Plant Cell Reports
Plant Cell Reports (1996) 16:196-199
© Springer-Verlag1996
Analysis of intra-specific somatic hybrids of potato (Solanumtuberosum) using simple sequence repeats d. Provan, A. Kumar, L. Shepherd, W. Powell, and R. Waugh Department of Cell and Molecular Genetics, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK Received 28 December 1995/Revised version received 9 May 1996 - Communicated by M. R. Davey
Abstract. We have utilised simple sequence
repeat (SSR) polymorphism to analyse two sets of potential intra-specific hybrids of potato. Two primer pairs were used and both showed that one set of fusion products could not be true heterokaryons. In the other set, one of the primer pairs showed that unique bands in each of the parents were present in all of the hybrids, unambiguously demonstrating hybridity. This simple and robust, high-resolution assay can be used at the callus level and is amenable to automation, making it possible to reduce greatly the time required to screen a large number of potential somatic hybrids. Key words: potato, simple sequence repeats,
somatic hybrids Introduction
Somatic hybridisation by protoplast fusion is a well established technique for gene introgression between sexually incompatible and compatible plant species (for review see Kumar et al. 1992). An important application of somatic hybridisation has been shown in potato (Solanum tuberosum) where the resynthesis of tetraploids from two dihaploid donor parents can be achieved without meiotic recombination (Ross 1986). The major limiting factor in the exploitation of somatic hybridisation is the lack of an appropriate method for unambiguous Correspondence to." J. Provan
identification of bona-fide nuclear hybrids. Recently, Baird et al. (1992) reported the use of randomly amplified polymorphic DNA (RAPD) markers to identify both inter- and intra-specific somatic hybrids of potato. The major drawbacks of using RAPDs for such analyses include problems with reliability and reproducibility, the co-migration of heterologous bands on agarose gels giving rise to misleading results and the relatively low levels of polymorphism associated with these dominant markers. Simple sequence repeats (SSRs) have become extensively used for genetic studies in both plants and animals (Litt & Luty 1989; Morgante & Olivieri 1993; Tautz 1989). In plants, they have been used for linkage mapping (Bell & Ecker 1994), population studies (Powell et al. 1995a; Powell et aL 1995b) and genotyping (Thomas etal. 1994). The high levels of polymorphism associated with SSR loci also makes them an attractive tool for diagnostic purposes at the molecular level. Here we report the analysis of intraspecific hybrids of potato using SSR polymorphism. Materials and Methods Plant material. Two sets of intra-specific
hybrids were used in this study. The first were the products of a somatic hybridisation between S. tuberosum dihaploid clones PDH 40 and PDH 417. The second set was derived from
197
(a)
e-. t--~7:~ ~ ~ i~ I~ ¢X,~
(b)
PDH40 x PDH727 I
PDH40 x PDH417 i
t-.- r--7: ~: ~Z ~ ~
PDH40 x PDH727
PDH40 x PDH417
,,,
Figure 1. Amplificationproducts in somatic hybrids PDH 40 (+) PDH 727 and PDH 40 (+) PDH 417 using primers STPRINPSG (a) and STIIKA (b). Arrowsshow polymorphicproducts in parental plants PDH 40 and PDH 417 using the STPRINPSG primers.
