Summary
The development of a technique for the identification of S alleles involved in self-incompatibility in Brassica oleracea which is based on the polymerase chain reaction (PCR) amplification of genomic DNA followed by restriction analysis is described. Primers homologous to conserved regions near to the 5′ and 3′ ends of the S coding sequence were used to amplify a number of members of the S multigene family. However, by designing a selective primer and using a higher temperature for annealing in the PCR, we were able to amplify certain members from the multigene family preferentially. These were considered to be the S-locus glycoprotein genes (SLG), since the patterns of restriction bands of the PCR products were shown to correspond to those of the SLG where sequence data were available. DNA samples from plants with certain S alleles were found not to amplify efficiently using the “selective” primers and high annealing temperature. This property, however, could be used as a means of distinguishing plants homozygous for these S alleles, as was demonstrated by an examination of a small F2 population that was segregating for the S5 and S29 alleles. In the investigation of the F2 population, it was found that preferential amplification of one of the alleles of the heterozygotes occurred when the “consensus” primers were used in the PCR. However, by using different primers, homologous to another region of the S sequence, we were able to amplify both alleles of the heterozygotes equally. The genotypes of the plants were determined by restriction analysis of PCR products and agreed with results based on pollen-tube growth tests.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21
Deveux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the Vax. Nucleic Acids Res 12:387–395
Feinberg AP, Vogelstein B (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6
Feinberg AP, Vogelstein B (1984) Addendum: A technique for radiolabelling DNA restriction c endonuclease fragments to high specific activity. Anal Biochem 137:266
Guilluy C-M, Trick M, Heizmann P, Dumas C (1991) PCR detection of transcripts homologous to the self-incompatibility gene in anthers of Brassica. Theor Appl Genet 82:466–472
Maeda M, Uryu N, Murayama N, Ishii H, Ota M, Tsuji K, Inoko H (1990) A simple and rapid method for HLA-DP genotyping by digestion of PCR-amplified DNA with allele specific restriction endonucleases. Hum Immunol 27:111–121
Nasrallah JB, Kao T-H, Goldberg ML, Nasrallah ME (1985) A cDNA clone encoding an S-locus-specific glycoprotein from Brassica oleracea. Nature 318:263–267
Nasrallah JB, Yu S-M, Nasrallah ME (1988) Self-incompatibility genes of Brassica oleracea: expression, isolation and structure. Proc Natl Acad Sci USA 85:5551–5555
Nasrallah JB, Nishio T, Nasrallah ME (1991) The self-incompatibility genes of Brassica: Expression and use in genetic ablation of floral tissues. Annu Rev Plant Physiol 42:393–422
Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF (1989) Analysis of any point mutation in DNA. The amplification refactory mutations system (ARMS). Nucleic Acids Res 17:2503–2516
Ockendon DJ (1974) Distribution of self-incompatibility alleles and breeding structure of open-pollinated cultivars of Brussels sprouts. Heredity 33:159–171
Olerup O (1990) HLA class II typing by digestion of PCR-amplified DNA with allele-specific restriction endonucleases will fail to unequivocally identify the genotypes of many homozygous and heterozygous individuals. Tissue Antigens 36:83–87
Sarkar G, Kapelner S, Sommer SS (1990) Formamide can dramatically improve the specificity of PCR. Nucleic Acids Res 18:7465
Scutt CP, Croy RRD (1992) An S5 self-incompatibility allele-specific cDNA sequence from Brassica oleracea shows high homology to the SLR2 gene. Mol Gen Genet 232:240–246
Scutt CP, Gates PJ, Gatehouse JA, Boulter D, Croy RRD (1990) A cDNA encoding an S-locus-specific glycoprotein from Brassica oleracea plants containing the S5 self-incompatibility allele. Mol Gen Genet 220:409–413
Stein JC, Howelett B, Boyes DC, Nasrallah ME, Nasrallah JB (1991) Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea. Proc Natl Acad Sci USA 88:8816–8820
Trick M, Flavell RB (1989) A homozygous S genotype of Brassica oleracea expresses two S-like genes. Mol Gen Genet 218:112–117
Trick M, Heizmann P (1992) Sporophytic self-incompatibility systems: Brassica S-gene family. Int Rev Cytol 140:485–524
Uryu N, Maeda M, Ota M, Tsuji K, Inoko H (1990) A simple and rapid method for HLA-DRB and -DQB typing by digestion of PCR-amplified DNA with allele specific restriction endonucleases. Tissue Antigens 35:20–31
Walsh PS, Erlich HA, Higuchi R (1992) Preferential PCR amplification of alleles: mechanisms and solutions. PCR Methods Applicat 1:241–250
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Brace, J., Ockendon, D.J. & King, G.J. Development of a method for the identification of S alleles in Brassica oleracea based on digestion of PCR-amplified DNA with restriction endonucleases. Sexual Plant Reprod 6, 133–138 (1993). https://doi.org/10.1007/BF00227658
Issue Date:
DOI: https://doi.org/10.1007/BF00227658