plant breeding programme (Davey and K u m a r 1982). For instance we have used somatic hybridisation methodology to introduce disease resistance genes from a sexually incompatible wild type, Solanum brevidens (a non-tuberising plant which has a potato leaf roll virus resistance gene), into the potato dihaploid line, PDH417 (a potato line derived from a tetraploid potato cultivar with good tuber shape and size properties). The somatic hybrid plants between S. brevidens and PDH417 have been characterised for their hybridity based on several easily identifiable morphological markers such as leaf
Cooper-BlandS, BairdE, Kumar A, De,MaineM, PowellW (1990) Annual Report of the Scottish Crop Research Institute, Dundee, Scotland: 31-33. Davey MR, KumarA (1982) Plant Protoplasts: IntemationalReview of Cytology Supp. 16: 219-263. Academic Press, New York. DevereuxJ, HaeberliP, SmithiesO (1984) NucleicAcids Research12: 387-395. Draper I, Scott RI, Armitage P, Walden R (ed) (1989) Plant genetic transformation and gene expression: A laboratory manual. Blackwell Scientific Publications. Feinberg AP, Vogelstein B (1983) Anal. Biochem. 132: 6-13. Hannapel DJ (1991) Physiol. Plant. 83: 568-573. Hendriks T, VreugdenhilD, Stiekema I (1991) Plant Mol. Biol. 17: 385-394. Hofgen R, WillmitzerL (1991) In: 2ridInternationalPotatoMolecular Biology Symposium, St Andrews, UK. Abstracts. KeflM, Sanchez-SerannoII, SchellJ-,WillmitzerL (1990) Plant Cell 2: 61-70. Paiva E, Lister RM, Park WD (1983) Plant Physiol 71: 161-168. ParkWD (1990) In: VaydaME, Park WD (eds)Molecularand cellular biology of the potato. CAB International, Oxford, UK: 43-56. PatersonAH, TanksleySD, SorreRsME (1991) Advancesin Agronomy 46: 39-90. Peterson RL, Barker WG, Howarth MI (1985) In: Li PH (ed) Potato Physiology. Academic Press, Orlando FI: 123-152. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratorymanual(secondedition).ColdSpringHarborLaboratory, NY. TaylorMA, DaviesI-IV,ScobieLA (1991) Physiol.Plant.81: 244-269. Added in proof: Kozak M (1986) Cell 44:283-292