PDH 40 and a second dihaploid, initially considered to be a third clone, designated PDH 727. The origin and agronomic properties of the first two dihaploids has been described by Cooper-Bland et al. (1994). Genomic DNA was isolated from fresh leaf material using the method of Edwards et al. (1991). Polymerase chain reaction. The following primers from Provan et al. (1996) were used in this study: STPRINPSGFOR 5'-TGTACTGGGGAGCCTCAAAG-3'; S T P R I N P S G R E V 5'-AATTTTAACCTCGTGACATGGG-3'; STIIKAFOR 5'-TTCGTTGCTTACCTACTA-3'; STIIKAREV 5'- CCCAAGATTACCACATTC-3'. STPRINPSG is a (TA)z~ repeat found in a proteinase inhibitor pseudogene, whilst STIIKA is a compound (T)12(A)9(TA)7 repeat from the IIK inhibitor gene intron. Annealing temperatures (T~) were 60°C for the STPRINPSG primers and 50°C for the STIIKA primers. PCR was carried out in a
total reaction volume of 10/tl containing 1 x PCR buffer (20 mM Tris-HC1 [pH 8.4], 50 mM KC1, 2.5 mM MgC12, 0.05% Wl [nonionic detergent]); 200 BM dATP/dGTP/dTI'P; 5/tM dCTP; 1 ,uCi [et-32P] dCTP (ICN); 10 pmol each primer; 0.05 U Taq DNA polymerase (Gibco BRL) and 20 ng template DNA. All reactions were carried out on a Perkin/Elmer 9600 thermal cycler using the following parameters: (i) 94°C for 3 min, [Tin] for 1 min, 72°C for 1.5 min x 1 cycle; (ii) 94°C for i min, [Tm] for I min, 72°C for 1.5 minx 29 cycles; (iii) 72°C for 5 min (for Tm values refer to Provan et al. (1996). PCR products were denatured by addition of 10/zl stop solution (95% formamide) and heating to 94°C for 2 min. Samples (2/A) were then loaded onto 6% polyacrylamide denaturing gels containing 8 M urea and separated for 3 h at 40 W constant power. Gels were wrapped in cling-film and exposed to X-ray film for 2-4 h without intensification screens.
198 Results and Discussion The parents (PDH 40, PDH 417 and PDH 727) and four of each of the hybrids (designated PDH 40 (+) PDH 727 and PDH 40 (+) PDH 417) were analysed using both single locus (STPRINPSG) and multi-locus (STIIKA) SSR polymorphism (Provan et al. 1996). Amplification products from these analyses are shown in Figure 1. From these it is apparent that the PDH 40 (+) PDH 727 fusion products do not represent somatic hybrids between the two lines. In both cases it can clearly be seen that two different genotypes are present and that samples 1 and 4 are different genotypes from samples 2 and 3. Using both primer pairs, samples 1 and 4 show the same genotype as PDH 40, whereas samples 2 and 3 have additional bands (alleles) not found in either of the parents. This demonstrates unequivocally that either the putative PDH 40 (+) PDH 727 somatic hybrids are not true heterokaryons or that the recently obtained PDH 727 material for inclusion in this study is 'different' from that used in the initial somatic hybridisation programme. The alleles observed in the PDH 40 (+) PDH 417 hybrids are consistent with the successful regeneration of true heterokaryons. In the case of the multi-locus primer (STIIKA - Figure lb), the alleles observed are identical to those from PDH 417, precluding the absolute determination of hybridity. This is, however, not the case with the STPRINPSG locus (Figure la), where each of the parents contain a single unique allele and both are present in all the hybrids. It is interesting to note that in the PDH 40 (+) PDH 417 hybridisation, although only four putative hybrids were analysed, all were shown to be nuclear hybrids. Baird et al. (1992) found that only 2 of 17 putative PDH 40 (+) PDH 727 hybrids of S. tuberosum analysed with RAPDs were actually nuclear hybrids. This may suggest that PDH 40 (+) PDH 417 heterokaryons are more likely to survive regeneration from protoplasts than those from the fusion of PDH 40 and PDH 727. Indeed, it was reported in the same
study that PDH 727 itself was difficult to regenerate. The results of the RAPD analysis suggest that the discrepancies between the PDH 40 (+) PDH 727 hybrids and the parental plants are due to lack of hybridisation rather than mutation at SSR loci during somatic organogenesis. Since the SSR assay is PCR-based, the analysis can be performed on small amounts of DNA. As with RAPDs, this means that putative hybrids can be analysed at a very early stage - indeed, it is possible to extract enough DNA from callus tissue to determine hybridity. Such PCR-based assays are amenable to automation, making it possible to screen large numbers of potential hybrids. The combination of these two factors provides an opportunity to greatly reduce the time required for the identification of true somatic hybrids. Unlike RAPDs, however, the high annealing temperatures involved in SSR-PCR means that the procedure is highly reproducible. In addition, multiple allelic forms can exist at a particular locus, increasing the probability of finding diagnostic alleles for the identification of heterokaryons. Baird et al. (1992) found that only 5 out of 36 RAPD primers detected polymorphic products unique to both parental plants. In conclusion, we have demonstrated here that SSRs provide a powerful, robust tool for the identification of intra-specific somatic hybrids in potato. Our previous work (Provan et al. 1996) has also shown that there is great potential for cross-species amplification of polymorphic SSRs within a genus. These results suggest that SSR polymorphism will also provide a means to examine somatic hybrids at both the inter- and intra-specific levels. Acknowledgements. The authors would like to
acknowledge Dr Stephanie Cooper-Bland who synthesised the somatic hybrids analysed in the communication. Louise Shepherd was funded by the European Social Fund. Jim Provan, Amar Kumar, Wayne Powell and Robbie Waugh are funded by SOAEFD.
