Registration of S alleles in Brassica campestris L by the restriction fragment sizes of SLGs

T. Nishio, M. Kusaba, M. Watanabe, K. Hinata

Research output: Contribution to journalArticlepeer-review

65 Citations (Scopus)

Abstract

Polymorphism of SLG (the S-locus glycoprotein gene) in Brassica campestris was analyzed by PCR-RFLP using SLG-specific primers. Nucleotide sequences of PCR products from 15 S genotypes were determined in order to characterise the exact DNA fragment sizes detected in the PCR-RFLP analysis. Forty-seven lines homozygous for 27 S-alleles were used as plant material. One combination of primers, PS5 + PS15, which had a nucleotide sequence specific to a class-I SLG, gave amplification of a single DNA fragment of approximately 1.3-kb from the genomic DNA of 15 S genotypes. All the DNA fragments showed different electrophoretic profiles from each other after digestion with MboI or MspI. Different lines having the same S genotype had an identical electrophoretic profile even between the lines collected in Turkey and in Japan. Another class-I SLG-specific primer, PS18, gave amplification of a 1.3-kb DNA fragment from three other S genotypes in combination with PS15, and the PCR product also showed polymorphism after cleavage with the restriction endonucleases. Genetic analysis, Southern- hybridization analysis, and determination of the nucleotide sequences of the PCR products suggested that the DNA fragments amplified with these combinations of primers are class-I SLGs. Expected DNA fragment sizes in the present PCR-RFLP condition were calculated from the determined nucleotide sequence of SLG PCR products. A single DNA fragment was also amplified from six S genotypes by PCR with a combination of primers, PS3 + PS21, having a nucleotide sequence specific to a class-II SLG. The amplified DNA showed polymorphism after cleavage with restriction endonucleases. The cleaved fragments were detected by Southern-hybridization analysis using a probe of S5 SLG cDNA, a class-IISLG. Partial sequencing revealed a marked similarity of these amplified DNA fragments to a class-II SLG, demonstrating the presence of class-I and class-II S alleles also in B. campestris. The high SLG polymorphism detected by the present investigation suggests the usefulness of the PCR-RFLP method for the identification of S alleles in breeding lines and for listing S alleles in B. campestris.

Original languageEnglish
Pages (from-to)388-394
Number of pages7
JournalTheoretical and Applied Genetics
Volume92
Issue number3-4
DOIs
Publication statusPublished - 1996 Mar

Keywords

  • Brassica campestris
  • DNA polymorphism
  • PCR-RFLP
  • S alleles
  • SLG

ASJC Scopus subject areas

  • Biotechnology
  • Agronomy and Crop Science
  • Genetics

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