Summary
To aid in the identification and analysis of somatic hybrids between potato (Solanum tuberosum, dihaploid line PDH 40) and the non tuber-bearing wild species S. brevidens, a series of species-specific repetitive DNA sequences have been isolated. This was accomplished by making libraries of HaeIII-digested total DNA of S. tuberosum and S. brevidens, by cloning fragments into the SmaI site of plasmid pUC18 and transforming them into E. coli (JM83). The S. brevidens library consisted of 1,000 recombinant clones, and that of S. tuberosum, 700. Nitrocellulose filters with recombinant clones were hybridised to nick-translated total DNA of S. brevidens and also S. tuberosum, and, following autoradiography, clones that hybridised strongly to the DNA of only one of the species were chosen. Two highly repeated S. brevidens clones (pSB1, 400 bp and pSB7,210 bp), one highcopy-number s. tuberosum clone (pST10, 200 bp) and one low-copy-number sequence of S. tuberosum (pST3, 1.5 kbp) were selected for further analysis by Southern hybridisation to digested total DNA. Clone pSB7 gave a ladder pattern on hybridisation to EcoR1-digested total DNA of S. brevidens, with signals at multiples of 200 bp DNA. Using these probes it was possible to verify the hybridity of putative hybrids of dihaploid S. tuberosum and S.brevidens, and to confirm by Southern analysis and by slot blots the parental genome dosage of hexaploid hybrids (two s. brevidens: one S. tuberosum, and vice-versa). The S. tuberosum-specific probe, pSTIO, hybridised with DNA of three other tuber-bearing wild species (S. hjertingii, S. capsicibaccatum and S. berthaultii). A squash-blot procedure was developed using the probes that would allow early identification of somatic hybrid callus. There are a number of useful applications of such species-specific probes in the identification and analysis of somatic hybrids.
Similar content being viewed by others
References
Austin S, Baer MA, Helgeson JP (1985) Transfer of resistance to potato leaf roll virus from Solanum brevidens into Solanum tuberosum by somatic fusion. Plant Sci 39:75–82
Austin S, Ehlenfeldt MK, Baer MA, Helgeson JP (1986) Somatic hybrids produced by protoplast fusion between S. tuberosum and S. brevidens: phenotypic varition under field conditions. Theor Appl Genet 71:628–690
Chen BY, Heneen WK, Simonsen V (1989) Comparative and genetic studies of isozymes in resynthesised and cultivated Brassica napus L., B. campestris L. and B. alboglabra Bailey. Theor Appl Genet 77:673–679
Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci USA 81:1991–1995
Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21
Feinberg AP, Vogelstein B (1983) A technique for radiolabelling DNA restriction fragments to high specific activity. Anal Biochem 132:6–13
Fish N, Karp A, Jones MGK (1987) Improved isolation of dihaploid S. tuberosum protoplasts and the production of somatic hybrids between dihaploid S. tuberosum and S. brevidens. In Vitro 23:575–580
Fish N, Karp A, Jones MGK (1988) Production of somatic hybrids by electrofusion in Solanum. Theor Appl Genet 76:260–266
Ganal MW, Lapitan NLV, Tanksley SD (1988) A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum). Mol Gen Genet 213:262–268
Gleba Y, Kolesnik NN, Meshkene IV, Cherep NN, Parokonny AS (1984) Transmission genetics of the somatic hybridisation process in Nicotiana. Theor Appl Genet 69:121–128
Gleddie S, Keller WA, Setterfield G (1986) Production and characterisation of somatic hybrids between Solanum melongena L. and S. sisymbriifolium Lam. Theor Appl Genet 71:613–621
Handley LW, Nickels RL, Cameron MW, Moore PP, Sink KC (1986) Somatic hybrid plants between Lycopersicon esculentum and Solanum lycopersicoides. Theor Appl Genet 71:691–697