199 References Baird E, Cooper-Bland S, Waugh R, De Maine M, Powell W (1992) Molecular and General Genetics 233:469-475 Bell CJ, Ecker JR (1994) Genomics 19: 137-144 Cooper-Bland S, De Maine MJ, Fleming MLMH, Phillips MS, Powell W, Kumar A (1994) Journal of Experimental Botany 45:1319-1325 Edwards K, Johnstone C, Thomson C (1991) Nucleic Acids Research 19:1349 Kumar A, Cooper-Bland S, Powell W (1992) In: Proceedings of the Symposium on Interactions Between Plants and Microorganisms, International Foundation for Science, Sweden, 475-490 Litt M, Luty JA (1989) Am J Hum Genet 44: 397-401
Morgante M, Olivieri AM (1993) Plant Journal 3:175-182 Powell W, Morgante M, Andre C, McNicol JW, Machray GC, Doyle JJ, Tingey SV, Rafalski JA (1995a) Current Biology 5: 1023-1029 Powell W, Morgante M, McDevitt R, Vendramin GG, Rafalski JA (1995b) Proc Natl Acad Sci USA 92:7759-7763 Provan J, Powell W, Waugh R (1996) Theoretical and Applied Genetics (in press) Ross H (1986) Potato breeding - Problems and perspectives - Advances in plant breeding 13. Suppl. J. Plant Breeding. Verlag Paul Parey, Berlin and Hamburg. Tautz, D (1989) Nucleic Acids Research 17: 6463-6471 Thomas MR, Cain P, Scott NS (1994) Plant Molecular Biology 25:939-949
Plant Cell Reports (1996) 16:196-199
© Springer-Verlag1996
Analysis of intra-specific somatic hybrids of potato (Solanumtuberosum) using simple sequence repeats d. Provan, A. Kumar, L. Shepherd, W. Powell, and R. Waugh Department of Cell and Molecular Genetics, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK Received 28 December 1995/Revised version received 9 May 1996 - Communicated by M. R. Davey
Abstract. We have utilised simple sequence
repeat (SSR) polymorphism to analyse two sets of potential intra-specific hybrids of potato. Two primer pairs were used and both showed that one set of fusion products could not be true heterokaryons. In the other set, one of the primer pairs showed that unique bands in each of the parents were present in all of the hybrids, unambiguously demonstrating hybridity. This simple and robust, high-resolution assay can be used at the callus level and is amenable to automation, making it possible to reduce greatly the time required to screen a large number of potential somatic hybrids. Key words: potato, simple sequence repeats,
somatic hybrids Introduction
Somatic hybridisation by protoplast fusion is a well established technique for gene introgression between sexually incompatible and compatible plant species (for review see Kumar et al. 1992). An important application of somatic hybridisation has been shown in potato (Solanum tuberosum) where the resynthesis of tetraploids from two dihaploid donor parents can be achieved without meiotic recombination (Ross 1986). The major limiting factor in the exploitation of somatic hybridisation is the lack of an appropriate method for unambiguous Correspondence to." J. Provan
identification of bona-fide nuclear hybrids. Recently, Baird et al. (1992) reported the use of randomly amplified polymorphic DNA (RAPD) markers to identify both inter- and intra-specific somatic hybrids of potato. The major drawbacks of using RAPDs for such analyses include problems with reliability and reproducibility, the co-migration of heterologous bands on agarose gels giving rise to misleading results and the relatively low levels of polymorphism associated with these dominant markers. Simple sequence repeats (SSRs) have become extensively used for genetic studies in both plants and animals (Litt & Luty 1989; Morgante & Olivieri 1993; Tautz 1989). In plants, they have been used for linkage mapping (Bell & Ecker 1994), population studies (Powell et al. 1995a; Powell et aL 1995b) and genotyping (Thomas etal. 1994). The high levels of polymorphism associated with SSR loci also makes them an attractive tool for diagnostic purposes at the molecular level. Here we report the analysis of intraspecific hybrids of potato using SSR polymorphism. Materials and Methods Plant material. Two sets of intra-specific
hybrids were used in this study. The first were the products of a somatic hybridisation between S. tuberosum dihaploid clones PDH 40 and PDH 417. The second set was derived from
197
(a)
e-. t--~7:~ ~ ~ i~ I~ ¢X,~
(b)
PDH40 x PDH727 I
PDH40 x PDH417 i
t-.- r--7: ~: ~Z ~ ~
PDH40 x PDH727
PDH40 x PDH417
,,,
Figure 1. Amplificationproducts in somatic hybrids PDH 40 (+) PDH 727 and PDH 40 (+) PDH 417 using primers STPRINPSG (a) and STIIKA (b). Arrowsshow polymorphicproducts in parental plants PDH 40 and PDH 417 using the STPRINPSG primers.
PDH 40 and a second dihaploid, initially considered to be a third clone, designated PDH 727. The origin and agronomic properties of the first two dihaploids has been described by Cooper-Bland et al. (1994). Genomic DNA was isolated from fresh leaf material using the method of Edwards et al. (1991). Polymerase chain reaction. The following primers from Provan et al. (1996) were used in this study: STPRINPSGFOR 5'-TGTACTGGGGAGCCTCAAAG-3'; S T P R I N P S G R E V 5'-AATTTTAACCTCGTGACATGGG-3'; STIIKAFOR 5'-TTCGTTGCTTACCTACTA-3'; STIIKAREV 5'- CCCAAGATTACCACATTC-3'. STPRINPSG is a (TA)z~ repeat found in a proteinase inhibitor pseudogene, whilst STIIKA is a compound (T)12(A)9(TA)7 repeat from the IIK inhibitor gene intron. Annealing temperatures (T~) were 60°C for the STPRINPSG primers and 50°C for the STIIKA primers. PCR was carried out in a
total reaction volume of 10/tl containing 1 x PCR buffer (20 mM Tris-HC1 [pH 8.4], 50 mM KC1, 2.5 mM MgC12, 0.05% Wl [nonionic detergent]); 200 BM dATP/dGTP/dTI'P; 5/tM dCTP; 1 ,uCi [et-32P] dCTP (ICN); 10 pmol each primer; 0.05 U Taq DNA polymerase (Gibco BRL) and 20 ng template DNA. All reactions were carried out on a Perkin/Elmer 9600 thermal cycler using the following parameters: (i) 94°C for 3 min, [Tin] for 1 min, 72°C for 1.5 min x 1 cycle; (ii) 94°C for i min, [Tm] for I min, 72°C for 1.5 minx 29 cycles; (iii) 72°C for 5 min (for Tm values refer to Provan et al. (1996). PCR products were denatured by addition of 10/zl stop solution (95% formamide) and heating to 94°C for 2 min. Samples (2/A) were then loaded onto 6% polyacrylamide denaturing gels containing 8 M urea and separated for 3 h at 40 W constant power. Gels were wrapped in cling-film and exposed to X-ray film for 2-4 h without intensification screens.