Jones MGK (1988) Fusing plant protoplasts. Trends Biotechnol 6:153–158
Junghans H, Metzlaff M (1988) Genome specific, highly repeated sequences of Hordeum vulgare: cloning, sequencing and squash dot test. Theor Appl Genet 76:728–732
Marchesi ML, Castiglione S, Sala F (1989) Effect of repeated DNA sequences on direct gene transfer in protoplasts of Nicotiana plumbaginifolia. Theor Appl Genet 78:113–118
McDonell WM, Simon MN, Studier FW (1977) Analysis of restriction fragments of T7DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol 110:119–196
Metzlaff M, Troebner W, Baldauf F, Schegel R, Cullum J (1986) Wheat-specific repetitive DNA sequences — construction and characterisation of four different genomic clones. Theor Appl Genet 72:207–210
Meyer P, Kartzke S, Niedenhof I, Heidmann I, Bussmann K, Saedler H (1988) A genomic DNA segment from Petunia hybrida leads to increased transformation frequencies and simple integration patterns. Proc Natl Acad Sci USA 85:8568–8572
Morikawa H, Kumashiro T, Kusakari K, Iida A, Hirai A, Yamada Y (1987) Interspecific hybrid plant formation by electrofusion in Nicotiana. Theor Appl Genet 75:1–4
Murashige T, Skoog A (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15:473–497
O'Connell MA, Hanson MR (1987) Construction and characterisation of somatic hybrids in Lycopersicon. Tomato Biotechnol: 71–80
Pehu E, Karp A, Moore K, Steele S, Dunckley R, Jones MGK (1989) Molecular, cytogenetic and morphological characterisation of somatic hybrids of dihaploid Solanum tuberosum and diploid S. brevidens. Theor Appl Genet 78:696–704
Pental D, Hamill JD, Pirrie A, Cocking EC (1986) Somatic hybridisation of Nicotiana tabacum and Petunia hybrida. Recovery of plants with P. hybrida nuclear genome and N. tabacum chloroplast genome. Mol Gen Genet 202:342–347
Pental D, Mukhopadhyay A, Grover A, Pradhan AK (1988) A selection method for the synthesis of triploid hybrids by fusion of microspore protoplasts (n) with somatic cell protoplasts (2n). Theor Appl Genet 76:237–243
Rosen B, Hallden C, Heneen WK (1988) Diploid Brassica napus somatic hybrids: characterisation of nuclear and organellar DNA. Theor Appl Genet 76:197–203
Saul MW, Potrykus I (1984) Species-specific repetitive DNA used to identify interspecific somatic hybrids. Plant Cell Rep 3:65–67
Schnabelrauch LS, Kloc-Bauchan F, Sink KC (1985) Expression of nuclear-cytoplasmic genomic incompatibility in interspecific Petunia somatic hybrid plants. Theor Appl Genet 70:57–65
Smith MA, Pay A, Dudits D (1989) Analysis of chloroplast and mitochondrial DNAs in asymmetric hybrids between tobacco and carrot. Theor Appl Genet 77:641–644
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–518
Vardi A, Breiman A, Galun E (1987) Citrus cybrids: production by donor-recipient protoplast-fusion and verification by mitochondrial-DNA restriction profiles. Theor Appl Genet 75:51–58
Viera J, Messing J (1982) The pUC plasmids and M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
Visser RGF, Hoekstra R, Leij FR van der, Pijnacker LP, Witholt B, Feenstra WJ (1988) In situ hybridisation to somatic metaphase chromosomes of potato. Theor Appl Genet 76:420–424
Author information
Authors and Affiliations
Additional information
Communicated by Yu. Gleba
Rights and permissions
About this article
Cite this article
Pehu, E., Thomas, M., Poutala, T. et al. Species-specific sequences in the genus Solanum: identification, characterization, and application to study somatic hybrids of S. brevidens and S. tuberosum . Theoret. Appl. Genetics 80, 693–698 (1990). https://doi.org/10.1007/BF00224231
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00224231