198 Results and Discussion The parents (PDH 40, PDH 417 and PDH 727) and four of each of the hybrids (designated PDH 40 (+) PDH 727 and PDH 40 (+) PDH 417) were analysed using both single locus (STPRINPSG) and multi-locus (STIIKA) SSR polymorphism (Provan et al. 1996). Amplification products from these analyses are shown in Figure 1. From these it is apparent that the PDH 40 (+) PDH 727 fusion products do not represent somatic hybrids between the two lines. In both cases it can clearly be seen that two different genotypes are present and that samples 1 and 4 are different genotypes from samples 2 and 3. Using both primer pairs, samples 1 and 4 show the same genotype as PDH 40, whereas samples 2 and 3 have additional bands (alleles) not found in either of the parents. This demonstrates unequivocally that either the putative PDH 40 (+) PDH 727 somatic hybrids are not true heterokaryons or that the recently obtained PDH 727 material for inclusion in this study is 'different' from that used in the initial somatic hybridisation programme. The alleles observed in the PDH 40 (+) PDH 417 hybrids are consistent with the successful regeneration of true heterokaryons. In the case of the multi-locus primer (STIIKA - Figure lb), the alleles observed are identical to those from PDH 417, precluding the absolute determination of hybridity. This is, however, not the case with the STPRINPSG locus (Figure la), where each of the parents contain a single unique allele and both are present in all the hybrids. It is interesting to note that in the PDH 40 (+) PDH 417 hybridisation, although only four putative hybrids were analysed, all were shown to be nuclear hybrids. Baird et al. (1992) found that only 2 of 17 putative PDH 40 (+) PDH 727 hybrids of S. tuberosum analysed with RAPDs were actually nuclear hybrids. This may suggest that PDH 40 (+) PDH 417 heterokaryons are more likely to survive regeneration from protoplasts than those from the fusion of PDH 40 and PDH 727. Indeed, it was reported in the same
study that PDH 727 itself was difficult to regenerate. The results of the RAPD analysis suggest that the discrepancies between the PDH 40 (+) PDH 727 hybrids and the parental plants are due to lack of hybridisation rather than mutation at SSR loci during somatic organogenesis. Since the SSR assay is PCR-based, the analysis can be performed on small amounts of DNA. As with RAPDs, this means that putative hybrids can be analysed at a very early stage - indeed, it is possible to extract enough DNA from callus tissue to determine hybridity. Such PCR-based assays are amenable to automation, making it possible to screen large numbers of potential hybrids. The combination of these two factors provides an opportunity to greatly reduce the time required for the identification of true somatic hybrids. Unlike RAPDs, however, the high annealing temperatures involved in SSR-PCR means that the procedure is highly reproducible. In addition, multiple allelic forms can exist at a particular locus, increasing the probability of finding diagnostic alleles for the identification of heterokaryons. Baird et al. (1992) found that only 5 out of 36 RAPD primers detected polymorphic products unique to both parental plants. In conclusion, we have demonstrated here that SSRs provide a powerful, robust tool for the identification of intra-specific somatic hybrids in potato. Our previous work (Provan et al. 1996) has also shown that there is great potential for cross-species amplification of polymorphic SSRs within a genus. These results suggest that SSR polymorphism will also provide a means to examine somatic hybrids at both the inter- and intra-specific levels. Acknowledgements. The authors would like to
acknowledge Dr Stephanie Cooper-Bland who synthesised the somatic hybrids analysed in the communication. Louise Shepherd was funded by the European Social Fund. Jim Provan, Amar Kumar, Wayne Powell and Robbie Waugh are funded by SOAEFD.
199 References Baird E, Cooper-Bland S, Waugh R, De Maine M, Powell W (1992) Molecular and General Genetics 233:469-475 Bell CJ, Ecker JR (1994) Genomics 19: 137-144 Cooper-Bland S, De Maine MJ, Fleming MLMH, Phillips MS, Powell W, Kumar A (1994) Journal of Experimental Botany 45:1319-1325 Edwards K, Johnstone C, Thomson C (1991) Nucleic Acids Research 19:1349 Kumar A, Cooper-Bland S, Powell W (1992) In: Proceedings of the Symposium on Interactions Between Plants and Microorganisms, International Foundation for Science, Sweden, 475-490 Litt M, Luty JA (1989) Am J Hum Genet 44: 397-401
Morgante M, Olivieri AM (1993) Plant Journal 3:175-182 Powell W, Morgante M, Andre C, McNicol JW, Machray GC, Doyle JJ, Tingey SV, Rafalski JA (1995a) Current Biology 5: 1023-1029 Powell W, Morgante M, McDevitt R, Vendramin GG, Rafalski JA (1995b) Proc Natl Acad Sci USA 92:7759-7763 Provan J, Powell W, Waugh R (1996) Theoretical and Applied Genetics (in press) Ross H (1986) Potato breeding - Problems and perspectives - Advances in plant breeding 13. Suppl. J. Plant Breeding. Verlag Paul Parey, Berlin and Hamburg. Tautz, D (1989) Nucleic Acids Research 17: 6463-6471 Thomas MR, Cain P, Scott NS (1994) Plant Molecular Biology 25:939-949
